Orally consumed cannabinoids provide long-lasting relief of allodynia in a mouse model of chronic neuropathic pain

Abstract

Chronic pain affects a significant percentage of the United States population, and available pain medications like opioids have drawbacks that make long-term use untenable. Cannabinoids show promise in the management of pain, but long-term treatment of pain with cannabinoids has been challenging to implement in preclinical models. We developed a voluntary, gelatin oral self-administration paradigm that allowed male and female mice to consume ∆⁹-tetrahydrocannabinol, cannabidiol, or morphine ad libitum. Mice stably consumed these gelatins over 3 weeks, with detectable serum levels. Using a real-time gelatin measurement system, we observed that mice consumed gelatin throughout the light and dark cycles, with animals consuming less THC-gelatin than the other gelatin groups. Consumption of all three gelatins reduced measures of allodynia in a chronic, neuropathic sciatic nerve injury model, but tolerance to morphine developed after 1 week while THC or CBD reduced allodynia over three weeks. Hyperalgesia gradually developed after sciatic nerve injury, and by the last day of testing, THC significantly reduced hyperalgesia, with a trend effect of CBD, and no effect of morphine. Mouse vocalizations were recorded throughout the experiment, and mice showed a large increase in ultrasonic, broadband clicks after sciatic nerve injury, which was reversed by THC, CBD, and morphine. This study demonstrates that mice voluntarily consume both cannabinoids and opioids via gelatin, and that cannabinoids provide long-term relief of chronic pain states. In addition, ultrasonic clicks may objectively represent mouse pain status and could be integrated into future pain models.

Fig. 1. Mice stably consume cannabinoid and opioid gelatin over 3 weeks.

a Schematic of study design for chronic neuropathic pain and gelatin consumption. b Average daily consumption of gelatin for the 4 groups (control, THC, CBD, and morphine, n = 10–17). Prior to pSNL, all groups consumed control gelatin. Mice consuming THC-gelatin ate significantly less than control mice (p = 0.003). c Total consumption of gelatin post-pSNL for control, THC, CBD, and morphine gelatins. Total THC-gelatin consumption was significantly lower than control mice (p = 0.005). d Comparisons within groups between the average consumption over the last 2 days prior to pSNL, days 6–7 after pSNL, and days 13–14 after pSNL. The top row represents animals who only received THC or CBD after pSNL. The bottom row represents animals that received THC or CBD before and after pSNL. Animals receiving drug gelatin prior to pSNL showed no differences in consumption after pSNL. e Serum levels of THC, CBD, and morphine collected either 7 or 24 h after the final gelatin insertion (n = 2–6). *p < 0.05, **p < 0.01, data represent the mean ± SEM.

Fig. 2. Microstructure of gelatin consumption.

a Schematic and signal flow of piezoelectric load cell that measures second-by-second consumption and disturbances of the gelatin cup. b Example of cumulative gelatin consumption and disturbances of the gelatin cup over 18 h from one mouse. c Correlation between the number of disturbances of the gelatin cup and the consumption of gelatin in 5 min windows (n = 126 points from 14 animals, p < 0.001). d Average cumulative consumption of the control, THC, CBD, and morphine gelatins over the 18 h measurement period. By 18 h, THC mice showed significantly less cumulative consumption than controls (n = 3–4 mice per group, p = 0.04). e Average number of bouts, defined as a set of disturbances preceded or succeeded by at least 2 min, during 2 h windows throughout the measurement period. There were no differences between the groups in the number of bouts (n = 3–4 mice per group). *p < 0.05, data represent the mean ± SEM.

Fig. 3. Cannabinoid consumption decreases measurements of neuropathic pain over 3 weeks.

a Paw withdrawal thresholds, plotted as a percentage of pre-pSNL response, are shown 1 day after pSNL, and on the 6 subsequent testing days after introduction of either control, THC, CBD, or morphine gelatin. THC and CBD groups showed long-lasting reduction in allodynia, while the effect of morphine lasted approximately 1 week (n = 10–19 per group). b Comparison of percentage of pre-pSNL response on day 22, the final day of pain testing, between the 4 treatment groups (n = 10–19 per group). c Latency to respond to hot plate, plotted as a percentage of pre-pSNL response, are shown 1 day after pSNL, and on the 6 subsequent testing days after introduction of either control, THC, CBD, or morphine gelatin. The morphine group showed an analgesic response in the first week, while no other groups were significantly different from control (n = 7–12 per group). d Comparison of percentage of pre-pSNL response on day 22. THC significantly reduced hyperalgesia while there was a trend effect of CBD (n = 7–12 per group). *p < 0.05, data represent the mean ± SEM.

Fig. 4. Measures of analgesia and body temperature in neuropathic pain-naïve mice.

a Change in body temperature 2 h after either vehicle, THC (30 mg/kg i.p.), or CBD (30 mg/kg i.p.) experimenter-administered dose in 7-day gelatin consuming animals or drug-naïve controls. THC injected mice showed the predicted decrease in body temperature in both groups, whereas CBD showed no change (n = 4 per group). b Change in locomotor activity after either vehicle, THC (30 mg/kg i.p.), CBD (30 mg/kg i.p.), or morphine (10 mg/kg i.p.) experimenter-administered dose in 7-day gelatin consuming animals or drug-naïve controls (n = 3–4 per group). c Change in latency to pain response 2 h after either vehicle, THC (30 mg/kg i.p.), CBD (30 mg/kg i.p.), or 30 min after morphine (10 mg/kg i.p.) experimenter-administered dose in 7-day gelatin consuming animals or drug-naïve controls. THC injected mice in both groups showed a similar increase in analgesic response, while CBD animals showed no change. Drug-naïve morphine injected animals showed analgesia, while there was no change in mice that have consumed morphine for 7 days (n = 4–8 per group). d Change in paw withdrawal threshold 2 h after either vehicle, THC (30 mg/kg i.p.), CBD (30 mg/kg i.p.), or 30 min after morphine (10 mg/kg i.p.) experimenter-administered dose in 7-day gelatin consuming animals or drug-naïve controls. THC injected mice in both groups showed a similar increase in paw withdrawal threshold, while CBD animals again showed no change. Drug-naïve morphine injected animals showed an increase in paw withdrawal threshold, while there was no change in mice that have consumed morphine for 7 days (n = 3–4 per group). *p < 0.05, **p < 0.01, data represent the mean ± SEM.

Fig. 5. Ultrasonic click vocalizations track pain state.

a Two representative ultrasonic clicks. Each is under 10 ms, wider than 10 kHz, with no appreciable power in the audible range. Dashed line represents 20 kHz, the upper limit of the human audible range. b Number of ultrasonic clicks per 5 min recording interval. Colors represent control (black), THC (red), CBD (blue), and morphine (green) gelatin mice (n = 4–7 per group). pSNL significantly increased the number of clicks (p < 0.001). After 4 days of gelatin, the number of clicks remained elevated in control mice, while THC, CBD, and morphine mice showed decreases similar to baseline counts. c Separated clicks for each treatment group. All groups showed a significant increase in clicks after pSNL, and in THC, CBD, and morphine consuming mice clicks significantly decreased after drug-gelatin consumption. *p < 0.05, **p < 0.01, data represent the mean ± SEM.