4.3. Seizure Induction

To examine the role of donepezil in neuronal death after pilocarpine-induced seizure, rats were intraperitoneally injected with lithium chloride (LiCl, Sigma-Aldrich Co., St. Louis, MO, USA, 127 mg/kg, i.p.) 19 hours before the injection of pilocarpine. Scopolamine (Sigma-Aldrich Co., St. Louis, MO, USA, 2 mg/kg, i.p.) was injected 30 min before pilocarpine injection, which was used to inhibit peripheral cholinergic properties. Pilocarpine (Sigma-Aldrich Co., St. Louis, MO, USA, 25 mg/kg i.p.) was intraperitoneally injected 30 min after scopolamine injection. Injection of pilocarpine induced status epilepticus (SE) [35]. SE classically occurred within 20–30 min of the pilocarpine injection [36]. The animals were placed at one animal per cage to observe seizure behavior. The seizure behavior was observed every 5 min according to the Racine process; Score 0 = no seizure behavior; Score 1 = stereotyped mouse and eye-blinking, facial exercise; Score 2 = head nodding; Score 3 = forelimb clonus; Score 4 = forelimb clonus and rearing; and Score 5 = rearing and falling [37]. Behavior changes from 0–5 were observed, and when the 5th step (falling) occurred, it was regarded as seizure onset. Diazepam (Valium, Hoffman-La Roche, Neuilly sur-Seine, France, 10 mg/kg, i.p.) was intraperitoneally injected 2 h after the start of SE. Some animals showed recurrent seizures after treatment with diazepam [38]. Despite the administration of diazepam, severe episodes of recurrent seizures were stopped by administering more diazepam (2 mg/kg, i.p.) [39].