NLRP1 inflammasome is activated in patients with medial temporal lobe epilepsy and contributes to neuronal pyroptosis in amygdala kindling-induced rat model

We collected the hippocampus samples of mTLE patients. Patients recruited in this study have a diagnosis of refractory epilepsy according to the definition of pharmacoresistant epilepsy [12]. The 24-h EEG monitoring indicated that widespread sharp or spike or slow-waves originated from unilateral temporal lobe. In addition, there was ipsilateral hippocampal sclerosis identified by MRI or low metabolism in ipsilateral mesial temporal lobe identified by 18F-FDG PET, without other pathological changes. Surgery was determined by a neurologist and a neurosurgery specialist in consultation. During the operation, cortical electrode monitoring confirmed that epilepsy-like waves originated from the inferior temporal lobe. All patients showed HS, with appreciable neuronal loss and reactive gliosis in CA1, CA3 and CA4. It was infeasible to obtain brain tissues of normal hippocampus cortex, so that normal temporal cortex tissues were used as negative control. Considering the difficulty in finding ‘real’ controls in human studies, in our experiments we used ‘healthy’ surgical samples from patients with other pathologies. Seizure absence was determined by the patient’s report to the neurologist during the scheduled visits. Therefore, for comparative purposes, we used six specimens of nonepileptic tissues from Histologically normal specimens (control samples) in accordance with all legal requirements. None of the patients in the control group had a history of systematic diseases. We also used autopsy hippocampal tissue as a second control tissue for these studies to avoid problems due to the localization of specific expression. Autopsy hippocampal tissues from six patients with no known history of epilepsy and other systematic diseases were used as controls. The average postmortem interval was within 12 h after death. Routine neuropathological examination of tissue sections of these control hippocampal samples showed no pathologic changes. The brain tissues were separated as needed and immediately preserved in liquid nitrogen. Clinicopathological data are presented in Table 1, Additional file 1: Figure S1, and Additional file 2: Figure S2. The study was approved by the Ethics Committee of Qingdao Municipal Hospital. Informed consent was obtained from the patients and their legal guardians on the use of their brain tissues in this research.

To avoid the interference of estrogen on neuroinflammation [13], only male rats were used in this study. Adult male Sprague-Dawley rats weighing 260 to 300 g were obtained from the Experimental Animal Center of Qingdao University. They were housed in a standard animal room on a 12 h light/dark cycle with a controlled temperature and humidity, and given free access to food and water. All experiments were performed in strict accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Animal care and sacrifice were conducted according to methods approved by the Qingdao University Animal Experimentation Committee. All efforts were made to minimize the number of animals used and their suffering.

Rats were positioned in a stereotaxic apparatus (Stoelting, USA, www.​stoeltingco.​com) under deep anesthesia (10% chloral hydrate, 3.5 mL/kg, i.p.). An electrode was implanted into the right basolateral amygdala (AP: -3.0 mm; L: -4.8 mm; V: -8.8 mm) [14,15]. The electrode was connected to a miniature receptacle, which were fixed to the skull using dental cement anchored with stainless steel screws. All rats underwent an identical surgical procedure, which were performed with the use of antiseptic technique.

After electrode implantation, the rats were allowed to recover for 2 weeks. Seizures were induced by a 20-minute amygdala stimulation (100 ms train of 1 ms, 60 Hz bipolar pulses, 400 μA, every 0.5 s) using a ML1101 electronic stimulator. Electroencephalograms of the right amygdala were recorded with a digital amplifier (AD Instrument, Racine, WI, USA). Following electrical stimulation, rats were video-monitored for 8 weeks. The rats with chronic TLE were identified by occurrence of frequent seizures (at least 2 times IV/V spontaneous seizures per week) from 1 week after electrical stimulation. Control rats were handled in the same manner but did not receive any electrical stimulation. Moreover, our preliminary experiments showed that this amygdala stimulation model is effective.

We prepared the Entranster in vivo- siRNA mixture according to the manufacturer’s instructions. Briefly, 50 μg NLRP1 siRNA (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) or caspase-1 siRNA (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) or control siRNA (Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) was resuspended in 50 μL RNAse-free water to make a siRNA solution. Then, 50 μL NLRP1 siRNA or control siRNA solution was mixed with 50 μL Entranster in vivo transfection reagent (Engreen, Inc., Beijing, China) and 100 μL artificial cerebrospinal fluid (aCSF, composition in mmol/L: NaCl 130, KCl 2.99, CaCl₂ 0.98, MgCl₂•6H₂O 0.80, NaHCO₃ 25, Na₂HPO₄•12H₂O 0.039, NaH₂PO₄•2H₂O 0.46) to get a 200-μL in vivo transfection mixture. NLRP1 siRNA or caspase-1 siRNA at this dose was well tolerated, and no signs of neurotoxicity including hind-limb paralysis, vocalization, food intake, or neuroanatomic damage were observed in the preliminary study.

We filled an osmotic pump (Model 2006; ALZET Inc., Cupertino, CA, USA) with the in vivo transfection mixture or aCSF alone. Meanwhile, rats were anesthetized with 10% chloral hydrate (0.3 mL/100 g, intraperitoneal) and were fixed in a stereotactic frame (Stoelting, USA, www.​stoeltingco.​com). A brain-infusion cannula (ALZET Inc., Cupertino, CA, USA) coupled via vinyl tubing to the osmotic pump was implanted into the dorsal third ventricle (AP: -1.8 mm; L: -0 mm; V: -5 mm) of rats with chronic TLE 8 weeks after electrical stimulation. Meanwhile, the osmotic pump was placed subcutaneously between the scapulae of rat, and the siRNAs were continuously infused into the brain at a flow rate of 0.15 μL/hour for 6 weeks by the osmotic pumps. All surgical procedures were ere performed with the use of antiseptic technique.

All animals were assessed for amygdala stimulation induced seizures during the first 24 h postsurgery. At the 8th week postsurgery, all animals were re-evaluated for behavioral progression of seizures 4 h/day for 5 consecutive days to record the spontaneous seizures and scored according to Racine’s classification [16]: 0, no reaction; 1, stereotypic mounting, eye blinking, and/or mild facial clonus; 2, head nodding and/or multiple facial clonus; 3, myoclonic jerks in the forelimbs; 4, clonic convulsions in the forelimbs with rearing; and 5, generalized clonic convulsions and loss of balance. Numbers and rates of animals with seizures (at least 2 times IV/V spontaneous seizures) during the first 24 h post-surgery and on the 6th week post-treatment were recorded.

Rats were sacrificed under deep anesthesia for the following biochemical assays:

Tissues samples were digested with RIPA lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% Nonidet-40, 0.5% sodium deoxycholate, 1 mmol/L EDTA, 1 mmol/L PMSF) with protease inhibitors (pepstatin 1 μg/mL, aprotinin 1 μg/mL, leupeptin 1 μg/mL) for 30 min and the protein concentration was determined using the Bradford assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Different samples with an equal amount of protein were separated using 8-12% SDS-polyacrylamide gels and transferred to PVDF membranes. After blocking with 10% non-fat milk at room temperature, the membranes were incubated with primary antibodies against NALP1 (1:1000; Novus Biologicals, Inc., Littleton, CO, USA), NeuN (1:500; Chemicon, www.​chemicon.​com.​br), cleaved caspase-1 (1:200; Cambridge, UK), cleaved IL-1β (1:500; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA), and β-actin (1:1000; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) at 4°C overnight. After rinsing, the membranes were appropriately incubated with horseradish peroxidase (HRP)-conjugated suitable secondary antibodies (1:5000; Zhongshan Inc., Beijing, China) for 2 h at room temperature. Cross-reactivity was visualized using ECL western blotting detection reagents and analyzed by scanning densitometry using a BioSpectrum Imaging System (UVP, Upland, CA, USA).

Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), using the protocol supplied by the manufacturer. Total RNA was reverse transcribed to cDNA by the Reverse Transcription System (Bio-Rad, Hercules, CA, USA). The reaction was performed at 42°C for 50 min, 95°C for 5 min, and 5°C for 5 min, then the cDNA was stored in -20°C. Amplification was carried out with the Stratagene Mx3000P real-time PCR system (Stratagene, LaJolla, CA, USA) with a SYBR Green PCR technology (Takara Bio, Inc., Shiga, Japan). Reverse transcription was performed in a final volume of 20 μL containing 2 μL cDNA, 10 μL SYBR Green, 0.4 μL ROX Reference Dye, 0.4 μL forward and reverse primer (1 mol/L), and 6.8 μL nuclease-free water. The optimal conditions were 40 cycles of 95°C for 30 s, 60°C for 32 s, and 72°C for 30 s. Relative quantification was given by the CT values, determined for triplicate reactions of SE samples and sham samples for each gene. Total RNA concentrations from each sample were normalized by quantity of β-actin mRNA, and the target genes’ expression was evaluated by ratio of the number of target mRNA to β-actin mRNA. Relative expression of genes was obtained by the 2^-△△CT method. Primers were purchased from Invitrogen as follows (name: forward primer, reverse primer): nlrp1: 5’-gccctggagacaaagaatcc-3’, 5’-agtgggcatcgtcatgtgt-3’; caspase-1: 5’-aaggtcctgagggcaaagag-3’, 5’-gtgttgcagataatgagggc-3’; β-actin: 5’-agggaaatcgtgcgtgac-3’, 5’-cgctcattgccgatagtg-3’.

Levels of secreted IL-1β were measured using commercially available ELISA kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. ELISA was conducted by technicians who were blinded to the experimental groups. The results are expressed in pg/mL.

Nissl staining was employed to detect surviving neurons. The brains were embedded in paraffin and cut into 7-μm sections. Next, the paraffin-embedded sections were dewaxed and rehydrated according to the standard protocols. Then, the sections were stained in 1% cresyl violet at 50°C for 5 minutes. After being rinsed with water, the sections were dehydrated in increasing concentrations of ethanol, mounted on the slides, and examined with a light microscope. Only the neurons with violet nucleus and the intact morphology were counted as surviving neurons. Besides, we used a cell death detection kit (In Situ Cell Death Detection Kit, POD; Roche, www.​roche.​com) for TUNEL assay to detect neuronal apoptosis. We employed TUNEL assay via a commercial kit according to the manufacturer instructions. Briefly, the paraffin-embedded sections of samples received deparaffinization and rehydration treatments, and then were incubated with proteinase-K for 15 min at room temperature followed by three washes in PBS. The TUNEL reaction mixture was added and incubated for 60 min at 37°C. Next, sections were washed with PBS and two drops of peroxidase-streptavidin conjugate solution in blocking buffer was applied. Then, the sections were incubated for 30 min at room temperature, washed again with PBS and exposed to 0.03% diaminobenzidine in 0.01% H₂O₂. Lastly, sections were counterstained with Mayer’s hematoxylin, dehydrated, mounted on the slides and examined with a microscope equipped with a charge-coupled device camera. We identified the neurons with deep black nuclei as TUNEL-positive neurons. Cell counting was performed on five randomly selected non-overlapping fields of per slide. The densities of surviving neurons or TUNEL-positive neurons in the hippocampus of the scanned digital images were calculated using Image-Pro Express software (Media Cybernetics, Silver Spring, MD, USA). The total cell counts were averaged from five sections per animal. The survival index was defined as follows: surviving index (%) = 100× (number of surviving neurons/total number of neurons). Furthermore, the TUNEL-positive neurons index was defined as follows: TUNEL-positive neurons index (%) = 100× (TUNEL-positive neurons/total neurons).

For immunofluorescence studies, 20-μm-thick sections were obtained by cryosectioning at -20°C, mounted on a glass slide, and incubated at room temperature for 1 hour. Next, the sections were fixed in ice-cold acetone for 10 minutes and then dried on a heater for 10 minutes at 40°C. Then, sections were blocked with 5% BSA and 0.1% TritonX-100 for 2 hours. After a single wash with PBS, sections were incubated overnight at 4°C with a rabbit polyclonal antibody against NALP1 (1:1000; Novus Biologicals, Inc., Littleton, CO, USA) combined with a mouse monoclonal antibody against NeuN (1:200; Chemicon, www.​chemicon.​com.​br). Afterward, sections were washed in PBS and incubated respectively with FITC conjugated anti-rabbit IgG (1:200; Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) and TRITC conjugated anti-mouse IgG (1:200; Zhongshan Inc., Beijing, China) in a dark and humidified container for 1 h at 37°C. After that, the sections were washed with PBS and sealed with a cover slip. The slides were analyzed with a fluorescence microscopy (Olympus, Inc, Tokyo, Japan). To ensure the specificity of the immunoblotting procedure, control experiments were performed in which the corresponding primary antibody was omitted. Under these conditions, no signal was observed.