Local administration of myrcene to adjuvant monoarthritic knees caused a dose-dependent reduction in secondary allodynia over the 3 h time-course (p < 0.05 two factor RMANOVA; n = 8 animals/group). The greatest effect of myrcene occurred at the 120 min timepoint where the 1 mg/kg dose improved nociception by 211.0 ± 17.93% and the 5 mg/kg dose 269.3 ± 63.27% (Figure 1A). Focusing on the 120 min timepoint (Figure 1B), the analgesic effect of myrcene was blocked by pre-treatment with either the CB₁-receptor antagonist AM281 (p < 0.001 one factor RMANOVA with Bonferroni’s post hoc test; n = 8 animals/group) or the CB₂-receptor antagonist AM630 (p < 0.0001). Neither antagonist alone had any effect on joint pain.

In day 7 arthritic knees, local administration of myrcene reduced leukocyte rolling with the maximal effect occurring at 60 min after topical application (p < 0.0001; n = 6–7 animals/group. Figure 2A). This anti-inflammatory effect was not dose-dependent. Myrcene had no effect on leukocyte adherence or joint blood flow (data not shown). Blockade of CB₂ receptors with AM630 attenuated the reduction in leukocyte rolling by myrcene (p < 0.05); however, AM281 did not affect the anti-inflammatory effects of myrcene (Figure 2B). Neither antagonist alone had any effect on joint inflammation.

In these experiments, a low dose of CBD, which in itself had no effect on pain and inflammation, was chosen to see if it could synergize with myrcene to augment antinociception and anti-inflammation. While myrcene alone reduced secondary allodynia and leukocyte rolling, the addition of CBD had no additional effect on these parameters (p > 0.05, Figure 3A,B).

Compared to vehicle, repeated administration of myrcene increased the hindpaw withdrawal threshold over 21 days of adjuvant-induced arthritis (p < 0.0001; n= 7–8; Figure 4). These measurements were carried out prior to myrcene administration on each day so cannot be attributed to any acute effects of the compound.

Chronic myrcene treatment had a wider effect on leukocyte trafficking and joint perfusion than acute administration (Figure 5). In addition to an effect on leukocyte rolling (p < 0.01), repeated topical myrcene was able to reduce leukocyte adherence (p < 0.05) and synovial blood flow (p < 0.01). In day 21 adjuvant monoarthritic knees, topical myrcene reduced leukocyte rolling from 72.6 ± 4.59 leukocytes/min to 47.9 ± 5.02 leukocytes/min; leukocyte adherence from 5 ± 0.8 to 3 ± 0.5 leukocytes/100 µm of venule; and perfusion from 225.8 ± 26.81 PU to 140.8 ± 6.53 PU.

Analysis of plasma taken from rats chronically-treated with myrcene revealed that the terpene had no significant effect (p > 0.05) on circulating cytokine levels compared to vehicle-treated animals (Table 1).

Twenty-one days following intra-articular injection of Freund’s complete adjuvant, rat knees showed moderate levels of joint damage including macrophage infiltration, pannus formation, cartilage damage and bone resorption (Figure 6). Multiple treatment of arthritic joints with myrcene had no effect on synovitis, pannus formation, cartilage destruction or bone resorption (Figure 6).

The main limitation of this study was the low number of doses of myrcene and CBD used. The solubility restrictions of myrcene meant that there was a finite concentration that could be achieved without the drug coming out of solution. A low dose of CBD was tested so as to maximize any synergistic effect of the cannabinoid; however, further experiments using higher doses of CBD would have helped to optimize the analysis of entourage. A further limitation was the restriction of drugs locally to the joint to evaluate peripheral effects only. The rationale for this was to provide a proof-of-concept analysis of myrcene as a potential topical treatment. Future experiments administering myrcene systemically will provide a more global effect of the terpene in modulating joint pain and inflammation. Finally, using other antagonists, for example against TRPV1, would elucidate alternative targets that myrcene could be acting through in the joint.

Deep anaesthesia was induced using isoflurane inhalation (2–5% in 100% O₂; 1 L/min) until absence of the flexor withdrawal reflex to noxious toe pinch and the corneal blink reflex. The right stifle (knee) joint was shaved and swabbed with 70% ethanol (Fisher Scientific Company, Ottawa, ON, Canada). The knee was flexed and 50 µL of Freund’s complete adjuvant was injected into the joint cavity using a 30 G needle. The joint was then gently flexed and extended for ten seconds to help disperse the adjuvant throughout the intra-articular space. The knee was again swabbed with ethanol and the animals were allowed to recover under a heat lamp.

Secondary mechanical allodynia was determined by von Frey hair algesiometry based on the procedure of Chaplan et al. [43]. Briefly, animals were acclimated to a quiet testing room for at least one hour prior to pain assessment. Animals were placed in a Plexiglass chamber (31 cm × 9.5 cm × 25 cm) with a wire mesh floor. Calibrated von Frey hairs (North Coast Medical, Gilroy, CA, USA) were sequentially applied to the volar surface of the ipsilateral hindpaw and held for two seconds. A withdrawal response was signified by retraction of the paw and/or licking of the paw area. If no response was observed, a thicker filament was applied up to a cut-off force of 5.18 g.

The 50% withdrawal threshold was calculated using the following equation:

where Xf is the bending force of the last von Frey filament used (in log units), κ is the tabular value for the pattern of the last six responses and δ is the mean difference (in log units) between stimuli.

Articular leukocyte trafficking and blood flow were quantified by intravital microscopy and laser speckle contrast analysis respectively. Animals were deeply anesthetized with 25% urethane (2 mL loading dose; 0.5–0.7 mL top-up doses every 20 min) until cessation of the ocular blink and pedal withdrawal reflexes. Internal body temperature was measured by a rectally inserted thermometer and core temperature was maintained at 37 ± 1 °C by a thermostatically controlled heating pad (TC-1000, CWE Inc., Ardmore, PA, USA). The anesthetized animal was placed supine on a surgical board and a longitudinal midline incision was made in the neck. The sternohyoid muscle was blunt dissected to expose the trachea, which was subsequently cannulated to ensure a clear, unobstructed airway.

The left carotid artery was then isolated and catheterized using a fine cannula containing warmed, 1% heparinized saline. The carotid artery cannulation was connected to a BLPR2 pressure transducer and thence to a calibrated bridge amplifier (BP-1, World Precision Instruments, Sarasota, FL, USA) to allow continuous measurement of mean arterial pressure. The left jugular vein was then isolated and cannulated with tubing also containing warmed 1% heparinized saline.

Finally, a small ellipse of skin was excised from over the ipsilateral knee joint and the underlying fascia removed to expose the joint microvasculature, which was kept moist by perfusion with 0.9% saline. For intravital microscopy, animals first received an i.v. injection of Rhodamine 6 G (0.1 mL; 5 mg/10 mL; Sigma Aldrich, St. Louis, Missouri, USA) to label circulating leukocytes. A Leica DM2500 microscope, (Leica Microsystems Canada, Inc., Richmond Hill, ON, Canada) with a HCX APOL 20X objective and HC Plan 10X eyepiece was used to observe capsular leukocyte trafficking under a 530 nm fluorescent light. Straight, unbranched post capillary venules (15–50 μm) located in the knee joint were selected and three, one-minute videos were recorded at a final magnification of 200× using a Leica DFC 3000 camera (Leica Microsystems Canada Inc., Richmond Hill, ON, Canada). The videos were then analyzed offline in a blinded manner, where rolling and adherent leukocytes were quantified. Rolling leukocytes were defined as cells moving slower than blood flowing past an arbitrary line perpendicular to the vessel of interest. Leukocytes were considered adherent if they remained immobile for more than 30 s within a 100 μm span of the postcapillary venule. The number of rolling and adherent leukocytes was quantified for each of the one-minute recordings.

Joint perfusion was carried out by laser speckle contrast analysis (LASCA) using the PeriCam PSI System (Perimed Inc., Ardmore, PA, USA). A one-minute recording was taken at a working distance of 10 cm, and a frame capture rate of 25 images/second using PIMSoft software (Version 1.5.4.8078; Perimed Inc., Ardmore, PA, USA). Post euthanasia, a “dead scan” recording was taken of the joint and subtracted from experimental perfusion values as a biological zero to account for any cellular Brownian motion or tissue optical interference. The speckle pattern was subsequently analyzed offline in a blinded manner and recorded in arbitrary perfusion units (PU) for each time-specific recording. Blood perfusion corresponding to the joint capsule was recorded.

Whole blood samples were collected by intracardiac puncture using a 5 mL syringe at endpoint on day 21. The sample was transferred immediately into a 6 mL EDTA-coated Eppendorf tube and placed on ice. Samples were centrifuged at 1000× g at 4 °C for 10 min. Plasma supernatant was then transferred into Eppendorf tubes and stored at −80 °C until use. Frozen aliquots were thawed on ice and centrifuged at 10,000× g for 5–10 min, and then diluted with 1X universal assay buffer. Antigen standards and a 4-fold serial dilution of the reconstituted standard were prepared as per the ProcartaPlex™ Mutliplex Immunoassay for Convenience and the Mix & Match Panels User Guide. The magnetic beads were vortexed for 30 s and 40 μL of the beads was added to each well, before subsequently removing the liquid and adding 150 μL of wash buffer. Standards and pre-diluted plasma samples (50 μL) were added to the bead-coated wells. Blank wells were filled with 50 μL of universal assay buffer. The plate was subsequently incubated at room temperature on a microplate shaker for 2 h. Post-incubation, the plate was washed a total of three times. Twenty microliters of the detection antibody mixture were added to each well and incubated at room temperature for 30 min. Next, 40 μL of Streptavidin-PE (SAPE) was added to each well and incubated at room temperature while shaking for a further 30 min. The plate was then prepared for analysis by adding 120 μL of Reading Buffer into each well and incubating for five minutes. A Bio-Plex 200 system (Bio-Rad, Hercules, CA, USA) was used to quantify specific cytokines (interleukin-1β (IL-1β), IL-10, IL-17A, IL-6 and tumor necrosis factor-α (TNF-α)).

At day 21 after FCA injection, four myrcene-treated and four vehicle-treated knees from the chronic experiments (see below) were assessed for joint pathology. Deeply anesthetized animals were perfused trans-cardially with saline, followed by 4% paraformaldehyde. The joint was isolated and immersed in 4% paraformaldehyde for 24 h before being washed with sterile water and transferred to 70% ethanol for storage. Joint histopathology was assessed by Bolder BioPATH Inc. (Boulder, CO, USA). Knee joint were initially decalcified for 4–5 days in 5% formic acid and then halved in the frontal plane and embedded in mounting paraffin. Joints were subsequently sectioned (8 µm sections) at 200 µm intervals across the whole knee. Sections were stained with 0.04% toluidine blue and assessed microscopically for signs of disease. The joint sections were then scored for four parameters (synovitis, pannus formation, cartilage damage, and bone resorption) using the previously described OARSI scoring system [44]. Each parameter was scored 0–5 to give a possible total summed score out of 20.

The acute effects of myrcene on adjuvant monoarthritic pain and inflammation were initially assessed on day 7 of the model. von Frey hair algesiometry was measured before (baseline, BL) and then over 3 h following administration of either vehicle (soybean oil, 50 µL s.c. over knee joint) or myrcene (1 and 5 mg/kg, 50 µL s.c. over knee joint). Dose was based on a systemic dose used in a previous study [7]. To examine cannabinoid receptor involvement in myrcene responses, separate cohorts of rats were pre-treated with either the CB₁ receptor antagonist AM281 or the CB₂ receptor antagonist AM630 (75 µg in 50 µL s.c. over the knee 10 min prior to 1 mg/kg myrcene injection). For joint inflammation experiments, a similar protocol was employed in separate groups of animals where intravital microscopy and LASCA were carried out over a 60 min period. The effect of myrcene on hindlimb mechanosensitivity and inflammation were also investigated in naïve animals as a further control.

To explore any synergistic effects between the phytocannabinoid CBD and myrcene, a combination regimen was employed. In day 7 adjuvant monoarthritic rats, low-dose CBD (200 µg) was co-administered with low-dose myrcene (1 mg/kg) in a single 50 µL bolus injected s.c. over the knee. This dose of CBD had no effect on joint inflammation or pain (data not shown). Following treatment, von Frey hair algesiometry was measured over 3 h while in separate animals intravital microscopy and LASCA were tested over 60 min.

For chronic studies, myrcene (5 mg/kg, 50 µL s.c. over knee joint) was administered on days 1, 2, 3, 7, 10, 14 and 21 after monoarthritis induction. Secondary allodynia was measured before (baseline: BL) and then prior to each injection of myrcene. Leukocyte trafficking and synovial blood flow were then tested in the same animals at day 21 after Freund’s adjuvant injection. Blood samples were then taken for cytokine analysis and ipsilateral knee joints harvested for histopathological assessment.

Myrcene (7-methyl-3-methylene-1,6-octadiene) in soy oil was purchased from Toronto Research Chemicals (Toronto, ON, Canada). CBD (2-[(1R,6R)-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol) was obtained from Tocris Bioscience (Bio-Techne, Abingdon, UK). AM281 (CB1 receptor antagonist; 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) and AM630 (CB2 receptor antagonist; 6-iodo-2-methyl-1-(2-morpholin-4-ylethyl)indol-3-yl]-(4-methoxyphenyl)methanone) were obtained from Cayman Chemicals (Ann Arbor, MI, USA). The selectivity of AM281 is 12 nM: 4200 nM for CB₁:CB₂, and for AM630 5µM: 31 nM for CB₁:CB₂. Rhodamine 6 G, cremophor, dimethyl sulphoxide (DMSO), urethane, and Freund’s complete adjuvant were obtained from Sigma Aldrich (St. Louis, MO, USA). Solutions of CBD, AM281, and AM630 were prepared in vehicle (1:1:18; DMSO:cremophor:saline) on the day of use. Rhodamine 6 G (0.05%) was dissolved in 0.9% saline.

All data were presented as an average of the percent baseline ± SEM. Normality was determined using the Kolmogorov–Smirnov test and compliant data were analyzed by parametric statistics, i.e., one- or two-factor repeated measures analysis of variance (RMANOVA) with Bonferroni’s post hoc multiple comparisons test, or Student’s t-test. Joint histopathology scores and cytokine concentrations were analyzed by the non-parametric Mann–Whitney U test. A probability value of p < 0.05 was considered statistically significant.

Data are available from the corresponding author upon request.