Fig. 5.

The effect and mechanism of action of P2Y₁₂R antagonists on intraplantar CFA injection (0.1 ml)-induced peripheral inflammation in rats. A, B, C. Robust elevation were detected in the levels of TNF-α, IL-1β and IL-6 in the hind paw 48 h after induction the inflammatory pain. The increase in the concentrations of cytokines was decreased by intrathecal (i.t.) PSB-0739 administration. D. Anti-inflammatory cytokine response: intraplantar injection of CFA (0.1 ml) significantly increased the level of IL-10 in the hind paw of rats 48 h after treatment. 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 (* < 0.05, ** < 0.01, *** < 0.001 one-way ANOVA followed by Neuman–Keuls-test). E. The effect of 6-OHDA pretreatment (40 mg/kg + 60 mg/kg + 60 mg/kg, i.p., in every 2nd day) on endogenous monoamine levels in the hind paw. Control rats received saline treatment in an identical manner. The level of noradrenaline (NA), normetanephrine (NM) and 3,4-dihydroxyphenylglycol (DOPEG) was measured by HPLC-EC and is expressed as pmol/mg protein. (*P < 0.05, Student t test with Welch correction, n = 6/group). F. Subdiaphragmatic vagotomy (VGX) and the α7 nAChR antagonist MLA, but not 6-OHDA pretreatment occludes the antihyperalgesic effect of PSB-0739 after CFA treatment. In these experiments, the α7 nACh receptor antagonist methyllycaconitine (MLA) or its vehicle (saline, SAL) was administered 45 min, while PSB-0739 (0.3 mg/kg)/saline (SAL) was injected i.t. 15 min before the post-CFA PWT determination, respectively. 6-OHDA pretreatment was performed as described in E. Vagotomy was induced after the determination of basal PWT values, and CFA was administered 10 days after surgery (* < 0.05, ** P < 0.01, one-way ANOVA followed by Neuman–Keuls-test, n = 5–10/group). G, H. The α7 nAChR antagonist MLA prevents the inhibitory effect of PSB-0739 on IL-1β (G) and TNFα (H) induction in the inflamed hind paw 48 h following CFA treatment. Experiments were performed according to the protocols described in F. After the experiments, IL-1β and TNF-α levels were quantified in the derived supernatants by multiplex cytokine assay. Data are given as the mean level of cytokines ± SEM of 5 independent experiments. Cytokine levels are expressed as pg/ml (n = 5/group). (* < 0.05, *** P < 0.001, ns, non-significant, one-way ANOVA followed by Neuman–Keuls-test, n = 5/group).