2.7. Intraperitoneal injection (i.p.)

Seventy‐two hours following subplantar injection of CFA and following Day 3 behaviour testing (see above), male and female adult Sprague–Dawley rats were given an i.p. injection of 1, 3 or 10 mg·kg⁻¹ Z944 or vehicle dissolved in 0.5% (weight/volume) of carboxymethylcellulose (Sigma Aldrich, USA). Z944 is highly bioavailable and CNS‐penetrant, with pharmacokinetic studies demonstrating that oral and systemic administration produces low micromolar concentrations of Z944 in rodent plasma (unpublished observations and Casillas‐Espinosa et al., ²⁰¹⁹). Moreover, i.p. injection of 10 mg·kg⁻¹ Z944 reduced brain epileptiform activity in adult rats (Tringham et al., ²⁰¹²), demonstrating CNS penetrance and actions on central excitability at the dose and route of administration of Z944 used here. Z944 was dissolved in DMSO to create a stock solution (100 mg·ml⁻¹ for animals receiving 10 mg·kg⁻¹, 30 mg·ml⁻¹ for animals receiving 3 mg·kg⁻¹, and 10 mg·ml⁻¹ for animals receiving 1 mg·kg⁻¹) that was then suspended in the carboxymethylcellulose solution (1‐part Z944 stock: 9‐part carboxymethylcellulose solution). Vehicle‐treated animals received DMSO without Z944 in 0.5% (weight/volume) of carboxymethylcellulose solution. All solutions were prepared the day of injection, and the experimenter was blinded to the treatment by a lab‐mate. All animals were allowed 20 min in their home cage between Day 3 testing and the administration of the i.p. injection. This allowed animals to eat or drink before being placed back in testing chambers for the time course. Animals received injections 1–1.5 min apart, with the exact time noted by the experimenter to ensure accurate readings during the time course. Intraperitoneal injection was performed by the experimenter alone using a surgical drape to restrain the animal. After i.p. injection, animals were placed in the behaviour testing chambers and allowed to acclimate for 20 min. During the time course, measurements of paw withdrawal threshold were taken every 15 min, starting 20 min after i.p. injection. The time of the test was measured from the third stimulus administration (five stimuli were presented in each trial, and the third, the middle stimulus, occurred at the given time interval). We found that i.p. injection of Z944 (1 to 10 mg·kg⁻¹) did not induce any behavioural signs of sedation or motor deficits. Moreover, a previous in‐depth analysis of potential non‐specific in vivo effects of Z944 demonstrated that i.p. injection of Z944 at a dose above that used in the present study (30 mg·kg⁻¹) did not induce any significant sedation or motor abnormalities (Tringham et al., ²⁰¹²).

The technique for measuring paw withdrawal threshold differed during the time course. Simplified up‐down method normally involves two trials of five stimuli that are then averaged to give paw withdrawal threshold. For the time course, only one trial was performed every 15 min to ensure that the animals were not overstimulated. A pilot study was used to determine an effective interval for the time course. One unsuccessful pilot was run using a testing interval of 10 min. This pilot was deemed unsuccessful because animals were displaying freezing behaviour: not reflexively withdrawing their paw at weights normally far‐above expected withdrawal threshold. We concluded that allowing animals more time to recover between trails was necessary and thus increased the testing interval by 50%. This decision, in turn, allowed more accurate timing of testing at each animal's given testing time. Animals were sacrificed immediately after the conclusion of the time course.