Fig. 4.

Time-dependent changes of P2Y₁₂R mRNA expression (A, B), IL-1β protein level (C, D) in the hind paw and lumbar spinal cord (L4-L6) induced by CFA; effect of P2Y₁₂R antagonists 96 h after CFA treatment on mechanical hypersensitivity (E, F) and on central cytokine response (G, H) in rats. A, B. Changes in mRNA expression level of P2Y₁₂ receptor in the hind paw (A) and L4-L6 spinal cord (B). Rats received intraplantar injection of 0.1 ml 50% CFA or saline (control), and were decapitated 48 h or 96 h after treatment. Quantitative SYBR Green real-time PCR was performed by using specific primers. The experiments were repeated two times with similar results. The expression level of the P2Y₁₂ receptors was normalized to the expression level of the distinct housekeeping gene 18S rRNA. Data are displayed as the mean ± S.E.M. Asterisks indicate significant differences from the corresponding control *P < 0.05, Student's t-test. C, D. Intraplantar injection of 0.1 ml CFA caused IL-1β production in the rat hind paw (C) and spinal cord (D) in a time-dependent manner (48 or 96 h). Data are given as the mean level of cytokines ± SEM of three independent experiments (**P < 0.01, ***P < 0.001, ns, non-significant, Student t-test). E, F. Effects of the P2Y₁₂R antagonist cangrelor (E) and PSB-0739 (F) on CFA mediated inflammatory pain behavior 96 h after injection. The graphs show the paw withdrawal threshold values (PWT) in g corresponding to doses indicated on the abscissa. Symbols indicate significant differences from the pre-CFA treatment (***P < 0.001) and post-CFA treatment PWT values (^##P < 0.01, ^###P < 0.001), respectively. One-way ANOVA followed by Neuman–Keuls post-hoc test, n = 6–12/group. G. H. Effect of P2Y₁₂R antagonists on intraplantar CFA (0.1 ml)-induced IL-1β (G) and IL-6 (H) levels in the spinal cord 96 h after treatment. The spinal inflammatory response was strongly inhibited by PSB-0739 (i.t.) and by systemic cangrelor administration. PSB-0739 (0.3 mg/kg) the selective P2Y₁₂ receptor antagonist was injected i.t. 15 min prior post-CFA PWT determination, while cangrelor (3 mg/kg) the P2Y₁₂/P2Y₁₃ antagonist was added i.p. 30 min prior to post-CFA PWT determination. The levels of cytokines were quantified in the derived supernatants by multiplex cytokine assay. Data are given as the mean level of cytokines ± SEM of 5 independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA followed by Neuman–Keuls test). N.d. indicates non-detectable level, which was regarded as 10^− 5 pg/ml in statistical analyses.