Pentylenetetrazol-induced SE in vivo

SE was induced in rats via intraperitoneal injection of Pentylenetetrazol (PTZ), a non-competitive antagonist of GABAaRs. PTZ (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in physiological saline before injections (10%). The rats were randomly divided into a normal control group (n = 6) and a PTZ-induced SE group (n = 6). Rats in the SE group were intraperitoneally injected with PTZ at an initial dose of 40 mg/kg, followed by 20 mg/kg every 10 min until SE development [38]. Rats in the control group were treated with physiological saline. The time of PTZ and saline injection was between 10:30 am to 12:30 am. Seizures were classified as follows [39, 40]: stage 0, no seizures; stage 1, ear and facial twitching; stage 2, strong myoclonus but no upright position; stage 3, strong myoclonus with upright position and bilateral forelimb clonus; stage 4, clonic–tonic seizures; and stage 5, generalized clonic–tonic seizures and loss of postural control. SE was defined as stage 4 or higher seizure, lasting at least 30 min with prolonged episodes of seizures interrupted by postictal phases of depression, but without regaining quadruped posture or consciousness [41]. The latent period of each sample was calculated based on the characteristics of an epileptic seizure, that is, the time between the first injection of PTZ and first seizure. The duration of episodes of stage 4 or higher seizure was recorded. Next, the rats were deeply anesthetized with isoflurane and their hippocampi were separated for biochemical experiments. In the in vivo model, a single animal was considered as the experimental unit.