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. Author manuscript; available in PMC: 2026 Feb 1.
Published in final edited form as: Pharmacol Rep. 2024 Dec 12;77(1):172–181. doi: 10.1007/s43440-024-00687-1

Select Terpenes from Cannabis sativa are Antinociceptive in Mouse Models of Post-Operative Pain and Fibromyalgia via Adenosine A2a Receptors

Caleb A Seekins 1,#, Alyssa M Welborn 1,#, Abigail M Schwarz 1, John M Streicher 1,2
PMCID: PMC11974608  NIHMSID: NIHMS2067124  PMID: 39663308

Abstract

Background:

Terpenes from Cannabis show promise for pain management. Our lab found that the terpenes geraniol, linalool, β-caryophyllene, and α-humulene relieve chemotherapy-induced peripheral neuropathy via Adenosine A2a receptors (A2aR). This suggests terpenes as potential non-opioid, non-cannabinoid therapeutics. In this study, we investigated post-operative and fibromyalgia pain, expanding potential terpene applications to different pain types.

Methods:

Male and female CD-1 mice had their baseline mechanical sensitivity measured via von Frey filaments and underwent either paw incision surgery or reserpine-induced fibromyalgia (0.32 mg/kg, sc). After pain was established, the mice received 200 mg/kg ip of a terpene, and their mechanical sensitivity was measured over three hours. To determine the potential mechanism of action, mice were given the A2aR antagonist istradefylline (3.2 mg/kg, ip) 10 minutes before terpene, with mechanical sensitivity measured after. Hot plate pain testing was performed as a control.

Results:

Terpene treatment caused time-dependent elevation of the mechanical thresholds of the mice from both pain models, strongest for geraniol, then linalool or α-humulene, indicating that these four terpenes are anti-nociceptive in post-surgical and fibromyalgia pain. Pretreatment with istradefylline blocked antinociception, suggesting the terpenes act via the A2aR in these pain models. Terpenes had no effect on hot plate latencies, ruling out non-specific motor effects.

Conclusions:

These results demonstrate that the terpenes geraniol, linalool, β-caryophyllene, and α-humulene may be a viable medication for post-operative and fibromyalgia pain relief. Their mechanism of action via the A2aR furthers our knowledge of its importance in pain processing and as a target of terpene drugs.

Keywords: Cannabis, Terpenes, Post-Operative Pain, Fibromyalgia, Adenosine A2a Receptors

Introduction

The use of Cannabis for pain management has become increasingly popular and more accessible within the United States in recent years. Nearly 30% of pain patients use Cannabis to mitigate their symptoms in states where medical Cannabis use is legal [1]. Cannabis contains a multitude of compounds, and much research has been done to establish the effects of phytocannabinoids like cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC). However, there have been fewer investigations into other classes of compounds from the plant, such as terpenes. Terpenes are small, aromatic hydrocarbons that we have shown to relieve acute thermal pain and chronic chemotherapy-induced peripheral neuropathy (CIPN) [2, 3]. Terpenes may thus offer a path for Cannabis pain relief without the negative psychotropic effects of phytocannabinoids such as THC. We found that the terpenes geraniol, linalool, β-caryophyllene (BCP), and α-humulene (α-Hum) are robustly antinociceptive in the CIPN pain model by acting on adenosine A2a receptors (A2aR) in the spinal cord [3]. However, it is not clear if the same terpenes could effectively treat other pain types, such as post-surgical and fibromyalgia pain.

Approximately 40 million Americans undergo major surgery each year; 30% of patients report moderate to severe pain, while 11% report severe pain within the first 24 hours post-operation [4, 5]. 9.2-13% of patients report postoperative chronic opioid use, which increases the likelihood of opioid use disorder [6]. However, the effective management of pain post-operation is essential and associated with shorter recovery times, reduced risks of post-surgery complications, and aids in the transition back to everyday life [5, 7]. Pain relief is also a human right [8]. Due to the risks associated with opioid use, many people are turning to alternative pain management strategies such as Cannabis. Cannabis, however, comes with its own set of side effects and has been reported as not effective for reducing opioid intake post-surgery [1, 9]. Some studies show Cannabis consumption post-surgery may even increase opioid consumption and lead to poorer postoperative outcomes, including increased reported pain [9, 10]. To our knowledge, no studies have tested the impact of terpenes on post-surgical pain.

Fibromyalgia is another common condition where the main symptom is pain (2-8% of the population, and more common in women); patients experience chronic pain, fatigue, and often depression [11]. Serotonin-Norepinephrine Reuptake Inhibitors (SNRI) such as duloxetine and milnacipran are often prescribed to manage fibromyalgia symptoms, as many patients have dysregulated monoamine neurotransmitters [12, 13]. However, a recent survey reported that almost half (49.5%) of responding patients with fibromyalgia have used Cannabis to manage their symptoms, primarily pain (98.9%) [14]. As previously stated, Cannabis use is often accompanied by unwanted psychotropic effects; it is therefore important to investigate therapeutic alternatives that have fewer side effects but are also effective for pain management, such as terpenes. Though it has previously been hypothesized that terpenes may play a role in the effectiveness of Cannabis in mitigating fibromyalgia symptoms, this is the first study to investigate isolated terpenes in an in vivo preclinical model of fibromyalgia.

This study continues our earlier work, which indicated that the terpenes geraniol, linalool, BCP, and α-hum relieve CIPN pain by acting on the A2aR receptor [3]. Here, we assessed the antinociceptive efficacy of the same terpenes in mouse models of post-surgical paw incision pain and reserpine-induced fibromyalgia. We further investigated A2aR as a potential mechanism of action of the terpenes in these models.

Methods

Drugs:

We diluted the terpenes geraniol (Alfa Aesar, Ward Hill, MA, Lot #10211653), linalool (Alfa Aesar, Lot #10216518), β-caryophyllene (Tokyo Chemical Industry, Lot #ARY4A-HD), and α-humulene (Tokyo Chemical Industry, Tokyo Japan, Lot #MTQZC-AI) in 10% DMSO, 10% Tween80 and 80% USP saline for injections. The A2aR antagonist istradefylline (Tokyo Chemical Industry, Lot #2PJUO-MT) was dissolved in 20% DMSO, 10% Tween80 and 70% USP saline. The opioid oxycodone (Mallinckrodt, St. Louis MO, #8865) was prepared in the same vehicle as the terpenes. Reserpine (Thermo Scientific, United Kingdom, Lot #5002J13A) was dissolved in 0.5% glacial acetic acid and 99.5% sterile USP water. Gentamicin (Fresenius Kabi, Lake Zurich, IL, Lot #6126509) was diluted in USP saline to 1 mg/mL for injection post-surgery. The antibiotic ointment Neosporin (Johnson and Johnson, Lititz, PA, Lot# 0193LZ) was used topically to prevent wound infection after surgery. All of the drugs were made less than an hour prior to the experiment. We used matched vehicle injections as a control.

Animals:

We obtained 5-8-week-old male and female CD-1 (a.k.a. ICR) mice from Charles River Laboratories for all experiments. Approximately equal numbers of male and female mice were used. Upon arriving at the University of Arizona vivarium, the mice were given at least five days to acclimate to the temperature and humidity-controlled space. We kept the mice on a 12-hour conventional light (7 AM-7 PM) and dark cycle and gave them ad libitum access to standard chow and water. Experiments were performed during the light cycle. Mice were acclimated to the experimental room for at least 30 minutes prior to any experimental manipulation. We then randomly assigned mice to treatment groups to which the experimenters were blinded via coded drug vials. Unblinding occurred after all of the data for an experiment had been collected. All of the experiments conducted were approved by the Institutional Animal Care and Use Committee at The University of Arizona (Protocol ID #16-078) and conducted according to the guidelines of the NIH Guide for the Care and Use of Laboratory Animals and the International Association for the Study of Pain Guidelines for the Use of Animals in Research. For the whole study, 374 animals were used in total. The target sample size for each group was N = 10 (5 male, 5 female); a few groups lost animals, resulting in a sample size of N = 9. For some experiments, a vehicle comparison was included in every cohort, so that the total vehicle sample size was higher (N = 50 for paw incision, N = 28 for fibromyalgia). These details are further noted in the Figure Legends.

Post-surgical pain:

We carried out the paw incision post-operative pain model as we have previously described [15]. We placed the mice under anesthesia with isoflurane (5% induction, 3% maintenance in standard air). The left hind paw was sterilized with iodine and 70% ethanol. We made a longitudinal incision through the skin and fascia with a no. 11 scalpel (~1 cm in length) to expose the plantar muscle with curved tweezers and made multiple shallow incisions parallel to the muscle fibers with the scalpel to cause tissue damage, with the origin and insertion intact. We then closed the incision with 5-0 Ethicon vicryl sutures and applied Neosporin to the closed wound followed by an injection of gentamicin (0.3 mg/kg, sc). The mouse was then returned to its enclosure for monitoring prior to returning to the animal colony. We allowed the mice to recover for 24 hours before drug treatment.

Fibromyalgia pain:

Reserpine was solubilized for injections as described above. Reserpine reduction of monoamines is a commonly used method for inducing fibromyalgia-like symptoms in rodents; we chose this model as it has been well-established and induces a majority of the symptoms associated with fibromyalgia [16]. We induced the pain state with 3 daily injections of 0.32 mg/kg sc reserpine on days 1-3 as reported in the literature [17, 18]. On day 4 we performed drug treatment and measurement.

von Frey assay:

We assessed allodynia for both post-surgical and fibromyalgia pain by collecting mechanical threshold data via the up-down method using von Frey filaments as we have previously described [3, 19]. First, we determined each mouse's mechanical threshold baseline before pain model treatment. We also measured the post-treatment baseline after the pain model was implemented but before drug treatment. Any mice that did not exhibit sufficient pain behavior (<0.5 g mechanical threshold) were excluded from the study. The mice were then administered terpene, istradefylline, or vehicle via ip injection, and mechanical thresholds were recorded over a three-hour time course.

Acute nociceptive hot plate pain assay:

A hot/cold plate device from Columbus Instruments (Columbus, OH) was used for this assay. The device was set to 55°C with a 30-second cutoff to prevent tissue damage. The mouse was placed on the plate, and the latency to withdraw and/or lick one of their hind paws was recorded. Baseline measurements were performed, followed by injection of terpene, oxycodone, or vehicle, followed by repeated measurements over a 120-minute time course.

Data analysis:

All of our data are reported as the mean ± SEM of raw threshold values, without normalization. Von Frey and hot plate time course data were analyzed via a repeated measures (RM) two-way ANOVA followed by Sidak’s post hoc test when 2 treatment groups were compared or Dunnett’s test for 3 or more groups. We considered p ≤ 0.05 a significant difference between groups for all statistical analyses. We completed an area under the curve (AUC) analysis for each von Frey time course using the AUC tool in GraphPad Prism 10.2.3. The treated groups were compared to the vehicle group via one-way ANOVA followed by a Dunnet’s post hoc test for the primary AUC pain data, or each istradefylline group compared to its own vehicle pre-treatment group by two-way ANOVA followed by Sidak’s post hoc test for the istradefylline experiments. All graphing and statistical analysis was completed using GraphPad Prism 10.2.3. Male and female mice were included in every experiment; no sex differences were found (p > 0.05), so both sexes were combined for analysis in the primary figures (separated male and female data are shown in the Supplement).

Results

As noted above, we sought to test 4 terpenes found in Cannabis sativa in post-surgical and fibromyalgia pain models. All experimental drugs were delivered by the ip route in male and female CD-1 mice. The terpene and istradefylline doses were established in our previous work, including the demonstration that our selected istradefylline dose does not alter mechanical thresholds [2, 3]. No sex differences were found, so the male and female groups were combined for primary analysis (separated data shown in the Supplement). Detailed statistical values for each data set are provided in the Figure Legends.

Terpenes relieve post-surgical pain

We tested the terpenes geraniol, linalool, BCP, and α-hum in a paw incision model to assess their antinociceptive efficacy in postoperative pain. We measured the pre-operation mechanical threshold using von Frey filaments. The mice immediately underwent paw incision surgery on their left hind paw, as described in the Methods. They recovered in their home cages for 24-hours, followed by measurement of their post-surgical mechanical threshold. After collecting the post-operative baseline, we injected the mice with 200 mg/kg of a terpene or vehicle as a control. Each terpene increased mechanical threshold significantly in comparison to the mice treated with vehicle (Figure 1A). The AUC was significantly different between geraniol vs. vehicle, and α-humulene vs vehicle (Figure 1B); linalool and BCP also appeared to be elevated but did not rise to the level of statistical significance. We did not identify significant differences between the mechanical thresholds between male and female mice treated with the same terpene (Figure S1). Taken together, the data suggests that all 4 terpenes are efficacious in relieving post-surgical pain.

Figure 1: Select terpenes are antinociceptive in a paw incision post-operative pain model.

Figure 1:

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Both male and female CD-1 mice had post-operative pain induced on their left hind paw via paw incision as described in the Methods. The data presented in these figures are shown as the mean ± SEM with a sample size of N = 10-50/group, as vehicle-treated mice were investigated in each technical replicate as a control group and thus have a higher number of mice. (A) Pre-Op BL = Pre-operation baseline, Post-Op BL = Post-operation baseline. After paw incision, the mice were given terpene (geraniol, linalool, β-caryophyllene or α-humulene) (200mg/kg, ip) as indicated or vehicle control, and the mechanical threshold was measured via von Frey filaments. RM two-way ANOVA: Time (F8,760 = 291.1, p < 0.0001); Treatment (F4,95 = 31.11, p < 0.0001); Interaction (F32,760 = 17.18, p < 0.0001). **** = p < 0.0001 vs. vehicle for all terpenes; ## = p < 0.01 vs. vehicle for 3 terpenes (excepting linalool); $, $$$$ = p < 0.05, 0.0001 vs. vehicle for geraniol; all by Dunnett’s post hoc test. (B) Area under the curve (AUC) values were calculated for each terpene and vehicle control and are shown here. One-way ANOVA: (F4,95 = 13.34, p < 0.0001). *, **** = p < 0.05, 0.0001 vs. vehicle group by Dunnett's post hoc test.

Terpenes relieve reserpine-induced fibromyalgia pain

We then assessed the same terpenes for their antinociceptive efficacy in a reserpine-induced fibromyalgia model of pain. After measuring the baseline mechanical threshold, we gave the mice reserpine (0.32 mg/kg, sc) over three days as per the Methods. On the fourth day, we measured their post-treatment mechanical threshold to establish that they had acquired pain behavior. Each mouse was then injected with a terpene (200 mg/kg, ip) or vehicle, and we measured their subsequent mechanical threshold over 3 hours. All four terpenes we tested, geraniol, linalool, BCP, and α-hum, significantly increased the mechanical threshold in comparison to vehicle-treated mice for at least two time points (Figure 2A). For the AUC analysis of the time course data, geraniol and linalool were statistically different from vehicle-treated mice; BCP and α-hum were elevated over vehicle but not statistically significant (Figure 2B). Similar to the paw incision model, we observed no differences in male and female mice in the measured pain behavior (Figure S2). These observations suggest that these four terpenes are also efficacious in a fibromyalgia pain model.

Figure 2: Select terpenes are antinociceptive in a reserpine-induced fibromyalgia pain model.

Figure 2:

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Male and female CD-1 mice were given injections of reserpine (0.32 mg/kg, sc) once a day over 3 days as described in the Methods. All of the data presented is shown as the mean ± SEM, with a sample size of N = 9-28/group, as vehicle-treated mice were investigated in multiple technical replications as a control group and thus have a higher number of mice. (A) Pre-Res BL = Pre-reserpine baseline, Post-Res BL = Post-reserpine baseline. After fibromyalgia-like pain was established, on day 4 the mice were given terpene (geraniol, linalool, β-caryophyllene or α-humulene) (200 mg/kg, ip) as indicated or vehicle control, and the mechanical threshold was measured via von Frey filaments. RM two-way ANOVA: Time (F8,416 = 72.57, p < 0.0001); Treatment (F4,52 = 9.246, p < 0.0001); Interaction (F32,416 = 6.246, p < 0.0001). *** = p < 0.001 vs. vehicle for 3 terpenes (excepting BCP); ## = p < 0.01 vs. vehicle for all terpenes; $$$$ = p < 0.0001 vs. vehicle for 3 terpenes (excepting α-Hum); &&& = p < 0.001 vs. vehicle for geraniol; all by Dunnett’s post hoc test. (B) The AUC values were calculated for each terpene and vehicle control. One-way ANOVA: (F4,62 = 5.939, p = 0.0004). **, *** = p < 0.01, 0.001 vs. vehicle control via Dunnett's post hoc test.

Terpene antinociception is mediated by the A2aR

Our previous work showed that the terpenes geraniol, linalool, β-caryophyllene, and α-humulene relieve CIPN by acting on A2aRs in the spinal cord [3]. We therefore sought to determine if a similar mechanism was at work with the pain reduction we observed after administering terpenes to mice with paw incision post-operative pain and reserpine-induced fibromyalgia. The mice had paw incision surgery as previously described, and after baseline measurements were taken, we administered the A2aR antagonist istradefylline (3.2 mg/kg, ip) or vehicle. We followed this ten minutes later with an injection of a terpene, and the mechanical threshold was measured. The mice that were given istradefylline exhibited no measurable antinociception and were significantly different from vehicle-pretreated terpene controls, suggesting the antinociceptive effects of the terpenes in a post-operative model of pain are A2aR mediated (Figure 3A-D). The AUC of each curve supported this conclusion (Figure 3E).

Figure 3: Terpenes alleviate post-operative pain via Adenosine A2a Receptors (A2aR).

Figure 3:

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Both male and female CD-1 mice underwent paw incision on their left hind paw as described in the Methods to induce post-operative pain. All data is presented as the mean ± SEM, and the sample size is N = 10/group. (A-D) Pre-Op BL = Pre-operation baseline, Post-Op BL = Post-operation baseline. After post-operative pain was established, the mice were given either the A2aR antagonist istradefylline (3.2 mg/kg, ip) or vehicle ten minutes before they were given terpene (A: geraniol, B: linalool, C: β-caryophyllene, or D: α-humulene) (200 mg/kg, ip). The mechanical threshold was then measured via von Frey filaments. A: RM two-way ANOVA: Time (F8,144 = 43.04, p < 0.0001); Treatment (F1,18 = 36.60, p < 0.0001); Interaction (F8,144 = 13.71, p < 0.0001). B: RM two-way ANOVA: Time (F8,144 = 70.06, p < 0.0001); Treatment (F1,18 = 34.08, p < 0.0001); Interaction (F8,144 = 17.51, p < 0.0001). C: RM two-way ANOVA: Time (F8,144 = 70.99, p < 0.0001); Treatment (F1,18 = 34.96, p < 0.0001); Interaction (F8,144 = 17.62, p < 0.0001). D: RM two-way ANOVA: Time (F8,144 = 138.6, p < 0.0001); Treatment (F1,18 = 56.27, p < 0.0001); Interaction (F8,144 = 29.38, p < 0.0001). ***, **** = p < 0.001, 0.0001 vs. same time point terpene + istradefylline group by Sidak’s post hoc test. (E) AUC values were calculated for istradefylline + terpene and vehicle + terpene control mice. Two-way ANOVA: Pre-Treatment (F1,72 = 48.24, p < 0.0001); Terpene (F3,72 = 1.030, p = 0.3847); Interaction (F3,72 = 1.026, p = 0.3863). *, **, **** = p < 0.05, 0.01, 0.0001 vs. same terpene istradefylline group by Sidak’s post hoc test.

We followed the same experimental design to investigate this potential mechanism in the fibromyalgia model. Similar to the post-operative mice, the fibromyalgia mice treated with istradefylline and terpene had no measurable antinociception, showing that the antagonist completely blocked terpene pain relief (Figure 4A-D). This suggests that the terpenes we tested act on the A2aR to induce antinociception in the reserpine-induced fibromyalgia model. The AUC analysis again supported this interpretation (Figure 4E). As for the experiments above, male and female mice showed no significant differences within treatment groups, so were combined for analysis. The disaggregated sex data for the istradefylline experiments is shown in Figures S3-S4.

Figure 4: Terpenes alleviate reserpine-induced fibromyalgia pain via the A2aR.

Figure 4:

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Male and female CD-1 mice were given injections of reserpine (0.32 mg/kg, sc) as per the Methods to induce fibromyalgia-like pain. The data presented in these figures is represented as the mean ± SEM, and the sample size is N = 9-10/group. (A-D) Pre-Res BL = Pre-reserpine baseline, Post-Res BL = Post-reserpine baseline. After fibromyalgia-like pain was established, the mice were given either the A2aR antagonist istradefylline (3.2 mg/kg, ip) or vehicle. Ten minutes later the mice were given terpene (A: geraniol, B: linalool, C: β-caryophyllene, or D: α-humulene) (200 mg/kg, ip). Mechanical thresholds were then measured via von Frey filaments. A: RM two-way ANOVA: Time (F8,144 = 27.78, p < 0.0001); Treatment (F1,18 = 16.25, p = 0.0008); Interaction (F8,144 = 9.113, p < 0.0001). B: RM two-way ANOVA: Time (F8,144 = 39.17, p < 0.0001); Treatment (F1,18 = 30.71, p < 0.0001); Interaction (F8,144 = 11.96, p < 0.0001). C: RM two-way ANOVA: Time (F8,136 = 29.48, p < 0.0001); Treatment (F1,17 = 30.83, p < 0.0001); Interaction (F8,136 = 4.953, p < 0.0001). D: RM two-way ANOVA: Time (F8,136 = 36.45, p < 0.0001); Treatment (F1,17 = 11.09, p = 0.004); Interaction (F8,136 = 4.963, p < 0.0001). **, **** = p < 0.01, 0.0001 vs. same time point terpene + istradefylline group by Sidak’s post hoc test. (E) AUC values were calculated for istradefylline + terpene and vehicle + terpene mice. Two-way ANOVA: Pre-Treatment (F1,70 = 35.21, p < 0.0001); Terpene (F3,70 = 0.4984, p = 0.6846); Interaction (F3,70 = 0.4836, p = 0.6947). *, ** = p < 0.05, 0.01 vs. same terpene istradefylline group with Sidak’s post hoc test.

Terpene antinociception is not due to motor suppression

One potential confounding factor in our analysis is that the von Frey assay requires a motor response, and our previous work showed that the terpenes are sedative and reduce motor activity [2]. To help rule out motor suppression as a confound we performed the acute nociceptive hot plate pain assay. In this assay, we found that the terpenes produced no antinociceptive response, the same as vehicle, while an oxycodone positive control showed robust antinociception (Figure 5). As above, males and females showed no sex differences, and the disaggregated sex data is shown in Figure S5. This experiment suggests that the mice can respond with normal motor function in a pain assay, and helps rule out sedation and/or motor suppression as the source of the von Frey responses above.

Figure 5: Terpenes are not antinociceptive in an acute thermal nociceptive hot plate pain assay.

Figure 5:

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Male and female CD-1 mice were tested using a 55°C Hot Plate assay (see Methods). The baseline (BL) response was recorded, followed by injection of terpene (200 mg/kg), oxycodone positive control (5 mg/kg), or vehicle control, all by the ip route. Nociceptive thresholds were measured over a time course. The data are represented as the mean ± SEM, with N = 9-10 mice/group. RM two-way ANOVA: Time (F7,371 = 8.768, p < 0.0001); Treatment (F5,53 = 15.21, p < 0.0001); Interaction (F35,371 = 7.442, p < 0.0001). *, **** = p < 0.05, 0.0001 vs. same time point vehicle group with Dunnett’s post hoc test. Oxycodone produced robust antinociception as expected, while terpenes produced no response; this observation suggests that the terpenes do not produce motor suppression which could confound our von Frey analysis above.

Discussion

This study was conducted as a continuation of our previous work investigating the use of terpenes in pain management. Previously, we found the terpenes geraniol, linalool, BCP and α-Hum to be effective in relieving CIPN and inflammatory pain in mice [3]. Their efficacy in other pain models, such as post-operative and fibromyalgia pain, had not yet been defined. Different types of pain often require different interventions for their mitigation. CIPN and post-operative pain are often managed with opioid analgesics, while a recommended treatment for fibromyalgia, as well as CIPN, includes SNIRIs [20-23]. Through this work, we found that these terpenes have significant antinociceptive effects in both the paw incision post-operative pain and the reserpine-induced fibromyalgia models in mice, reaching similar mechanical thresholds to those we observed with terpene treatment for CIPN pain [3]. This suggests that terpenes could be potential novel therapeutics for all of the above pain types.

Our previous work showed that the terpenes we tested act on A2aR in the spinal cord [3]. We did not explicitly test the terpenes in the spinal cord for the fibromyalgia and post-operative pain models, though this is likely the mechanism of action. We previously observed that these terpenes have a weak analgesic effect in an acute thermal pain model. Mice given terpenes had only a slight increase in tail flick latency after being injected with one of the terpenes, and this effect was blocked by a cannabinoid 1 receptor (CBR1) inverse agonist [2]. Similarly, in our study here, we showed the terpenes have absolutely no efficacy in the acute nociceptive hot plate assay. The overlapping mechanism involving the A2aR in post-operative, fibromyalgia, and CIPN pain models but not acute thermal pain suggests these terpenes act on a shared central mechanism specific to pathological pain states. The shared mechanism of A2aR in pathological pain suggests that the A2aR may be upregulated in the spinal cord in chronic pain states and leads to pain relief when the terpenes we tested act upon it. One limitation, however, is our sole use of von Frey mechanical testing as a pain assay in this study. Future work should use other pain modalities such as thermal and non-evoked assays such as burrowing or home cage monitoring to fully demonstrate terpenes’ efficacy in pathological but not acute nociceptive pain. In addition, our studies do not rule out terpenes acting through other receptor systems, such as the closely related A3R, which has been shown to decrease pain via both immune cell modulation and direct neuronal modulation of spinal and descending midbrain nociceptive circuits [24].

It is also possible the tested terpenes may affect serotonergic and/or dopaminergic signaling, leading to the observed pain-relieving behaviors. Serotonin and dopamine signaling is known to have a profound effect on pain perception; SSRIs and SNRIs are prescribed to treat many types of chronic pain [25-27]. The reserpine model of fibromyalgia, which we used in the present study, causes a depletion of monoamines leading to key fibromyalgia symptoms. The role of serotonin and dopamine in pain perception is CNS region and receptor-specific; elevated serotonin and dopamine receptor (D1R) activation in the dorsal horn of the spinal cord are associated with increased nociception [28, 29]. This contrasts with D2R receptor activation in the spinal cord, which is antinociceptive, specifically in mechanonociceptive assays [30]. Other higher-order CNS regions in the brain and brainstem, such as the rostral ventral medulla (RVM) and anterior cingulate cortex (ACC), contribute to top-down pain regulation via serotonergic signaling while the hypothalamus seems to regulate pain via both serotonergic and dopaminergic signaling [27, 28, 31-34].

A2aR activation can attenuate dopamine release in the striatum and alter serotonin release in the hippocampus [35, 36]. One study suggests an indirect mechanism where an increase in adenosine neuromodulators decreases serotonin release; their results do not suggest A2aR involvement, though only A2aR selective antagonists, and not agonists were tested [37]. Nonetheless, this literature provides a potential mechanism whereby terpenes modulate dopamine and serotonin signaling via A2aR activation, leading to antinociception. Further research is needed to establish alterations in serotonergic and/or dopaminergic signaling as an effect of the terpenes tested.

Our future work will determine if A2aR is upregulated in the spinal cord in chronic pain states. The spinal cord is critical for processing nociception; primary sensory afferents extend from the periphery and synapse in the dorsal horn of the spinal cord before traveling to the brain, leading to pain perception. A2aR are Gαs coupled GPCRS; since the terpenes we tested act via A2aR to elicit pain relief behavior, it is likely that if A2aR is upregulated in chronic pain states, they would be found increasingly on inhibitory interneurons. This would explain the high levels of pain relief we observed in chronic pain models while only moderate to low pain relief in acute pain models with terpene treatment.

A2aR is found in the spinal cord and has been identified explicitly in lamina II of the dorsal horn in projection neurons, an essential region for relaying pain information [38, 39]. Inhibitory interneurons are important in regulating pain signals; they comprise about 25% of cells in laminae I and II and about 40% in lamina III in mice [40]. Though the expression of A2aR on spinal interneurons has yet to be investigated, A2aR agonists are effective in neuropathic and chronic pain when administered via it injection [3, 41]. However, the pan adenosine receptor antagonist, caffeine, has also been shown to relieve neuropathic pain [42, 43]. Adenosine receptors also have mixed results in acute pain, which matches the data we collected on the terpenes and pain relief [2, 3]. Other studies suggest that A2aR may alleviate nociceptor sensitization by interacting with NMDA receptors. One study showed that A2aR agonists inhibit NMDA receptors in lamina II neurons in the dorsal horn [39]. Hussey et al. showed that the deletion of A2aR in the spinal cord makes mice less sensitive to formalin pain and alters the glutamate binding to NMDA receptors [44, 45]. These results suggest that A2aR interacts with NMDA receptors in the spinal cord to alter pain perception. Terpenes may activate A2aR on inhibitory interneurons or influence NMDAR signaling in the spinal cord to attenuate pain signaling, which provides one possible explanation for how terpenes act on A2aR to lead to decreased pain sensation in chronic pain models.

In conclusion, this work suggests that the terpenes geraniol, linalool, BCP, and α-Hum relieve post-operative and fibromyalgia pain by acting on A2aRs. As mentioned in our previous work, terpene administration has translational limitations. Oral and inhalation administration methods had limited bioavailability, which will need to be addressed if these terpenes are to be used in a clinical setting for pain management [3]. Further work with terpene formulations could overcome this hurdle [46]. This work further strengthens the case for the translational potential of Cannabis and its individual components to determine if they could be effective in relieving post-operative and fibromyalgia pain in patients while causing more tolerable side effects than current standard medications for these ailments.

Supplementary Material

Supplementary Data

Funding statement

This work was funded by a grant from the National Institutes of Health (R01AT011517). We would like to acknowledge the University of Arizona Comprehensive Center for Pain and Addiction for supporting our work. Our graphical abstract was created using BioRender.

Abbreviations:

α-Hum

α-Humulene

A2aR

Adenosine A2a Receptor

ACC

Anterior Cingulate Cortex

AUC

Area Under the Curve

BCP

β-Caryophyllene

CBD

Cannabidiol

CBR1

Cannabinoid 1 Receptor

CIPN

Chemotherapy-Induced Peripheral Neuropathy

DMSO

Dimethylsulfoxide

GPCR

G-Protein Coupled Receptor

ip

Intraperitoneal

it

Intrathecal

NMDA

N-Methyl-D-Aspartate Receptor

RVM

Rostral Ventral Medulla

sc

Subcutaneous

SNRI

Serotonin Norepinephrine Reuptake Inhibitor

THC

Δ9-Tetrahydrocannabinol

Footnotes

Conflict of interest statement

JMS is an equity holder in Teleport Pharmaceuticals, LLC and Botanical Results, LLC, a local cannabidiol company. He is further a consultant for Black Rock Nutraceuticals, LLC. None of these companies were involved in this study in any way. The authors have no other relevant conflicts of interest to declare.

Data availability

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

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data
NIHMS2067124-supplement-Supplementary_Data.docx (1.5MB, docx)

Data Availability Statement

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

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