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Abstract

Background

This study investigates the therapeutic potential of a combined dose of cannabidiol (CBD) and tetrahydrocannabinol (THC) at a 100:1 ratio (100 mg/kg CBD and 1 mg/kg THC) in mitigating central calcitonin gene-related peptide (CGRP)-induced migraine symptoms in a mouse model.

Methods

The 100:1 ratio of CBD to THC was administered intraperitoneally, 60 minutes prior to starting all the assays, followed by intracerebroventricular CGRP administration, 30 minutes later, with behavior assays conducted 30 minutes after CGRP injection. To determine whether pretreatment of CBD:THC could counteract CGRP-induced light aversion, we utilized the light/dark assay, which also recorded motility behavior. To investigate whether CBD:THC pretreatment could alleviate CGRP-induced spontaneous pain, we used the automated squint assay.

Results

Our findings show that pretreatment with 100:1 CBD:THC rescued light aversion caused by centrally administered CGRP in CD1 mice. Additionally, CBD:THC pretreatment rescued the increased resting time in darkness, decreased transitions between light and dark zones, and partially rescued the decreased rearing behavior induced by centrally administered CGRP. Moreover, an open field assay confirmed that centrally administered CGRP did not induce anxiety in a light independent assay. Finally, our findings from the automated squint assay indicate that pretreatment with 100:1 CBD:THC partially rescued centrally administered CGRP-induced spontaneous pain.

Conclusions

Collectively, these results demonstrate that a combination of CBD and THC can alleviate light aversion and pain symptoms induced by a centrally-acting migraine trigger.

Graphical abstract

This is a visual representation of the abstract.

Keywords

cannabidiol cannabinoids headache light sensitivity migraine pain squint tetrahydrocannabinol

Introduction

Migraine is a complex neurological disease characterized by reoccurring headache and other debilitating symptoms such as photophobia and pain.¹ According to the World Health Organization, migraine affects around 15% of the global population and women are up to three times more likely to be affected.² Indeed, around one billion people worldwide are affected making migraine the leading cause of disability in individuals less than 50 years old, significantly affecting work productivity and overall quality of life.³ The current treatment options available for migraine includes drugs such as triptans and ditans, which act on serotonin (5-HT_{1B, 1D, 1F}) receptors.^4–6 More recently, medications targeting the calcitonin gene-related peptide (CGRP) pathway have emerged, including small-molecule antagonists known as gepants, and monoclonal antibodies. While these medications are effective, it is estimated that only 50% of migraine patients respond adequately to these medications.^7,8 This highlights an urgent need for research into novel therapeutics such as cannabinoids, aiming to address the unmet needs of patients who do not respond well to current treatment options.

Pre-clinical studies have shown that intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) CGRP administration can cause migraine-like symptoms such as light aversion, a surrogate for photophobia in humans, and pain in mice.^9–13 Similarly, clinical studies have revealed that CGRP levels are elevated in migraine patients^14–19 and that injection of CGRP is sufficient to cause migraine-like headaches.²⁰ Additionally, neuroimaging studies have revealed distinct brain activity patterns across different migraine phases,²¹ highlighting the complexity of migraine pathogenesis.

The likely involvement of both peripheral and central mechanisms in migraine pathophysiology presents a challenge in developing therapeutics that effectively target both the central nervous system (CNS) and peripheral nervous system (PNS) without causing significant adverse effects. For example, CGRP monoclonal antibodies are primarily limited to the PNS due to their large molecular size, which restricts their ability to cross the blood–brain barrier (BBB) and act on CNS targets.²² Despite being 1500 times smaller than antibodies, gepants also have limited ability to cross the BBB.^22,23 Ditans such as lasmiditan can cross the BBB. However, there are some adverse side effects because some patients report dizziness up to 8 h after administration.⁴ Similarly, due to vasoconstrictive properties, there is concern with use of triptans in patients with or at high risk for cardiovascular disease.²⁴

The medicinal properties of the plant Cannabis can be traced back thousands of years, with some of its earliest records dating to Chinese medicine around 2700 BC,^25,26 and Indian medicine around 900 BC.²⁷ During the late 19th to early 20th centuries, many Western physicians considered Cannabis to be the “most satisfactory” remedy for migraine,²⁸ and, for eight decades, it was the primary treatment for migraine in mainstream Western medical literature.²⁹ Despite its prevalence, the use of medicinal cannabis declined in approval and was later banned by the Controlled Substances Act in 1970.^30,31 Recent findings are now supporting the use of cannabis as a migraine treatment. A retrospective chart review revealed that migraine patients who consumed cannabis reported a decrease in headache frequency.³² An additional study showed that cannabis resulted in long-term reduction in migraine frequency with lower antimigraine medication usage.³³ Several cannabinoid drugs, such as nabilone,³⁴ Sativex³⁵ and Epidiolex³⁶, have been approved for human use, although, to date, none are specific for treating migraine. In addition to phytocannabinoids, such as cannabidiol (CBD) and tetrahydrocannabinol (THC), there are also endogenous cannabinoids (endocannabinoids)^,³⁷ that comprise the complex endogenous endocannabinoid system (ECS).³⁸ Clinical observations led to the clinical endocannabinoid deficiency theory, which proposes that dysfunction in the ECS contributes to neurological disorders, including migraine.^32,39,40 Supporting this idea, a clinical study found that chronic migraine patients exhibited increased CGRP levels and decreased anandamide (AEA) levels in their cerebrospinal fluid.⁴¹ Another study found that female migraine patients had higher levels of fatty acid amide hydrolase, the enzyme responsible for degrading AEA in their blood.⁴² Together, this suggests that ECS dysfunction is present in migraine pathology, supporting further research into cannabis-based therapies.

A hallmark of the present study is that we have used central administration of CGRP to trigger migraine-like symptoms in mice. This builds on our recent report that cannabinoids could rescue symptoms following peripheral administration of CGRP.⁴³ In the peripheral CGRP study, a 100:1 CBD:THC combination, but not either CBD or THC on their own, rescued light aversion and pain behaviors. This combination also rescued light aversion and pain caused by sodium nitroprusside, a nitric oxide donor, and rescued a pain response triggered by optogenetic induction of cortical spreading depression (CSD).⁴³ Light aversion was not measured after CSD induction in that previous study. Consequently, we chose to focus on light aversion and pain in this study following a central CGRP trigger. Importantly, the dose of 100:1 CBD:THC produced no adverse effects in various behavior assays assessing memory, anxiety, motor function and depression-like symptoms.⁴³ The present study aimed to investigate whether a combination of CBD and THC at a 100:1 ratio could mitigate centrally induced CGRP effects in a migraine mouse model.

Methods

Animals

Male and female wild-type CD1 (Charles River, USA) mice were used. Mice were 8–9 weeks of age upon arrival and allowed to acclimate for a week before use. Mice were housed in groups of four per cage, under a 12:12 hour light/dark photocycle with food and water available ad libitum. Lights were turned on at 06.00 hours and turned off at 18.00 hours. For all experiments, investigators were blinded to drug treatment and animals randomized (block randomization) to each treatment group prior to commencement of experiments. For each assay, mice were brought to the experimental room one hour prior to the beginning of the experiment for acclimation. During the testing, all animals were continuously monitored for movement, behavior and overall wellbeing. If any mouse were not moving and/or showed signs of distress, the protocol was to remove the animal from the study; however, no animals were removed. Animal procedures were approved by the University of Iowa Animal Care and Use Committee (IACUC Title: CGRP-induced Light Aversion in a Preclinical Migraine Model; Protocol #: 2071836; Protocol Approval Date: 08/17/2022 Expiration Date: 08/17/2025) and performed in accordance with the standards set by the National Institutes of Health and the ARRIVE guidelines.

Surgical implantation of cannula

The cannulas were manually constructed from 304 stainless steel 24-gauge hypodermic tubing, cut to 8 mm in length as previously described.¹⁰ Obturators were created by soldering a 3-mm cannula to a 12-mm piece of 30-gauge wire tubing to seal the cannula opening. Cannula implantation was performed using a stereotaxic frame, with target coordinates set at (0.3 mm anteroposterior, 1.1 mm mediolateral and 2.35 mm dorsoventral). After surgery, mice were housed individually to minimize the risk of cannula displacement and allowed to recover for approximately two weeks before undergoing the first behavioral assays.

Drug administration

A combined dose of sunflower oil (negative control – vehicle) or CBD (100 mg/kg) and THC (1 mg/kg) prepared in vehicle sunflower oil (Sigma-Aldrich, St Louis, MO, USA) were administered via intraperitoneal (i.p.) injection. Thirty minutes later, mice received an intracerebroventricular injection of 1X phopshate-buffered saline (PBS) (negative control – vehicle) or CGRP (1 μg/μl) at a volume of 2 μl into the right lateral ventricle through a cannula. To ensure slow delivery, the treatments were delivered through PE-20 tubing and the injection rate was maintained at 0.5 μl per minute for four minutes. During injections, the animals were gently restrained but not anesthetized. They were given 30 minutes to recover in their home cages before testing.

The dosing rationale began with selecting a low dose of THC (1 mg/kg) known to have minimal adverse effects, which served as a fixed point for adjusting CBD concentrations. The selection of 100:1 ratio was then based on our previous study with peripheral CGRP administration.⁴³

Light aversion assay

The light aversion assay was performed to measure the animal's aversion to light, which is a surrogate for photophobia. Briefly, CD1 mice are placed individually in light/dark boxes that have two compartments: one dimly lit (55 lux) and the other not lit, black and fully enclosed. While we previously reported that C57BL/6J mice require bright light (25–27 K lux) to detect CGRP-induced light aversion¹¹ and an earlier study with CD1 mice also used bright light,¹⁰ we have subsequently found that CD1 mice show CGRP-induced light aversive behavior even with dim light (55 lux).⁴³ The increased light sensitivity is consistent with the CD1 mice being an albino strain. Mice could move freely in between the chambers. The mouse activity was collected with infrared beam tracking and Activity Monitor software (Med Associates, Dubuque, IA, USA), as previously described.^11,13 Mice were pre-exposed to the chamber once to reduce exploratory drive, then tested with 48 hour later. Data were collected over a 30-minute period and analyzed in sequential five-minute intervals. The time in light was reported as the mean ± SEM of all the mice at each interval and as the mean ± SEM of the average time per five-minute interval for each individual mouse. Depending on the assay, a repeated measures (RM) two-way analysis of variance (ANOVA) or restricted maximum likelihood (REML) mixed effects model followed by Tukey's multiple comparisons test were used to analyze the longitudinal data plotted over the course of 30 minutes. A sample size of 16 mice per group was determined to be sufficient to detect the desired effect size in the light aversion assay, as detailed in the statistical analysis. Any mice that spent greater than or equal to 90% of the total time resting were excluded from the analysis. The total number of mice used, including those excluded, along with all statistical analyses for the light aversion assay, are provided in the supplementary material (Tables S1 to S8).

Open field assay

The open field test was used to evaluate center avoidance as a measure of anxiety-like behavior. The test was conducted using the same equipment and light intensity (55 lux) as the light/dark assay, but without the dark insert. The chamber was configured with a center area of 19.05 × 19.05 cm and a 3.97-cm border along the edges, as previously described.⁴³ Mice were placed in the center of the area and observed for 20 minutes following either vehicle or cannabinoid injection. The time each mouse spent in the center was recorded and the results are reported as the mean ± SEM for each group. The differences in time spent in the center between the groups tested was analyzed using an unpaired t-test. Based on power calculations described in the statistical analysis section, a sample size of 16 mice per group was determined to be sufficient to detect the desired effect size in the open field assay. The total number of mice used, including those excluded, along with all statistical analyses for the open field assay, are provided in the supplementary material (Table S7).

Automated squint assay

The purpose of this test was to assess spontaneous pain by measuring the pixel areas of the right eye recorded by a camera. To reduce stress and movement during the test, mice were acclimated to a customized collar restraint for 20 minutes per session for three sessions, as previously described.^9,12,43 On the test day, the mouse was habituated to the testing room for one hour and then placed in the restraint. Two separate five-minute recordings were conducted (baseline and treatment), which were carried out under normal room light conditions as previously described.^12,13 Facial detection software (FaceX; LLC, Iowa City, IA, USA) was used to calculate the pixel area of the right eye every 0.1 seconds (10 frames per second) in the recordings. A custom MATLAB script (MathWorks Inc., Natick, MA, USA) was then used to compile the resulting values. Any frames with a tracking error rate greater than 15% were excluded from the analysis. The difference between baseline and treatment recordings was analyzed using a paired t-test. A sample size of 12 mice per group was determined to be sufficient to detect the desired effect size in the automated squint assay, as detailed in the statistical analysis section. The total number of mice used, including those excluded, along with all statistical analyses for the automated squint assay, are provided in the supplementary material (Table S9).

Statistical analysis

Statistical analysis was performed using Prism, version 10 (GraphPad Software Inc., San Diego, CA, USA). Given the inherent variability of mouse behavior, measurements for all behavioral tests (the light aversion assay, the open field test and the squint assay), were accomplished with at least two independent cohorts of CD1 mice. The effect sizes of experiments involving CD1 mice were based on comparable data from previous studies in the same laboratory.⁹^11–13^,44 With an α of 0.05 and power at 0.80, the sample size suitable for the desired effect size was estimated at 16 per group in the light aversion and open field assays, and 12 per group in the squint assay. Because we analyzed male and female mice together and separately by sex, groups of 32 and 24 were required. To allow for dropout of mice for various reasons, such as complications following surgeries and loss of cannulas, about six additional mice were included. Based on whether the data were paired and distributed, data were compared differently. One-way ANOVA, two-way RM ANOVA or the REML mixed effects model, followed by Tukey's multiple comparisons tests, were used. Data are presented as individual values and mean ± SEM. p < 0.05 was considered statistically significant. Our criteria for defining a partial rescue require that the CBD:THC treatment group is not statistically different from either the CGRP group or the vehicle control. Similarly, we define a full rescue as being statistically different from the CGRP group, but not the vehicle. The results of all statistical analyses are available in the supplementary material (Tables S1 to S9).

Results

Central CGRP-induced light aversion is rescued by 100:1 CBD:THC

The experimental design to evaluate the efficacy of the 100:1 CBD:THC (100 mg/kg CBD, 1 mg/kg THC) combination in rescuing central CGRP-induced light aversion is illustrated in Figure 1A. In mice pretreated with vehicle, CGRP injection led to a significant reduction in time spent in the light chamber compared to vehicle-only controls, indicating a strong light aversion phenotype. In contrast, mice pretreated with the 100:1 CBD:THC combination showed a complete rescue from CGRP-induced light aversion, with differences observed at multiple time points throughout the testing period (Figure 1B). The behavior of individual mice shown as the average time spent in the light across the entire testing period also showed that the CGRP group differed significantly from both the vehicle and CGRP + 100:1 CBD:THC groups (Figure 1C). Notably, there was no difference between the vehicle and CGRP + 100:1 CBD:THC groups, indicating that the 100:1 CBD:THC combination fully rescued the light aversion behavior. When separated by sex, female mice pretreated with 100:1 CBD:THC combination showed a full rescue from light aversion (Figure 1E) compared to a partial rescue seen in male mice (Figure 1D). These results demonstrate that a peripheral administration of 100:1 CBD:THC (100 mg/kg CBD, 1 mg/kg THC) combination effectively prevents central CGRP-induced light aversion. Further details of the statistical analyses including a breakdown by sex are provided in the supplementary material (Tables S1 and S2).

Figure 1.

Injection of 100:1 CBD:THC rescues central CGRP-induced light aversion. (A) Experimental design of the light aversion assay testing the effect of 100:1 CBD:THC (100 mg/kg CBD: 1 mg/kg THC). (B) Longitudinal representation of time in the light over the course of 30 minutes. The positive control CGRP was statistically different than PBS + Veh and CGRP + 100:1 CBD:THC at all the time points. Two-way RM ANOVA, Tukey's multiple comparisons test. (C) Average time spent in the light of individual mice. CGRP + Veh group was significantly different from the PBS + Veh (****p < 0.0001) and the CGRP + 100:1 CBD:THC group (**p = 0.001). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns, p = 0.6513). One-way ANOVA (****p < 0.0001), Tukey's multiple comparisons test. (D) Average time spent in the light of male mice. CGRP + Veh group was significantly different from the PBS + Veh (*p = 0.0103) but not from the CGRP + 100:1 CBD:THC group (ns, p = 0.4156). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns, p = 0.1532). One-way ANOVA (*p = 0.0131), Tukey's multiple comparisons test. (E) Average time spent in the light of female mice. CGRP + vehicle group was significantly different from the PBS + Veh (**p = 0.0047) and the CGRP + 100:1 CBD:THC group (***p = 0.0002). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns, p = 0.6204). One-way ANOVA (***p = 0.0001), Tukey's multiple comparisons test. For all data sets, the mean ± SEM was used. Number of mice: PBS + veh: n = 34 (1 excl.); CGRP + veh: n = 38 (1 exc.); CGRP + 100:1: n = 33, male mice (open circles) PBS + veh: n = 18; CGRP + veh: n = 16; CGRP + 100:1: n = 18, and female mice (closed circles) PBS + veh: n = 16 (1 excl.); CGRP + veh: n = 22 (1 excl.); CGRP + 100:1: n = 15. Statistical analyses are further described in the supplementary material (Tables S1 and S2). CBD = cannabidiol; CGRP = calcitonin gene-related peptide; THC = tetrahydrocannabinol; PBS = phosphate-buffered saline; RM ANOVA = repeated measures analysis of variance; Veh = vehicle.

Central CGRP-induced resting time in dark is rescued by 100:1 CBD:THC

During the light aversion experiments, we examined the effect of cannabinoid treatment on resting behavior in both dark and light environments. Similar to our previous findings,^10,11,45 following a CGRP injection, mice spent more time resting in the dark, but not the light (Figure 2). Notably, the increased resting in the dark induced by CGRP was significantly reduced by pretreatment with the 100:1 CBD:THC combination, as seen across several time points throughout the 30-minute observation period (Figure 2A). The behavior of individual mice shown as the average percentage resting in the dark, revealed that the CGRP group had an increased time spent resting in the dark compared to the vehicle and CGRP + 100:1 CBD:THC groups (Figure 2C). No significant difference was observed between the vehicle and CGRP + 100:1 CBD:THC groups, further supporting the efficacy of the combination treatment in reversing CGRP-induced resting behavior. When analyzed by sex, female mice pretreated with a 100:1 CBD:THC combination showed complete rescue from CGRP-induced increased in percentage resting time in the dark zone (Figure 2G), whereas male mice exhibited only partial rescue (Figure 2E).

Figure 2.

Injection of 100:1 CBD:THC rescues central CGRP-induced resting behavior. (A) Longitudinal representation of percentage resting in dark over the course of 30 minutes. The CGRP + Veh group was statistically different than the CGRP + 100:1 CBD:THC (100 mg/kg CBD: 1 mg/kg THC) group during the first 25 minutes and the PBS + Veh group at all time points. Mixed-effects model (REML), Tukey's multiple comparisons test. (B) Longitudinal representation of percentage resting in light over the course of 30 minutes. None of the treatment groups were statistically different in any of the time points. Mixed-effects model (REML), Tukey's multiple comparisons test. (C) Average percentage time resting in dark of individual mice. CGRP + Veh group was significantly different from the PBS + Veh (****p < 0.0001) and the CGRP + 100:1 CBD:THC group (***p = 0.0009). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns, p = 0.2102). One-way ANOVA (****p < 0.0001), Tukey's multiple comparisons test. (D) Average percentage time resting in the light zone of individual mice. CGRP + Veh group was significantly different from the PBS + Veh (*p = 0.0303) but not the CGRP + 100:1 CBD:THC group (ns, p = 0.134). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns. p = 0.8067). One-way ANOVA (*p = 0.0293), Tukey's multiple comparisons test. (E) Average percentage time resting in dark of male mice. CGRP + Veh group was significantly different from the PBS + Veh (*p = 0.0112) but not from the CGRP + 100:1 CBD:THC group (ns, p = 0.3858). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns, p = 0.2022). One-way ANOVA (*p = 0.0149), Tukey's multiple comparisons test. (F) Average percentage time resting in the light zone of male mice. CGRP + Veh group was not significantly different from the PBS + Veh (ns, p = 0.0861) or the CGRP + 100:1 CBD:THC group (ns, p = 0.8667). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns, p = 0.2104). One-way ANOVA (ns, p = 0.0812), Tukey's multiple comparisons test. (G) Average percentage time resting in dark of female mice. CGRP + Veh group was significantly different from the PBS + Veh (****p < 0.0001) and the CGRP + 100:1 CBD:THC group (***p = 0.0006). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns, p = 0.8126). One-way ANOVA (****p < 0.0001), Tukey's multiple comparisons test. (H) Average percentage time resting in the light zone of female mice. CGRP + Veh group was not significantly different from the PBS + Veh (ns, p = 0.4384) or from the CGRP + 100:1 CBD:THC group (ns, p = 0.0565). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns, p = 0.5520). One-way ANOVA (ns, p = 0.0686), Tukey's multiple comparisons test. For all data sets, the mean ± SEM was used. Number of mice: PBS + veh: n = 34 (1 excl.); CGRP + veh: n = 38 (1 exc.); CGRP + 100:1: n = 33, male mice (open circles) PBS + veh: n = 18; CGRP + veh: n = 16; CGRP + 100:1: n = 18, and female mice (closed circles) PBS + veh: n = 16 (1 excl.); CGRP + veh: n = 22 (1 excl.); CGRP + 100:1: n = 15. Statistical analyses are further described in the supplementary material (Tables S2 to S4). ANOVA = analysis of variance; CBD = cannabidiol; CGRP = calcitonin gene-related peptide; THC = tetrahydrocannabinol; PBS = phosphate-buffered saline; REML, restricted maximum likelihood; Veh = vehicle.

In terms of resting behavior in the light, none of the treatment groups showed significant differences across the time points (Figure 2B), indicating that the 100:1 CBD:THC combination did not affect resting in light behaviors. However, the CGRP group did spend a small, but statistically significant, reduced time resting in the light compared to vehicle controls (Figure 2D), similar to previous findings.¹¹ The 100:1 CBD:THC combination partially rescued the resting behavior because there was no significant difference from either vehicle or CGRP. Collectively, these results suggest that pretreatment with the 100:1 CBD:THC combination can rescue the CGRP-induced increased resting in the dark without significantly affecting resting time in the light. Additionally, there were no sex-specific responses when broken down by sex (Figure 2F, H). Further details of the statistical analyses including a breakdown by sex are provided in the supplementary materials (Tables S2 to S4).

Central CGRP-induced rearing behavior in dark is rescued by 100:1 CBD:THC

We also examined the effect of cannabinoid treatment on CGRP-induced rearing behavior. Mice injected with CGRP and vehicle displayed a significant decrease in rearing behavior in the dark compared to vehicle controls at several time points during the 30-minute observation period (Figure 3A). Specifically, CGRP decreased rearing at 10, 15 and 20 minutes compared to the vehicle group. CGRP caused a significant decrease in the average rearing behavior of the individual mice compared to vehicle (Figure 3C). The reduced rearing caused by CGRP was partially rescued by the 100:1 CBD:THC combination (Figure 3A, C). The rescue was considered to be partial because, although there was no significant difference between CGRP + 100:1 CBD:THC group from vehicle, there also was no significant difference from the CGRP group. By contrast to behavior in the dark, rearing behavior in the light was decreased by CGRP only at the 10-minute time point compared to vehicle (Figure 3B) and there was no significant difference overall between individual mice (Figure 3D). There was no significant effect of 100:1 CBD:THC combination on rearing behavior in the light. Additionally, there were no sex-specific responses when broken down by sex (Figure 3F, H). Further details of the statistical analyses including a breakdown by sex are provided in the supplementary material (Tables S5 to S7).

Figure 3.

Peripheral cannabinoid injections of 100:1 CBD:THC partially rescues reduced rearing in the dark caused by central CGRP. (A) Longitudinal representation of rearing in dark over the course of 30-minute testing the effect of 100:1 CBD:THC (100 mg/kg CBD: 1 mg/kg THC). The CGRP + Veh group was statistically different than PBS + Veh group at 10, 15 and 20 minutes. Mixed-effects model (REML), Tukey's multiple comparisons test. (B) Longitudinal representation of rearing in light over the course of 30 minutes. The CGRP + Veh group was statistically different than PBS + Veh group at 10 minutes. Mixed-effects model (REML), Tukey's multiple comparisons test. (C) Average rearing in dark of individual mice. The CGRP + Veh group was significantly different from the PBS + Veh (*p = 0.0185) but not the CGRP + 100:1 CBD:THC group (ns, p = 0.5162). The PBS + Veh and CGRP + 100:1 CBD:THC groups were not statistically different from each other (ns, p = 0.2424). One-way ANOVA (*p = 0.0245), Tukey's multiple comparisons test. (D) Average rearing in the light zone of individual mice. The CGRP + Veh group was not significantly different from the PBS + Veh (ns, p = 0.7952) or the CGRP + 100:1 CBD:THC group (ns, p = 0.8478). The PBS + Veh and CGRP + 100:1 CBD:THC groups were not statistically different from each other (ns, p = 0.9950). One-way ANOVA (ns, p = 0.7820), Tukey's multiple comparisons test. (E) Average rearing in dark of male mice. The CGRP + Veh group was not significantly different from the PBS + Veh (ns, p = 0.8429) or the CGRP + 100:1 CBD:THC group (ns, p = 0.8514). The PBS + Veh and CGRP + 100:1 CBD:THC groups were not statistically different from each other (ns, p = 0.4987). One-way ANOVA (ns, p = 0.5307), Tukey's multiple comparisons test. (F) Average rearing in the light zone of male mice. The CGRP + Veh group was not significantly different from the PBS + Veh (ns, p = 0.7662) or the CGRP + 100:1 CBD:THC group (ns, p = 0.8459). The PBS + Veh and CGRP + 100:1 CBD:THC groups were not statistically different from each other (ns, p = 0.9878). One-way ANOVA (ns, p = 0.7673), Tukey's multiple comparisons test. (G) Average rearing in dark of female mice. The CGRP + Veh group was significantly different from the PBS + Veh (**p = 0.0040) but not the CGRP + 100:1 CBD:THC group (ns, p = 0.1027). The PBS + Veh and CGRP + 100:1 CBD:THC groups were not statistically different from each other (ns, p = 0.4605). One-way ANOVA (**p = 0.0047), Tukey's multiple comparisons test. (H) Average rearing in the light zone of female mice. The CGRP + Veh group was not significantly different from the PBS + Veh (ns, p = 0.1506) or the CGRP + 100:1 CBD:THC group (ns, p = 0.3417). The PBS + Veh and CGRP + 100:1 CBD:THC groups were not statistically different from each other (ns, p = 0.8961). One-way ANOVA (ns, p = 0.1419), Tukey's multiple comparisons test. For all data sets, the mean ± SEM was used. Number of mice: PBS + veh: n = 34 (1 excl.); CGRP + veh: n = 38 (1 exc.); CGRP + 100:1: n = 33, male mice (open circles) PBS + veh: n = 18; CGRP + veh: n = 16; CGRP + 100:1: n = 18, and female mice (closed circles) PBS + veh: n = 16 (1 excl.); CGRP + veh: n = 22 (1 excl.); CGRP + 100:1: n = 15. Statistical analyses are further described in the supplementary material (Tables S5 to S7). ANOVA = analysis of variance; CBD = cannabidiol; CGRP = calcitonin gene-related peptide; THC = tetrahydrocannabinol; PBS = phosphate-buffered saline; REML, restricted maximum likelihood; Veh = vehicle.

Central CGRP-induced transitions behavior is rescued by 100:1 CBD:THC

The effects of the 100:1 CBD:THC on central CGRP-induced transitions behavior were assessed over a 30-minute period. Mice treated with CGRP and vehicle exhibited a significant reduction in transitions compared to the vehicle group. However, pretreatment with the 100:1 CBD:THC combination effectively rescued this behavior at various time points (Figure 4A). Specifically, the CGRP group displayed significantly fewer transitions at 10, 15, 20, 25 and 30 minutes compared to the vehicle group. By contrast, mice pretreated with the 100:1 combination exhibited a recovery in transitions behavior, showing improved performance at 15 and 25 minutes compared to the CGRP group.

Figure 4.

Peripheral cannabinoid injection of 100:1 CBD:THC rescues central CGRP-induced transitions. (A) Longitudinal representation of the total number of transitions over the course of 30-minute testing the effect of 100:1 CBD:THC (100 mg/kg CBD: 1 mg/kg THC). The positive control CGRP was statistically different than CGRP + 100:1 CBD:THC group at 15 and 25 minutes and the PBS + Veh group at 10, 15, 20, 25 and 30 minutes. Two-way RM ANOVA, Tukey's multiple comparisons test. (B) Average total number transitions of individual mice. The CGRP + vehicle group was significantly different from the PBS + Veh (**p = 0.0031) and the CGRP + 100:1 CBD:THC group (*p = 0.0395). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns, p = 0.6603). One-way ANOVA (**p = 0.0029), Tukey's multiple comparisons test. (C) Average total number transitions of male mice. The CGRP + Veh group was not significantly different from the PBS + Veh (ns, p = 0.5201) or the CGRP + 100:1 CBD:THC group (ns, p = 0.987). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns, p = 0.5997). One-way ANOVA (ns, p = 0.4876), Tukey's multiple comparisons test. (D) Average total number transitions of female mice. The CGRP + Veh group was significantly different from the PBS + Veh (**p = 0.0019) and the CGRP + 100:1 CBD:THC group (**p = 0.0060). The PBS + Veh and CGRP + 100:1 CBD:THC group were not statistically different from each other (ns, p = 0.9273). One-way ANOVA (***p = 0.0007), Tukey's multiple comparisons test. For all data sets, the mean ± SEM was used. Number of mice: PBS + veh: n = 34 (1 excl.); CGRP + veh: n = 38 (1 exc.); CGRP + 100:1: n = 33, male mice (open circles) PBS + veh: n = 18; CGRP + veh: n = 16; CGRP + 100:1: n = 18, and female mice (closed circles) PBS + veh: n = 16 (1 excl.); CGRP + veh: n = 22 (1 excl.); CGRP + 100:1: n = 15. Statistical analyses are further described in the supplementary material (Tables S7 and S8). CBD = cannabidiol; CGRP = calcitonin gene-related peptide; RM ANOVA = repeated measures analysis of variance; THC = tetrahydrocannabinol; PBS = phosphate-buffered saline; Veh = vehicle.

The behavior of individual mice shown as the average number of transitions during the 30-minute testing period also indicated that the CGRP + vehicle group was significantly different from both the vehicle and CGRP + 100:1 CBD:THC groups (Figure 4B). Importantly, there was no significant difference between the vehicle and CGRP + 100:1 CBD:THC groups, suggesting that the 100:1 CBD:THC combination successfully restored transitions behavior to levels comparable to the control group. When analyzed by sex, male mice showed no response to CGRP (Figure 4C). Interestingly, female mice pretreated with the 100:1 CBD:THC combination exhibited a full rescue from CGRP-induced reductions in transitions (Figure 4D). These findings demonstrate that the peripheral administration of the 100:1 CBD:THC dose rescues the reduction in transitions caused by central CGRP injection, supporting its efficacy in mitigating CGRP-induced behavioral impairments. Further details of the statistical analyses including a breakdown by sex are provided in the supplementary material (Tables S7 and S8).

Central administration of CGRP does not cause anxiety in the open field assay

To determine whether anxiety was driving the light aversion caused by central administration of CGRP, we conducted the open field assay. Briefly, CGRP was injected 30 minutes before placing mice in the open field chamber as seen in Figure 5A. Consistent with our previous findings,^10,11 there was no significant difference in the time spent in the center between mice treated with i.c.v. CGRP and vehicle (Figure 5B). Although this experiment was not sufficiently powered for sex differences, the responses appeared similar for male and female mice. Anxiety alone is not likely contributing to the CGRP-induced light aversion, although there remains the possibility of an anxiogenic contribution. Further details of the statistical analyses including a breakdown by sex are provided in the supplementary material (Table S7).

Figure 5.

Central administration of CGRP does not cause anxiety in the open field assay. (A) Open field assay experimental paradigm. (B) Average time spent in the center of individual mice. Central administration of CGRP does not cause anxiety in the open field assay as shown in percentage time in center. Using an unpaired t-test, the CGRP group was not significantly different from the PBS + Veh group (ns, p = 0.2127). Mean + SEM was used. Number of mice: PBS + veh: n = 19; CGRP + veh: n = 18, male mice (open circles) PBS + veh: n = 11; CGRP + veh: n = 9, and female mice (closed circles) PBS + veh: n = 8; CGRP + veh: n = 9. Statistical analyses are further described in the supplementary material (Table S7). CGRP = calcitonin gene-related peptide; PBS = phosphate-buffered saline; Veh = vehicle.

Central CGRP-induced squint is partially rescued by 100:1 CBD:THC

The experimental paradigm to assess the efficacy of a 100:1 combination of CBD and THC on squinting behavior is shown in Figure 6A. The effect of the 100:1 CBD:THC ratio on CGRP-induced squint behavior was assessed by measuring the average mean pixel area over five-minute intervals before and after treatment. Cannabinoid treatment partially rescued CGRP-induced squint behavior in CD1 mice (Figure 6B). Both the vehicle and CGRP + 100:1 CBD:THC groups showed no significant difference between baseline and treatment recordings, indicating no impact on squint behavior. In contrast, the CGRP group exhibited a significant reduction in pixel area from baseline to treatment, indicating squinting behavior induced by CGRP. When comparing the baseline treatments to each other, there was no significant difference between the groups. When comparing the treatment groups to each other, there was only a significant difference between the vehicle and CGRP groups. These results demonstrate that the 100:1 CBD:THC combination partially rescues the squinting behavior caused by central CGRP administration because there was no significant difference between CGRP + 100:1 CBD:THC group from vehicle, or from the CGRP group. Further details of the statistical analyses including a breakdown by sex are provided in the supplementary material (Table S9).

Figure 6.

Central CGRP-induced squint is partially rescued by 100:1 CBD:THC. (A) Automated squint assay testing paradigm testing the effect of 100:1 CBD:THC (100 mg/kg CBD: 1 mg/kg THC). (B) Average mean pixel area over 5 min (baseline and treatment). Cannabinoid injection (i.p.) of 100:1 CBD:THC rescues CGRP (i.c.v.) induced squint in CD1 mice. Using a two-way RM ANOVA, the baseline and treatment recordings of PBS + Veh group (ns, p > 0.9999) and CGRP + 100:1 CBD:THC (ns, p = 0.9908) were not significantly different. The baseline of CGRP + Veh group was significantly different from treatment recording (*p = 0.0208). When comparing the baseline (BL) recordings across the treatment groups, there was no significant difference between the PBS + Veh (BL) vs. CGRP + Veh (BL) (ns, p = 0.9934), PBS + Veh (BL) vs. CGRP + CBD:THC 100:1 (BL) (ns, p = 0.9981), and CGRP + Veh (BL) vs. CGRP + CBD:THC 100:1 (BL) (ns, p > 0.9999). When comparing the treatment (Tx) recordings across the treatment groups, there was no significant difference between the PBS + Veh (Tx) vs. CGRP + CBD:THC 100:1 (Tx) (ns, p = 0.889), and CGRP + Veh (Tx) vs. CGRP + CBD:THC 100:1 (Tx) (ns, p = 0.1275); however, there was a significant difference between PBS + Veh (Tx) vs. CGRP + Veh (Tx) (**p = 0.0063). Number of mice: PBS + veh: n = 20 (1 excl.); CGRP + veh: n = 23; CGRP + 100:1: n = 20, male mice (open circles) PBS + veh: n = 11; CGRP + veh: n = 13; CGRP + 100:1: n = 10, and female mice (closed circles) PBS + veh: n = 9 (1 excl.); CGRP + veh: n = 10; CGRP + 100:1: n = 10. Statistical analyses are further described in the supplementary material (Table S9). CBD = cannabidiol; CGRP = calcitonin gene-related peptide; THC = tetrahydrocannabinol; PBS = phosphate-buffered saline; RM ANOVA = repeated measures analysis of variance; Veh = vehicle.

Discussion

In the present study, we have demonstrated that CD1 mice pretreated with an i.p. injection of a CBD:THC mixture at a combined dose of 100:1 (100 mg/kg CBD and 1 mg/kg THC) effectively prevented light aversion and pain behaviors induced by centrally-administered (i.c.v.) CGRP. The CBD:THC rescue of migraine-like symptoms caused by the central actions of CGRP is consistent with our prior finding that the same treatment of 100:1 CBD:THC rescued light aversion and pain behaviors induced by peripherally (i.p.) administered CGRP and a nitric oxide donor.⁴³ Importantly, that study also showed that the same dose of 100:1 CBD:THC produced no adverse effects in various behavior assays assessing memory, anxiety, motor function and depression-like symptoms.

Over the last several decades, the neuropeptide CGRP has emerged as a key player in migraine pathophysiology.⁴⁶ CGRP is a potent vasodilator that is widely expressed in the PNS and CNS.^22,46 In the PNS, CGRP release is active in the trigeminovascular system where it acts on receptors on the vasculature, immune cells in the meninges and trigeminal neurons and glia.⁴⁷ CGRP is also produced in the CNS where it has sites of action throughout regions involved in pain and sensory processing, such as the posterior thalamus, cortices and the cerebellum.^13,48,49 Both peripheral and central administration of CGRP into mice causes several migraine-like symptoms of light aversion, touch sensitivity and grimace/squint.^9–11^,13 Indeed, a side note of this study is the finding that i.c.v. administered CGRP-induced light aversion without an anxiety-like phenotype extends our previous studies using C57BL/6J mice¹⁰ to now include the outbred CD1 strain. Although the relevant sites of action of CGRP in migraine patients are still not known, it has been speculated that both central and peripheral sites of CGRP action may be important.⁵⁰ Thus, a significant point of the present study is that it provides evidence that cannabinoids, specifically CBD and THC, can prevent the central actions of CGRP, even when administered peripherally.

A possible explanation for the efficacy of the 100:1 CBD:THC ratio compared to either cannabinoid alone or other tested ratios may lie in their overlapping ability to act on multiple receptors implicated in migraine pathogenesis. Due to their lipophilic nature, CBD and THC can readily cross the BBB and act on multiple migraine-relevant sites in the PNS and CNS,⁵¹ including cannabinoid receptors CB1 and CB2 expressed in migraine relevant regions,^52,53 and in meningeal immune cells,^47,54 which have been implicated in migraine pathogenesis.⁵⁵ For example, activation of CB1 receptors in the trigeminal ganglion has been shown to suppress CGRP release^47,56 while activation of CB2 receptors in meningeal immune cells have shown to reduce inflammatory cytokine levels⁴⁷ supporting the mechanistic basis for the effects observed in the current study. Additionally, cannabinoid receptors are present in several brain regions which are considered to be implicated in migraine pathogenesis such as the thalamus,⁵⁷ cortex⁵⁸ and the cerebellum.⁵⁹

In addition to their effects on the CGRP pathway, both CBD and THC possess potent neuro-modulatory and anti-inflammatory properties. CBD has been shown to modulate serotonin (5-HT_1A) and transient receptor potential vanilloid-1 (TRPV1) receptors, both of which play roles in pain signaling and migraine pathophysiology,^60–62 while THC's partial agonism at CB1 and CB2 receptors⁶⁰ contributes to central and peripheral modulation of nociceptive pathways. However, although a combination of CBD and THC was needed in our CGRP models, Sturaro et al.⁶³ reported that head sensitivity caused by peripheral CGRP administration could be blocked by CBD alone in mice. The reason for this difference is not known. The pharmacokinetics of CBD and THC, both individually and in combination, represent another important consideration, as they are influenced by factors such as dose, timing and route of administration. Both cannabinoids are primarily metabolized in the liver by cytochrome P450 enzymes⁶⁴ and, in rodents, i.p. administration of CBD produces a pharmacokinetic profile similar to THC.^65,66 Importantly, both are substrates of CYP3A4, and due to their structural similarities, CBD may competitively inhibit the metabolism of THC, potentially reducing the formation of the active metabolite 11-OH-THC. To fully dissect the underlying mechanisms, future studies will require a combination of pharmacological antagonists and knockout mice, which are beyond the scope of the present study.

Importantly, our preclinical results are consistent with results from a recently completed clinical trial that showed migraine patients had improved symptoms of pain, nausea and photophobia, after consuming vaporized cannabis containing a combination of CBD and THC, as opposed to cannabis with predominantly one or the other cannabinoid.⁶⁷ However, on the flip side, cannabis has been associated with medication overuse headache in people,⁶⁸ and with latent sensitization to stress-induced allodynia in a rat medication overuse headache model.⁶⁹ The possibility of this adverse effect warrants further investigation. Future studies should aim to further characterize the use of CBD and THC across different dosing ratios, and routes of administration to optimize therapeutic efficacy.

Although, overall, CBD:THC rescued light aversion and squint to similar extents following either central or peripheral CGRP administration, there were sex-specific differences in response to 100:1 CBD:THC treatments. In the light aversion assay for both time in light and percentage resting in the dark zone, pretreatment with the 100:1 ratio led to a partial rescue in male mice, whereas female mice showed a full rescue (Table S2). This differs from peripheral CGRP actions, which were fully rescued in both sexes with comparable efficacy.⁴³ On the other hand, CBD:THC partially rescued squint in both male and female mice following central CGRP administration, whereas, after peripheral CGRP, CBD:THC rescued squint only in male mice.⁴³ The reason for these sex differences between CBD:THC rescue of peripheral and central CGRP actions is not known but is consistent with the mechanisms of peripheral and central CGRP being similar, but not identical, as previously noted.¹⁰ In addition, we did note that female mice had significantly reduced transitions in response to i.c.v. CGRP, whereas males showed no significant response to CGRP in this assay (see supplementary material, Table S7), suggesting a sex-dependent sensitivity to CGRP. These results align with prior preclinical reports^70–72 that suggest females may be more sensitive to CGRP. A sex-specific effect of CBD:THC is consistent with preclinical and clinical studies on sex-based variations in the ECS. For example, female rats have lower levels of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the periaqueductal gray, a brain region critical for pain modulation.⁷³ Similarly chronic migraine patients have lower 2-AG levels compared to healthy individuals.⁷⁴ Among those with migraine, females exhibit even lower 2-AG levels than males.⁷⁴ Additionally, CBD inhibits the degradation of AEA,⁷⁵ which has been shown to decrease excitability in the trigeminovascular system,⁵⁶ enhance serotonin 5-HT_1A receptor activity and inhibit 5-HT2A receptors, contributing to its anti-nociceptive effects in rodents.⁷⁶ Recent studies have revealed that female migraine patients have increased activity of AEA hydrolase, an enzyme that degrades AEA.⁴² This suggests that AEA is broken down more rapidly in the platelets, leading to lower AEA levels in the bloodstream, which is assumed to contribute to a reduced pain threshold and could help explain the higher sensitivity to CGRP seen in female migraine patients. Future studies should aim to integrate these variables to better understand the underlying the molecular mechanisms driving sex-specific responses to cannabinoid treatment.

In conclusion, the present study highlights the potential of cannabinoid-based therapies to alleviate pain and sensory disturbances linked to central actions of CGRP. These promising results highlight the therapeutic benefits of CBD and THC as a non-invasive alternative for treating migraine-like symptoms. Future research should focus on further exploring the precise mechanisms through which cannabinoids exert their effects. Expanding our understanding of these therapeutic properties could lead to the development of novel, effective treatments for chronic pain, sensory hypersensitivity and other disorders.

Conclusions

Our preclinical findings indicate that cannabinoids may offer therapeutic potential for alleviating migraine symptoms induced by central actions of CGRP. These results align with previous research suggesting that cannabinoids could be effective in managing pain and migraine. Further investigations into CBD and THC combinations are necessary to establish their significance in a clinical setting.

Article highlights

A 100:1 CBD:THC combination alleviated light aversion, resting in the dark zone and motility behavior (rearing, transitions) induced by central administration of CGRP in mice.

A 100:1 CBD:THC combination partially rescued centrally-administered CGRP-induced squint in mice.

Combined administration of CBD and THC shows potential for migraine therapy.

Supplemental Material

sj-docx-1-cep-10.1177_03331024251392103 - Supplemental material for Cannabinoids rescue migraine symptoms caused by central CGRP administration in mice

Supplemental material, sj-docx-1-cep-10.1177_03331024251392103 for Cannabinoids rescue migraine symptoms caused by central CGRP administration in mice by Erik Zorrilla, Thomas L. Duong, Cassandra L. Piña and Andrew F. Russo in Cephalalgia

Footnotes

Acknowledgments

We thank Julien Masson,Romina Nasser and Emilie Partridge for help with experiments. The panels illustrating experimental designs were created with BioRender.com.

Author contributions

EZ,TLD and AFR generated the study concept and design. EZ,TLD and CLP collected and analyzed data. EZ wrote the first draft. EZ and AFR revised and wrote the final manuscript.

Data availability statement

The datasets used and/or analyzed during the current study are available upon request from the corresponding author.

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: AFR is a consultant and has stock in Delphian Therapeutics. He is also a consultant for Lundbeck,Abbvie,Pfizer,Vedana Therapeutics,Arrowhead Pharmaceuticals,Omeros Corporation,and holds patents on the CGRP monoclonal antibody for photophobia and diarrhea,on the PACAP monoclonal antibody for photophobia,and on the CGRP HO enhancer element. Delphian Therapeutics is interested in the development of cannabis-derived migraine therapeutics and provided the commercially available cannabinoids used in the study. Delphian Therapeutics had no influence on the study design,data analysis,manuscript preparation or decision to publish,although the company was shown a preprint prior to submission. The other authors (EZ,TLD,and CLP) declare no potential conflicts of interest with respect to the research,authorship,and/or publication of this article. The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

Ethical considerations

Ethical approval was obtained for experiments from the University of Iowa Animal Care and Use Committee and carried out in accordance with the standards set by the US National Institutes of Health.

Funding

This work was supported by a research grant from the National Institutes of Health R01NS075599 (AFR).

ORCID iDs

Erik Zorrilla

Thomas L. Duong

Cassandra L. Piña

Andrew F. Russo

Supplemental material

Supplemental material for this article is available online.

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