cbd fatty joint

December 15, 2021 By admin Off

Treatment of OA knees with CBD during the acute inflammatory phase of the MIA model (days 0–3 of MIA) inhibited saphenous nerve demyelination on day 14 compared with vehicle-treated knees ( P < 0.05; n = 6-8; Fig. ​ Fig.8 8 B).

After baseline recordings were completed on day 1 post-MIA induction, topical administration of CBD (300 μg) significantly decreased rolling and adherent leukocytes when compared with vehicle over the 3-hour time course ( P < 0.0001; n = 6; Figs. ​ Figs.5A 5 A and B). Cannabidiol had a moderate inhibitory effect on synovial hyperaemia ( P < 0.05; n = 6; Fig. ​ Fig.5C). 5 C). The MAP was unaffected by CBD treatment over the 3-hour time course (VEH: 70.7 ± 2.33 mm Hg; CBD: 68.8 ± 2.38 mm Hg), confirming a lack of any systemic effect on blood pressure.

It has previously been shown that the pain associated with the MIA model of OA is mediated in part by the sensitisation of joint afferent fibres. 35,37 Peripheral administration of CBD dose-dependently decreased joint afferent firing on day 14 after MIA injection. These electrophysiology data confirm that CBD has a peripheral site of action in knee joints. Because all recordings were made from Aδ or C fibres during noxious movement of the knee, this suggests that CBD can inhibit the mechanosensitivity of joint nociceptors.

Intravital microscopy was used to assess leukocyte-endothelial interactions within the microcirculation of the knee joint, as described previously. 2 The synovial microcirculation was visualised under incident fluorescent light using a Leica DM2500 microscope with a HCX APO L 20X objective and an HC Plan 10X eyepiece giving a final magnification of ×200. In vivo leukocyte staining was achieved by intravenous administration of 0.05% rhodamine 6G (in saline). Straight, unbranched postcapillary venules (15-50 μm in diameter) were chosen for visualisation and 3 fluorescent videos (per time point) were captured for 1 minute each by a Leica DFC 3000 camera (Leica Microsystems Canada Inc, Richmond Hill, ON, Canada). Two measures of leukocyte-endothelial interactions were used to assess articular inflammation: (1) the number of rolling leukocytes to pass an arbitrary line perpendicular to the venule in 1 minute were counted and (2) the number of adherent leukocytes within a 100-μm portion of the venule. Rolling leukocytes were defined as positively stained blood cells travelling slower than the surrounding blood flow, and adherent leukocytes were defined as positively stained cells that remained stationary for a minimum of 30 seconds.

Cannabidiol is the main noneuphoria producing component of the cannabis plant. 26 Pharmacologically, CBD has a complex signalling mechanism whereby it can both activate and silence classical cannabinoid receptors as well as modulate noncanonical cannabinoid receptor pathways. In in vitro studies, CBD has been shown to be an inverse agonist at CB 2 receptors, 40 and a full antagonist at CB 1 receptors 40 and G protein-coupled receptor-55 (GPR55). 33 In vitro, CBD was found to be an agonist at TRPV1 3 and transient receptor potential ankyrin 1 (TRPA1), 9 which play a central role in the development of OA. 27 In musculoskeletal disease models, systemic administration of CBD suppressed the progression of collagen-induced arthritis by reducing inflammatory cytokine production. 20 Although these preliminary findings indicate a possible role for CBD in relieving joint inflammation, the local effect of articularly applied CBD on OA and joint pain has not been investigated.

Table 1.

The inhibitory effect of CBD on leukocyte trafficking was blocked by the TRPV1 antagonist SB-366791. Opening of TRPV1 ion channels causes the peripheral release of inflammatory neuropeptides which promote neurogenic inflammation and enhanced leukocyte trafficking in joints. 19,41 Thus, the anti-inflammatory effects of CBD observed here could be due to desensitisation of TRPV1 ion channels as has been shown elsewhere. 14 The anti-rolling effect of CBD on joint leukocytes was also blocked by AM630 suggesting that CB 2 receptors may be involved in opposing leukocyte capture in day 1 MIA joints. Zhao et al. showed that activation of CB 2 receptors can inhibit the expression of P-selectin which is the adhesion molecule responsible for leukocyte rolling. 43 Whether CBD inhibits joint P-selectin activity by a CB 2 receptor mechanism requires further investigation.

Dose-dependent effect of CBD on pain-related measures in established OA. Intra-articular injection of MIA produced secondary allodynia and weight-bearing deficits in the ipsilateral hind paw and hind limb, respectively, 14 days after MIA injection (**** P < 0.0001, 1-way ANOVA with Dunnett post hoc test; n = 24). Cannabidiol (100, 200, or 300 μg i.artic. at BL]) improved hind paw withdrawal threshold (A) and hind limb weight bearing dose-dependently, over 4 hours. (**** P < 0.0001, ** P < 0.01, * P < 0.05 2-way ANOVA with Bonferroni post hoc test; n = 8). Data are mean values ± SEM. ANOVA, analysis of variance; BL, baseline, CBD, cannabidiol; MIA, sodium monoiodoacetate, OA, osteoarthritis; VEH, vehicle.

On day 14 post-MIA induction, 3 sets of noxious rotations, each lasting 5 seconds, were applied 5 minutes apart as a baseline measurement of afferent activity. After close i.a. infusion of CBD (100, 200, or 300 μg in 100 μL) or vehicle (100 μL), joint mechanosensitivity was assessed for an additional 15 minutes. To minimise the use of animals, multiple doses of CBD or vehicle were assessed in each fibre. A washout period of at least 50 minutes was observed between the administration of varying doses of CBD or vehicle to allow afferent firing to return to baseline levels. The percentage change in afferent activity before and after administration of CBD or vehicle was calculated offline using Spike2 software (Cambridge Electronic Design, Cambridge, United Kingdom). All recorded fibres fired in response to close i.a. administration of potassium chloride (KCl; 1 mM, 0.1 mL) at the conclusion of the experiment, confirming that administered drugs had reached the mechanosensory nerve endings and that the recorded fibre was still viable.

Prophylactic treatment of MIA-injected knee joints with CBD (on days 0–3 of MIA) significantly attenuated the development of MIA-induced tactile allodynia during both the acute and late phase of OA development ( P < 0.0001; n = 8; Fig. ​ Fig.7A). 7 A). Conversely, early treatment with CBD had no effect on hind limb weight bearing, when compared with vehicle-treated animals ( P > 0.05; n = 8; Fig. ​ Fig.7 7 A).

Availability of data and materials: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Animals were deeply anaesthetised by an intraperitoneal injection of urethane (25% solution; 2g/kg i.p.). A longitudinal incision was made along the ventral skin of the neck to expose the trachea which was cannulated with PE-200 tubing to permit unrestricted breathing. The right carotid artery was also cannulated with PE-30 tubing filled with heparinised saline (1 U/mL) to allow for continuous monitoring of the mean arterial pressure (MAP).

Contribution of cannabinoid and noncannabinoid receptors to the analgesic effects of CBD. Both hind paw withdrawal threshold (A) and hind limb weight bearing (B) were unaltered compared with control after local administration of the CB 1 receptor antagonist AM281 (75 μg) or CB 2 receptor antagonist AM630 (75 μg). Hind paw withdrawal threshold (A) was reduced compared with control after local administration of the TRPV1 antagonist SB-366791 (30 μg), but hind limb weight bearing (B) was unaffected. (* P < 0.05, ** P < 0.01 1-way ANOVA with Fisher post hoc test; n = 6-8). Data are mean values ± SEM. ANOVA, analysis of variance; CBD, cannabidiol; VEH, vehicle.

Author contributions: H. T. Philpott conducted the pain behaviour experiments, the inflammation measurements (IVM and LASCA), performed the G-ratio measurements, analysed data, and helped draft the manuscript. M. O’Brien conducted all electrophysiology experiments, analysed the data, and helped draft the manuscript. J. J. McDougall conceived the study, participated in its design and coordination, helped analyse data, and helped draft the manuscript. All authors read and approved the final manuscript.

This work was supported by an operating grant provided by The Arthritis Society.

The initial aim of this study was to assess the effect of locally administered CBD on joint pain in animals with end-stage OA. Since acute inflammation can contribute to the long-term development of OA joint pain, 32 the ability of CBD to reduce acute OA synovitis and prevent the subsequent progression of persistent OA pain was also investigated. Finally, the effect of prophylactic CBD treatment on OA joint neuropathy was assessed.

2.8. Statistical analysis.

On days 14 to 19 post-MIA induction, close i.a. administration of CBD rapidly reduced noxious movement-evoked firing of knee afferent fibres (Fig. ​ (Fig.1A) 1 A) in a dose-dependent manner ( P < 0.0001; n = 8, Fig. ​ Fig.1B). 1 B). The desensitising effect of 300 μg CBD during noxious joint rotation was significant at 3 minutes after drug application and reached a maximum anti-nociceptive effect at 7 minutes (29.3% ± 7.4% change compared with baseline). Although all doses of CBD significantly decreased the mean afferent firing over the course of the 15 minutes assessed, the 300 μg dose was the most effective, decreasing firing by 22.8% ± 1.2% overall ( P < 0.0001, n = 8, Fig. ​ Fig.1 1 C).

Male Wistar rats (150-175 g; Charles River Laboratories, Senneville, QC, Canada) were housed in ventilated racks at 22°C ± 2°C on a 12:12 hours light:dark cycle (light-on from 7:00 to 19:00). After arrival at the animal care facility, all rats were permitted at least 1 week to acclimate to their environment. Animals were housed in pairs, cages were lined with woodchip bedding, and animals were provided with environmental enrichment. Standard laboratory chow and water were provided ad libitum. All experimental protocols were approved by the Dalhousie University Committee on the Use of Laboratory Animals, which acts in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) and the standards put forth by the Canadian Council for Animal Care.

Cannabidiol (2-[(1 R ,6 R )-3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-pentyl-1,3-benzenediol) was obtained from Tocris Bioscience (Bio-Techne, Abingdon, United Kingdom). AM281 (CB 1 receptor antagonist; 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide) and AM630 (CB 2 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). SB-366791 (N-(3-methoxyphenyl)-4-chlorocinnamide), rhodamine 6G, cremophor, dimethyl sulphoxide (DMSO), urethane, and MIA were obtained from Sigma-Aldrich (St. Louis, MO). Solutions of CBD, AM281, AM630, and SB-366791 were prepared in vehicle (1:1:18; DMSO:cremophor:saline) on the day of use. Rhodamine 6G (0.05%) and MIA were dissolved in saline. Physiological buffer (135 mM NaCl, 20 mM NaHCO3, 5 mM KCl, 1 mM MgSO4*7H2O, pH = 7.4) was prepared in the laboratory.

Osteoarthritis (OA) is a multifactorial joint disease, which includes joint degeneration, intermittent inflammation, and peripheral neuropathy. Cannabidiol (CBD) is a noneuphoria producing constituent of cannabis that has the potential to relieve pain. The aim of this study was to determine whether CBD is anti-nociceptive in OA, and whether inhibition of inflammation by CBD could prevent the development of OA pain and joint neuropathy. Osteoarthritis was induced in male Wistar rats (150-175 g) by intra-articular injection of sodium monoiodoacetate (MIA; 3 mg). On day 14 (end-stage OA), joint afferent mechanosensitivity was assessed using in vivo electrophysiology, whereas pain behaviour was measured by von Frey hair algesiometry and dynamic incapacitance. To investigate acute joint inflammation, blood flow and leukocyte trafficking were measured on day 1 after MIA. Joint nerve myelination was calculated by G-ratio analysis. The therapeutic and prophylactic effects of peripheral CBD (100-300 μg) were assessed. In end-stage OA, CBD dose-dependently decreased joint afferent firing rate, and increased withdrawal threshold and weight bearing ( P < 0.0001; n = 8). Acute, transient joint inflammation was reduced by local CBD treatment ( P < 0.0001; n = 6). Prophylactic administration of CBD prevented the development of MIA-induced joint pain at later time points ( P < 0.0001; n = 8), and was also found to be neuroprotective ( P < 0.05; n = 6-8). The data presented here indicate that local administration of CBD blocked OA pain. Prophylactic CBD treatment prevented the later development of pain and nerve damage in these OA joints. These findings suggest that CBD may be a safe, useful therapeutic for treating OA joint neuropathic pain.

When compared with vehicle control, low dose CBD (100, 200 μg) had no effect on withdrawal threshold or hind limb weight bearing ( P > 0.05; n = 8; Figs. ​ Figs.2A 2 A and B). The 300 μg dose of CBD, however, significantly increased hind paw withdrawal threshold and hind limb weight bearing over the time course tested ( P < 0.0001; n = 8; Figs. ​ Figs.2A 2 A and B). All subsequent experiments used the 300 μg dose of CBD.

A total of 17 afferent fibres were recorded in this study. Fibres were characterised based on mechanical and electrical threshold, and conduction velocity (summarised in Table ​ Table1 1 ).

Animals were deeply anaesthetised (2%-4% isoflurane; 100% oxygen at 1 L/min) until cessation of all sensory reflexes. The right knee joint was shaved, swabbed with 100% ethanol and 50 μL of sodium monoiodoacetate (MIA) (3 mg in saline) was injected into the joint space (intra-articular; i.artic.). The knee was then manually extended and flexed for 30 seconds to disperse the solution throughout the joint.

3.3. Effect of acute administration of cannabidiol on sodium monoiodoacetate–induced inflammation.

Anti-inflammatory action of CBD on day 1 MIA-induced inflammation. When compared with naïve controls, intra-articular MIA significantly increased rolling (A) and adherent (B) leukocytes, and caused synovial hyperaemia (C) (**** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, P > 0.05, unpaired t test; n = 6-12). Over a 3-hour time course, CBD (300 μg) significantly decreased leukocyte rolling (A) leukocyte adherence (B) and knee joint blood flow (C) when compared to vehicle. (**** P < 0.0001, ** P < 0.01, * P < 0.05 2-way ANOVA with Bonferroni post hoc test; n = 6). Data are mean values ± SEM. ANOVA, analysis of variance; CBD, cannabidiol; MIA, sodium monoiodoacetate; PU, perfusion unit; VEH, vehicle.

To perform dynamic weight bearing (DWB) measurements, animals were placed in a Perspex chamber (model BIO-DWB-AUTO-R; Bioseb, Boulogne, France) with a pressure-sensitive floor and allowed to move freely. Hind limb weight bearing was tracked and recorded over a 3-minute period. Weight borne on the ipsilateral hind paw was calculated as a percentage of the total weight borne on the hind limbs.

Animals underwent baseline von Frey hair mechanosensitivity and DWB testing. Separate cohorts were treated on day 14 post-MIA with an i.artic. injection of either vehicle (50 μL) or CBD (100-300 μg/50 μL). In other experimental cohorts, day 14 OA rats were treated with the highest dose of CBD (300 μg/50 μL) and either the CB 1 receptor antagonist, AM281 (75 μg/50 μL), the CB 2 receptor antagonist, AM630 (75 μg/50 μL), or the TRPV1 receptor antagonist, SB-366791 (30 μg/50 μL) administered locally (subcutaneously; s.c.) over the joint 10 minutes before i.artic. CBD administration. Behavioural pain measurements for these experiments were conducted at 30, 60, 120, 180, and 240 minutes after drug administration. To investigate the prophylactic effects of CBD on OA pain and peripheral neuropathy, a separate cohort of rats was treated with CBD (300 μg/50 μL) or vehicle (50 μL) s.c. over the knee joint 30 minutes before i.artic. injection of MIA (3 mg/50 μL) and once daily on each of the subsequent 3 days; behavioural pain measurements were conducted on days 0, 1, 2, 3, 7, 10, and 14.

The most prominent form of synovial joint disease, osteoarthritis (OA), is characterised by joint degeneration, pain, and in some patients, articular neuropathy. 21 Chronic pain associated with OA is a major concern for which there are few viable treatments. The first-line therapy used to treat OA pain is nonsteroidal anti-inflammatory drugs; however, with long-term use their efficacy declines and they can lead to major adverse gastrointestinal and cardiovascular events. Historically, OA has been classified as noninflammatory arthritis; however, there is now overwhelming evidence that synovitis can occur in response to pro-inflammatory mediators being released into the joint. 10,11,13,29,32 It is believed that this low-level inflammation contributes to degenerative changes that affect the entire joint leading to the development of peripheral sensitisation and nociceptive pain. 18,22,37 In addition to structural defects, there is growing evidence to suggest that approximately 30% of patients with OA have neuropathic pain. 1,34 Thus, a therapeutic which can block inflammation, neuropathy, and pain is sorely needed.

Pain and disease progression are poorly managed in many patients with OA because of the multifactorial nature of the disease. Intra-articular injection of MIA produces monoarthritis with several features that resemble human OA, including joint pain, intermittent inflammation, and joint nerve damage. This study aimed to address, for the first time, whether the inflammatory and neuropathic pain associated with MIA could be blocked by local administration of the noneuphoria producing phytocannabinoid CBD.

Characterisation of the recorded fibres in the electrophysiology experiments.

The copper wire grids containing the saphenous nerve sections were inserted into a JEOL JEM 1230 transmission electron microscope (JEOL Corp Ltd, Tokyo, Japan). The microscope was set at a voltage of 80.0 kV, and images were captured at ×2500 using a Hamamatsu ORCA-HR digital camera (Hamamatsu Photonics, Hamamatsu City, Japan). One nerve cross-section image was visually partitioned into 9 quadrants and 3 images were captured (from quadrants 1, 5, and 9). All fibres were assessed using the G-ratio plugin in ImageJ processing software. The G-ratio was calculated using the equation where, a is the internal axonal area and A is the total axonal area of the fibre. The higher the G-ratio the higher the degree of demyelination.

After OA development (14-19 days after MIA), animals were deeply anaesthetised using urethane (25% solution; 2 g/kg i.p.). Core body temperature was measured by a rectally inserted thermometer and maintained at 37°C ± 1°C by a thermostatically controlled heating blanket (CWE Inc, Ardmore, PA). After loss of the pedal withdrawal reflex, the trachea was cannulated to allow for artificial ventilation with a Harvard rodent respiratory pump (Harvard Apparatus, Holliston, MA) with 100% O 2 (stroke volume: 2.5 mL; breath frequency: 52 breaths/min). The left carotid artery was cannulated to allow for continuous measurement of the mean arterial blood pressure. The cannula was attached to an in-line pressure transducer (Kent Scientific Corp, Torrington, CT) attached to a differentially amplified blood pressure monitor (World Precision Instruments, Sarasota, FL). The jugular vein was cannulated for administration of the muscle relaxant gallamine triethiodide (50 mg/kg), which eliminated neural interference from hind limb musculature, and the distal saphenous artery was cannulated for close intra-arterial (i.a.) administration of CBD or vehicle to the knee joint (100 μL injection volume). A specialised clamp was fixed to the mid-shaft of the isolated right femur and attached to a stereotaxic frame to prevent movement of the proximal aspect of the rat hind limb. The right hind paw was then placed in a shoe-like holder that was connected to a force transducer and torque meter (Data Track 244-1-R; Intertechnology, ON, Canada) to standardise the amount of rotational force being applied to the knee joint. A longitudinal skin incision was made along the medial aspect of the hind limb and the reflected skin was sutured to a metal “O” ring to create a pool which was filled with warm mineral oil to prevent tissue desiccation. The medial articular branch of the saphenous nerve was isolated and transected in the inguinal region to prevent spinal reflexes. The epineurium was removed and the nerve teased to isolate fine neurofilaments which were then placed on a platinum recording electrode to measure single-unit activity. To identify a joint afferent fibre and its receptive field, the knee joint was gently probed with a blunt glass rod. The mechanical threshold of each recorded joint afferent was determined by gradually increasing the torque applied to the joint until the fiber elicited an action potential. The conduction velocity of the fibres were determined by electrically stimulating the receptive field with a pair of silver bipolar stimulating electrodes (0.6 Hz, 2 ms pulse width, 1-15 V). The mechanosensitivity of the joint fibre was assessed by applying noxious outward rotations to the knee and counting the number of action potentials elicited during the rotation. Noxious rotation refers to torque occurring outside the normal range but not severe enough to cause soft tissue injury.

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