Experimental groups

Adult male Wistar rats (220–250 g) were divided randomly into sham-operated (SHAM; n = 20), epilepsy (EP; n = 20), and epilepsy plus DL-NBP treatment (EP+NBP; n = 20) groups. Each rat was housed separately during postoperative status epileptics, and then each group was fed in separate cages (five rats per cage) after status epileptics. All animals were housed in standard humidity and temperature conditions in a 12 h light/dark cycle, with ad libitum access to food and water. All experimental procedures were approved by the Animal Care and Use Committee of China Medical University. All animal experiments complied with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were carried out in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China.

TLE model

TLE model animals were established by injection of kainic acid (KA) into the right lateral ventricle [10]. Rats were anesthetized using a mixture of 70% nitrous oxide and 3% isoflurane in 30% oxygen, and maintained with 1.5% isoflurane. A solution of 0.4 μg/μL KA (2 μL total volume) in sterile saline was injected into the right ventricle using a Hamilton microsyringe (stereotactic coordinates: −1.5 mm mediolateral, −4.0 mm dorsoventral, and +0.8 mm anteroposterior; depth determined from the cerebral surface) and an infusion pump (0.5 μL/min). The needle was left in place for 5 min after the injection and then removed slowly. The first seizures began at 15–30 min after KA injection. The animals were removed the stereotaxic frame and then allowed to recover from anesthesia. A total of 47 rats received KA injection, with seven deaths (mortality = 14.9%).

Seizure severity was graded using five levels according to the Racine scoring system. Grade IV animals with more severe attack severity and animals with an attack duration >2 h were used in this study. After approximately 2 weeks of recovery, animals developed focal SRS accompanied by interictal spikes and waves (chronic phase). SHAM animals were injected with normal saline using the same procedures described above, with no deaths.

DL-NBP administration

The purity of DL-NBP was >99.5% (CSPC-NBP Pharmaceutical Co., Ltd., Hebei Province, China). DL-NBP was diluted to 7 mg/mL in vegetable oil and administered orally at a dose of 70 mg/kg/day from the 7th day post-surgery for 2 weeks.

Water maze test

Rats were subjected to the water maze test on days 31–33 after seizure initiation. As we reported, the match-to-place version was used to evaluate spatial learning [10]. On the last day of testing (postoperative day 33), the 60 s probe test without a platform was performed to estimate memory of the platform position (number of passes through the previous platform location).

Electroencephalograph

Electroencephalograph (EEG) was recorded from rats in the EP group and the EP+NBP group on days 34–36 after surgery. Intracranial EEG electrodes were placed into the right hippocampus (n = 5 per group). The electrodes were embedded within the CA3 region of the right hippocampus (2.4 mm lateral to midline, 3.4 mm ventral to dura, 3.8 mm posterior to bregma) and the regio nasalis using coordinates derived from the Paxinos and Watson atlas. After two days of recovery, the total duration of spontaneous EEG seizures over 2 h was recorded from freely moving animals. Digital acquisition software (LabScribe2; iWorx, USA) was used to record the EEG signals, which were stored for off-line analysis.

Tissue preparation

On day 37 of recovery, animals were perfused transcardially with 4% paraformaldehyde in phosphate-Buffered Saline. The tissues were post-fixed and stored in 30% sucrose solution at 4 °C. The brains were cut into coronal sections from 2.8 to 4.3 mm posterior to bregma (30 μm thick) on a cryotome (Thermo Electron, Waltham, MA, USA), when they were in the bottom. For each animal, five from every series of ten sections were used for immunofluorescent staining.

Immunofluorescent staining

Brain sections were incubated with primary antibodies against guinea pig anti-doublecortin (DCX; 1:400; Abcam) at 4 °C overnight, followed by incubation in Alexa Fluor 488 goat anti-rabbit secondary antibodies (1:200; Invitrogen, USA) for 2 h. Images were collected by microscopy (Olympus, Japan) using a ×20 or ×40 objective. The number of DCX-positive cells was counted using NIH ImageJ software (https://imagej.nih.gov/ij).

Nissl staining

Brain tissues were fixed with paraffin and then cut into a series of coronal sections (10 μm thick). Brain sections were then incubated in toluidine blue solution for 20 min at 56 °C, quickly washed in distilled water, incubated in 95% alcohol for 5 min, dehydrated in increasing alcohol concentrations for 5 min, cleared for 5 min with xylene, and then mounted in neutral gum solution. The number of CA3 pyramidal neurons in the hippocampus (per 1 mm2) in both cerebral hemispheres was counted by microscopy with a high magnification objective.

Timm staining

Animals in the three groups (n = 5 per group) were deeply anesthetized and then perfused intracardially with sodium sulfide solution (1.2% Na2S·9H20, 1% NaH2PO4·H20) and normal saline. The brains were then removed and post-fixed in paraformaldehyde solution. Coronal sections were stained with Timm staining solution for 90 min at room temperature. Images were taken using an Olympus BX51 microscope.

Statistics

All statistical analyses were performed using statistical software (SPSS v25). Water maze data were analyzed by repeated-measures or one-way analysis of variance. The remaining data were analyzed using the Kruskal–Wallis or Mann–Whitney U non-parametric tests. All data are presented as mean ± standard error of the mean. Significance was set at P < 0.05.

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