Experimental Animals
In this experiment, 45 clean male SD rats aged 6 to 8 weeks were selected from Shandong Jinan Pengyue Experimental Animal Breeding. The animals were reared in a controlled environment with the temperature of (22 ± 2) ℃, humidity of 50%-60% and alternating light/dark for 12 h. Adequate drinking water and standard feed were provided and relevant experiments were carried out after 1 week of adaptation. All animal experiments were approved by the Ethics Committee of the Affiliated Hospital of Binzhou Medical College.
Rat Chronic Epilepsy Model
The chronic epilepsy model was produced by injection of PTZ(35 mg/kg, i.p.; Sigma-Aldrich, St. Louis, MO, USA), a drug that is widely used to simulate clinical epilepsy[12]. The rats were divided into 3 groups with 15 rats each using the random number table method:(i) Control group-rats received normal saline(NS). (ii) PTZ group: rats received intraperitoneal injection of PTZ (35mg/kg) once every other day. (iii) CQ group:rats received CQ (40mg/kg) intraperitoneal injection 30 minutes before PTZ injection. Rats were observed 30min after injection, and the rats with 3 consecutive seizures above grade iii were recognized to be completely ignited[13]. The seizure intensity was scored as follows[14]: Stage 0: no response; Stage 1: facial rhythmic spat, ear or facial muscle twitch; Stage2: nodding with more severe facial muscle twitching; Stage 3: forelimb clonus, but not upright; Stage 4 : forelimb clonus with upright posture; Stage 5 : tonic clonus with posture out of control; Stsge 6 : death.
Behavioral Observation and EEG Recording
The seizure grades, seizure latency and seizure times within 30 minutes were observed after the last intraperitoneal injection. Abnormal brain discharges after drug intervention were recorded with an electroencephalogram (EEG) apparatus (Nicolet EEG V32 recorder, CareFusion, San Diego, USA) for 30 minutes. The rats were anesthetized with 1.5% pentobarbital sodium(30mg/kg) before being fixed on the stereotaxic brain locator. Subcutaneous needle-shaped electrodes, 2.0 mm in front of the anterior fontanelle and 2.0 mm on both sides of the sagittal line, were implanted at the top center. The electrodes were sutured with skin needle. Binaural electrode was used as the reference electrode. The recording parameters were as follows: filter at 40 Hz, sensitivity of 70 μV/mm, and a paper speed of 30 mm/s. EEG of rats in each group was recorded and EEG amplitude and SWI were analyzed. SWI was used to quantify and evaluate the percentage of rats with at least one SWI[15].
Electrophysiological Recording and Analysis
Electrophysiological recordings were performed as previously described[16]. Rats (n=6 per group) were anesthetized with 1.5% pentobarbital sodium(30mg/kg) after the last drug injection. The brains were quickly taken after decapitation and placed in an ice water combination (254 mmol/L sucrose, 3 mmol/L KCl, 2 mmol/L MgCl2, 2 mmol/L, 1.25 mmol/L NaH2PO4, 10 mmol/L D-glucose, and 24 mmol/L NaHCO3) for 2 min. Coronal brain sections containing hippocampal bodies (350 μm) were prepared in artificial cerebrospinal fluid (124 mmol/L NaCl, 3 mmol/L KCl, 1.3 mmol/L MgSO4, 25 mmol/L NaHCO3, 2 mmol/L CaCl2, 1.25 mmol/L NaH2PO4, and 10 mmol/L D-glucose). After that, the brain slices were placed in artificial cerebrospinal fluid at 30°C and continuously perfused with 95% O2 + 5% CO2 for 2 hours.. Pyramidal neurons in the hippocampal CA1 region were observed by a 40 X water-immersion lens. The physiological characteristics of the action potentials(e.g. the frequency, threshold, latency, duration, amplitude, input resistance, and resting potential) of pyramidal neurons in the hippocampal CA1 region were recorded in current clamp mode (Multiclamp 700A system; Molecular Devices, USA). Data were exported and further analyzed (Clampfit v10.6).
Nissl Staining
For the remaining animals, the brains were removed, immediately placed in a neutral formalin buffer, dehydrated, made transparent, paraffin-embedded, and then sectioned continuously (3 μm-thick). The slices were conventional dewaxed with xylene and washed with distilled water,and then put into Nissl dyeing solution (toluidine blue method). After that, the dyeing tank was placed in an incubator at 50-60℃ for 25-50 minutes. Sections were dehydrated with 95% and 100% ethanol solutions, made transparent with xylene, and coverslipped. The color of nissl body in the neurons is hyacinthine, and it’s clearly visible in normal neurons, however, the nissl bodies in the pathological nerve cells are reduced and lightly stained, the normal nerve cells has been recgnized as positive cells. The number of positive cells was counted using a microscope.
Evaluation of MDA, Glutathione, and Iron Levels in the Hippocampus
The level of MDA was detected using a lipid oxidation detection kit (Beyotime Technology Institute, Shanghai, China), which utilizes the spectrophotometric measurement of red products produced during the reaction of thiobarbituric acid with MDA as a marker of lipid peroxidation. The concentration of iron in hippocampal tissues was measured by using a tissue Iron Assay Kit (Solarbio Biochemical Assay Division, Beijing, China). The level of Glutathione (GSH) were evaluated by using the GSH test kit (BEOTIME Technology Institute).
Immunohistochemistry
Brain tissues were cut into sections, dewaxed, rehydrated, treated with 3% hydrogen peroxide, and then the sections were incubated with rabbit anti-LC3B monoclonal antibody (1:500; Abcam), rabbit anti-P62 monoclonal antibody (1:500; Abcam), mouse anti-Keap1 monoclonal antibody (1:100; Abcam), rabbit anti-Nrf2 monoclonal antibody (1:100; Abcam), rabbit anti-GPX4 monoclonal antibody (1:100; Abcam), rabbit anti-COX2(PTGS2) monoclonal antibody (1:100; Abcam), at 4 °C overnight. Sections were then washed in PBS and incubated in goat antirabbit IgG (1:400; Affinity Biosciences, OH, USA) for 40 minutes at 37 °C, Subsequently, the sections were displayed using DAB kits (Zhongshan Golden Bridge Biotechnology, Beijing, China) and restained with hematoxylin. Images were captured on HPLAS-1000 pathological image analysis system, and 10 hippocampal fields were randomly selected in each section. The expression levels of LC3, P62 KEAP1, NRF2 gpX4 and PTGS2 were observed, and the mean optical density (MOD) was measured by ImageJ software.
Western Blot
After ice grinding, the hippocampal brain tissue was fully lysed by adding RIPA containing 0.1mmol/L PMSF cell lysate. The supernatant was taken after centrifugation. Proteins were quantified by BCA method, and samples were separated by SDS-polyacrylamide gel, and transferred to polyvinyl fluoride membrane by electrophoresis. TBST buffer containing 8% skim milk powder was sealed at normal temperature for 2 h. Primary antibody (rabbit anti-GPX4 monoclonal antibody, 1:1000), (rabbit anti-Nrf2 monoclonal antibody, 1:500), (mouse anti-Keap1 monoclonal antibody, 1:1000), (rabbit anti-PTGS2 monoclonal antibody, 1: 1000), P62(rabbit anti-P62 monoclonal antibody, 1:2000), LC3(rabbit anti-LC3 monoclonal antibody, 1:200), were used to incubate the membrane at 4 ℃ overnight, and it was washed by TBST buffer for 3 times, and then incubated at normal temperature for 1 h with secondary antibody (horseradish peroxidase labeled goat anti-rabbit IgG, 1∶5000). After washing, immunoassay was performed using an enhanced chemiluminescence kit. β-actin was used as internal reference to analyze the protein bands.
Statistical Analysis
All experimental data were analyzed using statistical software (Prism v8.0; GraphPad). Data are expressed as mean ± standard deviation. A statistical significance was identified between three or more groups by one-way ANOVA analysis of variance with the Tukey HSD test based on data in this study. But the data of the number of neuronal action potentials (APs) were analyzed by multiple-way ANOVA analysis. P-value < 0.05 was used to indicate statistical significance.