Tail-Flick Antinociception: Dose-Response Analysis

The hot water tail-immersion test was used to assess the latency to withdraw the tail from a 53–54°C water bath in the absence of paclitaxel treatment. The distal 2 cm of the tail was immersed in the water bath and the latency to elicit a ‘flick’ response was measured as previously described (Bohn et al., 1999; Kim et al., 2015). Prior to injection, three different baseline values were recorded (i.e., with a 10-min interval between successive stimulations). A cut-off of 15 s was applied to avoid tissue damage. A within subjects dose-response curve was calculated using escalating doses of morphine (0, 1, 3, 10, 30, 100 mg/kg i.p.) administered 30 min apart. Approximately 24 h after the last morphine injection, mice were randomized to receive once daily chronic treatments with either vehicle, GAT211 (20 mg/kg i.p.), morphine (10 mg/kg i.p.), or GAT211 (20 mg/kg i.p.) + morphine (10 mg/kg i.p.) for seven consecutive days (days 2–8). Mice were then tested for tail-flick withdrawal latencies 30 min following injection of the aforementioned pharmacological treatments on days 2, 4, and 6 of chronic dosing. On day 9, mice received the same escalating doses of morphine as delivered on day 1. Values were converted to %MPE to compare antinociceptive effects of morphine following acute (i.e., day 1) and chronic (i.e., day 9) drug treatments. See Figure 4A for time course of the experimental protocol.

Co-treatment of GAT211 with morphine reduces tolerance to morphine antinociception in the tail-flick test. Schematic shows timing of experimental procedures (A); vertical arrows show time of assessment of tail flick latencies, which were measured 30 min following drug administration (i.p.) on days 2, 4, and 6 of repeated dosing (A). Ascending doses of morphine (0, 1, 3, 10, 30, 100 mg/kg i.p.) produced dose-dependent increases in tail-flick antinociception. Repeated injections of vehicle or GAT211 (20 mg/kg i.p. × 7 days) did not reliably shift the morphine dose response curve (B,C). Repeated injection of morphine (10 mg/kg i.p. × 7 days) produced a right-ward shift in the dose-response curve of morphine in producing antinociception in the tail-immersion test (D,E). The combination of GAT211 (20 mg/kg i.p.) + morphine (10 mg/kg i.p.) also produced a right-ward shift in the dose-response curve for morphine to produce tail-flick antinociception albeit to a lesser degree (D,E). Tolerance to morphine-induced antinociception in the tail immersion test was delayed by co-treatment with GAT211 (20 mg/kg i.p.) (F,G). GAT211+ morphine cotreatment produced heightened antinociception on day 4 but not on day 6 of repeated injections compared to all other groups (F,G). Vehicle and GAT211 do not elicit tail-flick antinociception when administered alone (F,G). Data are expressed as tail-flick latencies in seconds (B,D,F) and values transformed to % MPE (C,E,G) values. ^XP < 0.05 morphine vs. Vehicle and GAT211, ⁺P < 0.05 GAT211 + morphine vs. Vehicle and GAT211 *P < 0.05 GAT211 + morphine vs. all other groups, two-way ANOVA followed by Bonferroni post-hoc. Mean ± SEM (n = 6 per group).