Pilocarpine-induced status epilepticus (SE) in rats

The present study was approved by the Institutional Ethical Review Boards of the Shengjing Hospital of China Medical University (Shenyang, China). Male Sprague-Dawley rats had access to food and water ad libitum. All the rats were used following university animal care and use protocols. A total of 40 male Sprague-Dawley rats (age, ~20 days; weight, 100–120 g) were purchased from the Department of Experimental Animals, Shengjing Hospital of China Medical University. Rats were housed in single cages with a 12-h light/dark cycle at 25°C and 50% humidity. Rats were divided into four different groups: Control group, SE group, CB2R agonist group and CB2R antagonist group. At 30 min prior to pilocarpine injections, rats were administered with methylscopolamine nitrate (S8502-1G; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; 1 mg/kg i.p.) to minimize the peripheral parasympathetic effects of pilocarpine treatment. Pilocarpine nitrate (P6503-5G; Sigma-Aldrich; Merck KGaA; 375 mg/kg i.p.) was subsequently administered. Onset of SE typically occurred 20–40 min following the pilocarpine injection when the animal displayed continuous moderate to severe behavioral seizures characterized by forelimb clonus, rearing, and falling. The CB2R agonist (JWH133, 1.5 mg/kg) was administered every 6 h following termination of seizure activity in the agonist group. The CB2R antagonist (AM630, 1 mg/kg) was administered every 6 h following termination of seizure activity in the antagonist group.

SE was defined as continuous seizure activity lasting ≥30 min or intermittent seizures without regaining consciousness between seizures lasting ≥30 min (24). The severity of convulsions was evaluated and only the rats that displayed behaviors consistent with ongoing SE were used in the present study. Seizure activity was terminated by consecutive diazepam (Shanghai Xudong Haipu Pharmaceutical Co., Ltd., Shanghai, China) injections (5 mg/kg i.p., solubilized in 10% ethanol, 45% propylene glycol and 45% H₂O) at 1, 3, and 5 h post onset of SE. Control groups were composed of naive and sham control rats that received methylscopolamine nitrate and diazepam injections only. Rats were sacrificed at 24 h after termination of SE. Hippocampus tissue was isolated and frozen at −80°C.