Corneal Kindling

In the corneal kindling experiments, KRM-II-81 was evaluated for its ability to block seizures in fully kindled mice. Adult male CF1 mice (n = 8 per group, 18–25 g) were kindled using methods previously described (Matagne and Klitgaard, 1998; Rowley and White, 2010). They were kindled to a criterion of five consecutive secondarily generalized seizures of stage 5 (Racine, 1972). Briefly, mice were exposed to twice daily corneal stimulation (3 mA at 60 Hz for 3 seconds). After reaching the first stage 5 seizure (generally after about 2 weeks), the twice daily stimulation regimen was continued until each mouse achieved the criteria of five consecutive stage 5 seizures, at which point the mouse was considered to be fully seizure kindled. Fully kindled mice continued to be stimulated every 2 to 3 days until all mice became fully kindled.

Testing of KRM-II-81 began at least 5 days after the last corneal stimulation. KRM-II-81 was dosed orally 2 hours prior to stimulation for the assessment of anticonvulsant efficacy. Doses of 1–30 mg/kg were tested in groups of eight mice. Doses were selected based on the efficacy of KRM-II-81 studied in other seizure models by this route, where 10 mg/kg was a minimal effective dose (Witkin et al., 2018). Oral dosing was used here to define oral efficacy based on the oral dosing exposure of KRM-II-81 (Poe et al., 2016; Witkin et al., 2019ba). The percentage of mice protected and the seizure severity score (Racine, 1972) were measured.

The effects of KRM-II-81 on seizure protection were assessed by Fisher’s exact probability test and the ED₅₀ and 95% confidence limits were determined by probit analysis. Effects of KRM-II-81 on seizure severity scores were analyzed by one-way ANOVA followed by Dunnett’s test. Probabilities of < 0.05 were set a priori as the accepted level of statistical significance.