Male ICR mice weighing 25–30 g were used in this study and were obtained from Oriental Bio Service Inc. (Nanjing, China). All the animals were housed in the Animal Core Facility of Nanjing Medical University under controlled conditions (temperature: 23 ± 2 °C; relative humidity: 55 ± 5%; 12/12 h light/dark cycle; free access to food and water). Each cage contained four to five mice. This study was approved by the Ethics Committee of Nanjing Medical University (No. IACUC2009007) and all animal experiments were performed in accordance with the Guidelines for Laboratory Animal Research set by Nanjing Medical University. Each experimental group contained nine mice (see Supplementary Methods).
Preparation of PISE model mice
Mice were first intraperitoneally (ip.) injected with methylscopolamine (1 mg/kg) to inhibit peripheral muscarinic activity. After 20 min, pilocarpine (300 mg/kg) was injected (ip.) to induce SE (Wang et al. 2019). Seizure severity was rated using the Racine scale (Racine, 1972). SE was defined as the onset of category 4–5 seizures and was terminated after 1 h using diazepam (10 mg/kg). Animals that did not develop category 4–5 seizures within 30 min of pilocarpine injection were excluded from subsequent parts of the study. Control mice were injected with the same volume of saline.
GSK1016790A (a TRPV4 agonist) and HC-067047 (a TRPV4 antagonist) were administered by intracerebroventricular (icv.) injection, and TAK-242 (a TLR4 antagonist) and BAY 11-7082 (an NF-κB inhibitor) by ip. injection, as previously described (Hua et al. 2015; Miyamoto et al. 2010; Wang et al. 2019). For icv. injection, mice were anesthetized with ketamine (100 mg/kg)/xylazine (10 mg/kg; i.p.) and then were placed in a stereotaxic device (Kopf Instruments, Tujunga, CA, USA). A guide cannula of 23-gauge stainless-steel tubing was implanted into the right lateral ventricle (0.3 mm posterior, 1.0 mm lateral, and 2.5 mm ventral to bregma) and anchored to the skull with stainless-steel screws and dental cement. Drugs were slowly injected into the right lateral ventricle using a 26-gauge stainless-steel needle (Plastics One, Roanoke, VA, USA) at the rate of 0.2 μL/min. GSK1016790A (1 μM) or HC-067047 (10 μM) was injected once daily for 3 consecutive days. To block TRPV4 following SE induction, HC-067047 (10 μM) was injected 1 h after SE was terminated and then once daily for 3 days. TAK-242 (3mg/kg) and BAY 11-7082 (5mg/kg) were injected 30 min before GSK1016790A injection and subsequently once daily for 3 days. The doses of the above drugs were chosen based on previous reports (Hua et al. 2015; Miyamoto et al. 2010; Wang et al. 2019).
Western blot analysis
Hippocampal samples were obtained 8 h after the last injection of GSK1016790A or HC-067047 or 3 days after SE induction. Total protein was extracted using the Whole Cell Lysis Assay Kit from Nanjing KeyGen Biotech Co., Ltd (Cat: KGP250; Nanjing, China) while nuclear and cytosolic proteins were extracted using the Nuclear/Cytosol Fractionation Kit (Cat: R0050, Beijing Solarbio Science & Technology Co., Ltd, Beijing, China) according to the manufacturers’ protocols. The protein concentrations were determined using a BCA Protein Assay Kit (Pierce, Rochford, IL, USA). Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking using 5% nonfat milk in Tris-buffered saline (TBS)/Tween20, the membranes were incubated with primary antibodies against TLR4 (Cat: sc-293072, 1:300, Santa Cruz Biotechnology Inc., Dallas, TX, USA); HMGB1 (Cat: 10829-1-AP, 1:1,000), IL-1β (Cat:16806-1-AP, 1:1,000), TNF (Cat: 17590-1-AP, 1:1,000), IL-6 (Cat: 21865-1-AP, 1:1,000), and Lamin B1 (Cat:12987-1-AP, 1:3,000) (all from Proteintech Group Inc., Wuhan, China); phosphorylated (p)-NF-κB p65 (Ser536) (Cat: AF2006, 1:1,000) and p-IκK (Ser180/Ser181) (Cat: AF3013, 1:1,000) (both from Affinity Biosciences, Zhenjiang, China); IκBα (Cat:4814, 1:1,000) and NF-κB p65 (Cat: 8242, 1:1,000) (both from Cell Signaling Technology, Beverly, MA, USA); p-IκBα (Ser36) (Cat: ab133462, 1:20,000), IκK (Cat: ab178870, 1:1,000), and NF-κB p105/p50 (Cat:ab32360, 1:5,000) (all from Abcam, Cambridge, MA, USA); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat: AP0063, 1:1,000, Bioworld Technology, Minneapolis, MN, USA) at 4 °C overnight. After washing with TBST, the membranes were incubated with an HRP-labeled secondary antibody, developed using an ECL Detection Kit (Amersham Biosciences, Piscataway, NJ, USA), and analyzed using ImageJ software (National Institutes of Health).
Anesthetized mice were transcardially perfused with ice-cold phosphate-buffered saline (PBS) and then with 4% paraformaldehyde 8 h after the last injection of GSK1016790A or HC-067047 or 3 days after the onset of SE. Excised brains were fixed at 4 °C overnight and paraffin-embedded. Coronal sections (5 μm) were cut at the level of the dorsal hippocampus and stained with toluidine blue. The surviving neurons were observed using a light microscope (Olympus DP70, Olympus Corporation, Tokyo, Japan) and quantified as previously described (Hong et al. 2015; Jie et al. 2016).
HC-067047 (Cat: HY-100208), TAK-242 (Cat: HY-11109), and BAY 11-7082 (Cat: HY-13453) were obtained from MedChemExpress (Shanghai, China); pilocarpine (Cat: 14487) and methylscopolamine (Cat: 23862), ketamine (Cat: 11630) and xylazine (Cat: 14113) were obtained from Cayman Chemical Company (Ann Arbor, MI, USA); unless otherwise stated, GSK1016790A (Cat: G0798) and all other chemicals were obtained from Sigma Chemical Company (St Louis, MO, USA).
Data are expressed as means ± SD and were analyzed with Stata 7.0 Software (STATA Corporation, College Station, TX, USA). Normality of the distribution was assessed using the Kolmogorov-Smirnov test and variance homogeneity was assessed using Levene’s test before statistical analysis. Unpaired t-tests or ANOVA followed by Bonferroni’s post-hoc test were used for statistical analysis and significance levels were set at P<0.05. The numbers of surviving cells or protein levels in mice injected with drugs were expressed as percentages of those values in vehicle-injected mice. The numbers of surviving cells or protein levels in mice with PISE were normalized to those of control mice. The protein levels in vehicle- or HC-067047-treated PISE model mice were normalized to those of vehicle-treated control mice. The numbers of surviving cells or protein levels in vehicle- or drug-treated GSK1016790A-injected mice were normalized to those of vehicle-treated control mice (see Supplementary Table 1).