All experimental procedures were approved by the Ethics Committee of the Army Medical University (Third Military Medical University), and was performed in accordance with the guidelines in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, and followed the ARRIVE guidelines. Two hundred and fifty-five adult male C57BL/6 mice weighing 22 to 28 g were provided by the Experimental Animal Center of the Army Medical University. Mice were acclimated in a reversed 12h dark/12h light cycle environment and provided with free access to water and food.
The study contained four experiments, which were designed as follows and in Supplemental Fig.1
To investigate the time course and cell location of NETs after SAH, 20 mice were randomly divided into the following five groups (n = 4/group): sham, 4h after SAH, 12h after SAH, 24h after SAH, 48h after SAH. CitH3 protein expression was detected by Western blot in cortex isolated from the ipsilateral/left hemisphere. Additionally, 6 mice from sham and 24h after SAH group (n=3/group), were used for double immunofluorescence staining to confirm the spatial distribution of NETs and microthrombi in the cortex. The time points were selected based on our pre-experimental results and previous studies report, in which the level of NETs peaked at 24 h post-SAH in the brain .
To elucidate the effect of neutrophil depletion treatment on EBI, 72 mice were randomly divided into three groups (n=24/group): sham, SAH +vehicle and SAH+anti-Ly6G antibody. For neutrophil depletion, anti-Ly6G antibody (1A8 Biolegend) was intravenously injected into mice at a dose of 5µg/g mouse at 24h before SAH [27, 28]. SAH severity, neurological scores (beam balance and modified Garcia tests), brain water content, Evans blue dye extravasation, Immunofluorescence staining and western blotting were evaluated at 24 h after SAH induction.
To elucidate the effect of DNase I treatment on EBI, 72 mice were randomly divided into three groups (n=24/group): sham, SAH +vehicle and SAH + DNase I. DNase I was dissolved in saline. DNase I (50μg in 250μL of saline intraperitoneally and a second dose of 10μg intravenously) was injected 1h after SAH induction . SAH severity, neurological scores (beam balance and modified Garcia tests), brain water content, Evans blue dye extravasation, Immunofluorescence staining and western blotting were evaluated at 24 h after SAH induction.
To study the effect of DNase I treatment on CSF flow, glymphatic function and cortical perfusion after SAH induction, 36 mice were randomly divided into the following three groups (n=12/group): sham, SAH +vehicle and SAH + DNase I. CSF EB spread assessment (cisterna magna injection) was used to evaluate CSF flow and glymphatic function at 24h post-SAH. Laser speckle blood monitor was used to evaluate cortical perfusion at 6h after SAH.
Mouse SAH Model
Endovascular perforation was used to establish the SAH model as previously described [29, 30]. Briefly, the animals were anesthetized with halothane (70% N2O and 30% O2; 4% for induction, 2% for maintenance, China). A midline incision was made in the neck to expose the left common carotid artery, external carotid artery, and internal carotid artery. A 5-0 monofilament nylon suture was inserted into the left internal carotid artery through the external carotid artery stump to perforate the artery at the bifurcation of the anterior and middle cerebral artery. Body temperature was kept constant 37°C during the operation. The mice in the sham group underwent the same procedures without the artery perforation.
The SAH severity grading score was blindly evaluated by two independent investigators as previously described. The basal cistern was divided into six segments, and each segment could be scored from 0 to 3 according to the amount of subarachnoid blood clotting in the segment. The total score was calculated by adding the scores from six segments (0–18 points). Mice that received a score <8 were excluded from the study.
The neurobehavioral test was blindly evaluated using the modified Garcia test and beam balance test at 24 hours after SAH as previously described[32, 33]. The modified Garcia scale included six measurements as follows: spontaneous activity, forepaw outstretching, symmetry of limb climbing, responses to body proprioception, and vibrissae touch. The mice could receive a total score ranging from 3 to 18. For the Beam Balance Score text, the mice were placed on the center of a wooden beam to assess the walking distance within 1 min and were subsequently assigned 0–4 points in total. Two blinded observers were employed for grading mean of the neurological score.
Brain Water Content
Brain edema was determined using the wet/dry method . Mice were decapitated under deep anesthesia at 24h after SAH induction. The brain samples were quickly removed from the skull, and were divided into the left and right cerebral hemispheres, the cerebellum, and the brain stem. These four parts of the brain were weighed (wet weight) respectively, and the brain samples were then dried at 55℃ for 72 h in an oven and weighed again (dry weight). The water content percentage formula was: ([wet weight - dry weight]/wet weight) ×100%.
Evans Blue Extravasation
Evans blue extravasation was performed evaluate BBB permeability as previously described [31, 34]. Mice were anesthetized by pentobarbital sodium (40 mg/kg) i.p. injection 24h post-SAH. Evans blue dye (2%, 5ml/kg Sigma–Aldrich, USA) was administered into the caudal vein and circulated for 1h. Under deep anesthesia, mice were sacrificed by intracardialiy perfused with phosphate-buffered solution (PBS).The brains were removed and quickly divided into the left and right cerebral hemispheres, weighed, homogenized in PBS, and centrifuged at 15,000g for 30min. Subsequently, the resultant supernatant was added with equal volume of trichloroacetic acid, incubated overnight at 4◦C and centrifuged at 15,000 g for 30 min. Next, the resultant supernatant was collected and spectrophotometrically quantified at 610 nm for Evans blue dye.
Western blotting was performed using the left cerebral cortex at 24 h after SAH as described previously [35, 36]. Equivalent (30µg) protein amounts were loaded in SDS-PAGE gels. After gel electrophoresis, protein was transferred onto nitrocellulose membrane, which was then blocked by blocking buffer for 2h at room temperature. Following primary antibodies were diluted to incubate with the membrane under gentle agitation at 4◦C overnight: anti-CitH3 antibody (1:2000; Abcam; ab5103). The membranes then were incubated with horseradish peroxidase-conjugated secondary antibodies for 2h at room temperature. GAPDH (1:10000; ZEN BIO; 200306-7E4) was blotted on the same membrane as a loading control.
Immunofluorescence staining was performed on fixed frozen brain sections as previously described . Briefly, the mice were deeply anesthetized and perfused with PBS and 4% PFA at 24h after SAH. Brain samples were isolated and post-fixed in 4% PFA for 24h, then soaked in 30% sucrose for 3 day. Coronal brain sections (10 µm) were obtained using a cryostat (Leica, CM1860UV, Germany) and treated with 0.3% Triton for 30 min. The brain sections were subsequently blocked with 5% normal goat serum for 2h and incubated with the primary antibodies overnight at 4◦C freezer. The primary antibodies are listed as follows: anti-Fibrinogen antibody (1:400; Abcam; ab119948), anti-CD31 antibody (1:200; Abcam; ab28364), anti-CitH3 antibody(1:200; Abcam; ab5103), anti-Ly6G antibody(1:200; Abcam; ab25377).Then the slices were incubated with corresponding secondary antibodies for 2h at room temperature, followed by staining with DAPI for 5 min. Neuronal injury was detected using Fluoro-Jade C (FJC) staining according to the manufacturer’s protocol (AG325, Millipore, Germany).The brain sections were successively incubated with 80% alcohol containing 1%NaOH for 5 min, 70% alcohol for 2 min, 0.06% potassium permanganate for 10 min, and 0.0004% FJC working solution for 20min. Finally, sections were washed and dried at 42℃ for 30min in an oven and cleared in xylene and coverslipped.
Images were obtained at basal cortex by confocal laser scanning microscope (Zeiss880, Germany). We selected at least 3 slices from each mouse and analyzed three fields of per slice from similar areas of the ipsilateral cortex at a magnification of × 200. For quantification of fibrinogen positive microthrombi, each recognizable thread-like microthrombi was counted as one regardless of its length. This resulted in a relatively lower number of total counts. All the procedures were performed by two investigators who were blinded to the experimental conditions.
CSF Flow and Glymphatic Function Assessment
Cisterna magna injection of EB dye was performed to observe the movement of CSF and glymphatic fluid at 24h after SAH as previously described[25, 38]. After anesthetization, mice were placed in the stereotaxic frame, and the atlantooccipital membrane was exposed by a midline incision. Needle (Hamilton/7803-05) was inserted 1-1.5 mm depth into the cisterna magna followed by an injection of 5μl of 2% EB dye (Sigma-Aldrich) at rate of 1μl/min. After injection, the needle was kept in place for another 10 min to allow EB dye to diffuse and then withdrawn.
EB dye was allowed to circulate for total of 1h from the start of cistern magna injection. The mice were then deeply anesthetized and perfused with PBS, brain and deep cervical lymph nodes (dcLNs) were collected. Two methods were used to quantify the EB dye distribution in the brain parenchyma and perivascular space. First, the ventral surface of the mouse brain was divided into six segments (R1-R6), each segment was given a score of 1 for dye present or 0 for no dye present, then the score was summed. Second, the concentration of EB dye in the dcLNs and in the forebrain parenchyma (R1–R3) was quantified. The dcLNs and forebrain parenchyma were homogenized in PBS, and centrifuged at 15,000g for 30min. Subsequently, the resultant supernatant was added with equal volume of trichloroacetic acid, incubated overnight at 4◦C and centrifuged at 15,000 g for 30 min. Next, the resultant supernatant was collected and spectrophotometrically quantified at 610 nm for Evans blue dye.
Cerebral cortical perfusion analysis
Cerebral cortical perfusion of the whole convexity was measured using a Laser speckle blood monitor (PeriCam PSI System, Sweden) at 6h after SAH as previously described[39, 40]. The mice were mounted on a stereotaxic frame, and a midline incision was made to expose the calvaria. 60 perfusion images were recorded at 1 picture per second.. After these measurements, the wound was then closed with sutures and anaesthesia was terminated. The perfusion data were evaluated using PIMSoft software. A mean image was calculated from the 60 perfusion images. The mean flux values were determined by evaluating a region of interest (ROI) of 7 mm2 placed over the perfusion territory of the left middle cerebral artery . Perfusion was evaluated by an investigator blinded to the treatment.
All statistical analyses were performed using GraphPad Prism 8 software. Quantitative data are expressed as the mean ± SEM. One-way ANOVA and Tukey’s multiple comparisons were employed for comparisons among the different groups. The Kruskal-Wallis test was employed for the analysis of the behavior scores p < 0.05 was considered statistically significant.