Ethics
Study participants were recruited from the Department of Neurosurgery, The First Affiliated Hospital of Wannan Medical College, Wuhu City, China. The study was performed in accordance with the Declaration of Helsinki. Written informed consent was received from participants or valid proxies (family or a professional not directly involved in the study) prior to inclusion in the study. All experiments were approved by the Ethics Committee of the First Affiliated Hospital of Wannan Medical College.
Study design
The aSAH patients included in the present study were admitted from May 2016 to March 2017. We collected plasma from 117 patients with aSAH within the 24 h after SAH, out of a total of 224 patients with SAH during this period. The mean age ± SD was 59.91 ± 9.65 years (range, 40–83 y); there were 72 women and 45 men. Exclusion criteria were as follows: admission later than 24 h after onset of bleeding; non-aneurysmal SAH; liver, kidney, heart or lung insufficiency or infectious diseases; rebleeding after admission and poor prognosis upon admission without any intervention. Age-matched health persons in the fasting state were used as controls (n = 40).
Initial clinical status was accessed was using World Federation of Neurological Surgeons (WFNS) grading [28]. The amount of blood in computed tomography (CT) was assessed using the modified Fisher’ scale.Hydrocephalus and DCI were classified as present or absent based on CT scans or magnetic resonance imaging (MRI). All patients were treated with endovascular coiling. Neurologic outcome was assessed using the Modified Rankin Scale (mRS) at 1-year post-aSAH using a structured telephone interview[29] . If no contact was obtained after this procedure, the patient was declared lost to follow-up. At the end of the follow-up period, patients with mRS scores of 0–2 were classified as having good outcomes, and those with mRS scores of 3–6 were classified as having poor outcomes. Tables 1 and 2 summarizes the basic characteristics of the population.
Sample processing
The blood samples were processed for plasma isolation within 2 h after withdrawal using BD EDTA-treated plasma separator tubes. Whole blood was centrifuged at 500 rpm and 4 °C for 10 min. The upper-layer was transferred into a RNase/DNase-free 1.5 mL EP tubes followed by further centrifugation at 3000 rpm and 4 °C for 10 min. Plasma was divided into aliquots and stored at -80 ºC for further analysis.
Isolation of NDSEVs from plasma samples
sEVs were isolated using miRCURY Exosome Kits (from Cell/Urine/CSF; Qiagen, Valencia, CA) following the manufacturer’s protocol. Briefly, 400 μL of precipitation buffer B was added to 1 mL of CSF samples and mixed well for 60 minutes at 4 ºC, followed by centrifugation at 10,000 g for 30 minutes at room temperature. The sEVs were harvested by removing the supernatant. The sEVs were resuspended in 100 μL of resuspension buffer. To enrich NDSEVs, total sEVs were harvested by immunoadsorption enrichment with mouse anti-human CD171 (L1CAM neural adhesion protein) biotinylated antibody (eBiosciences, San Diego, CA, USA), as described[11] .
Then NDSEVs were lysed by 1% Triton X-100 lysis buffer (Beyotime, P0013) that contained protease and phosphatase inhibitor cocktails. The lysates were stored at -80 ºC.
Western blot analysis
Molecular weight maker (5 μl /lane; Thermos Scientific, Waltham, MA, USA) and protein samples (20 lg/lane) were separated using a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and electrophoretically transferred onto polyvinylidene difluoride membranes (PVDF, Immobilon-PSQ, Millipore Corporation, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature. Blots were incubated with primary antibodies in 5% BSA (in PBS + 0.1% Tween 20) overnight at 4 °C. The primary antibodies used were as follows: mouse anti-CD63 (1:1,000, Abcam), and rabbit anti-Alix (1:1,000, Abcam). Corresponding HRP-conjugated anti-rabbit, or anti-mouse (1:10,000, Pierce) secondary antibodies were incubated for 2 h at room temperature. Bands were visualized using an enhanced chemiluminescence (ECL) kit (Beyotime).
Transmission electron microscopy (TEM)
NDSEVs were adsorbed on carbon-coated nickel grids for 1 h, subsequently washed three times with PBS for 5 minutes and fixed with 2% formaldehyde for 10 min. Samples were contrasted using uranyl acetate and lead citrate (Sigma-Aldrich). After three washings in deionized water, grids were dried for several minutes and finally examined with a JEOL JEM-1400 transmission electron microscope at 80 kV.
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IL-6, and tetra-spanning exosome marker CD81 in NDSEVs were quantified using specific ELISA kits (Elabscience Biotechnology, China) according to the manufacturer’s instructions. The mean value for all determinations of CD81 in the patient cohort was set at 1.00 and relative values for each specimen were used to normalize their recovery. The final concentration of cytokines was measured using OD values. One laboratory technician measured all ELISAs without knowledge of the clinical information.
Statistical analysis
All data were analyzed using MedCalc version 15.0.0 (Medcalc Software bvba, Ostend, Belgium). Data were presented as mean ± standard deviation (SD). The Mann–Whitney U test was used to assess the differences between two groups and the Kruskal–Wallis test was used for differences between more than two groups. The correlations among the variables were calculated using Spearman rank correlation coefficient analysis. For comparison of levels of IL-6 over time between patients with poor and good outcome, a generalized linear relationship model with Spearman's correlation coefficient was used. Receiver operating characteristic (ROC) curves were constructed to determine the optimal thresholds of IL-6 for aSAH. A multivariable logistic regression model was analyzed to determine factors that predicted the mRS after adjusting for risk factors that reached P <0.1 in the univariate analyses. A P-value of <0.05 was considered significant.