Animals
A total of 90 male Sprague-Dawley rats (age, 2 months; weight, 200–250 g) were raised under controlled conditions (temperature 24–25°C, humidity 50–60% and a 12-h light/dark cycle). The animals had ad libitum access to food and water. All procedures were implemented according to the guidelines of the Animal Care Committee of Huazhong University of Science and Technology and the study protocol was approved by the Ethics Committee of Huazhong University of Science and Technology.
Drugs
The following drugs were used in the present study: Pilocarpine hydrochloride (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), lithium chloride (Sigma-Aldrich; Merck KGaA), atropine sulfate (Shanghai Harvest Pharmaceutical Co., Ltd., Shanghai, China), anti-P2RX7 antibody (Abcam, Cambridge, UK), anti-glial fibrillary acidic protein (GFAP) antibody (Abcam), anti-glutamate (GLU) antibody (Sigma-Aldrich; Merck KGaA) and diazepam (Tianjin Jinyao Group Co., Ltd., Tianjin, China). Lithium chloride and pilocarpine hydrochloride were dissolved in physiological saline (0.9% sodium chloride).
Animal model of TLE
First, the rats were administered an intraperitoneal (IP) injection of lithium chloride (127 mg/kg). After 18 h, atropine sulfate (1 mg/kg, IP) was injected, followed by pilocarpine hydrochloride (15 mg/kg, IP) after a further 30 min. The seizure severity was graded according to Racine's standard classification as follows: Stage 1, movement of the mouth and face; stage 2, nodding of the head; stage 3, clonus of the unilateral forelimb; stage 4, clonus and rearing of the bilateral forelimbs; and stage 5, stage 4 seizure with episodes of falling. Pilocarpine hydrochloride injection (15 mg/kg, IP) was repeated every 30 min if there was no seizure, or if seizure activity did not reach stage 4 on the Racine scale. The maximum dose of pilocarpine hydrochloride was 60 mg/kg. When status epilepticus persisted for 1 h, it was terminated by an injection of diazepam (10 mg/kg, IP). The present experiment only used rats graded at stage 4 or 5.
Behavioral observation
A total of 2 weeks after the onset of status epilepticus, all rats underwent uninterrupted video monitoring throughout the daytime to observe whether any spontaneous repeated seizures occurred over 7 weeks. Behavioral parameters, including latency period, frequency of spontaneous recurrent seizure, duration of chronic epilepsy and mortality were analyzed during the entire study period.
Electroencephalogram monitoring
Rats were anesthetized with 10% chloral hydrate (0.3 ml/100 g, IP) to undergo an electroencephalography (EEG). The rats were fixed and two electrodes were inserted underneath the scalp on both sides in the temporal region (0.65 cm in front of the connection of the external ear gate and 0.4 cm beside the center line). The reference electrode was inserted underneath the scalp at the frontal pole midpoint (1.2 cm in front of the external ear gate). EEG waveforms filtered <0.53 Hz and >30 Hz were subjected to analog-to-digital conversion by a dynamic electroencephalography device (Beijing Symtop Instrument Co., Ltd., Beijing, China). When the rats entered the spontaneous recurrent seizure period, they underwent EEG monitoring for 4 h once per week to record EEG waveforms and spike-wave discharges. Some rats with spontaneous recurrent seizures were injected with brilliant blue G (BBG) or saline for 2 weeks and EEG waveforms were recorded 30 min before and 60 min after the injection.
Animal grouping and treatment
This study used a total of 90 male Sprague-Dawley rats, among which 6 were given an IP injection of saline solution to serve as the control group, whereas the remaining 84 were used to establish models of epilepsy. A total of ~21 rats had no epileptic seizures, among which 5 were randomly selected to comprise the no seizure group. The remaining 63 rats were eventually affected by status epilepticus, among which 5 were randomly selected to comprise the acute seizure group. A total of 13 rats died after the onset of status epilepticus, 8 rats died during the latency period, 9 rats were in the spontaneous recurrent seizures group and 28 rats were in a chronic phase of spontaneous recurrent seizures. These 28 rats were randomly divided into two groups: The BBG group (21 rats) and the saline group (7 rats). The BBG group was further randomly divided into 3 subgroups (7 rats each) that were separately administered IP injections of BBG at different doses (50, 100 or 200 mg/kg) for 2 weeks.
Patients with TLE
From June 2017 to June 2018, a total of 25 patients with TLE (15 men and 10 women; age range, 18–55 years) from the Wuhan Union Hospital in China were included in the present study. All the patients had characteristic EEGs and representative clinical features (mean number of seizures, 5.5/month), and were on the highest dose of ≥3 antiepileptic drugs (AEDs), such as valproic acid, oxcarbazepine, ethosuximide, lamotrigine, and clonazepam. All patients underwent presurgical assessment including medical history review, detailed neurological examination and ictal or normal EEG studies; they also underwent magnetic resonance imaging (MRI), video EEG, and sphenoidal electrode EEG prior surgery. All brain MRI scans demonstrated no progressive disease in the central nervous system. All patients underwent surgery to remove the epileptogenic zone in the temporal neocortex. For comparison purposes, from June 2017 to June 2018, 6 patients with head trauma (3 men and 3 women; age range, 12–60 years) whose neocortices were histologically normal and who had no history of epilepsy or use of AEDs were examined. All procedures adhered to the conduct of research involving human subjects established by the National Institutes of Health of China and the Committee on Human Research of Huazhong University of Science and Technology. All subjects provided written informed consent to participate in this study.
Tissue processing
The rats were anesthetized with 10% chloral hydrate (0.3 ml/100 g, IP) and then quickly perfused with 4% paraformaldehyde in PBS. After embedding in paraffin, the brain tissue was sliced into 4-µm sections. Similarly, 4-µm sections were prepared from the 4% paraformaldehyde-fixed and paraffin-embedded human brain tissue samples.
Immunohistochemical staining
First, rat hippocampal sections were incubated with 5% bovine serum albumin (Cell Signaling Technology, Inc., Danvers, MA, USA) in PBS for 30 min at room temperature. Subsequently, the sections were incubated with anti-P2X7 immunoglobulin G (cat. no. ab48871; 1:100), anti-GLU IgG (cat. no. G9282; 1:100), or anti-GFAP IgG (cat. no. ab33922; 1:100) overnight at 4°C. The sections were washed in PBS for three times for 10 min each time and then incubated with an Alexa® 594-conjugated secondary antibody (cat. no. ab150160; 1:100; Abcam) for 40 min at 4°C. Then, the sections were visualized with 3,3′-diaminobenzidine for 10 min at room temperature in the dark, rinsed in distilled water and dehydrated in a graded ethanol series. Negative control sections were incubated with PBS instead of the primary antibody. Images were captured via fluorescence microscopy at the same magnification (×200) and grayscale was measured using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
Western blotting
The tissues were harvested, and total cell lysates were prepared using a protein extraction kit (Wuhan Servicebio Technology Co., Ltd., Wuhan, China), following the manufacturer's protocol. Protein concentrations were quantified using a bicinchoninic acid kit (Beyotime Institute of Biotechnology, Haimen, China). Subsequently, 50 µg samples were boiled in gel-loading buffer and separated by 10% SDS-PAGE. The proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and blocked at 37°C with 5% skimmed milk powder in Tris-buffered saline for 1 h. Then, the blots were incubated for 6 h at 37°C with anti-P2RX7 (cat. no. ab48871; 1:1,000; Abcam) and β-actin (cat. no. ab8226; 1:2,000; Abcam) antibodies independently. The membranes were then incubated for 1 h at 37°C with goat anti-rat horseradish peroxidase-conjugated secondary antibody (cat. no. ab150160; 1:100; Abcam). Chemiluminescent signals were detected using the Enhanced Chemiluminescent Plus kit (KPL, Inc., Gaithersburg, MD, USA) and the signal intensity was measured by densitometry analysis using the Versa-Doc™ Imaging system (version 4.0; Bio-Rad Laboratories, Inc.). β-actin was used as the internal control to normalize the samples.
RNA extraction and polymerase chain reaction (PCR)
Hippocampi from rats in each group were treated with TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) reagent to extract total RNA following the manufacturer's recommended protocol. Subsequently, genomic DNA was digested using an RNase-free DNase. A total of 4–6 independent RNA samples were used in duplicate in reverse transcription-quantitative (RT-q) PCR analysis. NanoDrop 2000 (Thermo Fisher Scientific, Inc.) was used to detect RNA concentration. Then, cDNA was transcribed from a total of 500 ng DNase I-treated RNA using a cDNA reverse transcription kit (DP304; Tiangen Biotech Co., Ltd., Beijing, China). Primers used in the study were as follows, P2X7R primers: 5′-GGCGGGGTGACGAAGTTAGTA-3′ (forward) and 5′-TTTGTGGGTCCATCCATCCTT-3′ (reverse); β-actin primers 5′-TGCTATGTTGCCCTAGACTTCG-3′ (forward) and 5′-GTTGGCATAGAGGTCTTTACGG-3′ (reverse). The temperature protocol was as follows: 37°C 15 min and 95°C 5 min. qPCR analysis was performed on an ABI Prism 7500 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using Thunderbird SYBR qPCR mix (Toyobo Life Science, Osaka, Japan) in 20 µl of reaction mixture. The thermocycling conditions were as follows: Initial denaturation at 95°C for 5 min, followed by 33 cycles of 95°C (15 sec) and 60°C (1 min). Data were analyzed using Sequence Detection Software 1.4 (Applied Biosystems; Thermo Fisher Scientific, Inc.). β-actin was selected as the endogenous reference and data were analyzed using the 2−ΔΔCq method (15).
Statistical analysis
All values were presented as the mean ± standard deviation of three separate experiments and were analyzed by the one-way analysis of variance, which was used to compare multiple groups, followed by a Bonferroni post-hoc test. An unpaired two-tailed Student's t-test was employed to compare the levels of P2X7 receptor and GFAP and GLU in patients with or without intractable epilepsy. Statistical analysis was performed using SPSS software, version 19.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.