Chemicals and reagents
The standard compounds of taurine and caffeic acid were obtained from Shanghai yuanye Bio-technology Co.,Ltd (Shanghai, China). Deionized water used in experiment was supplied by a Milli-Q Academic ultra-pure water system (Milford, Millipore, USA). Acetonitrile and methanol were obtained from Merck (Chromatographic, Germany); formic acid was obtained from Tedia (Chromatographic, USA). Edaravone injection was from China national medicines Guorui pharmaceucital Co., Ltd (Anhui, China; lot number:2005018). Lactate dehydrogenase (LDH) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) and cysteine sulfinic acid decarboxylase (CSAD) was obtained from Abcam (Cambridge, England).
Animals and middle cerebral artery occlusion/reperfusion (MCAO/R) model
Adult male specified-pathogen-free (SPF) C57BL/6J mice weighing 18 - 22 g were obtained from the Experimental Animal Research Centre of Yangzhou University (Yangzhou, China; certificate no SCXK 2017 - 0007). All experimental protocols were performed according to the National Institutes of Health (NIH) guidelines and the research was approved by the Institutional Animal Care and Use Committee of the Animal Ethics Committee of the School of Chinese Materia Medica, China Pharmaceutical University. All mice were housed with a 12:12 h light-dark cycle at 23±1℃. Prior to experiments, mice were split randomly into three groups: sham, MCAO/R and MCAOR+EDA. Stroke was induced by MCAO/R model in mice as reported previously [10]. Briefly, the right middle cerebral artery was occluded with a blunt-tip 6-0 nylon monofilament for 1 h followed with reperfusion for 24 h. Longa’s method was used for evaluating neurological deficit [10]. Sham operated control mice underwent the same surgical procedures except for the occlusion by nylon monofilament. EDA was administrated intraperitoneally to mice with 3 mg/kg (refer to the clinical dose) after 1 h of ischemia, the remaining model mice were given an equal volume of normal saline.
Haematoxylin and Eosin (H&E) Staining
H&E staining was used for histomorphological analysis. In short, brain slices were put into haematoxylin and eosin solution, redehydrated in gradient ethanol solution again, treated with dimethylbenzene and covered with coverslips. The pathological images were scanned with a digital pathological section scanner (Hamamatsu, Japan) and analyzed with NDPView2 software.
TTC staining
After I/R, mice were euthanized and perfused by normal saline. Then, the whole brains were taken out, freezed at -20 degrees followed by cut into 1mm thick slices rapidly. These brain slices were incubated in 1 % TTC for 10 min at 37 °C. The infarcted areas were analyzed with Image J software (NIH, Bethesda, MD).
Transmission electron microscopy
After I/R, mice were euthanized and perfused by normal saline followed by perfusion with the fixative (2% glutaraldehyde and 2% lanthanum nitratein in 0.1M sodium cacodylate pH 7.4 - 7.5) at room temperature, as previously described [11]. 1 mm3 sample obtained from the region encompassing ischemic infarction of removed brains were kept in the same fixative overnight at 4℃. The samples were post-fixed in 1% osmium tetroxide for 1h followed with embedded in Epon 812. After polymerization, three blocks were randomly selected from each brain sample. An Ultratome (Nova, LKB, Bromma, Sweden) was used for cutting ultrathin sections. Then, ultrath insections were mounted on mesh grids (6-8 sections/grid) and stained with uranyl acetate and lead citrate. Finally, the prepared samples were examined under a transmission electron microscope (JEOLLtd., Tokyo, Japan).
Untargeted metabolomics analysis
Sample pretreatment
Serum and urine of mice were collected after 24 hours reperfusion. After standing for about 60 minutes, the blood was centrifuged with 3500 r/min for 10 min at 15 °C. The obtained serum samples were sub-packed and stored in -80°C until the analysis. Urine samples were collected at 4 °C and kept at −80 °C until the analysis. 200 μL of serum and urine were used for untargeted metabolomics analysis and 600 μL of methanol was added into samples for precipitating protein. Samples were subsequently centrifuged (13,000 rpm, 15 min) at 4 °C followed by swirling 60 s. The supernatant was transferred to a tube and dried under a gentle stream of nitrogen at room temperature. Then, the residue was dissolved with 200 μL methanol and centrifuged (13,000 rpm, 15 min) at 4 °C for further analysis.
HPLC-Q-TOF/MS analysis
The detection of metabolites in urine and serum samples was performed on an Agilent Technologies 6540 Accurate-Mass Q-TOF LC/MS (USA) with an electrospray ionization (ESI) source and the data were collected by mass hunter workstation. The eluent A and B were deionized water (0.1 % formic acid) and acetonitrile (0.1 % formic acid), respectively. Serum analyses were achieved on a SynergiTM Fusion-RP C18 column (50×2 mm i.d., 2.5 μm) with a gradient elution program: 0 - 5 min, 5 - 5% B; 5 - 10 min, 5 - 30% B; 10 - 15 min, 30 - 60% B; 15 - 20 min, 60 - 70% B; 20 - 22 min, 70 - 80% B; 22 - 25 min, 80 - 95% B; 25 - 30 min, 95 - 95% B. Urine analyses were achieved on a TSK-GEL Amide-80 column (150 × 2.0 mm i.d., 5 μm) with a gradient elution program: 0 - 7 min, 90 - 90% B; 7 - 9 min, 90 - 75% B; 9 - 11 min, 75 - 75% B; 11 - 13 min, 75 - 50% B;13 - 20 min, 50 - 50% B. Both of the flow rates were set at 0.2 mL/min with the injection volume of 10 μL. The Q-TOF/MS operating parameters were set as follows: fragment voltage, 120 V; nebulizer gas, 35 psig; capillary voltage, 4000 V; drying gas flow rate, 9 L/min; temperature, 325℃; detection range, m/z 50 - 1500 in full scan mass spectra. The MS data acquisition was carried out in positive and negative ionization mode.
Validation of system stability
Repeatability and robustness of the experiment were validated with the pooled quality control sample (QC) [12]. The QC sample was prepared to mix equal volumes (30 μL) of each test sample, and treated with the same method as the test samples. QC sample was randomly injected throughout the sequence list.
Data analysis of metabolomics strategies
Before multivariate analysis, the data format (.mzdata) files obtained by MassHunter Workstation Software (Version B.06.00, Agilent Technologies) were processed by XCMS software performing on the R+ package (R Foun-dation for Statistical Computing, Vienna, Austria), and the data pretreatment procedures include nonlinear retention time alignment, peak discrimination, filtering, alignment, and matching. All detected peaks were tabulated with tR-m/z pairs and outputted for statistical analyses. In order to screen the significant compounds that were responsible for the difference between model and model + EDA, metabolomics strategies were subsequently used to dispose the data. Principal component analysis (PCA), orthogonal partial least-squares discriminant analysis (OPLS-DA), volcano Plot and heatmap developed by Metaboanalyst (https://www.metaboanalyst.ca/) were adopted to do the preliminary screening. PCA is a multivariate technique which can select the typical variables from a data table by several linear transformations and OPLS-DA is a supervised machine learning model. The online database including HMDB (http://www.hmdb.ca/), METLIN (http://metlin.scripps.edu/), and MassBank (http://www.massbank.jp/) were performed to identify the potential metabolites by matching with the message of ion fragments.
Targeted analysis for taurine by HPLC-QQQ-MS/MS
Targeted analysis was performed on a triple quadrupole tandem high performance liquid chromatography-mass spectrometry (HPLC-QQQ-MS/MS) system (Agilent, 6465) with caffeic acid as the internal standard. Chromatographic separation was performed on a TSK-GEL Amide-80 column (150 × 2.0 mm i.d., 5 μm) with a gradient elution program: 0 - 1 min, 75 - 75% B; 1 - 2 min, 75 - 60% B; 2 - 3min, 60 - 60% B; 3 - 5 min, 60 - 50% B. The mobile phase system consists of deionized water containing 0.1 % formic acid (A) and acetonitrile containing 0.1 % formic acid (B) at a flow rate of 0.2 mL/min. Multiple reaction monitoring transitions in negative mode were performed at m/z124→79.9 for the target analyte taurine, and m/z 179→135 for internal standard compound. MS parameters for the LC-MS/MS system including fragment and voltage collision energy of taurine and internal standard were 110 V, 21 V and 90 V, 17 V, respectively.
Western blot analysis
The RIPA buffer supplemented with protease inhibitor cocktail was adopted for lysing ischemic penumbra of the brain tissues, and obtained samples were used for western blotting as described previously [13]. Protein concentration of tissues were determined by bicinchoninic acid (BCA) protein assay kit (Bi yuntian Biotech. Co., Ltd., China) after centrifuging (12,000 rpm, 10 min, 4℃). The supernatant was diluted by loading buffer to 1 μg/μL followed by heating at 100℃ for 5 min. Equal protein amounts of different groups were electrophoresed on SDS-PAGE gels and transferred to a polyvinylidene fluoride (PVDF) membrane. Then, the obtained PVDF membrane were blocked with 5 % BSA solution for 2 h and incubated with specific primary antibodies overnight at 4℃followed by suitable secondary antibodies at room temperature for 2 h. Protein signals were detected with ECL plus system and imaged by the Gel Imaging System (BioRad, Hercules, CA, USA). The protein levels were calculated by protein signals to correlative GAPDH or β-actin.
Immunofluorescence staining
After perfusion with PBS and 4% paraformaldehyde, brain tissues were picked up and put into 4% paraformaldehyde. After 24 h, brain tissues were dehydrated with 40% sucrose for 5 days, embedded in OTC and frozen at −80℃. Sectioned brain tissues into slices of 10 μm thickness with a cryotome (Leica, Mannheim, Germany). Brain sections were fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 in PBS, blocked with 5% bovine serum albumin, and incubated with specific primary antibodies overnight at 4℃. Next day, tissue sections were incubated with appropriate fluorescence-conjugated secondary antibodies at room temperature, and cell nucleus was stained with DAPI. The immunofluorescence TUNEL assay was performed according to the instructions of the manufacturer. Fluorescent images were observed by the confocal laser scanning microscopy (CLSM, LSM700, Zeiss, Germany).
Statistical analysis
Student’s t test and one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test operating on the Graph Pad Prism 8.0 (Graph Pad Software, La Jolla, CA, USA) were used for analyzing two group comparisons and multiple comparisons, respectively. Differences were considered significant at P <0.05.