Ethics
All experiments were performed in accordance with Guidelines for the Care and Use of Laboratory Animals formulated by the National Institutes of Health. The Second Xiangya Hospital Animal Care Committee of Central South University approved this study. Rats were housed in standard cages with ambient temperature of 22 ± 1 °C, humidity of 40–50%, a 12-hour dark/light cycle, and free access to food and tap water.
Animals And Groups
Adult male Sprague-Dawley (SD) rats weighing 220 to 260 g were provided by the HUNAN SJA LABORATORY ANIMAL CO., LTD [license no. SCXK (Xiang) 2019-0004]. 96 SD rats were divided randomly into four groups: Sham group(24 rats), MCAO group (24 rats), RSG group(24 rats), and RSG + daidzein group (24 rats). RSG (Sigma-Aldrich) was dissolved in dimethyl sulfoxide (DMSO) and diluted to a final concentration of 2 mg/mL. In the RSG group, rats received RSG immediately after MCAO at a dose of 10 mg/kg, q12 h. In the RSG + daidzein group, intraperitoneal administration of Diadzein (Sigma-Aldrich) was given along with RSG immediately after the onset of stroke at a dose of 0.6 mg/kg.
Animal Model Of Mcao
Animal model of MCAO was established in SD rats as Longa et al. described[14]. Briefly, after the rats were injected with 10% (w/v) chloral hydrate (300 mg/kg), a silicone-coated nylon monofilament with a heatblunted tip was passed through the bifurcation of the common carotid artery to the internal carotid artery until the tip reached the origin of the right middle cerebral artery. After 120 minutes of occlusion, the monofilament was withdrawn slowly to allow for 22 hours of reperfusion. The body temperature of rats should be maintained at 37 ± 0.5 °C until recovery. Rats in the sham operation group were subject to the same surgery except for the filament insertion.
Neurological Deficits Score
The neurological defects were assessed by two independent examiners who were blind to each experiment treatment before the killing[14]. 0: no symptoms of neurological deficits; 1: left forepaws cannot be fully extended ; 2: circling or rotating to the contralateral side; 3: falling to the contralateral side; 4: No spontaneous activity with depressed levels of consciousness. Those with a score of 1–3 were included in the experiment, and those who were not satisfied were excluded. Other excluded conditions included subarachnoid hemorrhage, and rats died within 24 hours.
Triphenyltetrazolium Chloride Staining
To assess the infarction areas, after the rats were fully anesthetized, they were sacrificed by rapid cervical dislocation. Then, the brain tissue were quickly harvested and placed in a refrigerator at -20 °C for 20 minutes. Then we removed the olfactory bulb and cerebellum, and cut the brain tissue into 5 continuous coronal sections with a thickness of 2 mm. The sections were incubated with 1% (w/v) 2,3,5-triphenyltetrazolium chloride (TTC; Sigma) in the dark at 37 °C for 30 minutes and were flipped every 10 minutes. Then the brain slices were fixed in 4% (w/v) paraformaldehyde solution at 4 °C overnight. Pictures were taken and infarct volume was calculated with Image J software (ImageJ, MD, USA).
Evans Blue Extravasation
Evans blue (EB; Sigma) was dissolved in saline and rats were injected with 2% EB (4 ml/kg ) via the tail vein. After 2 hours, rats were perfused with 0.9% (w/v)sodium chloride to flush away the intravascular dye from the blood vessels. Then the nonischemic and ischemic brain tissues were quickly harvested, weighed, and homogenized and proteins were precipitated with formamide solution at 60℃ for 24 hours. After centrifuging at 5000 g for 5 minutes, the absorbance at 620 nm was measured with a multifunctional microplate reader (Eppendorf, Germany) in the supernatant. The extravasation of EB was evaluated with a standard curve and expressed as microgram per gram of brain tissue.
Immunofluorescence Staining
At 24 hours after MCAO, the brains were removed after perfusion with 0.9% sodium chloride and 4% paraformaldehyde. Then the brain tissue were immersed in 4% paraformaldehyde solution for post fixation, dehydrated in gradient sucrose solutions of 15% and 30% at 4 °C, embedded in optimal cutting temperature compound and cut into 20 µm thick serial coronal sections. Sliced tissues were then blocked with buffer containing 1% goat serum, and 0.2% Triton in PBS and incubated with the following primary antibodies: rabbit polyclonal anti-MMP-9 (1: 500, Abcam), rabbit polyclonal anti-caveolin-1 (1: 500, Abcam), and rabbit monoclonal anti-occludin antibody (1:100, Abcam). After overnight incubation at 4 °C, sections were washed four times, secondary fluorescent antibodies were added and incubated for 1 hour in the dark at room temperature, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). The sections were observed using a fluorescence microscope (Olympus, Japan).
Western Blot
After 22 hours of reperfusion, rats in each group were intraperitoneally injected with 10% chloral hydrate. After deep anesthesia, the ischemic brain tissues were quickly removed and were homogenized with RIPA lysis buffer. The mixtures were incubated on ice for 30 minutes and then centrifuged for 15 min at 4 °C. After collecting the supernatant and denaturing it, equal amounts of protein samples were electrophoretically seperated in 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels and then transferred to polyvinylidene fluoride (PVDF) membranes. PVDF membranes were placed in an incubation box containing 5% BSA and blocked for 2 h at room temperature on a shaker. Then the membranes were incubated with primary antibodies against MMP-9 (1:5000, Abcam), caveolin-1 (1:5000, Abcam), and occludin (1:1000, Abcam) at 4 °C overnight. After washing the membranes with tris-buffered saline and 0.1% Tween-20 (TBS-T) for 7 min × 4 times, they were incubated with the corresponding secondary antibodies (1:3000, Santa Cruz) at room temperature for 2 hours. Then membranes were washed four times with TBS-T for 7 minutes each, and were developed with the enhanced chemiluminescence (ECL) (Servicebio, China). The membranes incubated with GADPH primary antibodies (1:4000, Sigma) were regared as the loading control. The bands were visualized using the Gel image analysis system (Bio-Rad, Italy) and quantified using the Image J software. The final results were standardized and expressed as the ratios of target proteins to GADPH.
Statistical analysis
Data were presented as mean ± SD. Statistical analysis was performed with GraphPad Prism software (version 8.3) and data were analyzed with one-way ANOVA or two-way ANOVA followed by Tukey's multiple comparisons test. P < 0.05 was considered statistically significant.