Pilocarpine-induced status epilepticus

A prolonged status epilepticus (SE) was induced using a ramping pilocarpine protocol [18–20] in male 28-to-34-day-old C57BL/6 mice (Charles River, Sulzfeld, Germany). All procedures were performed according to national and international guidelines on the ethical use of animals (European Council Directive 86/609/EEC, approval of local authority, State Office for Agriculture, Food Safety and Fisheries Mecklenburg-Vorpommern, Germany; LALLF M-V/TSD/7221.3–1.1-013/10 and LALLF M-V/TSD/7221.3–1.1-047/12). Peripheral cholinergic effects were reduced by pre-application of methyl-scopolamine nitrate (Sigma-Aldrich, 1 mg/kg, i.p.) 30 min prior to pilocarpine treatment. Then, pilocarpine hydrochloride (Sigma-Aldrich, 100 mg/kg, i.p.) or saline as control was administered. The pilocarpine injection (100 mg/kg, i.p.) was repeated every 20 min until the animals developed SE (up to 800 mg/kg; on average 457±113 mg/kg, mean ± SD). During this procedure, all animals were carefully observed in individual cages. Control animals received saline instead of pilocarpine. Ten percent of animals were randomized to get saline instead of pilocarpine as control, but due to overt behavioral seizure activity the experimenter could not be blinded to whether the animal was injected with pilocarpine or with saline. The onset of SE was determined when an animal had a stage 4 or 5 seizure [22], which was followed by continuous epileptic motor activity. After 90 min of SE duration, all animals received a 100 μl bolus injection of diazepam solution (Ratiopharm, Ulm, Germany, 5 mg/ml, i.p.). Occasionally, diazepam had to be re-injected in order to stop seizure activity (up to 200 μl altogether). Using this ramping pilocarpine protocol, 54% of animals developed and survived status epilepticus. At last, all animals were given 100 μl of 0.9% saline solution and kept in separate cages.