Combined effects of cannabidiol and Δ9-tetrahydrocannabinol alleviate migraine-like symptoms in mice

Abstract

Background: The therapeutic use of cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) to treat migraine has been understudied. Using three mouse models, we examined the impact of CBD and THC on migraine-relevant behaviors triggered by: 1) calcitonin gene-related peptide (CGRP), 2) sodium nitroprusside (SNP), and 3) cortical spreading depolarization (CSD).

Methods: Both male and female CD1 mice were treated with CBD (100 mg/kg) or THC (1 mg/kg) alone or in combinations of CBD (1, 30 or 100 mg/kg) and THC (1 mg/kg) prior to injection of CGRP or SNP. The mice were assessed for light aversion (photophobia), squint (non-evoked pain), and periorbital tactile hypersensitivity, as well as possible adverse effects. In a separate set of experiments, CSD events were optogenetically induced in familial hemiplegic migraine 1 (FHM1) mutant and wildtype littermates (WT) mice (C57BL/6 background), followed by grimace and motor assessments with and without combinations of CBD (30 or 100 mg/kg) and THC (1 mg/kg).

Results: In CD1 mice, a 100:1 CBD:THC combination mitigated light aversion induced by CGRP and SNP in males and females. Rescue of CGRP- and SNP-induced squint was observed only in male mice with 100:1 CBD:THC. None of the treatments rescued periorbital tactile hypersensitivity in either sex. In FHM1 mutant and WT mice, the 100:1 CBD:THC ratio did not affect CSD characteristics but did reduce CSD-induced grimace features (i.e., head pain mimic). No adverse effects of any of the cannabinoid treatments were observed using cognitive, emotional, or motor tests.

Conclusions: A 100:1 ratio of CBD:THC has a beneficial effect on some of the most bothersome migraine-related symptoms in three mouse models. Our findings support a potential therapeutic efficacy of combined CBD and THC treatments.

Keywords: CGRP; CSD; SNP; cannabinoids; headache; photophobia.

Figure 1.

Schematic overview of the animal models, treatments, and behavioral tests. The left panel illustrates a migraine without aura model in CD1 mice. Mice received intraperitoneal (i.p.) injections of cannabinoids or vehicle, followed by either CGRP or SNP to induce migraine-like symptoms. Behavioral assessments included light aversion, eye squinting, and tactile hypersensitivity. Adverse effects were evaluated using anxiety-related tests (light aversion and open field), spatial memory (Y-maze), motor coordination (rotarod), and depression-like behavior (tail suspension test) in both naïve mice and mice previously tested for migraine-like symptoms. No significant differences were observed between naïve and previously tested mice. The right panel depicts a migraine with aura model using transgenic FHM1 mutant (ChR2/FHM1) mice and wild-type littermates (ChR2/WT). Cannabinoids or vehicle were administered i.p. prior to optogenetically induced cortical spreading depolarization (CSD). Behavioral assessments included the mouse grimace scale for pain scoring. Adverse effects were evaluated using grip performance (wire grip test) before, during, and after CSD induction, as well as muscle endurance (four-limb hanging mesh test).

Figure 2.

A 100:1 CBD:THC combination effectively reduced both CGRP- and SNP-induced light aversion in CD1 mice. (a) Experimental design of the light aversion assay. (b) CGRP treatment. CD1 mice were pretreated with sunflower oil as vehicle (Veh) or cannabinoids at the indicated doses (mg/kg) and then injected with CGRP or PBS vehicle. Time spent in the light zone of individual mice is shown as the mean (± SEM) per 5-min interval. Mice given CGRP + 100:1 CBD:THC spent significantly more time in light than mice given CGRP + vehicle (*p= 0.0498) and were not significantly different from the negative control group (PBS + Veh) (p = 0.2917). For the 30:1 CBD:THC combination, there was a trend towards more time in the light, but no significant difference from either CGRP + vehicle (p = 0.3291) or from PBS + vehicle (p = 0.0962). One-way ANOVA, Tukey’s multiple comparisons, ****p < 0.0001. Groups were PBS + Veh (male n = 24, female n = 27), CGRP + Veh (male n = 24, female n = 27), CGRP + CBD 100 mg/kg (male n = 10, female n = 10), CGRP + THC 1 mg/kg (male n = 10, female n = 11), CGRP + CBD:THC 1:1 mg/kg (male n = 11, female n = 10), CGRP + CBD:THC 30:1 mg/kg (male n = 15, female n = 18), CGRP + CBD:THC 100:1 mg/kg (male n = 20, female n = 21). (c) SNP treatment. Time spent in the light of CD1 mice injected with SNP or PBS following pretreatment with cannabinoids or sunflower oil, as in panel b. Mice given SNP + 100:1 CBD:THC spent significantly more time in light than mice given SNP + vehicle (***p = 0.0005) and were not significantly different from the negative control group (PBS + Veh) (p = 0.9758). One-way ANOVA, Tukey’s multiple comparisons, ****p < 0.0001. Groups were PBS + Veh (male n = 10, female n = 12), SNP + Veh (male n = 11, female n = 10), SNP + CBD 100 mg/kg (male n = 11, female n = 12), SNP + THC 1 mg/kg (male n = 10, female n = 10), SNP + CBD:THC 1:1 mg/kg (male n = 11, female n = 9), SNP + CBD: THC 100:1 mg/kg (male n = 10, female n = 11). For all panels, open and closed symbols represent males and females, respectively. See Online Supplementary Figure 1a and b for longitudinal data over the 30-min testing period for CGRP and SNP treatments, respectively, and Online Supplementary Table 1 for full statistical data.

Figure 3.

A 100:1 CBD:THC combination rescued SNP- but not CGRP-induced resting in the dark zone in CD1 mice. Data were collected at the same time as light aversion data from the same CD1 mice shown in Figure 2. (a) CGRP treatment. Resting time (percentage ± SEM) in the dark of individual mice following CGRP treatment. CGRP significantly increased resting time under all conditions except for pretreatment with THC, which was not significantly different from CGRP + vehicle (p = 0.1983) or from PBS + vehicle (p = 0.0842). One-way ANOVA, Tukey’s multiple comparisons, ****p < 0.0001. Resting time (percentage ± SEM) in the light zone of individual mice following CGRP treatment. There was no significant effect by any of the treatments. One-way ANOVA, Tukey’s multiple comparisons, p = 0.1319. Groups were PBS + Veh (male n = 24, female n = 27), CGRP + Veh (male n = 24, female n = 27), CGRP + CBD 100 mg/kg (male n = 10, female n = 10), CGRP + THC 1 mg/kg (male n = 10, female n = 11), CGRP + CBD:THC 1:1 mg/kg (male n = 11, female n = 10), CGRP + CBD:THC 30:1 mg/kg (male n = 15, female n = 18), CGRP + CBD:THC 100:1 mg/kg (male n = 20, female n = 21). (b) SNP treatment. Resting time (percentage ± SEM) in the dark zone of individual mice following SNP treatment. SNP significantly increased resting time under all conditions except for the 100:1 CBD:THC combination, which rescued SNP-induced resting behavior against SNP + vehicle (**p = 0.0033) and PBS + Veh (p = 0.9633). One-way ANOVA, Tukey’s multiple comparisons, ****p < 0.0001. Resting time (percentage ± SEM) in the light zone of individual mice following SNP treatment. There was no significant effect by any of the treatments. One-way ANOVA, Tukey’s multiple comparisons, p = 0.1110. Groups were PBS + Veh (male n = 10, female n = 12), SNP + Veh (male n = 11, female n = 10), SNP + CBD 100 mg/kg (male n = 11, female n = 12), SNP + THC 1 mg/kg (male n = 10, female n = 10), SNP + CBD:THC 1:1 mg/kg (male n = 11, female n = 9), SNP + CBD:THC 100:1 mg/kg (male n = 10, female n = 11). For all panels, open and closed symbols represent males and females, respectively. See Online Supplementary Figure 2a and b for longitudinal data over the 30-min testing period for CGRP and SNP treatments, respectively. See Online Supplementary Table 3 for full statistical data.

Figure 4.

Cannabinoids rescued CGRP- and SNP-induced squint in male but not female CD1 mice. (a) Experimental design of the automated squint assay. (b) CGRP treatment. Average mean pixel area over 5-min (baseline and treatment). Male CD1 mice treated with a 100:1 CBD:THC combination showed a rescue of CGRP-induced squint. 100:1 was not significantly different from the baseline recording paired t-test 100:1 (p = 0.0856 against baseline 100:1 group). One-way ANOVA ****p < 0.0001. Average mean pixel area for female CD1 mice over 5 min. None of the treatment groups rescued CGRP-induced squint. One-way ANOVA ****p < 0.0001. (c) CGRP treatment delta (baseline – treatment) values of male and female CD1 mice squint values. Difference between male and female mice treated with the 100:1 ratio (*p = 0.0472, unpaired t-test). There are no other differences between sexes across all treatment groups. One-way ANOVA ****p < 0.0001. For panels b and c, the groups were PBS + Veh (male n = 19, female n = 16), CGRP + Veh (male n = 18, female n = 18), CGRP + CBD 100 mg/kg (male n = 10, female n = 11), CGRP + THC 1 mg/kg (male n = 12, female n = 12), CGRP + CBD:THC 1:1 mg/kg (male n = 9, female n = 11), CGRP + CBD:THC 30:1 mg/kg (male n = 12, female n = 12), CGRP + CBD:THC 100:1 mg/kg (male n = 15, female n = 16). (d) SNP treatment. Average mean pixel area over 5-min. Male CD1 mice treated with a 100:1 CBD:THC combination showed a rescue of SNP-induced squint. 100:1 was not significantly different from the baseline recording (p = 0.1910 paired t-test). Ordinary one-way ANOVA **p = 0.0030. For female CD1 mice, none of the treatment groups rescued SNP-induced squint. One-way ANOVA, ***p = 0.0004. (e) SNP treatment delta values of male and female CD1 mice squint values. There were no differences between sexes across all treatment groups. For the 100:1 CBD:THC combination, there was a trend towards rescue, but no significant difference between males and females (p = 0.4074 unpaired t-test). One-way ANOVA **p = 0.0026. For panels d and e, the groups were PBS + Veh (male n = 15, female n = 15), SNP + Veh (male n = 14, female n = 14), SNP + CBD:THC 100:1 mg/kg (male n = 15, female n = 14).For all panels, open and closed symbols represent males and females, respectively. See Online Supplementary Table 6 for full statistical data.

Figure 5.

Cannabinoids did not rescue CGRP- or SNP-induced periorbital tactile hypersensitivity in CD1 mice. (a) Experimental design of the periorbital tactile hypersensitivity assay. (b) Sensitivity to von Frey filaments in CGRP-injected CD1 mice. The positive control CGRP was different from the vehicle group (****p < 0.0001) but there were no significant effects with any of the treatments. One-way ANOVA, Tukey’s multiple comparisons, ****p < 0.0001. Groups were PBS + Veh (male n = 16, female n = 16), CGRP + Veh (male n= 16, female n = 16), CGRP + CBD 100 mg/kg (male n = 15, female n = 14), CGRP + THC 1 mg/kg (male n = 16, female n = 16), CGRP + CBD:THC 1:1 mg/kg (male n = 16, female n = 16), CGRP + CBD:THC 30:1 mg/kg (male n = 16, female n = 15), CGRP + CBD: THC 100:1 mg/kg (male n = 16, female n = 17). (c) Sensitivity to von Frey filaments in SNP-injected CD1 mice. There was no significant effect by any of the treatments and the positive control SNP was different from the vehicle group (****p < 0.0001). One-way ANOVA, Tukey’s multiple comparisons, ****p < 0.0001. Groups were PBS + Veh (male n = 17, female n = 16), SNP + Veh (male n = 15, female n = 16), SNP + CBD 100 mg/kg (male n = 15, female n = 16), SNP + THC 1 mg/kg (male n = 14, female n = 16), SNP + CBD:THC 1:1 mg/kg (male n = 14, female n = 17), SNP + CBD:THC 100:1 mg/kg (male n = 15, female n = 17). For all panels, open and closed symbols represent males and females, respectively. See Online Supplementary Table 7 for full statistical data.

Figure 6.

Cannabinoids did not induce adverse effects in CD1 mice. (a) Average time spent in the light during the light aversion assay in CD1 mice. None of the cannabinoid treatments changed the time spent in the light compared to vehicle. One-way ANOVA, Tukey’s multiple comparisons, p = 0.2732. Groups were Veh (male n = 11 (2 excl.), female n = 11), CBD 100 mg/kg (male n = 11, female n = 11), THC 1 mg/kg (male n = 10, female n = 12), CBD:THC 1:1 mg/kg (male n = 12, female n = 10), CBD:THC 100:1 mg/kg (male n = 11, female n = 11). (b) Average time spent in the center during the open field assay. None of the cannabinoid ratios the time spent in the center compared to vehicle. One-way ANOVA, Tukey’s multiple comparisons, p = 0.1764. Groups were Veh (male n = 8, female n = 7), CBD 100 mg/kg (male n = 8, female n = 8), THC 1 mg/kg (male n = 8, female n = 8), CBD:THC 1:1 mg/kg (male n = 8, female n = 9), CBD:THC 30:1 mg/kg (male n = 8, female n = 8), CBD:THC 100:1 mg/kg (male n = 7, female n = 8). (c) Total entries in novel arm of Y-maze assay. None of the cannabinoid ratios changed the number of entries of the novel arm compared to vehicle. One-way ANOVA, Tukey’s multiple comparisons, p = 0.5549. Groups were Veh (male n = 18, female n = 17), CBD 100 mg/kg (male n = 11, female n = 11), THC 1 mg/kg (male n = 9, female n = 11), CBD:THC 1:1 mg/kg (male n = 12, female n = 8), CBD:THC 100:1 mg/kg (male n = 11, female n = 9). (d) Average time to fall in the rotarod assay. None of the cannabinoid ratios changed the average time to fall compared to vehicle. The positive control diazepam (DZP) was significantly different from the vehicle group (**p = 0.0093). One-way ANOVA, Tukey’s multiple comparisons, ***p = 0.0001. Groups were Veh (male n = 17, female n = 15), CBD 100 mg/kg (male n = 11, female n = 11), THC 1 mg/kg (male n = 10, female n = 12), CBD:THC 1:1 mg/kg (male n = 11, female n = 10), CBD:THC 30:1 mg/kg (male n = 8, female n = 8), CBD:THC 100:1 mg/kg (male n = 11, female n = 10), DZP (male n = 7, female n = 8). (e) Average immobility time measured in the tail suspension assay. None of the cannabinoid ratios changed the average immobility time compared to vehicle. The positive control diazepam (DZP) was significantly different from the vehicle group (****p < 0.0001). One-way ANOVA, Tukey’s multiple comparisons, ****p < 0.0001. Groups were Veh (male n = 16, female n = 16), CBD 100 mg/kg (male n = 7, female n = 8), THC 1 mg/kg (male n = 8, female n = 8), CBD:THC 1:1 mg/kg (male n = 8, female n = 8), CBD:THC 30:1 mg/kg (male n = 8, female n = 8), CBD:THC 100:1 mg/kg (male n = 8, female n = 8), DZP (male n = 7, female n = 8). For all panels, open and closed symbols represent males and females, respectively. See Online Supplementary Table 8 for full statistical data.

Figure 6.

Cannabinoids did not induce adverse effects in CD1 mice. (a) Average time spent in the light during the light aversion assay in CD1 mice. None of the cannabinoid treatments changed the time spent in the light compared to vehicle. One-way ANOVA, Tukey’s multiple comparisons, p = 0.2732. Groups were Veh (male n = 11 (2 excl.), female n = 11), CBD 100 mg/kg (male n = 11, female n = 11), THC 1 mg/kg (male n = 10, female n = 12), CBD:THC 1:1 mg/kg (male n = 12, female n = 10), CBD:THC 100:1 mg/kg (male n = 11, female n = 11). (b) Average time spent in the center during the open field assay. None of the cannabinoid ratios the time spent in the center compared to vehicle. One-way ANOVA, Tukey’s multiple comparisons, p = 0.1764. Groups were Veh (male n = 8, female n = 7), CBD 100 mg/kg (male n = 8, female n = 8), THC 1 mg/kg (male n = 8, female n = 8), CBD:THC 1:1 mg/kg (male n = 8, female n = 9), CBD:THC 30:1 mg/kg (male n = 8, female n = 8), CBD:THC 100:1 mg/kg (male n = 7, female n = 8). (c) Total entries in novel arm of Y-maze assay. None of the cannabinoid ratios changed the number of entries of the novel arm compared to vehicle. One-way ANOVA, Tukey’s multiple comparisons, p = 0.5549. Groups were Veh (male n = 18, female n = 17), CBD 100 mg/kg (male n = 11, female n = 11), THC 1 mg/kg (male n = 9, female n = 11), CBD:THC 1:1 mg/kg (male n = 12, female n = 8), CBD:THC 100:1 mg/kg (male n = 11, female n = 9). (d) Average time to fall in the rotarod assay. None of the cannabinoid ratios changed the average time to fall compared to vehicle. The positive control diazepam (DZP) was significantly different from the vehicle group (**p = 0.0093). One-way ANOVA, Tukey’s multiple comparisons, ***p = 0.0001. Groups were Veh (male n = 17, female n = 15), CBD 100 mg/kg (male n = 11, female n = 11), THC 1 mg/kg (male n = 10, female n = 12), CBD:THC 1:1 mg/kg (male n = 11, female n = 10), CBD:THC 30:1 mg/kg (male n = 8, female n = 8), CBD:THC 100:1 mg/kg (male n = 11, female n = 10), DZP (male n = 7, female n = 8). (e) Average immobility time measured in the tail suspension assay. None of the cannabinoid ratios changed the average immobility time compared to vehicle. The positive control diazepam (DZP) was significantly different from the vehicle group (****p < 0.0001). One-way ANOVA, Tukey’s multiple comparisons, ****p < 0.0001. Groups were Veh (male n = 16, female n = 16), CBD 100 mg/kg (male n = 7, female n = 8), THC 1 mg/kg (male n = 8, female n = 8), CBD:THC 1:1 mg/kg (male n = 8, female n = 8), CBD:THC 30:1 mg/kg (male n = 8, female n = 8), CBD:THC 100:1 mg/kg (male n = 8, female n = 8), DZP (male n = 7, female n = 8). For all panels, open and closed symbols represent males and females, respectively. See Online Supplementary Table 8 for full statistical data.

Figure 7.

A 100:1 CBD:THC combination ameliorated acute head pain mimics following optogenetically induced CSD in freely behaving ChR2/WT mice. (a) Schematic representation of the locations of the recording electrodes and optic fiber used for light stimulation. CSDs were induced in the visual cortex (V1) in a minimally invasive manner by optogenetic (LED pulse) stimulation through the intact skull. Three silver ball-tip electrodes were connected to the skull overlaying the visual (V1), somatosensory (S1) and motor (M1) cortex for direct current (DC-potentials) recording of the CSD-related DC shifts. (b) Experimental design of mouse grimace scale (MGS) scoring following optogenetic induction of three CSD events. Post-CSD MGS scores were assessed using a longitudinal design, organized into four test days with at least 48 h of resting time in between. The first test day served as control without cannabinoid pretreatment. The remaining test days were randomly assigned between vehicle, 30:1 or 100:1 CBD:THC pretreatment. Injections (i.p.) were performed at 30 min prior to the start of the first CSD induction. On each test day, after a 30-min baseline recording to ensure stability of DC signals and allow for acclimatization of the mouse in the video-setup for MGS scoring, three CSDs were induced optogenetically within a 10-min timeframe. Behavioral monitoring was performed 10 min before CSD (as a baseline) and after CSD, i.e., at 30 min (1), 24 h (2) and 48 h (3) after the last CSD. (c) Representative DC-potential shifts during optogenetic induction of three CSDs in a freely behaving ChR2/WT mouse recorded 30-min after vehicle, 30:1 and 100:1 CBD:THC pretreatment. (d) MGS scores were assessed 30 min after CSD induction and presented as % calculated of pre-CSD baseline values (taken as 100%). For the raw values and time series data including the 24- and 48-h MGS scores see Online Supplementary Figure 4. Pretreatment with the 100:1 CBD:THC combination reduced MGS scores at the 30-min time post-CSD point in comparison to vehicle (Veh) pretreatment (*p = 0.0158). Effects of pretreatment with a 30:1 CBD:THC combination were less pronounced and not different against vehicle (p = 0.2542). Kruskal-Wallis test, Dunnett’s multiple comparisons, *p = 0.0123. Groups of mixed males and females were Veh (n = 6), CBD:THC 30:1 mg/kg (n = 6), CBD:THC 100:1 mg/kg (n = 6). For all panels, open and closed symbols represent males and females, respectively. See Online Supplementary Table 9 for full statistical data.

Figure 8.

A 100:1 CBD:THC combination ameliorated acute head pain mimics following optogenetically induced CSD in freely behaving ChR2/FHM1 mutant mice. (a, b) Configurations of DC-potential electrodes, optic fiber placements for stimulation (using LED pulses), and experimental design for post-CSD MGS assessment were similar to those used for ChR2/WT mice (and described in Figure 7). (c) Representative DC-potential shifts during optogenetic induction of three CSDs in a freely behaving ChR2/FHM1 mutant mouse recorded 30 min after vehicle, 30:1 and 100:1 CBD:THC pretreatment. (d) MGS scores were assessed 30 min after CSD induction and presented as percentage of pre-CSD baseline values (taken as 100%). For the raw values and time series data including the 24- and 48-h MGS scores, see Online Supplementary Figure 4 a–c. Pretreatment with a 100:1 CBD:THC combination reduced MGS scores at the 30-min post-CSD time point in comparison to vehicle (Veh) pretreatment (*p = 0.0103). Effects of pretreatment with a 30:1 CBD:THC combination was less pronounced and only showed a trend-significant reduction compared to vehicle pretreatment (p = 0.06). Kruskal-Wallis test, Dunnett’s multiple comparisons, **p = 0.0037. Groups of mixed male and female males were Veh (n = 7), CBD:THC 30:1 mg/kg (n = 7), CBD:THC 100:1 mg/kg (n = 7). For all panels, open and closed symbols represent males and females, respectively. See Online Supplementary Table 9 for full statistical data.

Figure 9.

Ratios of CBD:THC did not aggravate CSD-related transient motor dysfunction in FHM1 mutant and C57BL/6 mice. (a) Grab duration of the left and right forepaws, as assessed by wire grip testing, was assessed following optogenetic CSD induction in V1 cortex. In ChR2/WT and ChR2/FHM1 mutant mice, when a CSD reached the M1 cortex, a transient impairment of left forepaw motor function was observed that was consistent across pretreatments with vehicle, 30:1 CBD:THC or 100:1 CBD:THC. The grey area in the graphs spanning from 5 to 6 min after the end of the M1 CSD wave represents the time period used for comparison across the different treatment groups. (b) In ChR2/WT mice, none of the cannabinoid treatments aggravated motor function of the left forepaw in relation to the right forepaw against vehicle (Veh): 30:1 CBD:THC (p = 0.2264) and at 100:1 CBD:THC (p = 0.4156). Kruskal-Wallis test, Dunnett’s multiple comparisons, p = 0.1657. Groups of mixed male and female mice were Veh (n = 4), CBD:THC 30:1 mg/kg (n = 4), CBD:THC 100:1 mg/kg (n = 4). (c) In ChR2/FHM1 mutant mice, none of the cannabinoid treatments aggravated motor function of the left forepaw in relation to the right fore paw against vehicle (Veh): 30:1 CBD:THC (p > 0.9999) and 100:1 CBD:THC (p = 0.0581). Kruskal-Wallis test, Dunnett’s multiple comparisons, *p = 0.0398. Groups of mixed male and female mice were Veh (n = 4), CBD:THC 30:1 mg/kg (n = 4), CBD:THC 100:1 mg/kg (n = 4). For all panels, open and closed symbols represent males and females, respectively. See Online Supplementary Table 13 for full statistical data.