The vasoactive effects of the synthetic cannabinoid (CB) arachidonyl-2-chloroethylamide (ACEA) was

The vasoactive effects of the synthetic cannabinoid (CB) arachidonyl-2-chloroethylamide (ACEA) was tested in the knee joints of urethane-anaesthetised rats. Adult male Wistar rats (240-430 g) were caged in pairs at room temperature under a 12/12 h (07:00/19:00) light-dark cycle and fed with standard rodent food. On the day of blood flow assessment animals were anaesthetised with urethane (1.5 mg kg?1 i.p.) and depth of anaesthesia was confirmed by an absence of the pedal withdrawal reflex. The hindlimb of the rat was shaved and the animal was then placed in dorsal recumbency on a thermostatically controlled heating pad (Fine Science Tools Inc. Vancouver Canada) to maintain a core body ASP3026 temperature of 37°C. All subsequent surgical and experimental procedures had received prior approval from the University of Calgary Animal Care Committee which is in accordance with the Canadian Council for Animal Care guidelines. Surgical preparation The fur covering the ventral aspect of the rat neck was lacquered using mineral oil and a longitudinal incision was made in the skin to expose the trachea which was cannulated to allow unrestricted breathing. The left carotid artery was then isolated and cannulated with a polyethylene catheter (0.5 mm internal diameter 1 mm outer diameter; Portex Kent ASP3026 U.K.) containing heparinised saline (50 U 100 ml?1). This carotid cannula was connected in series to a pressure transducer (Stoelting Co Illinois U.S.A.) and mean arterial ASP3026 pressure (MAP) was continuously recorded on a differentially amplified blood pressure monitor (World Precision Instruments Florida U.S.A.). The skin covering the anteromedial region of the rat stifle (knee) joint was excised and all superficial fascia removed to allow unrestricted access to joint capsular blood vessels. Following joint exposure warmed (37°C) physiological saline (0.9% NaCl) was intermittently superfused over the surface of the knee to prevent tissue desiccation. A number of studies have consistently shown that topical ASP3026 application of saline in this manner has no discernible effect on joint blood flow (McDougall observations. Drugs AM251 AM281 AM630 and capsazepine were all supplied by Tocris Cookson Inc. (Ellisville MO U.S.A.). ACEA capsaicin and urethane were Plscr4 obtained through Sigma-Aldrich Ltd (Ontario Canada). Hypnorm was purchased from Janssen Pharmaceutica (Beerse Belgium) and diazepam from Sabex Inc. (Boucherville Canada). ACEA and all CB antagonists were dissolved in dimethylsulphoxide (DMSO) and cremophor before diluting to the working doses alliquoting and storing at either 4°C or ?20°C as required. The final concentration of vehicle was maintained at 2% DMSO and 1% cremophor for each dose and this vehicle was found to have no significant effect on joint blood flow (see Figure 1a). Figure 1 (a) Time course of vehicle (2% DMSO/1% cremophor) on knee joint blood flow. Vehicle was found to have no significant effect on synovial perfusion over the 5 min test period (TRPV1 receptors located on capsaicin-sensitive joint afferents although the evidence presented here is somewhat indirect. Elsewhere nerve recordings from rat knee joint afferents exposed that anandamide sensitised nociceptors present on capsaicin-sensitive nerves and that this effect was clogged by capsazepine (Gauldie fibres to cause the secondary launch of inflammatory neuropeptides which in turn generate vascular clean muscle relaxation (Zygmunt TRPV1 receptors located on capsaicin-sensitive unmyelinated and thinly myelinated joint afferents. These findings suggest that as with other peripheral cells endocannabinoids are likely endogenous candidate ligands for synovial TRPV1 receptors and may contribute to the development and progression of joint neurogenic swelling. Acknowledgments We say thanks to Chunfen Zhang for her technical assistance with this project. Financial support from your Arthritis Society of Canada the Alberta History Basis for Medical Study (AHFMR) and the Canadian Institutes of Health Study (CIHR) are gratefully acknowledged. JJMcD is an AHFMR Scholar and an Arthritis Society/CIHR New Investigator. Abbreviations.