Correlation analysis of plasma HOCl level and infarct sizes and neurological deficits in acute ischemic stroke patients

We performed a clinical study at Sun Yat-sen Memorial Hospital, Sun Yat-sen University. The protocol was approved by the medical ethics committee of the university (No. SYSEC-KY-KS-059). All patients have been informed of the background and procedures of the study and signed the informed consent before enrollment. First-ever stroke patients aged 40–80 years old were included regardless of gender. The recruitments of ischemic stroke patients had the following inclusive criteria: (a) having a primary confirmed diagnosis of ischemic stroke caused by the MCAO after 6–72 h of symptoms onset (NIHSS > 0 and CT/MRI detected acute infarct area); (b) having competent to give written informed consent. The exclusion criteria for the patients include (a) with cancer; (b) with transient ischemic attack (TIA) or intracranial hemorrhage or subarachnoid hemorrhage (SAH) within 6 months; (c) with hepatic, renal, hematologic diseases; (d) with mental disorder, serious dementia; (e) with major surgery in the previous 6 months; (f) with pregnancy; (g) with epilepsy. Venous blood samples were obtained from the recruited patients immediately at admission and temporarily stored at 4 °C in EDTA-K2 coated tubes (aged between 40–80 years, N = 41). The NIHSS [69] was routinely used to evaluate the neurological deficits of each patient at admission by an investigator who was blinded to the experimental setting. The infarct volume was determined by the manual tracing technique on CT scanning image as previously described [70, 71]. The basic clinical data including body temperature, blood leukocyte, highly sensitive C-reactive protein (Hs-CRP) were also collected to exclude patients with symptoms of peripheral infection that might affect plasma HOCl level. Healthy individuals aged between 40–80 years without any parenchymal organ ischemia or inflammatory disease in the past three months were recruited as the control group (matched with similar ages and sex, N = 12).

All blood samples were assayed with HKOCl-3 probe immediately upon collection (< 60 min) to minimize the degradation of HOCl ex vivo. Briefly, blood samples were centrifuged at 1800g for 10 min at 4 °C, then the extracted plasma was co-incubated with the HKOCl-3 probe (2 μM) at 37 °C in darkness for 30 min and then examined using a multimode reader (SpectraMax M5, Molecular Devices). An equal volume of PBS was taken as a negative control. Each experiment was performed independently at least three times. All results were adjusted by subtracting the negative control to avoid background interference.

Animals

Animals were obtained from the Laboratory Animal Unit, the Faculty of Medicine, the University of Hong Kong, which is an AAALAC International accredited service unit. Animal experiments were approved by the Committee on the Use of Live Animals in Teaching and Research, the University of Hong Kong (CULATR number: 5062-19). All animals were housed in 14-h day/light cycles with free feeding. All the studies followed ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Cerebral I/R injury and drug treatment

Male Sprague–Dawley (SD) rats were randomly assigned into groups: sham-operation (sham), cerebral I/R, I/R plus 4-ABAH (I/R + 4-ABAH), I/R plus taurine (I/R + taurine) and sham-operation plus 4-ABAH (4-ABAH). We adopted a transient MCAO model to mimic ischemic stroke as previously described with modification [72]. Briefly, male SD rats (250–300 g) were anesthetized with 10% chloral hydrate. An MCAO monofilament (Beijing Cinontech Co., Ltd; China; 2838-A4) was inserted into the internal carotid artery via the external carotid artery until mild resistance. After 2 h of ischemia, the monofilament was withdrawn to allow reperfusion. The success of the MCAO model was confirmed by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Sham control rats were subjected to similar MCAO operation procedures but without occlusion. MPO-specific inhibitor 4-ABAH (20 mg/kg, Sigma, 5351-17-7) was intraperitoneally injected at the onset and 12 h of reperfusion, respectively (I/R + 4-ABAH group) [8]. Taurine (50 mg/kg, Aladdin, T103831, China) was intravenously administered at the onset of reperfusion (I/R + taurine). An equal volume of normal saline was used to replace 4-ABAH or taurine in the I/R group.

Behavioral test

A modified neurological severity score (mNSS) was performed at 24 h of reperfusion to evaluate the neurological deficit of the rats as previously described [73]. The mNSS is a composite of motor, sensory, reflex, and balance tests, graded on a scale of 0 to 18 (normal score, 0; maximal deficit score, 18).

Infarct volume

Infarct volume was measured at 24 h of reperfusion or at the same time points of sham operation. The rat brain was removed and sliced into 2.0-mm-thick slices by a brain sectioning matrix (Beijing Cinontech Co., Ltd; China; 300–600 g). Coronal brain slices were incubated with 2% TTC (Sigma, 298-96-4) at 37 °C for 20 min. The stained slices were photographed and the area of infarct in each slice was evaluated using the software Image-Pro Plus 6.0 (Media Cybernetics, USA). The percentage of infarct volume was measured according to a previously described method [74]: infarct volume (%) = [(volume of the contralateral hemisphere – the red volume of the intact ipsilateral hemisphere)/ 2 × contralateral hemisphere volume] × 100%.

BBB integrity

The BBB leakage was assessed by Evans blue (EB) assay as previously reported [72]. The rats were intravenously administered with 2% EB solution (4 mg/kg, Biosharp) through the femoral vein at 24 h of reperfusion or sham operation. Two hours later, the rats were killed by cardiac perfusion with PBS. After rat brains were removed, the ischemic hemispheres were weighed and homogenized in 50% trichloroacetic acid. The samples were then incubated at 4 °C in darkness overnight and centrifuged at 12,000 g for 30 min. The supernatant was spectrophotometrically quantified at 620 nm wavelength (SpectraMax M5, Molecular Devices) to determine the amount of extravasated EB dye.

Immunofluorescence

Brains were removed and fixed with 4% paraformaldehyde at 4 °C overnight. Frozen sectioning was performed after sucrose gradient dehydration followed by blocking with 10% goat serum containing 0.5% Triton X-100 at room temperature for 30 min. Brain sections were incubated with different primary antibodies at 4 °C overnight, including anti-MPO (Abcam, ab90812, 1: 1000), anti-NeuN (Abcam, ab177487, 1: 1000), anti-CD31 (Abcam, ab222783, 1: 1000), anti-Iba-1 (Abcam, ab153696, 1: 1000), anti-GFAP (Abcam, ab7260, 1: 1000) antibodies. The sections were washed with PBS 3 times and incubated with the mixture of Alexa Fluor 488 conjugated goat-anti-mouse IgG (Beyotime, A0428, 1: 500) or Alexa Fluor 555 conjugated donkey-anti-rabbit IgG (Beyotime, A0453, 1: 500) for 1 h in darkness at room temperature, followed by staining with DAPI for 5 min. After being washed with PBS 3 times, sections were mounted with fluorescent mounting medium (Dako, S3023), and examined by a confocal laser scanning microscope (LSM 780, Carl Zeiss).

TUNEL assay

Brain-frozen sections were incubated with the primary antibodies at 4 °C overnight as described above, including anti-NeuN, anti-CD31 or anti-Iba-1. The sections were washed 3 times with PBS and then incubated with the TUNEL reaction mixture (Roche, 11684795910) and Alexa Fluor 555 conjugated donkey-anti-rabbit IgG for 1 h at room temperature, followed by staining with DAPI for 5 min. After washing with PBS, sections were mounted with fluorescent mounting medium (Dako, S3023), and then examined by using a confocal laser scanning microscope (LSM 780, Carl Zeiss). The number of apoptotic cell death was calculated by counting the TUNEL-positive cells per mm2 from all images taken in the same cortical regions with HKOCl-3 staining sessions. Six images per region and five sections per brain were taken according to a similar experimental method [75]. Cell counting was carried out by an investigator who was blinded to the experimental designs.

HOCl detection

We determined the level of HOCl by using HKOCl-3, a novel fluorescent probe with high selectivity and sensitivity and a rapid turn-on response [38]. In cellular experiments, the cultured cells were co-incubated with HKOCl-3 (2 μM) in 12-well plates in dark for 30 min. After washing with PBS, the samples were mounted, and the fluorescent image was observed and taken under a fluorescent microscope (l × 71, OLYMPUS). In animal experiments, the blood samples were obtained from femoral veins before the animals were killed and the plasma HOCl level was detected by using similar methods as human blood samples. Then, brain tissues were collected for fresh frozen sections, and brain slices were incubated with HKOCl-3 (10 μM) at 4 °C in darkness for 30 min, followed by staining with DAPI for 5 min. After washing with PBS 3 times, the sections were mounted with fluorescent mounting medium (Dako, S3023) and examined by a fluorescent microscope (BX51, OLYMPUS). Cell counting was carried out by an investigator blinded to the experimental settings.

Preparation and quantification of HOCl

HOCl was prepared by the dilution of the concentrated stock solution of sodium hypochlorite. We determined the concentration of HOCl by using the iodometric titration method.

Cell culture

BV2 microglia, PC12 neuronal cells and brain microvascular endothelial bEND-3 cells were purchased from Shanghai Aolu Biotechnology Co., Ltd, China. BV2 cells were cultured in DMEM/ F12 medium (Gibco, 11330-032) supplemented with 10% fetal bovine serum (FBS, Gibco, 10099-141) and 1% penicillin/streptomycin (HyClone, SV30010). PC12 or bEND-3 cells were cultured in DMEM medium (HyClone, SH30243.01) supplemented with 10% FBS and 1% Penicillin/ Streptomycin. Before treatment with HOCl, the medium was removed and washed with Hanks’ buffered salt solution (HBSS) to prevent reactions of the medium with the HOCl. PC12 cells were then exposed to different concentrations of HOCl (0–100 μM) diluted in HBSS for 15 min.

MPO-knockdown BV2 cell line construction

The shRNA against mouse MPO was purchased from the Guangzhou IGE biotechnology Co., Ltd (Guangzhou, China) to stably knock down MPO in BV2 cells. Briefly, the sequence of shRNA was annealed and ligated into a pLKO.1 lentiviral vector. A scrambled shRNA was used as a control. Lentiviral particles were prepared by co-transfecting HEK293T cells with a package plasmid containing the MDLg, MD2g and RSV genes and shRNA vectors using Lipofectamine 2000 reagent according to the manufacturer’s instructions. After 48 and 72 h transfection, the medium was collected, and the supernatant was used to infect the BV2 cells with polybrene. Two days after infection, the cells were selected with 2.5 μg/ml puromycin until no cells died, and knockdown efficiency of MPO in the cells was evaluated by western blot.

Transwell co-cultured cell systems

For the co-culture experiments, we used a transwell culture system which mimicked the in vivo conditions as previously described [76]. Briefly, PC12 cells (2.5 × 105 cells/well) were plated in 6-well dishes or inserts. The BV2 cells (1.5 × 105 cells/well) were plated in the transwell inserts on top of the wells or in the wells. The co-cultured cell systems were incubated in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin. For comparison, the same densities of PC12 cells were plated without transwell chambers.

Oxygen–glucose deprivation/ reoxygenation (OGD/R) and drug treatment

Cells seeded in 6, 12 or 96-well plates or the co-cultured transwell systems were incubated with glucose-free DMEM medium (Gibco, 11966-025) in a hypoxic incubator (MIC-101, Billups-Rothenberg Inc.) containing the gas mixture of 95% N2 and 5% CO2 for 4 h at 37 °C, and then returned to normal culture conditions. 4-ABAH (50 μM), taurine (2 mM) or PBS was added into the medium at the onset of reoxygenation. For the normal control group, cells were incubated under NC without OGD/R exposure.

Exosome isolation and labeling

After 4 h of OGD, the BV2 cells were incubated with refreshed DMEM plus 10% exosome-depleted FBS (Gibco, A2720803) and 1% penicillin/streptomycin for 24 h. We isolated exosomes from the cultured medium using ultracentrifugation according to previous studies [77]. Briefly, the medium was collected after the cells were exposed to 24 h of reoxygenation and centrifuged at different speeds. Firstly, the supernatant was collected and centrifuged at 300 g at 4 °C for 10 min to remove living cells. Then, the dead cells and cell debris were sequentially removed by centrifugation at 3000 g and 10,000 g for 30 min at 4 °C. Followed by filtering through a 0.22 μm membrane (Merck Millipore), the supernatant was transferred to a 70 ml ultracentrifuge tube (Beckman, 355622) and ultracentrifuged at 100,000 g (Beckman, Ti70) for 70 min at 4 °C. The bottom pellet was resuspended in PBS and collected by another ultracentrifugation at 100,000 for 70 min. Finally, the exosome pellet was dissolved in 100 μL PBS for further studies.

Isolated exosomes were labeled with CM-Dil, a fluorescent dye. Briefly, exosomes were incubated with 20 μM CM-Dil for 15 min with regular mixing. Excess dye from the labeled sample was removed by washing 3 times with PBS. After purifying and labeling, the Dil-labeled exosomes were co-cultured with PC12 cells for 4 h at 37 °C. Then PC12 cells were washed with PBS and fixed in 4%PFA. The uptake was observed by fluorescence microscopy.

Western blot analysis

Samples from the cultured cells or fresh brain tissues were prepared for protein lysates using total protein lysis buffer (Beyotime, P0013) and analyzed by SDS-PAGE or non-reducing SDS-PAGE. The membrane was incubated with primary antibodies against HMGB1 (Abcam, ab79823, 1:1000, 25 kDa), MPO (Abcam, ab208670, 1: 1000, 59 kDa), CD63 (Abcam, ab216130, 1:1000, 26 kDa), MMP-2 (Abcam, ab92536, 1: 1000, 65 kDa), MMP-9 (Abcam, ab76003, 1: 1000, 92 kDa latent and 83 kDa active form), Claudin-5 (Abcam, ab15106, 1: 1000, 24 kDa) and GADPH (Cell Signaling Technology, #2118S, 1: 5000, 37 kDa) at 4 °C overnight, followed by incubation with anti-rabbit or anti-mouse IgG (MultiSciences (LiankeBio), GAR007, 1:5000) for 1 h at room temperature. The immune bands were visualized using the ECL kit (KeyGEN BioTECH, KGP1126) and photographed with ChemiDoc XRS + (Bio-Rad, Hercules, CA, USA). Each experiment was performed independently 5 times.

MTT assay

We used a 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide (MTT) kit (Sigma, M5655) to detect cell viability. Briefly, cells were plated in 96-well dishes and incubated with MTT solution (final concentration: 0.5 mg/ ml) at 37 °C in darkness for 4 h followed by dimethyl sulfoxide (DMSO, 150 μl/ well). Dishes were gently stirred in a gyratory shaker for 5 min. The O.D. value was examined by a multimode reader (SpectraMax M5, Molecular Devices) at a wavelength of 490 nm. Each experiment was performed independently 5 times.

Flow cytometry

We used the Annexin V-FITC/PI apoptosis detection kit (KeyGEN BioTECH, KGA107) to examine apoptotic cell death. Briefly, isolated single-cell suspensions were surface-stained with Annexin-V and PI at room temperature in darkness for 15 min. The cell populations were determined by flow cytometry (LSR II, BD). Each experiment was performed independently 5 times.

Nuclear/cytosolic fractionation

Cytoplasmic localization of HMGB1 was detected by density fractionation of cytoplasmic homogenates. Resting or HOCl-stimulated PC12 cells in 6-well plates were harvested. Nuclear and cytosolic extractions were collected by using Nuclear and Cytoplasmic Protein Extraction kit (Thermo Scientific, 78835) according to the manufacturer’s protocols.

Statistical analysis

Image Pro Plus 6.0 (Media Cybernetics, lnc., USA) software was used to analyze the optical density of Western blot’s results and calculate the number of fluorescence-positive cells. Statistical analysis was performed with SPSS 19.0 (SPSS Inc., USA) software. Data were presented as means ± SEM. Pearson correlation coefficients were used to analyze the correlation of the plasma HOCl level with NIHSS as well as infarct volume in the patients. Independent t-test or Pearson Chi-square test was used to compare the difference between the two groups. Kruskal–Wallis test was used for multiple comparisons among the data from HKOl-3 positive cells counting without the homogeneity of variance. Two-way ANOVA was used to compare the results among multiple groups from flow cytometry assay followed by Bonferroni's post hoc test. One-way ANOVA was used to compare the results among multiple groups from other assays followed by Bonferroni's post hoc test. P < 0.05 was considered statistically significant. Statistical tests were two-tailed.