Animals

All experimental protocols were evaluated and approved by the institutional animal care committee of Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Diseases (HHLL-2023-025) and complied with the ARRIVE guidelines. Male Sprague–Dawley (SD) rats (grade SPF; 8–9 weeks old, 270–320 g) were housed in the animal care facility (four rats per cage) on a constant 12-hour light/dark cycle with controlled temperature at Tianjin Huanhu Hospital (Tianjin, China). All SD rats had free access to food and water.

Experimental DesignExperiment 1

Rats were randomly divided into six groups consisting of a sham group and five SBI subgroups (at 6 h, 12 h, 1 d, 3 d, and 7 d post-SBI). Plasma and samples of brain tissues were collected for the enzyme-linked immunosorbent assay (ELISA) (n = 6/group) and western blot (n = 6/group) detection respectively. Brain edema was evaluated using brain water content in six groups (n = 6/group). The surrounding brain tissues of injured foci and neutrophils isolated from the peripheral blood were observed by scanning electron microscopy (n = 5/group). Furthermore, brain tissues were obtained for immunofluorescence at 3 days post-modeling (n = 6/group).

Experiment 2

The rats were randomly divided into four groups: sham, SBI + vehicle (saline), SBI + Cl-amidine, and SBI + DNase I. Plasma and brain tissues of rats were obtained at 1 d and 3 d post-SBI respectively. ELISA (n = 6/group), western blot (n = 6/group), immunofluorescence (n = 6/group), brain water contents (n = 6/group), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) (n = 6/group) were performed. The modified Garcia scores were used to assess neurological deficits in different groups at 1 day, 3 days, and 7 days (n = 8/group).

Experiment 3

Rats were randomly divided into two groups: SBI + vehicle (1% DMSO diluted in corn oil) group, and SBI + RU.521 group. The plasma samples were collected for ELISA (n = 6/group) at 1 day post-SBI. Brain tissues were obtained at 3 days post-SBI for ELISA (n = 6/group), brain water content (n = 6/group), and TUNEL analyses (n = 6/group) respectively. Neurological function was assessed by the modified Garcia scores at 1 day, 3 days, and 7 days after SBI (n = 8/group).

Experiment 4

Rats were randomly divided into four groups: sham, SBI + vehicle (saline), SBI + DNase I, and SBI + DNase I + cGAMP. Brain tissues were collected for western blot 3 days post-SBI (n = 6/group). Furthermore, rats were randomly divided into two groups: SBI + DNase I + vehicle (saline), SBI + DNase I + cGAMP. Brain tissues were collected 3 days post-SBI for immunofluorescence, TUNEL, and brain water content analyses respectively (n = 6/group). Neurological function was evaluated at 1 day, 3 days, and 7 days post-SBI (n = 8/group).

Experiment 5

Rats were randomly divided into three groups including a sham group, an SBI + vehicle (saline) group, and an SBI + DNase I group, and two groups containing SBI + DNase I + vehicle (saline) group, SBI + DNase I + cGAMP group. Neutrophils isolated from peripheral blood were obtained at 1 day after SBI. Primary rat microglia were cocultured with neutrophils isolated from different groups. Immunofluorescence (n = 6/group) and ELISA (n = 6/group) were performed.

Experiment 6

Rats were randomly divided into four groups: SBI + vehicle (saline) group, SBI + vitamin C (100 mg/kg) group, SBI + vitamin C (200 mg/kg) group, and SBI + vitamin C (500 mg/kg) group. The ROS levels of neutrophils isolated from peripheral blood were measured in different intervention groups (n = 6/group). Plasma and samples of brain tissues were collected for the ELISA (n = 6/group) and western blot detection (n = 6/group). Neutrophils isolated from normal healthy rats were pretreated with different concentrations of vitamin C (100 nM, 500 nM, 1µM) and then stimulated with phorbol 12-myristate 13-acetate (PMA). Live-cell-forming NETs were visualized by extracellular DNA (SYTOX Green) and intracellular DNA (Hoechst 33342) on laser confocal microscopy (n = 5/group).

SBI Model

The SBI rat model was performed as previously described (Jadhav et al. 2007). Rats were anesthetized by intramuscular injection of a mixture of tiletamine/zolazepam (Zoletil 50, Virbac, France, 20 mg/kg) and xylazine hydrochloride (#T1500, TargetMol, China, 5 mg/kg). The skin and subcutaneous tissue were incised, and the right frontal skull was exposed. A bone flap was removed, the dura was incised, and a partial right frontal lobectomy was performed by making 2 incisions: 2 mm lateral to the sagittal suture and 1 mm proximal to the coronal suture, with depth extending to the skull base. Sham-operated animals were only subjected to craniotomy without any dural incisions. Then they were closely observed and transferred to cages. The rats were sacrificed at the indicated time points after surgery.

Drug Administration

For drug administration, Cl-amidine (#T10831L, TargetMol, China) and DNase I (#11284932001, Roche Diagnostics, Germany) were diluted in sterile saline. As previously reported (Vaibhav et al. 2020; Feng et al. 2021), Cl-amidine (50 mg/kg) was administered via intraperitoneal injection 10 min after SBI, and DNase I (5 mg/kg) was administered via the tail vein at 1 h post-SBI. SBI rats of the vehicle groups were treated with a corresponding dose of saline. The administrations of Cl-amidine, DNase I, and saline were performed daily respectively until the rats were euthanized.

RU.521 (selective cGAS inhibitor) and 2′3′-cGAMP (STING agonist) were used in this study. Based on previous studies (Wang et al. 2021; Gamdzyk et al. 2020), RU.521 (#T5486, TargetMol, China, 450 μg/kg, dissolved in 1% DMSO + corn oil) or vehicle (1% DMSO diluted in corn oil) was intranasally administered at 2 h, 24 h, and 48 h after SBI, and 2′3′-cGAMP (#B8362, ApexBio Technology, USA, 500 μg/kg) or vehicle (saline) was injected intravenously through the tail vein 10 min before SBI, and this was repeated at 24 and 48 h after SBI. Furthermore, the groups used for neurological function assessment were treated for 7 days.

To assess the effective dose of vitamin C (#A4034, L-ascorbic acid, Sigma–Aldrich, USA), rats received intravenous administrations of low (100 mg/kg), medium (200 mg/kg) and high (500 mg/kg) doses of vitamin C one hour after SBI based on the previous literature (Chang et al. 2020). Vehicle-treated rats were injected intravenously with an equal volume of saline vehicle one hour after SBI. The procedures were repeated once a day until the animals were killed.

Evaluation of Brain Water Content

The rats were anesthetized by the method described and euthanized by cervical dislocation. The fresh brains were quickly removed and separated into separate hemispheres. The right brain hemispheres were weighed immediately to determine the wet weight, and the samples were dried at 100 °C for 48 h to obtain the dry weight. Then the brain water content was determined as [(wet weight-dry weight)/wet weight] × 100%.

Enzyme-Linked Immunosorbent Assay (ELISA)

Blood samples were collected using EDTA as an anticoagulant. Plasma was prepared by centrifugation at 1500 × g for 15 min at 4 °C. The plasma supernatant was removed, split into 0.5 mL aliquots, and assayed immediately or stored at − 80 °C until analysis. The levels of interleukin-6 (IL-6, #ml064292), tumor necrosis factor (TNF, #ml603709), Citrullinated histone H3 (CitH3, #ml002859), Myeloperoxidase (MPO)-DNA (#ml085663-J), interferon-β (IFN-β, #ml102842-J), and vitamin C (#ml003052) in plasma, brain tissue, or cell supernatant were measured using ELISA kits (Mlbio, Shanghai, China) according to the manufacturer’s guidelines. The absorbance at 450 nm was measured immediately with a multimode microplate reader (SpectraMax 190, Molecular Devices, USA).

Neutrophil Isolation

Fresh whole blood was collected from the abdominal aorta of the rat and stored in EDTA anticoagulant tubes. Next, neutrophils were purified from the peripheral blood at room temperature by using a blood neutrophil isolation kit (#P9200, Solarbio, China) according to the manufacturer’s manual.

Scanning Electron Microscopy

Neutrophils isolated from rat peripheral blood were seeded onto polylysine-coated coverslips. The coverslips and rat brain tissues were fixed with 2.5% glutaraldehyde for 24 h and with 1% osmium acid for 1 h. Then, they were dehydrated with a graded ethanol series in turn (30, 50, 70, 80, 90, and 100%). After drying, these specimens were coated with platinum and observed under scanning electron microscopy.

Western Blot Analysis

Brain tissue samples were collected from the injured cortex and extracted with RIPA lysis buffer (#R0010, Solarbio, China). The protein concentration was determined by a BCA Protein Assay Kit (#PC0020, Solarbio, China). Protein samples (20 µg) were separated by SDS–PAGE and transferred to PVDF (#88518, Thermo Fisher Scientific, USA) membranes. Then the PVDF membranes were blocked in 5% skim milk for 2 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: CitH3 (AB_304752, #ab5103, Abcam, 1:1000), GAPDH (AB_2107436, #60004-1-Ig, Proteintech, 1:50000), β-actin (AB_2687938, #66009-1-Ig, Proteintech, 1:20000), STING (AB_10665370, #19851-1-AP, Proteintech, 1:1000), β-Tubulin (AB_2881629, #66240-1-Ig, Proteintech, 1:20000), pTBK1 (AB_2840252, #AF8190, Affinity, 1:500), TBK1 (AB_2882504, #67211-1-Ig, Proteintech, 1:500). Then the membranes were incubated with an HRP-labeled secondary antibody at room temperature for 1 h. The protein bands were visualized using an ECL detection kit (#310209, ZETA LIFE, USA). All primary antibodies were validated and reported in published literature or by companies. ImageJ software was used to analyze the immunoblot images.

Immunofluorescence

Neutrophils isolated from the peripheral blood of rats and primary rat microglia were seeded onto polylysine-coated coverslips in 24-well plates. After washing with PBS, the coverslips were fixed with 4% paraformaldehyde for 20 min. Then the coverslips were blocked with 1% BSA and incubated at 4 °C overnight with primary antibodies against CitH3 (AB_304752, #ab5103, Abcam, 1:1000), MPO (AB_2892996, #sc-390109, Santa Cruz, 1:100), Iba-1(AB_2820254, #17198, CST, 1:50) and IL-1β (AB_629741, #sc-52012, Santa Cruz, 1:100).

Rats were anesthetized deeply and transcardially perfused with ice-cold PBS followed by whole brains removed quickly, fixed in 4% paraformaldehyde overnight at 4 °C and dehydrated with 30% sucrose at 4 °C until sinking to the bottom. The brain samples were embedded in the optimal cutting temperature (O.C.T.) compound and stored at − 80 °C. The damaged cerebral hemispheres were then sliced into 8 µm-thick coronal sections using a freezing microtome (CM1950, Leica, Germany). The frozen sections were blocked for 1 h and incubated overnight at 4 °C with antibodies against CitH3 (AB_304752, #ab5103, Abcam, 1:1000), MPO (AB_2892996, #sc-390109, Santa Cruz, 1:100), Iba-1 (AB_667733, #sc-32725, Santa Cruz, 1:100), cGAS (AB_2770305, #A8335, ABclonal, 1:50), STING (AB_10665370, #19851-1-AP, Proteintech, 1:1000), and GFAP (AB_561049, #3670, CST, 1:400). Then the corresponding fluorescence-conjugated secondary antibodies were added and incubated for 2 h at room temperature. Nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI, #ZLI-9557, ZSGB-BIO, China) for 10 min. All primary antibodies were validated in published literature or by companies. Finally, images of cells and brain sections were captured with laser confocal microscopy (LSM 800, Zeiss, Germany). We selected three sections per rat from similar areas which were around the injured frontal and parietal cortices, and analyzed three fields per section at a magnification of × 20 or × 40. Images were analyzed with ImageJ software. The relative immunofluorescence intensity of CitH3 or MPO was calculated by the ratio of immunofluorescence intensity of CitH3 or MPO relative to the immunofluorescence intensity of CitH3 or MPO in sham group respectively. The percentage of Iba-1-positive cells was calculated using the formula: (number of Iba-1-positive cells /number of total cells)*100, total cells were counted by DAPI. The positive cell results in each group were averaged.

TUNEL Assay

A TUNEL kit (#C1088, Beyotime, China) was used to detect neuronal cell death according to the manufacturer’s protocol. Briefly, brain sections were incubated with NeuN (AB_2651140, #24307, CST, 1:50) at 4 °C overnight. Then, the sections were washed with PBS and incubated with TUNEL reaction mixture for 1 h at 37 °C, followed by staining with DAPI for 10 min. The TUNEL-positive neurons were observed by laser confocal microscopy. We selected three sections per rat. The average number of TUNEL-positive neurons was counted in four fields per section at a magnification of × 20, and quantified with the ImageJ software by the experimenter blinded to grouping. TUNEL-positive neurons were expressed as a percentage of the total neuron count.

Neurological Function Evaluation

We used the modified Garcia test to assess neurological function on days 1, 3, and 7 after SBI, including seven evaluations: spontaneous activity, axial sensation, vibrissae touch, limb symmetry, lateral turning, forefoot movement, and climbing. The maximum score was 21 points, and each test received a score ranging from 0 (worst performance) to 3 (best performance). Details of the modified Garcia test are provided in the Supplementary Information. The evaluation was performed by two evaluators in a blinded manner.

Primary Rat Microglia Culture and Coculture with Neutrophils

The primary rat microglia culture has been described in the previous literature (Bahrami et al. 2018). Briefly, 1–3-day-old neonatal SD rats were sterilized in 75% alcohol, and then cerebral cortices were dissociated and stripped of their blood vessels and meninges carefully under a dissecting microscope. After digestion of the clipped cerebral cortices using 0.25% trypsin-EDTA (#25200056, Gibco, USA), an equal volume of DMEM (#11965092, Gibco, USA) was added to terminate the digestion.

The single cell suspension was obtained by centrifugation and resuspension after filtration through a 75-µm mesh filter, and cultured in a 75 cm2culture flask at 37 °C in a 5% CO2 atmosphere. The culture medium was changed on days 3 and 7. After 12–14 days, primary rat microglia were obtained by shaking the mixed glial cultures at 200 rpm for 1 h. Then isolated microglia were treated in DMEM with 10% fetal bovine serum (#16140071, Gibco, USA), 1% penicillin/streptomycin (#15140122, Gibco, USA) and cultured in 5% CO2 at 37 °C.

Then, neutrophils isolated from rats in different groups were treated with DMEM-conditioned media (DCM) for 1 h as previously described (Zeng et al. 2022). Next, DCM-treated neutrophils (5 × 105/well) were cocultured with primary rat microglia for 8 h using a Transwell coculture device (pore size 3 μm). Primary rat microglia were placed in the lower chambers embedded with cover slips, and neutrophils were localized in the upper chambers. Then microglia were observed under a confocal laser-scanning microscope, and the levels of inflammatory factors produced by activated microglia in the cell supernatant were measured by ELISA.

Intracellular Reactive Oxygen Species (ROS) Measurement

Neutrophils were isolated from the peripheral blood of rats in different groups, including sham-operated, vehicle, and low-, medium-, and high-dose vitamin C-administered groups. Next, neutrophils (3 × 104 cells/mL) were seeded into 96-well plates and loaded with 10 µM DCFH-DA (#S0033S, Beyotime, Shanghai, China) fluorescent probe at 37 °C for 30 min. After washing with PBS, the fluorescence intensity was measured with a multimode microplate reader (SpectraMax 190, Molecular Devices, USA).

Live-cell Imaging

Purified neutrophils (1 × 105 cells/mL) isolated from healthy rats were plated on a confocal dish and incubated with different concentrations of vitamin C (#A4034, Sigma–Aldrich, USA) for 30 min at 37 °C in a 5% CO2 atmosphere. Then the cells were stimulated with 100 nM PMA (#HY-18,739, MedChemExpress, USA) for 3 h at 37 °C and 5% CO2 atmosphere and imaged under a laser confocal microscopy (LSM 800, Zeiss, Germany). The cell-permeable fluorescent DNA dye Hoechst 33342 (#C0030, Solarbio, China) was mixed with cell-impermeable SYTOX Green (#S7020, Invitrogen, USA) to detect NETs.

Statistical Analysis

All statistical analyses were performed with GraphPad Prism 9.0 (San Diego, CA, USA). All data are presented as the mean ± standard deviation (mean ± SD) or median (interquartile range). The normality of the data was assessed by Shapiro–Wilk test, and variance homogeneity was assessed by the F test and Brown–Forsythe test. The Mann–Whitney’s test and Kruskal–Wallis test were used to analyze differences for two groups and multiple groups respectively. Associations between variables were analyzed using Spearman correlation. A P-value < 0.05 was indicated as statistically significant.