Patients and clinical characteristics
Sixty first-ever AIS patients (34 females and 26 males; average age, 62.2 ± 8.2 years) were recruited from January 1, 2017, to September 31, 2017, at The Pudong New Area Gongli Hospital, Shanghai, China. All patients were diagnosed as having an acute cerebral infarction according to the World Health Organization criteria [18] and had symptoms within 72 h. Age- and sex-matched healthy individuals were also selected from the Pudong New Area Gongli Hospital (32 males and 28 females; average age, 58.4 ± 6.9 years). Body weight and height were recorded for subsequent calculation of body mass index (BMI) using a standard formula: BMI (kg/m2) = body weight (kg)/height (m2). Stroke severity was assessed using the National Institutes of Health Stroke Scale.
Blood samples (20 ml) were taken from the cubital vein within 24 h of symptom onset and used for further analysis. Whole blood was used for assessment of white blood cells (WBC), red blood cells (RBC), platelet (PLT) count, hemoglobin (Hb) concentration, and erythrocyte sedimentation rate (ESR).
Serum concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) were analyzed using Randox kits (Randox Laboratories Ltd., Crumlin, UK) with an automated RX Imola biochemical analyzer (Randox Laboratories, Ardmore, UK); and the resulting atherogenic index was calculated using a standard formula: AI = (TC-HDLC)/HDLC (AI, atherogenic index; TC, total cholesterol; HDLC, high-density lipoprotein cholesterol). The serum expression levels of the inflammatory factors, interleukin 33 (IL-33), interleukin 6 (IL-6), interleukin 1β (IL-1β), and tumor necrosis factor-α (TNF-α), interleukin 10 (IL-10), were measured with commercially available Enzyme-Linked Immuno Sorbent Assay (ELISA) kits (Sen-Xiong Company, Shanghai, China).
Animals and ethics statement
Male Sprague–Dawley rats (230–280 g, 36 rats) were purchased from Shanghai Sippr Bk Laboratory Animals Co., Ltd., (Shanghai, China). All rats were allowed free access to food and water under controlled conditions (12/12 h light/dark cycle with humidity of 60 ± 5%, and a temperature of 22 ± 3 °C). All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals, and all experiments were approved and performed according to the guidelines of the Ethics Committee of Pudong New Area Gongli Hospital. All surgical procedures were performed under anesthesia, and every effort was made to minimize suffering. Rats were anesthetized by intraperitoneal injection of pentobarbital sodium (30 mg/kg).
Murine models of middle cerebral artery occlusion
Animals were anesthetized by intraperitoneal injection of sodium pentobarbital. Body temperature was monitored and maintained at 36.5 °C to 37.5 °C. A modified model of middle cerebral artery occlusion (MCAO) was used to induce permanent focal ischemia, as previously described [19]. Briefly, the right middle cerebral artery (MCA) was occluded by inserting a monofilament nylon suture (diameter 0.24–0.26 mm) with a heat-rounded tip into the internal carotid artery, which was advanced further until it closed the origin of the MCA. Sham-operated rats underwent the same surgical procedure without insertion of the filament. No mortalities of rats were observed.
Animal treatments
Cerebral ischemia and sham-operated animals were randomly assigned to either vehicle (12 rats) or celastrol groups (24 rats). Twelve rats from celastrol groups were administered with 1 mg/kg celastrol dissolved in 0.9% NaCl and 1% dimethylsulfoxide (DMSO) (InvivoGen, San Diego, CA, USA) immediately and 1 day post-operation through intraperitoneal injection. Rats were re-anesthetized and sacrificed 72 h (control: MCAO: MCAO+celastrol = 6: 6: 6) or 10 days (control: MCAO: MCAO+celastrol = 6: 6: 6) post-MCAO. Behavioral tests were performed 3, 5, 7, and 10 days after MCAO.
Measurement of infarct volume
Measurements of infarct volumes were performed as previously described [13]. Briefly, infarct volume was determined with 2, 3, 5-triphenyltetrazolium chloride (TTC) 72 h post-MCAO (n = 6 in each group). Brain tissue was sliced into thick sections (1-mm-thick coronal sections) and stained with a 2% solution of TTC for 20 min at 37 °C, followed by fixation with 4% paraformaldehyde. TTC-stained sections were imaged and analyzed using Image Pro-Plus 5.1 analysis system (Media Cybernetics, NY, NY, USA). Lesion volumes were calculated using the following formula: [total infarct volume − (the volume of intact ipsilateral hemisphere–the volume of intact contralateral hemisphere)]/contralateral hemisphere volume × 100%.
Behavioral tests
Sensorimotor functional recovery after stroke was measured before MCAO and also 3, 5, 7, and 10 days post-procedure. All behavioral tests were performed by an investigator blinded to the experimental groups. The rotarod (IITC Life Science, NY, NY, USA) test was performed to determine sensorimotor coordination. Briefly, rats were placed on an accelerated rotating rod with an increasing speed from 4 to 120 rpm within 5 min. Rats were tested three times daily with an intermission of 5 min, and latency to fall off the rotating rod was recorded. The data were expressed as mean values from three trials. The adhesive removal test was also employed [20]. In brief, a rat was placed in a cage for 1 min and an adhesive tape (50 mm2) was applied to the distal radial region of the right forelimb as a tactile stimulus. The time to contact and the time to remove the tape were both recorded. Each animal was tested three times with a cutoff time of 120 s per trial. The data are presented as the mean time to contact and the mean time to remove the tape on each testing day.
Immunofluorescence
Cells or post-fixated brain slices with 4 μm thickness were incubated with ST2 (cat. nos. 6131; 1:200; mouse original), Iba-1 (cat. nos. 6756; 1:400), CD206 (cat. nos. 53206; 1:400), CD16 (cat. nos: 6641; 1:400), and NeuN (cat. nos. 6745; 1:400) antibodies (all from InvivoGen, San Diego, CA, USA) overnight at 4 °C, then incubated with conjugated secondary antibody for 1 h at room temperature in the dark. After several washes with phosphate-buffered saline (PBS), the slides were incubated with DAPI for 3 min and then mounted in glycerol. Slides were then visualized using fluorescence microscopy (excitation range from 490 to 495 nm and optimal emission range from 515 to 525 nm), and ten random fields in each section were analyzed. Then further statistical analysis in each independent experiment was performed.
Primary neuron-microglia co-cultures and treatments
Primary neurons and microglia were prepared from the brains of mixed-sex embryonic day 17 Sprague–Dawley rats, as previously described [21]. The co-culture system was established by inserting a transwell chamber (Millipore, Bedford, MA, USA) with 3 μm pores into 12-well plates (Costar, NY, USA). Primary neurons were seeded (Costar) at a density of 1 × 103 cells/well, and an equal amount of microglia were seeded in the transwell chamber. The chamber was inserted into the wells of the plate to allow communication between the two cell types. Co-culture systems were subjected to 3 h oxygen-glucose deprivation (OGD) or control treatment, after which 12.5, 25, 50, or 100 ng/mL IL-33 (Enzo Life Science, NY, USA) or PBS were added to transwell chambers for 24 h. In addition, celastrol (0.25, 0.5, 1, or 2 μM) or PBS were added 3 h after OGD or control treatment for 24 h to assess possible neuroprotective effects. Neurons were subsequently collected for cell viability assays.
Primary microglia-enriched cultures and treatments
Primary microglia-enriched cultures were prepared from whole brains of 1-day old Sprague–Dawley rat pups as previously described [21]. Microglia were transfected with a ST2 interference vector before pretreatment with OGD for 3 h, then treated with 50 ng/mL IL-33, 1 μM celastrol, or PBS for 24 h. Cells were collected for microglial polarization analysis using immunofluorescent staining.
Primary neuronal cultures and treatments
Primary neuronal cultures were prepared from 1-day old Sprague–Dawley rat pups as previously described [21]. Neurons underwent OGD for 3 h after which they were treated with different concentrations of IL-33 (12.5, 25, 50, or 100 ng/mL), celastrol (0.25, 0.5, 1, and 2 μM) or PBS for 24 h. Cell viability was assessed with the CCK8 assay.
ELISA for soluble inflammatory cytokines
The expression of inflammatory factors IL-33, IL-6, IL-10, IL-1β, and TNF-α in cell supernatants or serum were measured using commercially available ELISA kits (Sen-Xiong Company). In accordance with the manufacturer’s instructions, supernatants were stored at − 80 °C before measurements and both standards and samples were run in triplicate. The optical density (OD)450 was calculated by subtracting the background, and standard curves were plotted.
Cell viability assay
Cell counting kit-8 (CCK8) was used to assess cell viability. Cells (1 × 104) were seeded into 96-well plates, and grown overnight and treated as described previously in this section. Medium was removed, and the cells were washed three times with PBS. DMEM (90 μL) and CCK8 (10 μL) were subsequently added to each well and incubated for 1.5 h at 37 °C, and a microplate reader was used to measure the OD450.
Apoptosis assay
Flow cytometry was used to determine the percentage of apoptotic cells. Apoptotic cells were differentiated from viable or necrotic cells by the combined application of annexin V (AV)-FITC (Roche, Indianapolis, IN, USA) and propidium iodide (PI) (Roche). Cells were washed twice and adjusted to a concentration of 1 × 106 cells/mL with cold D-Hanks buffer. AV-FITC (10 μL) and PI (10 μL) were added to 100 μL of cell suspension and incubated for 15 min at room temperature in the dark. Finally, 400 μL of binding buffer was added to each sample without washing and analyzed with flow cytometry. Each experiment was performed at least in triplicate.
To detect apoptotic neurons, post-fixated brain slices (4 μm thickness) were measured with TUNEL detection kit (Roche) according to the manufacturer’s instructions. The nuclei were stained with DAPI, and TUNEL staining was then assessed. Nuclei that were double-labeled with DAPI and TUNEL were considered apoptotic.
Western blotting analysis
Cells were harvested and lysed in Triton X-100 lysis buffer containing 25 mM Tris–HCl (pH 7.5), 137 mM NaCl, 2.7 mM KCl, 1% Triton X-100, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 4 °C. Purified proteins were then detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% separating gel and a 5% stacking gel followed by staining with Coomassie blue to determine the purity of the enzyme preparations. Western blotting analysis was performed as follows: the proteins were transferred from SDS-PAGE gels onto nitrocellulose membranes and blocked in TBS-T buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl and 0.2% Tween-20) for 1 h at room temperature with 5% nonfat milk. Membranes were then probed with primary antibodies, including ST2 and GAPDH, followed by incubation in respective secondary antibodies (Santa Cruz Biotechnologies Inc., Santa Cruz, CA, USA). Membranes were then incubated with Lumi-PhosTM WB (Pierce, Rockford, IL, USA) chemiluminescent substrate for 5 min. Protein expression levels were measured by densitometry using ImageJ 1.48u4 software (National Institutes of Health, NY, USA), and the data were normalized against the corresponding loading control (GAPDH).
RNA extraction and qRT-PCR
Total RNA was extracted from cells using TRIzol reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Total RNA was eluted with RNase-free water and stored at − 80 °C. RNA concentrations were determined with Epoch spectrophotometry. Quantification of ST2 and endogenous control mRNA U6 was performed using TaqMan assays with the supplied assay-specific RT primers (Applied Biosystems, Foster City, CA, USA). The data were analyzed using the 2−ΔΔCt method.
Construction and transfection of siRNAs
Specific siRNA against ST2—with the target sequence: 5′-CACTGGGAACTTATGTTGAATGG-3′—was synthesized by GenScript (Piscataway, NJ, USA). Microglia cells were dispersed into collagen I-coated six-well plates at a density of 5 × 104 cells/well and cultured overnight under normal conditions. The following day, cells were transfected with siRNA in serum-free medium using oligofectamine reagent (Invitrogen) for 48 h at 37 °C with 5% CO2. Cells were then collected for further analysis.
Statistical analysis
Data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA with post hoc tests for comparisons between two groups using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P values ≤ 0.05 indicate statistically significant differences.
