Animals
Male-specific pathogen-free (SPF) C57BL/6 mice (weight: 20–25 g, age: 6–8 weeks) were purchased from the Experimental Animal Center of Southern Medical University (Guangzhou, China). The IRAK-M−/− mice were obtained from the Jackson Laboratories (Bar Harbor, ME, USA). Heterozygous (IRAK-M+/−) mating pairs were maintained to produce wild-type (WT) and homozygous (IRAK-M−/−) littermates for use in the study. Genotyping results for the IRAK-M−/− mice were confirmed by 1% agarose protein gels (Supplementary Fig.1). The experimental study design is outlined in Figure 1.
Lithium-pilocarpine-induced SE model
SE was characterized by continuous limbic seizures, which were induced as reported elsewhere[21]. Briefly, the mice were injected intraperitoneally (i.p.) with lithium chloride (127 mg/kg, Sigma–Aldrich, St. Louis, MO, USA). After 20 h, the mice received subcutaneous injections of pilocarpine hydrochloride (30 mg/kg, i.p., Sigma–Aldrich) to induce SE. To reduce the peripheral side-effects of pilocarpine, methyl scopolamine nitrate (1 mg/kg, i.p., Tokyo Chemical Industry, Tokyo, Japan) was administered 30 min before pilocarpine administration. Racine's scoring was performed to assess the severity of behavioral seizures, which ranged from 0 to 5 stages[22]. Seizure latency was considered the time from pilocarpine injection to the first stage 3 seizure. SE was defined as a seizure of >4 stages that lasted for at least 30 min. Finally, the mice were intraperitoneally administered with diazepam (15 mg/kg, i.p., King York, Tianjin, China) after 2 h to stop the seizure, and the mice were kept warm. The mice recovering from SE received intraperitoneal injections of sodium chloride with glucose (5%, i.p., Kangjier, Zhejiang, China) once a day for energy supplementation. The mice recovering from SE were given a bolus of saline (5%, Kangjier) subcutaneously in the scruff to increase the survival chances. The mice in the control group were treated similarly, albeit they received an equal volume of saline instead of lithium-pilocarpine.
Monitoring for seizures
After injection with pilocarpine to induce SE, the seizures of the mice were recorded continuously for 2 h by using a camera equipped with infrared night vision (Xiaomi Technology, Shenzhen, China). For 28 days after SE, the behavior of the mice (n = 12) was videotaped for 24 h every day. The number of seizure episodes during 28 days and the behavioral score according to the Veliskova scoring system per seizure episode was recorded for every mouse. Finally, all video recordings were tallied by individuals who were independently blinded to this study.
Slice Preparation
The brain slices were prepared according to the procedures described previously[24, 25] Briefly, the experimental mice were deeply anesthetized with pentobarbital sodium (75 mg/kg, i.p.) and intracardially perfused with ice-cold oxygenated high-sucrose artificial cerebrospinal fluid (slice ACSF, in mM: 220 sucrose, 26 NaHCO3, 10 D-glucose, 2 KCl, 12 MgSO4, 1.3 NaH2PO4, and 0.2 CaCl2). The mice were sacrificed via decapitation, and their brains were rapidly removed and chilled in ice-cold oxygenated slice ACSF. Multiple coronal slices (300-μm thick) containing the hippocampal CA1 were prepared using a vibratome (Leica, VT1200S). The slices were transferred to a holding chamber containing continuously oxygenated ACSF (recording ACSF, in mM: 124 NaCl, 3 KCl, 26 NaHCO3, 10 D-glucose, 1 MgSO4, 1.25 NaH2PO4, and 2 CaCl2, at pH 7.4, 305 mOsm) at 34°C for 30 min and then kept at room temperature until required. After an hour of the recovery period, the slices were transferred to the recording chamber that was continuously perfused with oxygenated ACSF at the rate of 2–3 mL/min. Whole-cell patch-clamp recordings were performed by using an upright microscope (ECLIPSE FN1, Nikon, Japan) equipped with a 40× water-immersion lens and infrared-sensitive camera (DAGE-MTI, IR-1000E). The patch pipettes were fabricated from filamented borosilicate glass capillary tubes (inner diameter, 0.84 μm) with a horizontal puller (Sutter Instruments, P-97). The recordings were obtained using a multiclamp 700B amplifier and pClamp software (Molecular Devices). The data were low-pass filtered at 2 kHz and sampled at 10 kHz with the Digidata 1550A device (Molecular Devices).
Electrophysiological Recordings
For spontaneous and miniature excitatory postsynaptic current (sEPSC and mEPSC, respectively) recording, the neurons were held at a potential of −70 mV in the presence of 20 µM bicuculline (BMI, the GABAA receptor antagonist) without or with 1 µM tetrodotoxin (TTX). The pipette resistance was typically 4–6 MΩ after it was filled with an internal solution containing (in mM) 130 K-gluconate, 20 KCl, 10 HEPES, 0.2 EGTA, 4 Mg-ATP, 0.3 Na-GTP, and 10 NaCreatine (pH 7.3, 285 mOsm). To determine the proportion of AMPA and NMDA components, the EPSCs were evoked at -70 mV (AMPA) and +40 mV (NMDA) in the presence of 20 µM BMI. The internal solution contained (in mM): 110 Cs2SO4, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 HEPES, 5 TEA, and 5 Mg-ATP (pH 7.3, 285 mOsm). The AMPA component was measured as the EPSC peak amplitude, while the NMDA component was determined by measuring the current amplitude at 50 ms after the EPSC onset (an average of 10 sweeps).
For each cell, the recordings were commenced after stabilization of the holding potential approximately 2–5 min after the break-in. Only the cells with a series resistance <30 MΩ and leak currents <100 pA were included. The synaptic currents were recorded in the voltage-clamp mode and analyzed with the Minianalysis (Synaptosoft Inc.) and Clampfit 10.7 (Molecular Devices).
Hematoxylin–Eosin (H&E) and Nissl Staining
The whole mouse brains were fixed in 4% paraformaldehyde (Leagene, Beijing, China) overnight at room temperature and dehydrated in a gradient alcohol series, and then embedded in paraffin. The coronal sections of the brain tissues were cut at a thickness of 5 μm for H&E staining (BaSO, Taiwan, China) or Nissl staining (Beyotime, Jiangsu, China). Two regions in the ipsilateral hippocampus (CA1 and CA3) were randomly selected in each mouse and photographed under a microscope (Olympus, Tokyo, Japan). The cell counts were performed using the ImageJ software, and a total of 3 mice per group were used for quantification (Supplementary Fig. 2).
Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from the hippocampus with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions. The concentration and quality of the mRNA extracts were determined by measuring the 260/280 and 260/230 ratios using a DS-C spectrophotometer (DeNovix, DE, USA). Then, RNA (1 μg) of each sample was reverse transcribed to cDNA with the PrimeScriptTM RT kit. (Takara Bio Inc., Shiga, Japan). Thereafter, RT-qPCR was performed with the SYBR® Premix Ex Taq™ (Takara Bio Inc.) using the LightCycler 96 instrument (Roche, Switzerland). Use the template (10 μL of the PCR mixture, 5 pmol of forward and reverse primers, 1 μL of the cDNA template, and an appropriate volume of water) in a 20-μL reaction volume. The PCR reactions are performed as follows: cycling was first performed at 95°C for 30 s with an initial DNA denaturation step, followed by 40 cycles at 95°C for 5 s, 60°C for 30 s, and 72°C for 30 s. Threshold cycle (CT) readings were collected. The relative expression of the target genes mRNA was calculated by the 2-ΔΔCT method,[26] and the mean value of the glycerol-3-phosphate dehydrogenase (GAPDH) served as an internal reference for normalization. Next, the expression level of mRNA was reported as the fold change when compared to the control group. The sequence of the primer pairs for the target gene is listed in Supplementary Table 1.
Western blotting
The hippocampal tissues were homogenized in the RIPA lysis buffer (Beyotime) containing phosphatase inhibitor (Sigma–Aldrich) and protease inhibitor (Sigma–Aldrich) on an ice bath for 30 min and centrifuged; the supernatant was aspirated for protein quantification and denaturation. The BCA Protein Assay Kit (Beyotime) was used to measure the protein concentration. Protein samples (approximately 40 µg) were separated by 7.5-15% PAGE Gel (EpiZyme, Shanghai, China) at 150 V, transferred onto a PVDF membrane (Millipore, MA, USA) at 375 mA for 120 min, and then blocked with 5% BSA (Biofroxx, Germany) for 2 h. The membranes were blotted overnight at 4°C with the primary antibodies, including anti-IRAK-M antibody (1:1000, Bioss, bs-16695R), anti-NMDAR2A antibody (1:1000, Bioss, bs-3507R), anti-NMDAR2B antibody (1:1000, Bioss, bs-0222R), anti-IRAK1 antibody(1:1000, ab238, Abcam, Temecula, CA, USA), anti-TRAF6 antibody (1:2000, Abcam, ab40675), anti-STAT1 antibody (1:1000, CST, 14994), anti-p-STAT1 antibody (1:1000, CST, 9167), anti-SOCS-1 (1:1000, Abcam, ab62584), and anti-GAPDH (1:50000, Proteintech, 60004-1-Ig). After the membranes were washed thrice with TBST, they were incubated with the corresponding IgG antibody for 2 h. Finally, the protein bands were visualized by using an ECL kit (Affinity, Cincinnati, OH, USA). Image J was performed to calculate the protein density.
Immunofluorescence
The brain slices were fixed in 4% paraformaldehyde (Leagene) for 24 h and then dehydrated in serial 15% and 30% sucrose solutions. The brain tissue was then sliced into 20-μm-thick coronal sections. After rinsing the brain sections with PBS for residual embedding medium (OCT), the sections were blocked with 5% goat serum (Beyotime) and 1% Triton-X 100 (Beyotime) for 1.5 h, followed by incubation with primary antibodies against NeuN (Rabbit, 1:200, Abcam) and Iba-1 (Rat, 1:500, Synaptic Systems, 234017) overnight at 4°C, followed by treatment with Alexa Fluor 594-conjugated IgG (1:200, Invitrogen) and Alexa Fluor 488-conjugated IgG (1:200, Invitrogen) secondary antibodies for 1 h at room temperature. Next, PBS was used to rinse the sections thrice, followed by DAPI staining (Solarbio, Beijing, China) for 10 min. Subsequently, a confocal microscope (Nikon) was employed for observation. All fluorescent image data was quantified by using Image J. For microglia morphology data collection, the fluorescence photomicrographs were converted into representative binary and skeletonized images and then analyzed using the ImageJ plugins AnalyzeSkeleton (2D/3D) and FracLac.
Flow Cytometry
The hippocampal tissues were ground with a grinding rod, digested with the StemPro Accutase Cell Dissociation Reagent (Gibco, Spain), and then filtered through a 70-μm filter (BD Falcon, USA) to obtain a cell suspension, which was centrifuged at 400 ×g for 5 min at 4°C. Pre-cooled pbs and the Debris Removal Solution (Miltenyi Biotec, Miltenyi) were used to remove myelin and impurities, followed by centrifugation at 4°C for 10 min at 3000 xg with full acceleration and full brake. Staining buffer (eBioscience, CA, USA) was used to resuspend the cells, at the density of 1 × 107 cells/mL in each tube. The tube was incubated at RT for 30 min in the dark, followed by the addition of 1 μL/mL of Fixable Viability Dye (eFluorTM 450, eBioscience) to label the dead cells and 100 μL of IC Intracellular Fixation (eBioscience) to fix the cells. Next, 2 mL of 1X permeabilization buffer (eBioscience) was added to the cells, and the mixture was centrifuged for 10 min at 400 xg, and the supernatant was removed. The antibody mixture (2 µL CD11b, 2 µL CD45, 2 µL CD86, and 2 µL CD163) was added to the tubes (detailed information on the flow cytometry antibodies is available in Supplementary Table 2), and the tube contents were mixed and incubated for 20–60 min at 2–8°C away from direct light. All samples were read on the CytoFLEX flow cytometer (Beckman, USA) and analyzed with the CytExpert software (Beckman). Positive cell percentages were calculated.
Statistical analysis
All statistical analyses were performed using the SPSS 20.0 (IBM, Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad, La Jolla, CA, USA) by an investigator who was blinded to the experiment. Unless otherwise specified, all data were presented as the means ±standard error of the mean (SEM). The significance is denoted with an asterisk. All experiments were performed with at least 3 independent mice. First, the Shapiro–Wilk test was performed to assess the normality of data distributions. The data were compared using the Student's t-test or one-way analysis of variance (ANOVA) with a non-parametric Kruskal–Wallis test, followed by Dunn's multiple comparison test for post-hoc group comparisons, t-test, Mann–Whitney U-test, or Fisher's least significant difference. Individual p-values are indicated in the figure legends, with p < 0.05 considered to indicate statistical significance.