Kaolin-carrageenan inflammation

All animals had an acute unilateral inflammation induced by injection of kaolin and carrageenan into the right knee joint. Animals were deeply anaesthetized (2 to 4% isoflurane; 100% oxygen at 1 L/min) and an acceptable plane of anaesthesia was confirmed by failure to produce a hindpaw withdrawal reflex. The right knee was shaved and the knee joint diameter was measured using a digital micrometer (Mituyoto Instruments, Tokyo, Japan). The knee area was then swabbed with 100% ethanol and 10 μl of 2% kaolin was injected into the intra-articular space. The knee was then manually extended and flexed for 10 min to disperse the kaolin throughout the joint and irritate the synovium. Next, 10 μl of 2% carrageenan was injected in the same manner and was followed by 30 s of hindlimb flexion and extension. Animals were returned to their cages for 24 hr. Knee joint diameter was then reassessed to confirm positive inflammation.

Surgical preparation for vascular assessment

The following preparation was conducted before intravital microscopy (IVM) and laser speckle contrast analysis (LASCA) of the inflamed knee. Animals were anaesthetized by an intraperitoneal injection of urethane (25% in saline; 0.3 to 0.4 ml) and a surgical plane of anaesthesia was confirmed by failure to elicit a hindpaw withdrawal reflex. A longitudinal incision was made in the skin of the neck to expose the trachea, left carotid artery and left jugular vein, which were individually cannulated. PE-60 tubing was used for the tracheal cannulation while PE-10 tubing, filled with heparinized saline (1U/ml), was used for the blood vessel cannulations. The capsular microcirculation of the right knee was exposed by surgically removing a small ellipse of overlying skin (<1 cm long; <0.5 cm wide), and the knee was immobilized. Physiological buffer (37°C) was immediately and continuously perfused over the exposed joint.

Intravital microscopy

IVM was used to assess leukocyte trafficking within the microcirculation of the knee joint, as previously described [26]. After surgical preparation, the synovial microcirculation was visualized under incident fluorescent light using a Leica DM2500 microscope with a HCX APO L 20X objective and a HC Plan 10X eyepiece (Leica Microsystems Inc., Richmond Hill ON, Canada; final magnification 200X). In vivo leukocyte staining was achieved by intravenous administration of 0.05% rhodamine 6G (0.05 ml). Straight, unbranched, postcapillary venules (15 to 50 μm in diameter) overlying the knee capsule were selected for visualization. Videos (1 min duration) of leukocyte kinetics were captured using a BC-71 AVT camera (Horn Imaging, Aalen, Germany).

Two measures of leukocyte-endothelial interactions were used to assess inflammation: (i) number of rolling leukocytes and (ii) number of adherent leukocytes per 50 μm of vessel. Rolling leukocytes were defined as rhodamine 6G-stained cells travelling slower than the surrounding flow of blood in the vessel of interest. The rolling leukocyte measure was obtained by counting the number of rolling leukocytes per minute to pass an arbitrary line perpendicular to the vessel of interest. Adherent leukocytes were defined as rhodamine 6G-stained cells that remained stationary for a minimum of 30 s. Total leukocyte adhesion was quantified by counting the number of adherent cells within a 50 μm portion of the vessel. A baseline measure was first taken to observe leukocyte kinetics before drug administration. Following this measure, either vehicle, URB597 (0.3 to 30 mg/kg), URB597 (0.3 mg/kg)+AM251 (CB1 antagonist, 0.2 mg/kg) or URB597 (0.3 mg/kg)+AM630 (CB2 antagonist, 0.2 mg/kg) was applied directly over the exposed knee joint. Doses were chosen based on effective dosing regimens as previously reported [18–20,25]. For all URB597+antagonist trials in this study, the antagonists were administered 10 min before URB597. Antagonist alone control trials were also conducted. Subsequent recordings were taken 5, 10, 20, 30 and 60 min after URB597 administration.

Laser speckle contrast analysis

In order to quantify microvascular perfusion, knee joint blood flow was assessed using a PeriCam PSI System (Perimed Inc., Ardmore, PA, USA). Recordings were taken at a working distance of 10 cm with a frame rate of 25 images per second, which were then averaged to create one perfusion image per second. Following a baseline recording of 1 min, a 100 μl bolus of URB597 (0.3 to 30 mg/kg) was applied directly to the knee joint, with each dose being applied in separate cohorts of animals. The effect of AM251 or AM630 (0.2 mg/kg) on URB597 perfusion responses was also tested in different groups of mice. Mean perfusion (1 min sampling time) was measured at 5, 10, 20, 30 and 60 min following drug treatment. A dead scan of the knee was taken post mortem and this value was subtracted from all measurements to account for any optical noise in the tissue.

Hindlimb incapacitance

Mice were habituated to the hindlimb incapacitance testing apparatus (Linton Instrumentation, Diss, UK) for three consecutive days prior to baseline measurement. Weight distribution measurements of one-second duration were taken while the animal was standing on both hindlimbs, with one hindpaw on each of the two force plates. Baseline weight-bearing measurements were taken before and 24 hr after induction of inflammation. Separate cohorts of mice were then treated with either vehicle, URB597 (3 mg/kg), URB597 (3 mg/kg)+AM251 (0.2 mg/kg), or URB597 (3 mg/kg)+AM630 (0.2 mg/kg), applied subcutaneously (s.c.) over the inflamed knee joint, or URB597 (3 mg/kg) applied s.c. over the contralateral knee joint. Test measurements (average of six measures per time point) were then taken at 30, 60, 90 and 180 min following drug administration.

von Frey hair mechanosensitivity

The same animals underwent concurrent secondary allodynia testing as a behavioural measure of referred pain. Ipsilateral hindpaw mechanosensitivity was assessed by plantar application of von Frey hair filaments using a modification of the Dixon up-and-down method [27]. Animals were placed in elevated Plexiglas chambers on metal mesh flooring allowing access to the paws. After allowing the animal to acclimate until exploratory behaviour ceased, a von Frey hair was applied perpendicular to the plantar surface of the hindpaw (avoiding the toe pads) until the hair just bent, and the hair was held in place for three seconds. If there was a positive response (that is withdrawal, shake or lick of the hindpaw), the next lower strength hair was applied; if there was no response, the next higher strength hair was applied up to a maximum cutoff level, which corresponded to a 4 g bending force. After the first difference in response was observed, four more measurements were made and the pattern of responses were converted to a 50% withdrawal threshold calculated using the following formula: 10[Xf+kδ]/10,000; where Xf=value (in log units) of the final von Frey hair used, k=tabular value for the pattern of the last six positive/negative responses, and δ=mean difference (in log units) between stimuli. A time course at 30, 60, 90 and 180 min following drug administration was constructed.

Drugs and reagents

URB597 (FAAH inhibitor; [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate), AM251 (CB1 receptor antagonist; 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-piperidin-1-ylpyrazole-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). Rhodamine 6G, cremophor, dimethyl sulphoxide (DMSO), urethane, carrageenan and kaolin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Solutions of URB597 (0.3 to 30 mg/kg), AM251 (0.2 mg/kg) and AM630 (0.2 mg/kg) were prepared in vehicle (1:1:8; DMSO:cremophor:saline) on the day of use. Rhodamine 6G (0.05%) was dissolved in saline. Physiological buffer (135 mM NaCl, 20 mM NaHCO3, 5 mM KCl, 1 mM MgSO4*7H2O, pH =7.4) was prepared in house. Solutions of carrageenan (2%) and kaolin (2%) were dissolved in saline and stored at 4°C.

Mean arterial pressure

During IVM and LASCA measurements, mean arterial pressure did not change in response to drug administration and was found to be comparable between all experimental groups.

Leukocyte trafficking

Over the course of one hour, URB597 (0.3 mg/kg) significantly decreased leukocyte rolling when compared to vehicle (P <0.0001; n=7 to 11; Figures 1 and ​and2A).2A). The most robust effect was observed 20 min after administration; therefore all subsequent measurements focused on this time point. Low-dose URB597 (0.3 mg/kg) caused an average of 68.0±6.8% (n =11) decrease in leukocyte rolling 20 min after administration (Figure 2B). This inhibition of rolling was significantly different from vehicle-treated animals (P <0.001; n =7 to 11; Figure 2B) and was blocked by both AM251 and AM630 (P<0.01; n =6; Figure 3A). Interestingly, higher doses of URB597 (3 to 30 mg/kg) had no inhibitory effect on leukocyte rolling (Figure 2B).

Figure 1

Intravital microscopy images showing circulating leukocytes in the anteromedial aspect of acutely inflamed mouse knees in response to either (A) vehicle or (B) the FAAH inhibitor URB597. Arrows indicate stained leukocytes. Scale bar =50 μm. FAAH, …

Figure 2

The effects of URB597 on leukocyte trafficking in the inflamed knee. (A)Time course of the effect of locally administered URB597 (closed circles) vs. vehicle (open circles) on leukocyte rolling. There was a significant reduction in the number of rolling …

Figure 3

The contribution of cannabinoid receptors to the effects of URB597 on leukocyte trafficking in the inflamed knee. (A) Number of rolling leukocytes per minute 20 min after administration of URB597 (0.3 mg/kg) to acutely inflamed knees. Local application …

At 20 min, URB597 (0.3 and 3.0 mg/kg) also significantly decreased the number of adherent leukocytes compared to vehicle (P <0.05; n =6; Figure 2C). Higher doses of URB597 (10 to 30 mg/kg), however, were less effective (n =5 to 6; Figure 2C). The inhibitory effect of URB597 (0.3 mg/kg) on leukocyte adhesion was not blocked by AM251 (P >0.05; n =6; Figure 3B) nor AM630 pretreatment (P >0.05; n =6; Figure 3B).

When administered alone, neither antagonist had any effect on leukocyte rolling or adherence, when compared to vehicle (P >0.05; n =6 to 7; data not shown).

Microvascular perfusion

Local administration of URB597 (3.0 mg/kg) decreased microvascular perfusion, when compared to vehicle treatment (P<0.01; n =6 to 10; Figure 4A). This effect was most prominent at 20 min, where URB397 (3.0 mg/kg) decreased microvascular perfusion by an average of 19.6±3.6% (n =7; Figure 4B). The inhibitory effect of URB597 on joint hyperaemia was blocked by both AM251 and AM630 (P <0.05; n =6 to 10; Figure 4C). High-dose URB597 (10 to 30 mg/kg) was ineffective at reducing inflamed joint hyperaemia (n =5 to 7; Figure 4B).

Figure 4

Laser speckle contrast analysis of the inflamed mouse knee joint. (A)Time course of the effect of locally administered URB597 (closed circles) vs. vehicle (open circles) on vascular perfusion. There was a significant reduction in local blood flow after …

Hindlimb weight bearing

Based on the results of the inflammation studies, a dose of 3.0 mg/kg was chosen for pain behaviour assessment. URB597 significantly improved inflamed hindlimb weight bearing during the initial 3 hr after administration (P <0.05; n =12; Figure 5A). The most profound effect of URB597 was observed at 30 min; therefore, subsequent experiments focused on this time point. In mice pretreated with the CB1 receptor antagonist AM251 (0.2 mg/kg; n =9), the anti-nociceptive effect of URB597 was abolished (P <0.05; Figure 5B). Conversely, pretreatment with the CB2 receptor antagonist AM630 (0.2 mg/kg; n =9) had no effect on URB597-induced anti-nociception (P >0.05; Figure 5B). In order to confirm that FAAH inhibition was occurring locally in the treated joint and not systemically, URB597 (3.0 mg/kg) was administered to the contralateral joint and, in this instance, the drug had no effect on incapacitance (P >0.05; n =9; Figure 5B).

Figure 5

Hindlimb weight bearing in mice with acute experimental joint inflammation. (A) Time course of the effects of URB597 (3.0 mg/kg) on inflamed hindlimb weight bearing (baseline (BL) values represent pre-inflammation induction). Over the course of 3 hr, …

When administered alone, neither antagonist had an effect on hindlimb weight bearing when compared to vehicle (P >0.05; n =6 to 7; data not shown).

The effects of URB597 on inflammation

Activation of the endocannabinoid system has been shown to decrease the development of inflammation in the intestine [29], central nervous system (CNS) [30] and hindpaw [31]. More specifically, cannabinoids are capable of reducing leukocyte proliferation and cytokine release [32,33]. In our study, inflammation was measured in vivo by visualizing the interactions between leukocytes and the vascular endothelium, as well as measuring changes in vascular perfusion in acutely inflamed mouse knee joints. When kaolin/carrageenan-inflamed knee joints were treated with low-dose URB597, there was a significant reduction in the number of rolling and adherent leukocytes, and inflammation-induced hyperaemia. A similar effect has been observed in the cerebral [34,35] and ocular [36] microcirculation, where synthetic cannabinoids decreased leukocyte rolling and adhesion. However, as dosing increased, the effects of URB597 on leukocyte kinetics and hyperaemia were less pronounced. One possible explanation for this effect is the downregulation and/or desensitization of articular cannabinoid receptors as a result of excessive endocannabinoid accumulation in the joint [37]. Alternatively, high doses of URB597 may be having off-target effects, which promote inflammation and pain thereby masking any beneficial responses. For example, high concentrations of anandamide are known to cause peripheral sensitization in joints by opening vanilloid-gated ion channels [38]. Furthermore, anandamide can be oxygenated via a cyclooxygenase-2 pathway leading to the formation of pro-inflammatory and algesic prostamides, which may offset the therapeutic benefits of FAAH inhibition [39,40]. Therefore, URB597 may need to be administered with selective prostamide antagonists in order to achieve greater pain relief and resolution of inflammation.

In the present study, the inhibitory effects of URB597 on leukocyte rolling and blood flow were blocked by both CB1 and CB2 receptor antagonism, while the reduction in leukocyte adhesion was CB receptor independent. The inhibitory effect of URB597 on inflammation could be due to a decrease in joint nerve activity following CB1 receptor activation. It has been shown that CB1 receptors are highly expressed on knee joint nerve terminals [17] and that selective activation of CB1 receptors can cause a decrease in the neuronal release of pro-inflammatory neuropeptides, such as calcitonin gene-related peptide (CGRP) [41]. Since CGRP promotes leukocyte migration [42] and vasodilatation [43], it is possible that the anti-inflammatory effects of URB597 described here are due to a CB1 receptor-dependent reduction in CGRP release although other pro-inflammatory neurogenic factors could also be involved.

The present investigation found that CB2 receptors are involved in endocannabinoid-mediated inhibition of rolling and hyperaemia in inflamed joints. This CB2-mediated inhibitory effect on leukocyte rolling has previously been observed in the retina [44]. The CB2 receptor agonist JWH-133 has been shown to inhibit the expression of P-selectin [44], an adhesion molecule responsible for the initiation and maintenance of leukocyte rolling [3]. It is, therefore, possible that URB597 decreased leukocyte rolling by promoting the inhibitory effect of the endocannabinoid system on selectin expression.

The inability of CB receptor antagonists to block the inhibitory effects of URB597 on leukocyte adhesion is intriguing. While cannabinoids classically bind to either CB1 or CB2 receptors, atypical cannabinoids have been found to bind to alternate G protein-coupled receptors such as GPR55 and GPR18 [45]. Both of these receptors have been found to have immunoregulatory functions [46] and, in the brain, GPR18 stimulation has been shown to inhibit leukocyte recruitment [47]. It is, therefore, possible that URB597 is causing the accumulation of atypical endocannabinoids in the joint whose activity reduces leukocyte adhesion through either or both of these atypical cannabinoid receptor subtypes.

The changes described here, concerning leukocyte adhesion, are in contrast to a study by Ni and colleagues [35], who found that cannabinoid-mediated leukocyte anti-adhesion was CB2 receptor-dependent and the inhibitory effect on leukocyte rolling was CB1 receptor-independent. The reason for the disparity between the two studies could be that Ni et al. were investigating leukocyte changes in response to a non-selective CB receptor agonist (WIN5512-2) and activity was assessed in the brain. Thus, there may be subtle differences in cannabinoid receptor properties between the microcirculation of the CNS versus peripheral tissues.

This study was funded by the Canadian Institutes of Health Research, and the Nova Scotia Health Research Foundation. We would also like to thank Dr. Christian Lehmann for use of his intravital microscopy equipment.