Study population
The derivation cohort was derived from the regional stroke database between March 2016 and May 2020. We selected SAH patients from this database based on the following inclusion criteria: 1) adult patients > 18 years old; 2) SAH due to ruptured aneurysm; 3) dense localized clot and/or vertical layer of blood greater than 1mm in thickness on computed tomography (CT); and 4) SAH patients who were treated with endovascular coil embolization. The exclusion criteria were: 1) non-aneurysmal SAH such as trauma, infection or perimesencephalic SAH, 2) patients treated with surgical clipping and 3) previous history of central nervous system disorder or mitochondrial diseases.
TEM was used to detect the autophagic vacuoles and morphological changes of mitochondria in CSF cells derived from SAH patients with DCI. We evaluated and compared the autophagy and mitophagy biomarkers in CSF cells of SAH patients with and without DCI using qRT-PCR. The markers included DAPK-1, BNIP3L, BAX, PINK1, ULK1 and NDP52. We further evaluated the expression of autophagy executor gene of BECN1 and autophagy adaptor protein of p62.26, 27 Confocal microscopy was used to identify colocalization of differentially expressed genes in vWF-positive CSF cells, which represented endothelial cell origin, and were increased in SAH patients with DCI.8
In our study, the diagnosis of DCI wad performed through the following criteria: 1) new developed neurological changes such as motor weakness, dysphasia, and sensory change; 2) decrease consciousness by more than 2 points via the Glasgow Coma Scale score or National Institutes of Health stroke scale; 3) fluctuation of symptoms lasing more than 1 hr; 4) cerebral infarction identified on CT or MRI, but not complications related to the endovascular coil embolization; 5) concomitant severe cerebral vasospasm with narrowing more than 50% compared to the initial radiological tests; and 6) excluding other causes that may neurological
changes such as re-bleeding, hydrocephalus, seizures or electrolyte imbalances.2,28 DCI was monitored daily based on the transcranial Doppler (TCD) velocity. When severe vasospasm was suspected based on a TCD greater than 200 cm/s in the middle cerebral artery or 85 cm/s in the basilar artery,29 catheter angiography was additionally performed to confirm the degree of vasospasm and chemical angioplasty, as needed. Poor outcome was defined by a modified Rankin scale (mRS) score of ≥ 3 or Glasgow Outcome Scale (GOS) of ≤ 4 at the 3-month follow-up.
After coil embolization, continuous lumbar drainage of CSF was maintained in the neurointensive care unit in every SAH patient for 1 week after ictus in our institution. Because difference in mitochondrial membrane potential of the CSF cells was most prominent on day 5 post ictus in SAH patients with and without DCI,8 we analyzed the CSF samples obtained from days 5 to 7. Clinical, laboratory, and radiological information was reviewed by the two investigator independently. Disagreements were resolved by the third investigator. The protocol of DCI diagnosis among reviewers are presented in detail in the Supplemental Data. Sample collection and study design were performed according to the principles of the Declaration of Helsinki and were approved by the Institutional Review Board of the Chuncheon Sacred Heart Hospital (No. 2017-9, 2018-6, and 2019-6). All methods were performed in accordance with the relevant guidelines and regulations in manuscript. Informed consent was received from the patients or their relatives.
Transmission electron microscopy
Previously, SAH patients with DCI showed depolarization of mitochondrial membrane potential, which triggers alteration in mitochondrial function and morphologies.8 In the present study, TEM was used to investigate the changes in subcellular ultrastructure of CSF cells in SAH patients with DCI. CSF samples were centrifuged at 4000 rpm for 10 min, and the pellets were analyzed by electron microscopy.8 The pellets were fixed overnight in 2% glutaraldehyde in cacodylate buffer (0.1 M sodium cacodylate, 2 mM MgCl2) at 4 ℃. After washing three times with cacodylate buffer at 4℃, the samples were post-fixed in 2 % osmium tetroxide for 1 h at 4℃. The samples were rinsed with deionized water and dehydrated through a gradient series of ethanol, ranging from 50% to 100% ethanol, 20 min each step. The samples were incubated with progressively concentrated propylene oxide dissolved in ethanol followed by infiltration with increasing concentration of Eponate 812 resin. Samples were baked in a 65°C oven overnight and sectioned using an Ultra microtome. Sections were viewed with a Field Emission TEM unit (JEM-2100F, JEOL) at the Korean Basic Science Institute, Chuncheon, South Korea.8
Western blot analysis
CSF cells obtained from SAH patients were lysed with RIPA buffer (50 mM Tris-base, 10 mM EDTA, 150 nM NaCl, 0.1 % SDS, 1 % Triton X-100, 1 % sodium deoxycholate, 1 mM PMSF). Protein lysates of the supernatant were quantified using the BCA protein assay kit (Thermo Scientific, USA). Equal amounts of protein were separated on 10 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Bio-Rad, USA). After blocking the membranes with 2 % BSA in TBS-T (Tris-buffered saline including 0.1 % Tween-20) for 1 h, the membranes were incubated with the primary antibodies overnight at 4℃. After extensive washing, the membranes were incubated with HRP-conjugated secondary antibodies, and developed using an enhanced chemiluminescence (ECL) kit (Thermo Scientific, USA). Antibodies used in this study were: BECN1 (#3495, Cell Signaling Technology, USA, dilution 1:1000), p62 (sc-48402, Santa Cruz Biotechnology, USA, dilution 1:1000), LC3 (#3868, Cell Signaling Technology, USA, dilution 1:1000), pBECN1Ser15 (#84966, Cell Signaling Technology, USA, dilution 1:1000), DAPK (PA5-14044, Invitrogen, USA, dilution 1:1000), PINK1 (ab23707, Abcam, UK, dilution 1;1000), BNIP3L/NIX (ab8399, Abcam, UK, dilution 1:1000), and actin (sc-47778, Santa Cruz Biotechnology, USA, dilution 1:1000)
Immunofluorescence staining
To evaluate the colocalization of autophagy and mitophagy markers with dysfunctional mitochondria in vWF-positive CSF cells, multi-color immunofluorescence staining was performed via confocal microscopy. CSF cells were fixed with paraformaldehyde (4 % w/v) and then washed with PBS. After blocking with 2.5% blocking solution (Vector, USA) for 1h, the cells were incubated with antibodies specific to DAPK1 (PA5-14044, Invitrogen, USA, dilution 1:100), PINK1 (ab23707, Abcam, UK, dilution 1;100), BECN1 (#3495, Cell Signaling, USA, dilution 1;100) and BNIP3L/NIX (ab8399, Abcam, UK, dilution 1:100) overnight at 4℃. After incubation with anti-rabbit Alexa Fluor 750-conjugated secondary antibody (ab175733, Abcam, UK, dilution 1:500), the coverslips were mounted in Fluoroshield™ with DABCO (ImmunoBioScience Corp. USA). vWF-FITC (von Willebrand factor–fluorescein; ab8822, Abcam, UK, dilution 1:500, Abcam) was used as an endothelial marker to confirm the endothelial origin of CSF cells.16 CD41 (GTX113758, GeneTex, USA, dilution 1:100) was used to differentiate endothelial and platelet, and it was confirmed that vWF was an endothelial specific marker in our study. To assess the dysfunctional mitochondria undergoing autophagy in vWF-positive CSF cells, autophagy- or mitophagy-related markers were subjected to co-immunofluorescence stained with MTDR (M22426, Invitrogen, USA, dilution 200 nM), which is widely used as a mitochondria-selective dye to determine mitophagy.30 Nuclei were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, D1306, Invitrogen, USA, dilution 300 nM). Fluorescence images were obtained via confocal microscopy at 400x magnification.
Real time qRT-PCR
The loss of mitochondrial membrane potential leading to mitochondrial dysfunction results in activation of mitochondrial autophagy. Therefore, autophagy and mitophagy markers such as DAPK1, PINK1, BAX, BNIP3L, and NDP52 were examined in CSF cells.17,18 Total RNAs were isolated from the CSF cells using TriZOL (Ivitrogen, USA) according to the manufacturer’s instructions. The cDNA was synthesized from 5 μg of RNA using Maxime RT PreMix Kit (iNtRON Biotechnology, Korea). The expression of mitophagy-related genes was measured by qRT-PCR using the 2x Rotor-Gene SYBR Green PCR Master Mix (Qiagen, Carlsbad, CA, USA) in the Rotor-Gene Q (Qiagen, USA). Primer sequences are presented in the Supplemental Table S1.
Immunogold staining
We further performed immunogold staining to confirm the location of the most significant differentially expressed markers within the mitochondria of CSF cells in SAH patients with DCI. CSF cells were fixed with 0.1% glutaraldehyde and 2% paraformaldehyde in phosphate buffer at pH 7.4 for 1 h at 4°C, followed by incubation with post-fixed osmium tetroxide for 30 min at 4°C. The samples were dehydrated in a graded series of ethanol. The samples were treated with graded ethanol series and embedded in LR white resin (EMS). The sections were then sliced into ultra-thin sections of 80 nm each, and placed on a copper grid. The sections were treated with 0.02 M glycine for 10 min to ensure quench-free aldehyde groups. Sections were then washed with deionized water, blocked in 1% BSA and incubated with rabbit anti-DAPK1 antibodies (PA5-14044, Invitrogen, USA, dilution 1:100) for 1 h. The grid was washed five times with 0.1% BSA in PBS, and incubated with a secondary antibody conjugated with 10 nm gold (AUR810.011, AURION, Netherlands, dilution 1:100) in 0.1% BSA in PBS. The final samples were stained with uranyl acetate and lead citrate, and then visualized under a transmission electron microscope (JEOL-2100F, USA) at 200 KV.
Statistical analysis
Continuous variables are expressed as the mean and ± standard deviation (SD). A chi-square or Student’s t test was carried out to identify meaningful differences between DCI and non-DCI patients. Comparative analysis via qRT-PCR was performed using the Mann-Whitney U test. The results were presented as the median and 25th -75th percentiles. Quantification of western blots using the relative optical densities with actin protein as the reference and presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed with SPSS V.21 (SPSS, Illinois, USA) and GraphPad Prism software (v.6.01; GraphPad Software Inc., San Diego, CA, USA) with a statistical significance indicated at p < 0.05.