1. Reagents and antibodies
Cell culture medium (DMEM), fetal calf serum, bovine serum albumin (BSA), and cocktail were obtained from GIBCO/Invitrogen. Lipofectamine 2000, Combined Protease and Phosphatase Inhibitors were from ThermoFisher Scientific Inc. Anti-Swell1, anti-FOXO3a, anti-phospho-FOXO3a (Ser316), anti-CREB and anti-Phospho-CREB (Ser133) were from Cell Signaling Technology. TUNEL Assay Kit, anti-TMEM119, anti-WNK1, anti-Phospho-WNK1 (Thr58), FITC-conjugated secondary antibodies, Cy5-conjugated secondary antibodies and DAPI Staining Solution were from Abcam. Anti-NeuN, anti-SGK1 and anti-phospho-SGK1 (Ser422) were purchased from Merck Millipore. Anti-SPAK, anti-OSR1 and anti-NKCC1 were from ThermoFisher. Anti-phospho-SPAK (Ser380), anti-phospho-OSR1(Ser315) and anti-phospho-NKCC1(Thr203) were from CUSABIO. Hyperfect Transfection Reagent (HiPerfect) was purchased from QIAGEN. Cell Counting Assay Kit-8 was purchased from Dojindo Molecular Technologies. Annexin V-PE/7-AAD Apoptosis Detection Kit and Cell Cycle Analysis Kit (PI) were purchased from Keygen Biotech. DCPIB was purchased from Tocris Bioscience, GSK65394 and WNK463 were purchased from MedChemExpress. Other chemicals, if not indicated, were all purchased from Sigma-Aldrich.
2. Animals and treatment
Male transgenic mice (C57BL/ Cx3cr1- Cre) weighing 18-20 g (3-4 weeks old) were purchased from Cyagen (Suzhou) Biotechnology. Mice were injected pAAV-CMV-DIO-EGFP-tWPA (100 μl, 1*1012 v.g./ml) or pAAV-CMV-DIO- LRRC8A-3×FLAG-tWPA (100 μl, 1*1012 v.g./ml) virus designed by OBiO Technology (Shanghai), through tail vein and housed in a temperature-controlled environment with a 12 h-light-dark cycle and allowed free access to food and water for 21 days. Prior to experiments, the mice were randomized into indicated experimental groups and the indices were measured by the researcher blinded to treatment.
3. In vivo focal ischemic stroke model
The mice were subjected to 1 h of middle cerebral artery occlusion (MCAO) with 24 h of reperfusion. In brief, the mice were subjected to 3% isoflurane for an anesthesia induction, thereafter with 1-1.5% isoflurane maintenance during the whole procedure. During the operation, rectal temperature was maintained at 37.0±0.5 °C by a heating pad (ALC-HTP homeothermic system, Alcott Biotech, Shanghai, China). Focal cerebral ischemia was induced by inserting a 0622-0624 monofilament (YUSHUN, Guangzhou, China) into the left internal carotid artery via the common carotid artery. After 1 h of occlusion, the filament was removed and the skin incision was sutured. Mice were returned to their cages and were sacrificed 24 h after reperfusion. All animals had free access to food and water following the procedure.
4. Evaluation of neurological score
Neurological score was assessed an investigator blinded to animal grouping at 24 h after cerebral ischemia according to a protocol (Longa Scoring Method) described 27. The scoring was based on the following tests: 0 point, the mice moved normally; 1 point, the mice could not fully stretch their left front legs; 2 points, the mice turned around in a circle; 3 points, the mice fell on their left side; 4 points, the mice could not move by themselves and lost consciousness.
5. Rotarod test
Animals were placed on an accelerating rotating rod (from 4 to 40 rpm over 300 s) and their latency to fall was recorded. Preoperative training was performed for 3 days with 3 daily trials (total 15min), and most of the mice could maintain on the rod for 260-300s, with the last time trial serving as a preoperative baseline. The pre-trained animals (n = 9) were subjected to tMCAO as described above, and then the animals were placed on the rotating bar to evaluate the time of permanence.
6. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining
After neurological evaluation, brains were removed and cut into five 1-mm serial coronal sections. The sections were stained with 1.5% TTC (T8877, Sigma, St. Louis, MO, USA) in PBS for 5 min at 37°C. The TTC stained sections were photographed and digitized. Using Image-Pro Plus, the areas of TTC negative and the ipsilateral hemisphere were measured. The volume of infarct was calculated and expressed as the sum area of infarct/the sum area of brain slice.
7. Cell culture and transfection
BV2 cells were purchased from ATCC. The cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin (P/S) at 37℃ in a humidified incubator gassed with 95% CO2. Swell1 overexpressing adenovirus that expressed mouse Swell1 (NM_177725.4) with an EGFP (green fluorescent protein) tag and its negative control were purchased from OBiO Technology. BV2 were transfected with the adenovirus according to a previously described method24. Swell1 small interfering RNA (siRNA) (5′GCCUGCAUUGGUUUGCCAATT3′) and Scrambled RNA (5′UUCUCCGAACGUGUCACGU-3′) were designed and synthesized by Qiagen. Scrambled RNA was used as a negative control. Plasmids with RFP tag were designed and produced according to these small interfering RNA chain sequence and used for cell labeling in patch clamp experiments. Swell1 siRNA and negative control siRNA (or Plasmids) were transfected into BV2 by using HiPerfect (or Lipofectamine 2000) transfection reagent according to a previously described protocol24.
8. Microglia electrophysiology
BV2 cells were seeded on glass coverslips (diameter 12 mm) and cultured for at least 24 h in DMEM until fully adherent. For hypotonicity-activated VRAC current recordings, BV2 cells were whole-cell patched in isotonic bath solution containing 90 mM NMDG-Cl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, 10 mM glucose, 100 mM mannitol (305 mOsm/kg, pH 7.3 adjusted with NMDG) or in a hypotonic solution that had the same ionic composition as isotonic bath solution except no mannitol (212 mOsm/kg, pH 7.3 adjusted with NMDG). Recording electrodes (3-5 MΩ) were filled with a standard internal solution containing 133 mM CsCl, 10 mM HEPES, 4 mM Mg-ATP, 0.5 mM Na3-GTP,2 mM CaCl2, 5 mM EGTA (pH 7.2 adjusted with CsOH and osmolality was 295-305 mOsm/kg). For the step protocol, cells were held at -60 mV and voltage step pulses (5 s interval, 500 ms duration) were applied from -100 to +100 mV in 20 mV increments. For the VRAC time course, constant voltage ramps (5 s interval, 500 ms duration) were applied from a holding potential of 0 to 100 mV.
9. Oxygen and glucose deprivation (OGD) and reoxygenation treatment
OGD was used to mimic ischemic conditions for microglia in vitro as described previously28. Briefly, BV2 cells were transfected and cultured for 24 h in DMEM, and were then incubated in a self-contained and sealed hypoxia incubator chamber (Billups-Rothenberg) (5% CO2 and 95%N2) for 1 h in DMEM without serum and glucose. After 1 h of exposure to OGD challenge, the cultures were removed from the anaerobic chamber, and the OGD medium was replaced with normal culture medium. The cells were then allowed to recover for 24 h before examinations of cell viability, flow cytometry or q-PCR.
For animal studies, anesthetized mice were perfused transcardially with PBS, followed by 4% PFA in PBS. Brains were removed and post-fixed in 4% PFA at 4°C overnight. After dehydration by 30% sucrose, brains were embedded in OCT (Tissue-Tek) and cut into 10 μm-thick sections on cryostat microtome (Leica). For cellular studies, the BV2 cells were post-fixed in 4% PFA at room temperature (RT) for 15 minutes after washing with PBS. The brain sections and BV2 cells were permeabilized with 0.2% Triton X-100 and 1% BSA in PBS for 45 min at room temperature (RT), washed three times with PBS, blocked in 10% BSA, and incubated with anti-Swell1, anti-TMEM119 or anti-NeuN (1:200) at 4°C overnight. After washing three times with PBS, the samples were incubated with Fluor-conjugated secondary antibody (1:500) for 1 h at RT, followed by DAPI staining. Images were taken under Zeiss LSM780 confocal microscope.
11. Western blot
Cultured BV2 cells were lysed in RIPA with 1% protease inhibitors cocktails and 1% phosphatase inhibitor cocktails. After centrifugation at 15,000 g for 15 min at 4 ◦C, the supernatant was obtained, and the protein concentration was measured with a BCA Kit (Beyotime). Fifty μg proteins from each sample were subjected to polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% milk (or 5% BSA for protein phosphorylation assay) in Tris-buffered saline buffer at RT for 1 h, and then add primary antibody (1:500 or 1:1000) and incubate overnight at 4°C. After wash with TBS buffer for 3 times, the membranes were incubated with HRP-conjugated secondary antibody (1:2000, Cell Signaling Technology) in the same TBS buffer for 1 h at RT. The blots were detected by a scanner (Bio-Rad) and analyzed with ImageJ (NIH).
12. Cytokine detection
Cytokine levels in mice serum and tissues of mice brain were measured using a multiplex cytokine/chemokine assay (Bio-Plex 23-plex Mouse cytokine assay; Bio-Rad), according to the manufacturer’s instructions. The concentrations of IL-1β, IL-6, IL-10, and TNF-α in BV2 cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Quantification of these cytokines was performed with the corresponding kits according to the manufacturer's protocol.
13. CCK-8 assay for cell viability
Cell viability was measured by the CCK-8 method according to a previously described protocol24. In brief, BV2 cells were seeded into 96-well plates and tested 24 h after transfection and OGD treatment. Cells growth was arrested by incubation in DMEM with 0.5% fetal bovine serum for 24 h before the OGD treatment. Then, 10 μl of CCK-8 solution was added to each well and incubated for another 2 h before measuring the absorbance at 450 nm with a microplate reader (Bio-Tek).
14. Quantification of apoptosis and cell cycle transition by Flow cytometry
BV2 cells were cultured in 6-well plates at 70% confluence and were transfected with Ad-Swell1 or Swell1 siRNA for 24 h before exposing to 1 h OGD and 24 h reoxygenation. For apoptosis assessment, the cells were harvested for Annexin V-PE and 7-AAD staining using the Annexin V-PE/7-AAD Apoptosis Detection Kit according to manufacturer’s instructions. The stained cells were counted by flow cytometry analysis (Coulter, Hialeah, FL). For data analysis, the cells in the lower right, upper right, lower left and upper left corners represented the early apoptotic cells, late apoptotic cells, the survival and necrotic cells, respectively. For cell cycle analysis, the cells were fixed in ice-cold 70% ethanol/PBS, and then treated with ribonuclease A (0.2 mg/mL) before staining with propidium iodide at 4°C overnight. Minimums of 10,000 events were recorded for each sample. The data were tabulated as the sum of cells in S, G0/G1 and G2 phase and normalized as a fold change of the normal controls.
15. Real-Time PCR
Total RNA was extracted from cultured BV2 cells using TRIZOL reagent (Invitrogen) according to the manufacturer’s guidelines. Then, cDNA was synthesized using PrimeScript RT reagent Kit (Takara), qPCR reactions were performed in triplicate with TB Green Premix Ex Taq II (Takara) on an Applied Biosystems 7500 Real-Time PCR Detection System (Applied Biosystems). Relative gene expression was normalized to the housekeeping gene GAPDH (or β-actin) and calculated using the 2−ΔΔCt method. The primers used for amplification are shown in supplement: Table S1.
16. 4D label-free phosphorylation modification proteomics
The proteomics experiment was done by PTM BioLab (Hangzhou, China) using a standard protocol29. Briefly, the BV2 cells were removed from -80°C storage, and processed through the steps of protein extraction, trypsin digestion, affinity enrichment of the phosphopeptides, and then were detected by LC-MS/MS analysis. The resulting MS/MS data were processed using the MaxQuant search engine (v22.214.171.124). Protein annotation and functional enrichment were analyzed by Gene Ontology (GO) annotation and KEGG pathway annotation. For phosphoproteome, software MoMo (motif-x algorithm) was used to analyze the model of sequences constituted with amino acids in specific positions of modify-21-mers (10 amino acids upstream and downstream of the site, respectively) and modify-13-mers for phosphorylation (6 amino acids upstream and downstream of the site) in all protein sequences.
17. Statistical Analysis.
Data analysis was performed using GraphPad Prism 9 (GraphPad software Inc). All data are represented as the mean ± S.E.M. Statistical significance was calculated by Tukey's multiple comparisons test, one- or two-way ANOVA followed by Bonferroni multiple comparisons test. n value represents the number of independent experiments. Statistical significance was set at *p ≤ 0.05, and denote p ≤ 0.01 or 0.001 as **p or ***p, respectively.