FIGURE 2.
A role for ASIC1b in acid-induced mechanical hyperalgesia. (A) Mechanical hyperalgesia of wild-type (Asic1b+/+, N = 7) and ASIC1b-knockout (Asic1b– /–, N = 8) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 1 and evaluated by 0.20-mN von Frey filament. Data were analyzed by two-way ANOVA (Interaction F(10,143) = 5.34, P < 0.0001; Time F(10,143) = 14.5, P < 0.0001; Genotype F(1,143) = 23.11, P < 0.0001), followed by Sidak post hoc test. ∗∗∗P < 0.001 for genotype difference at specific times. (B) Mechanical hyperalgesia of Asic1b+/+ (N = 10) and Asic1b– /– (N = 7) mice was induced by intramuscular injections of pH-4.0 saline on days 0 and 5. Data were analyzed by two-way ANOVA [Interaction F(12,195) = 5.933, P < 0.0001; Time F(12,195) = 21.28, P < 0.0001; Genotype F(1,195) = 59.07, P < 0.0001], followed by Sidak post hoc test. ****P < 0.0001; ∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05 Asic1b+/+ vs. Asic1b– /–. For comparison, the effect of mambalgin-1 on acid-induced hyperalgesia in Asic1b– /– mice was plotted. (C) Effect of mambalgin-1 (MB-1) on acid-induced mechanical hyperalgesia (vehicle, N = 7; MB-1 [3 pmol], N = 6; MB-1 [15 pmol], N = 7; MB-1 [30 pmol], N = 7). Data were analyzed by two-way ANOVA [Interaction F(21,168) = 4.014, P < 0.0001; Time F(7,168) = 26.61, P < 0.0001; Drug dose F(3,168) = 29.32, P < 0.0001], followed by Sidak post hoc test. ###P < 0.001, ####P < 0.0001, MB-1 (30 pmol) vs. vehicle; $$$$P < 0.0001, MB-1 (15 pmol) vs. vehicle. (D) Cumulative withdrawal response after second acid injection 5 days before acid + MB-1 injection is shown as the area under the receiver operating characteristic curve (AUC) calculated by the trapezoidal method. Data were analyzed by one-way ANOVA [F(3,21) = 4.719, P = 0.0114], followed by Dunnett post hoc test. ∗∗P < 0.01, ∗P < 0.05 vs. vehicle. (D’) While 30 pmole mambalgin-1 was applied in the second acid injection, it blocked the development of the acid-induced chronic hyperalgesia. (E) Effect of PcTx1 on acid-induced mechanical hyperalgesia. Data were analyzed by two-way ANOVA (vehicle, N = 4; PcTx1 [120 nmol], N = 6) Interaction F(7,64) = 0.2935, P = 0.9541; Time F(7,64) = 31.29, P < 0.0001; Drug dose F(1,64) = 0.054, P = 0.8169). (F) Cumulative withdrawal response after a second acid injection 5 days before acid + PcTx1 injection is shown as the AUC calculated by the trapezoidal method. Data were analyzed by unpaired t-test [F(3,5) = 2.191, P = 0.4149). Black arrows, the time mice received intramuscular injection of pH 4.0 saline. Red arrows, the time mice received intramuscular injection of pH-4.0 saline mixed with mambalgin-1 or PcTx1. B, baseline. Data are mean ± SEM.