Thirty-six Sprague–Dawley male rats (weighing 320–340 g) and 12 E16–18 pregnant Sprague–Dawley rats were obtained from the Vital River Laboratory Animal Technology Co., Ltd., Beijing, China. The animals were housed at 20–25°C and 50 ± 5% humidity with a 12:12-h light/dark cycle and were given ad libitum access to food and water. All animal experiments complied with ethical requirements and were approved by the ethics committee of the Beijing Tiantan Hospital of Capital Medical University.

Transient middle cerebral artery occlusion (MCAO) was used as the rat model of ischemic stroke, and the procedure was based on a modification of previously described methods [(21), p. 84–91]. Briefly, rats were anesthetized with 5% isoflurane delivered in air for 2 min. Anesthesia was maintained with 2–3% isoflurane delivered in air at 0.5 L/min during surgery. Body temperature was continuously monitored with a rectal probe and maintained at 36.5–37.0°C using a temperature-controlled blanket. Then, 0.1 mg/kg buprenorphine was given pre-emptive to reduce the animal's pain, suffering, and distress. Humane endpoints were met if the animal's excessive blood loss or drop in body temperature or more than 20% of its body weight lost within 8 h post-operation. The right common carotid artery (CCA), external carotid artery (ECA), internal carotid artery (ICA), and vagus nerve were exposed and isolated carefully under a dissecting microscope. Flows through the CCA and ICA were temporarily stopped using a slipknot, and the distal end of the ECA was electrocauterized and resected. Then, a small hole was cut on the proximal end of the ECA, and a 3-0 silicon rubber-coated monofilament was inserted via the ECA into the lumen of the ICA. After loosening of the slipknot on the ICA, the filament was advanced 18–20 mm beyond the carotid bifurcation to the middle cerebral artery (MCA). After 2 h of occlusion, the filament was withdrawn, the hole in the ECA was sealed by electrocauterization, and the slipknot on the CCA was loosened to allow reperfusion.

Sham animals were anesthetized with isoflurane, and the CCA, ECA, lCA, and vagus nerve were exposed and isolated carefully, and then the incision was closed. Model animals were randomly divided into two groups and kept in different cages with clear labels: Model and Model + DCS. For rats in the Model + DCS group, one dose of 0.125 mg/gbw DCS (Avanc Pharmaceutical Co., Ltd., Jinzhou, China) was injected intraperitoneally immediately after reperfusion, and the brains were collected after 24 h [(22), p. 105–110, (23), p. 256–266, (24), p. 1638-018-20095–9]. There were 12 animals per group, and the animals were monitored every 8 h with food and water accessible. Animals that do not show clinical signs of focal cerebral ischemia (i.e., hemiparesis or circling), indicating insufficient vessel occlusion, were excluded. Rats in the Model group only received an injection of the same amount of saline (vehicle).

Neurological examinations were performed at 24 h after reperfusion by two observers who were blinded to the animal grouping. The assessment of neurological dysfunction (neurological deficit score) was made using the modified Bederson's method [(25), p. 472–476].

Each rat was sacrificed after the neurological examination had been completed, and the brains of 18 animals (6 per group) were removed and cut into 2-mm thick coronal sections (the remaining animals were used for Western blot testing). The brain sections were incubated in a solution of 1% triphenyltetrazolium chloride (TTC) in normal saline for 20 min at 37°C. The slices were photographed using a Leica microscope (Leica, USA), and the infarct size was determined.

Primary neurons were cultured from E18 Sprague–Dawley rat fetuses as previously described [(26), p. 28–40]. The bilateral cortices were dissected from 10 E18 rat embryo brains, the meninges were carefully removed, and the tissue was placed in a conical tube containing a mixture of Dulbecco's modified Eagle medium and Ham's F-12 medium (DMEM/F12 medium) on ice. The tissue was allowed to settle at the bottom of the tube, and the supernatant was then carefully removed to leave only the tissue covered by the medium. The tissue was enzymatically digested with 0.25% trypsin containing 0.5 mg/ml DNase for 20 min at 37°C, with gentle shaking of the tube every 5 min. Then, complete DMEM/F12 medium containing 10% fetal bovine serum (FBS) was added to the tube to terminate the digestion, and the mixture was passed through a cell strainer to obtain single cells and then centrifuged for 5 min at 1,000 rpm. The supernatant was removed, and the cells were re-suspended in complete DMEM/F12 medium containing 10% FBS, 1% penicillin/streptomycin, and 0.5 mmol/L l-glutamine by gentle pipetting. The cells were counted using trypan blue and a cell counter and then seeded into 24-well culture plates pre-coated with poly-l-lysine (0.1 g/L). Once the cells had attached ~4 h later, the plating medium was replaced with cell culture medium consisting of neurobasal medium, B27 (2%), bovine serum albumin (1%), and penicillin/streptomycin (1%). The cultures were maintained in an incubator at 37°C with 5% CO₂.

Cell purity was assessed by immunostaining for neurofilament (NF), which is expressed in neurons, and glial fibrillary acidic protein (GFAP), which is expressed in astrocytes. After the primary neurons were cultured for 7 days, the cells were washed with cold phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 30 min at room temperature. Then, the cells were rinsed three times with PBS, blocked with 5% donkey serum for 1 h at room temperature, and then incubated with (1) mouse polyclonal anti-NF (1:200; Beijing Guan Xing Yu Science and Technology Co., Ltd., Beijing, China) and (2) rabbit polyclonal anti-GFAP (1:500; Beijing Guan Xing Yu Science and Technology Co., Ltd.) at 4°C overnight. After removal of the primary antibodies and rinsing, the cells were incubated with Alexa-Fluor 488-conjugated goat anti-mouse antibody (1:500; Invitrogen, Thermo Fisher Scientific, Inc.) and Alexa-Fluor 568-conjugated goat anti-rabbit antibody (1:500; Invitrogen, Thermo Fisher Scientific, Inc.) for 1 h at room temperature. The nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole, 1:500, D1306; Invitrogen, CA, USA). An LSM-780 laser-scanning confocal microscope (Carl Zeiss Micro Imaging GmbH, Jena, Germany) was used to capture the images.

Confluent cultures grown for 7 days on 24-well culture plates were subjected to hypoxia (1% O₂, 94% N₂, and 5% CO₂) in glucose-free medium for 2 h. The cultures were then re-oxygenated in neurobasal medium with 2% B-27 Serum-Free Supplement under normal conditions (5% CO₂) (26).

Cultured neuron cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R) were treated with a range of DCS concentrations (0, 0.2, 0.4, 0.6, 0.8, or 1.0 μg/μl) for 4 h; cells were reoxygenated; and the blank control group (not exposed to OGD/R) was also included. Cell viability was detected used a cell counting kit (Cell Counting Kit-8; Dojindo, Kumamoto, Japan) in accordance with the manufacturer's instructions. Cell Counting Kit-8 solution was added to the medium at a dilution of 1:10, and the plate was incubated for 4 h. The absorbance at 450 nm was measured using SpectraMax M5 (Molecular Devices, Sunnyvale, CA, USA).

Apoptosis was detected by using phycoerythrin (PE)–Annexin V Apoptosis Detection Kit I (BD559763; BD Biosciences, San Jose, CA, USA). Briefly, primary neuronal cells were washed twice with cold PBS, trypsinized, and re-suspended in 1× binding buffer at a concentration of 1 × 10⁶ cells/ml. A 100-μl volume of cell suspension (1 × 10⁵ cells) was transferred to a 5-ml FACS tube, and then 5 μl each of PE–Annexin V and 7-amino-actinomycin D (7-AAD) was added. The cells were gently vortexed and incubated for 15 min at 25°C in the dark. Then, 400 μl of 1× binding buffer was added to each tube, and the fluorescence was measured within 1 h at 520 nm (SpectraMax M5). For plotting of the results, PE–Annexin V was set as the horizontal axis and 7-AAD as the vertical axis: the upper-left quadrant indicated mechanically damaged cells, the upper-right quadrant apoptotic or necrotic cells, the lower-left quadrant normal cells, and the lower-right quadrant early apoptotic cells. The level of apoptosis was normalized to that observed in cells not exposed to OGD/R.

In order to detect apoptosis, TUNEL staining was performed using the in situ Cell Death Detection kit for Fluorescein (Roche, China). After reoxygenation for 24 h, cortical neurons were stained with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) according to the manufacturer's recommendations, and DAPI was used as counterstain. Negative controls were treated in the same way but were incubated without the TdT enzyme, while positive controls were treated with DNase I. In microscopic observation, TUNEL-positive showed green, and DAPI-positive showed blue. Six view fields were chosen randomly to calculate the percentage of apoptotic cells. The counts of positive stained cells from five observed fields were then averaged.

Western blotting was performed as previously described with modifications [(27), p. e1025]. Cells were lysed in whole-cell lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EGTA, 10 mM sodium pyrophosphate, 1.5 mM MgCl₂, 100 mM NaF, 10% glycerol, and 1% Triton X-100, pH 7.2) containing an inhibitor cocktail (1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 1 mM sodium orthovanadate) to extract total protein. Protein concentrations were determined using a standard bicinchoninic acid assay (23225; Thermo Fisher Scientific Inc., Waltham, MA, USA), and 50 μg of total protein was subjected to 10% SDS-PAGE followed by electrotransfer onto nitrocellulose membranes. The membranes were blocked in 5% skimmed milk and then incubated overnight at 4°C with primary antibodies against rat BCL-2 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), rat Bax (1:1,000; Cell Signaling Technology), or rat β-actin (1:20,000, TA-09; Zhongshan Golden Bridge Co., Ltd, Beijing, China). The membranes were then washed three times (10 min each) with Tris-buffered saline/Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibodies in 5% skimmed milk for 1 h at room temperature. After washing three times (10 min each) with TBST, immunoreactive signals were detected using enhanced chemiluminescence (Pierce, Rockford, IL, USA). Band intensity was quantified using ImageJ software (NIH). Relative protein expression was normalized to β-actin.

For the detection of intracellular ROS level, we used ROS-sensitive probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, D-399; Invitrogen). H2DCFDA was dissolved in DMSO to obtain 10 mM stock solution and further diluted before use. Cells were incubated with 5 μM staining solution in PBS in the dark for 30 min at 37°C, then harvested with 0.05% trypsin–EDTA solution, suspended in fresh medium, and immediately analyzed with flow cytometer (Epics XL; Beckman Coulter, USA; 488 nm laser).

Statistical analyses were performed by Prism software. Data were presented as mean ± standard error of the mean (SEM). Statistical significance was evaluated by one-way analysis of variance (ANOVA) or Student's t-test. A two-tailed p-value of < 0.05 was considered statistically significant (^*p < 0.05, ^**p < 0.01, ^***p < 0.0005, ^****p < 0.0001).

In animals exposed to transient MCAO, infarcted tissue was found in the hemisphere ipsilateral to the occlusion (right side), and the infarcted region was consistent with the area supplied by the MCA (Figure 1A). Infarct volume percentage of contralateral brain volume in rats administered 0.125 mg/gbw DCS was 16.43 ± 4.64%, infarct volume percentage of rats treated with vehicle was 36.25 ± 4.63% compared with the vehicle group, and the infarct volume size was significantly reduced by DCS (^***p < 0.005; Figure 1B).

In rats that had received transient MCAO, neurological deficit scores of animals treated with 0.125 mg/gbw DCS and vehicle were 6.67 ± 2.4 and 12.17 ± 1.9, respectively (^***p < 0.0005; Figure 1C). The animal behavior test was rescued by DCS very much significantly.

According to our isolation protocol, after 7 days culture in conditional medium, the purity of the primary neurons was tested by immunofluorescence. Immunostaining for NF and GFAP revealed that the purity of the primary neuronal cells cultured from E18 embryo rats exceeded 95% (Figure 2A). The viability of cultured primary neurons unexposed to OGD/R was set as 100%, and the viability of primary neurons exposed to OGD/R was 64.4% to cells that were not exposed to OGD/R (^***p < 0.0005; Figure 2B), which indicated that the OGD was successful. In cultured neurons subjected to OGD/R, cell viability in neurons treated with DCS at concentration of 0.2, 0.4, 0.6, 0.8, or 1.0 μg/μl was 76.7, 85.3, 75.9, 73.5, and 75.6%, respectively. Although all groups of DCS treatment showed improved cell viability compared with the OGD/R group, 0.4 μg/μl DCS had no significant changes compared with untreated cells (p > 0.05), but had significantly higher cell viability than cells not administered DCS (^*p < 0.05; Figure 2B). Based on these results, subsequent experiments utilized a DCS concentration of 0.4 μg/μl.

Primary neurons exposed to OGD/R exhibited a significantly higher level of apoptosis than cells not exposed to OGD/R, which was validated by 7AAD–PE–Annexin V staining and TUNEL staining. 7AAD–PE–Annexin V staining results showed that the apoptosis rate of OGD/R was 4.33 ± 0.34 times on untreated cells; however, the 0.4 μg/μl DCS group was 2.53 ± 0.34 times on untreated cells (Figure 3A). TUNEL staining results showed that the apoptosis rate of OGD/R was 8.13 ± 0.96 times on untreated cells, and the 0.4 μg/μl DCS group was 4.37 ± 0.96 times on untreated cells (Figure 3B).

Overall, 7AAD–Annexin V staining and TUNEL staining showed that the apoptosis level in cells exposed to OGD/R was rescued by the administration of 0.4 μg/μl DCS (^*p < 0.05; ^**p < 0.01; ^***p < 0.0005; Figure 3).

Relative level of intracellular ROS release caused by OGD/R increased from 1 to 1.34 ± 0.08 times higher in cultured neurons. Notably, treatment with 0.4 μg/μl DCS reduced the ROS release relative level to 1.04 ± 0.08 in cells subjected to OGD/R (^*p < 0.05; Figure 4). DCS may protect the primary neuron via inhibiting the ROS release.

The Bcl2 family proteins are key regulators of apoptosis cell death. Bcl2-like subgroup, such as Bcl2, Bcl-xL, Mcl-1, Bcl-w, and A-1, suppresses cell death, and pro-apoptotic Bax-like subgroup, such as Bax, Bak, Bok, and Bik, promotes cell death. Bcl-2 and Bax were employed to check the role of DCS in cell apoptosis.

Primary neurons subjected to OGD/R showed 2.21 ± 0.13 times higher level of Bax protein expression and 0.62 ± 0.13 lower level of Bcl-2 protein expression than cells not exposed to OGD/R. In cells subjected to OGD/R, treatment with 0.4 μg/μl DCS downregulated the expression of Bax protein from 2.21- to 0.71-fold compared with the untreated cells. Expression of Bcl-2 protein was upregulated by DCS from 0.62- to 1.04-fold compared with untreated cell at the mean time (^*p < 0.05; ^****p < 0.0001; Figure 5).