Data shown are means SEM; = 8 (total rats used =24). reversed MIA\induced weight\bearing asymmetry (MIA/vehicle: 68 6?g; MIA/MJN110: 35 4?g) and lowered ipsilateral PWTs (MIA/vehicle: 7 0.8?g; MIA/MJN110: 11 0.6?g), via both CB1 and CB2 receptors. Repeated treatment with MJN110 (5?mgkg?1) resulted in anti\nociceptive tolerance. A lower dose of MJN110 (1?mgkg?1) acutely inhibited pain behaviour, which was maintained for 1?week of repeated administration but had no effect on joint histology. MJN110 significantly inhibited expression of membrane\associated PGE synthase\1 in the ipsilateral dorsal horn of the spinal cord of MIA rats, compared with vehicle\treated MIA rats. Both doses of MJN110 significantly elevated brain levels of the endocannabinoid 2\arachidonoylglycerol. Conclusions and Implications Our data support further Vandetanib (ZD6474) assessment of the therapeutic potential of MAG lipase inhibitors for the treatment of OA pain. Abbreviations2\AG2\arachidonoylglycerolAAarachidonic acidGFAPglial fibrillary acidic proteinMAG lipasemonoacylglycerol lipaseMIAmonosodium iodoacetatemPGES1membrane\associated PGE synthase\1OAosteoarthritis Tables of Links for 15?min at 4C), and the resulting supernatants were evaporated to dryness. Tissue extracts were reconstituted in 200?L of acetonitrile?:?water (1:1), and 10?L was injected for analysis by LC\MS/MS using an Agilent 1100 series Vandetanib (ZD6474) (Agilent Technologies, Waldbronn, Germany) coupled to a Micromass Quattro Ultimatm triple quadrupole mass spectrometer (Waters, Manchester, UK) equipped with electrospray negative ionization. The column used was an ACE Excel 3?m particle diameter, Super C18, 150 2.1?mm at 40C, and the mobile phase was a linear gradient of water +0.02% formic acid (mobile phase A) and acetonitrile?:?methanol (4:1) +0.02% formic acid (mobile phase B). Flow rate was 300?Lmin?1, and analysis time was 15?min. Quantification of the PGE2, AA and 2\AG was done using fully extracted calibration standards for each of the analytes and was performed using QuanLynx v 4.1, Waters, Manchester, UK. Data and statistical analysis The data and statistical analysis in this study comply with the recommendations on experimental design and analysis in pharmacology (Curtis values were less than 0.05. For data that did not pass normality testing, non\parametric statistics were used (KruskalCWallis one\way ANOVA). Correlations between rat spinal gene expression and pain behaviour were determined with a Pearson correlation. To evaluate whether the anti\nociceptive efficacy of MJN110 was reduced over time, regression analysis determined whether there was a significant difference (< 0.05) from zero in Mouse monoclonal to CTNNB1 the slope of the pain behaviour in the presence of repeated MAG lipase inhibition; a significant difference indicated a reduction in inhibition of pain behaviour. Materials Vandetanib (ZD6474) MJN110 was a kind gift from Micah Niphakis and Benjamin Cravatt. MIA, Emulphore and Ethanol was purchased from Sigma (Gillingham, Dorset, UK) were purchased from Sigma, UK. Rimonabant and SR144528 were from Cayman Chemical Ann Arbor, Michigan, USA. Results Acute MAG Vandetanib (ZD6474) lipase inhibition reverses established MIA\induced pain behaviour As previously described (Sagar = 6 saline groups; = 8 rats MIA groups; total rats used = 22. *< 0.05, significant difference between MIA + vehicle and saline + vehicle; #< 0.05, significant difference between MIA + vehicle versus MIA + MJN110; two\way ANOVA and Bonferroni test. Contributions of CB1 and CB2 receptors to MJN110\mediated inhibition The ability of CB1 or CB2 receptor antagonists to block the anti\nociceptive effects of MJN110 on pain behaviour was investigated. The anti\nociceptive effects of MJN110 (5?mgkg?1) on weight\bearing asymmetry were blocked by the CB1 receptor antagonist SR141716A (Figure?2A, C). The CB2 receptor antagonist SR144528 also reduced the anti\nociceptive effects of MJN110 on weight\bearing asymmetry (Figure?2A, C), but the magnitude of the reversal was significantly less than that achieved by SR141716A. MJN110\mediated reversal of MIA\induced lowering of ipsilateral hindpaw withdrawal thresholds was significantly blocked by the CB2 receptor antagonist SR144528, but not the CB1 receptor antagonist (Figure?2B, D). Open in a separate window Figure 2 Effect of CB receptor blockade on MJN110\mediated analgesia. (A) Intra\articular injection of MIA was associated with a significant increase in weight\bearing asymmetry, which was reversed by a single administration of 5?mgkg?1 MJN110; this effect was blocked by the CB1 receptor antagonist SR141716A (3?mgkg?1) and the CB2 receptor antagonist SR144528 (3?mgkg?1). Data are mean + SEM; = 10 rats per group. *< 0.05, significant difference between MJN110 + vehicle group pre\ and post\drug on day 21; + < 0.05, significant difference between MJN110 + SR144528 pre\ and post\drug on day 21; NS = no significant difference between MJN110 + SR141716A pre\ and post\drug on day 21; two\way ANOVA and a Bonferroni test. (B) Intra\articular injection of MIA was associated with a significant decrease in ipsilateral paw withdrawal thresholds, which was reversed by a.