C57BL/6 mice (all males, 3 months of age) were used in this study. All mice were maintained under a 12:12 h light:dark cycle and had free access to food and water. All experiments were performed in accordance with international guidelines of animal experimentation (ARRIVE guidelines) and were approved by the Animal Care Committees of The Second Affiliated Hospital of Nanchang University.
Induction of SE
SE was induced in mice according to a previous report . Briefly, mice were injected with a low dose of pilocarpine hydrochloride (100 mg/kg) (Sigma Aldrich, St. Louis, MO, USA) by intraperitoneal (i.p.) administration every 20 min until the onset of SE. Methylscopolamine nitrate (1 mg/kg; i.p., Sigma Aldrich) was injected 30 min before the injection of pilocarpine to alleviate peripheral cholinergic side effects. A similar volume of 0.9% (w/v) sterile saline was injected into control mice. SE was defined as continuous stage 4 or 5 seizure activities according to the Racine scale . The first seizure usually occurred after three injections. If SE was not induced after five injections, no further attempts were made to avoid death of the mouse. All mice that developed SE lasting for several hours received diazepam (10 mg/kg) after 90 min to terminate seizure activity. Control mice were also injected with the same dose of diazepam. Mice that did not develop SE and died after pilocarpine injections were excluded. Mice were distributed into three groups: the developed SE group (SE group); Saracatinib (Sar) treatment group (Sar + SE group); and the control group (CON group).
Mice cognitive functions was assessed using the Morris water maze (MWM) and novel object recognition (NOR) behavioral tests. The investigator was blinded to the genotype during testing. Data were analyzed using Smart v2.5.21 from Panlab. [n = 9 in CON group; n = 8 each in the SE group and Sar + SE groups. The experimental scheme is shown in Figure 1.
Mice were tasked to find a target platform within 90 s from different starting positions in each trial. Four trials were performed each day for five consecutive days. Escape latencies (time spent in swimming from the start point to the target platform) and path length (the distance from start point to the target platform) before reaching the target platform were recorded on these five consecutive days. On day 6, the probe trial was performed to assess memory consolidation by removing the target platform. The number of area crossings with the target platform placed in previous training and the staying time in the quadrant of the target platform placed in previous training were recorded.
Novel object recognition test
Mice were exposed to two identical objects placed at a distance of 10 cm from the sidewalls in two opposite corners of the apparatus for 10 min in the training session. After 90 min, the mice were allowed to explore in the presence of one familiar and one novel object for 10 min. The preference index (PI) was defined as the time exploring one of the identical objects/total time exploring two of the identical objects. Recognition index (RI) was defined as the time exploring the novel object/total time exploring the novel object and the familiar object.
Sarscatinib was administered (25 mg/kg) orally starting 2 h after diazepam injection and repeated twice daily for the first three days followed by a single dose each day for the next 11 days during the two weeks after SE.
Electroencephalogram (EEG) recording
Mice that received continuous video-EEG monitoring were implanted with silver wire electrodes (0.125 mm in diameter) into the hippocampal dentate gyrus (DG) region after anesthesia with pentobarbital (Sigma Aldrich, 50 mg/kg, i.p.). The electrode implantation site used the following coordinates with the bregma as the reference: bregma: -2.3 mm, lateral: 1.8 mm and depth: 2.0 mm. The reference electrode was placed in the frontal cortex. All implanted surgery was performed at seven days before the induced SE. EEG activity was recorded 12 h every day for up to 7 days at 14 days after induced SE using PowerLab 8/35 software (ADInstruments, Sydney, Australia). Epileptic spikes were detected and scored by the Gotman spike using PowerLab software. Mice behavior was monitored using video and reviewed by an investigator who was blinded to the identity of the groups.
One month after induced SE, the mice were anesthetized with pentobarbital (Sigma Aldrich; 50 mg/kg, i.p.) and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M PBS. The brain was dissected and fixed in 4% paraformaldehyde overnight at 4oC and then cryoprotected in 30% sucrose in PBS for 72 h at 4oC. Coronary sections (8 m) were cut with a cryostat and mounted onto glass slides. Sections were washed in PBS and rehydrated in ethanol of decreasing concentrations. After washing with PBS, the sections were incubated with anti-NeuN antibody (ab104224, 1:500; AB_10711040; Abcam, Cambridge, UK) overnight at 4oC and washed with PBS. The sections were then incubated with secondary antibody and visualized using 3,3-diaminobenzidine.
Data are expressed as the mean + SEM. One-way or two-way analysis of variance was used to assess differences between two groups and the least significant difference test was used to compare multiple groups. A value of p < 0.05 was considered statistically significant.