Animals and experiments

Forty male Sprague Dawley (SD) rats (6–8 weeks old) obtained from the Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd. (animal production license no: SCXK (Zhe) 2019-0001), weighing 180–220 g, were kept under constant temperature (20–24 °C), humidity (55%), 12 h light and dark cycle, and wind change times 15–20 times/h. The animal research was approved by the Ethics Committee of the Animal Center of Zhejiang Eyong Pharmaceutical Research and Development Center (animal use license number: SYXK (Zhe) 2021–0033).

Establishment of middle cerebral artery occlusion (MCAO) combined CRF model

Forty rats were divided into five groups of eight rats each: control, sham, MCAO + CRF, cilostazol, and aspirin. In the treatment group, cilostazol (30 mg/kg; 73963-72-1, Sigma, China) or aspirin (10 mg/kg; 50-78-2, Sigma, China) was administered daily by gavage for 7 days. In the sham group, the perirenal fat was removed from the kidney, and no nephrectomy was performed. Based on previous studies [12, 13], CRF rats underwent a complete nephrectomy on the right kidney and a two-third nephrectomy on the left kidney, simultaneously. Antibiotics were used to prevent incision infection in each group during the procedure. After the CRF rats were anesthetized, as described in a previous study [14], a nylon thread was prepared, and the external and common carotid arteries were ligated using a silk thread. The prepared nylon cord was then inserted along the common carotid artery into the left internal carotid artery, the vascular clamp was released, the threaded plug was advanced to obstruct the ipsilateral middle cerebral artery, and the threaded plug was fixed above the incision of the left common carotid artery to complete the modeling of MCAO in rats. Neither ligation nor obstruction was performed in the sham group.

Body weight, urine volume, and 24-h urinary protein level

After 7 days of administration, body weight, urine protein content, and total urine volume in 24 h were measured.

Modified neurological severity score (mNSS)

The mNSS is frequently used for neurobehavioral evaluation after MCAO. The score range is 0–18; the higher the score, the more severe the neurological impairment is. Each group of rats was trained before testing.

Sample collection

On the seventh day, the drug was administered for 30 min, and the mNSS score was evaluated. Blood was collected from the submaxillary vein, and brain and kidney tissues were collected and quickly placed on an ice plate to determine the brain water content. The remaining samples were used for molecular and biochemistry experiments, and some were stained in sections.

Determination of brain water content

The brain tissue was weighed, and after diluting the excess water and blood stains on the surface using disposable sterile gauze, the brain tissue was weighed using a balance, and the weight was noted as wet mass. Subsequently, the brain tissue was incubated in the oven at 60 °C for 48–96 h, and the brain tissue was repeatedly weighed until the mass no longer changed (termed as the dry mass). Brain water content = (wet mass − dry mass)/wet mass × 100%.

Triphenyltetrazolium chloride (TTC) staining

Brain tissues were coronally sectioned with a thickness of 2 mm. They were stained in 1% TTC at 37 °C for 30 min and fixed in 10% formaldehyde solution for 6 h. The infarct volume was calculated using the Medical Image Processing System software.

Nissl staining

Nissl staining was performed according to the manufacturer’s instructions. After deparaffinizing and rehydrating coronal slices, the slides were stained for 5 min at 37 °C in Nissl Staining Solution (C0117, Beyotime, Jiangsu, China). The ImageJ software was used to count the cells.

Hematoxylin–eosin (H&E) staining

The brain tissues were fixed, dehydrated, and immersed in wax. The tissues were cut into 5 μm slices and affixed to the anti-peeling slides. The slices were treated at 60 °C for 1–2 h, dewaxed, hydrated with xylene and gradient ethanol, and stained with H&E staining (G1005, Servicebio, Wuhan, China). Finally, ethanol in increasing concentrations was added for dehydration. After vitrification with xylene, the slices were sealed with neutral balsam and observed under a microscope.

Quantitative real-time polymerase chain reaction PCR (qRT-PCR)

Pure brain tissue RNA was extracted using TRIzol (B511311; Sangon Biotech, Shanghai, China) and transcribed into cDNA using a reverse transcription kit (CW2569; Jiangsu Cowin Biotech). The primers, diethypyrocarbonate (DEPC), cDNA, and SYBR Green (RR820A; Takara, Beijing, China) were used to prepare the corresponding system for amplification products in the PCR instrument. The primer sequences are listed in Table 1. The fold changes of mRNA were calculated using the 2−ΔΔCT method.

TUNEL assay

The TUNEL assay was performed following the manufacturer’s instructions. Deparaffinized tissue slices was treated with Proteinase K (G1205, Servicebio, Wuhan, China) for 15 min in a humid environment, followed by incubation sections in 3% hydrogen peroxide for 10 min and terminal deoxynucleotidyl transferase (G1501, Servicebio, Wuhan, China) labeling buffer at 37 °C for 1 h. TUNEL-positive cells were stained red; nuclei were stained with diamidinylphenyl indole (DAPI) to observe the TUNEL-positive cells.

Evans blue (EB) staining

One hour before anesthetization with isoflurane, EB physiological saline solution (E8010, Solarbio, Beijing, China) was injected into the femoral vein of the mice to ascertain the EB content. The conjunctiva of the eyes and limbs turned blue after the injection, and after 1 h of circulation, heart perfusion was performed. The brains of the mice were promptly sectioned, collected, and placed under an inverted fluorescence microscope with blue excitation light to observe EB leakage. The amount of EB present in the brain tissue was determined using a fluorescence spectrophotometer.

Cerebral blood flow (CBF) evaluation

At 7 days post-treatment, the dynamic blood flow value of the wound was measured using a laser Doppler blood flow meter (SKK-1100, Shenzhen Reward Life Technology Co., LTD, China). Before testing, the skin was excised to expose the skull, and a MSP200XP surface probe was placed on the wound surface of the brain. Three points were chosen on each wound surface; each point was recorded for 30 s, and the PowerLab Chart5 v5.2.2 image analysis software was used for analysis. Color-coded images represented different perfusion levels. The average value of the measurements from these three points was considered as the blood flow value of the intraoral wound.

Enzyme-linked immunosorbent assay (ELISA) assay

The serum levels of endothelin-1 (ET-1) (MM-0560R1; MEIMIAN, Jiangsu, China), nitrous oxide (NO) (MM-20607R1; MEIMIAN, Jiangsu, China), interleukin-1β (IL-1β) (MM-0047R1; MEIMIAN, Jiangsu, China), IL-6 (MM-0190R1; MEIMIAN, Jiangsu, China), tumor necrosis factor-α (TNF-α) (MM-0180R1; MEIMIAN, Jiangsu, China), endothelial nitric oxide synthase (eNOS) (RX300651R; RUIXING, Fujian, China), malonaldehyde (MDA) (S0131S; Biyuntian Biotechnology Co., LTD. Jiangsu, China), glutathione (GSH) (S0053; Biyuntian Biotechnology Co., LTD. Jiangsu, China), and superoxide dismutase (SOD) (S0101S; Shanghai Biyuntian Biotechnology Co., LTD. China) were tested using the ELISA kits following the manufacturer’s instructions. The serum creatinine (Scr) and blood urea nitrogen (BUN) levels of cilostazol in MCAO rats were determined using an automatic biochemical analyzer.

Immunofluorescence assay

Brain tissues were fixed and then stabilized in 0.5% Triton X-100. After blocking with blocking buffer, the tissues were incubated with occludin (DF7504, Affinity, Jiangsu, China), LC3 (AF5402, Affinity, Jiangsu, China), and zonula occludens-1 (ZO-1) (21773-1-AP, proteintech, Shanghai, China) overnight at 4 °C. The tissues were then incubated with an anti-rabbit antibody and counterstained with DAPI. The cells were observed under a microscope.

Immunohistochemistry (IHC) assay

The brain tissue sections were dewaxed with xylene, followed by addition of ethanol in decreasing concentrations for tissue rehydration, and the addition of antigen repair solution. Subsequently, the sections were washed with hydrogen peroxide to block endogenous peroxidase, sealed with bovine serum, and incubated overnight at 4 °C with transforming growth CD31 (AF6191, affinity, Jiangsu, China), vascular endothelial growth factor (VEGF) (ab72807, abcam, Shanghai, China), and Caspase 3 (Ab184787, abcam, Shanghai, China). On the next day, the sections were incubated in HRP secondary antibodies, and DAB (G1212, Servicebio, Wuhan, China) was added. The positive expression of DAB was brown-yellow, and the nuclei were stained with hematoxylin. Finally, ethanol in increasing concentrations was added for dehydration, and vitrification was performed with xylene. The slices were sealed with neutral balsam and observed under a microscope.

Western blotting

Pure protein was extracted from the brain tissue, and the protein concentration was measured using the bicinchoninic acid (BCA) method. After adding the loading buffer, the protein was denatured via boiling. The total protein was separated by electrophoresis, and the corresponding proteins were transferred to a polyvinylidene fluoride membrane (PVDF) membrane. The non-specific antigen was blocked with 5% milk, and the proteins on the membrane were incubated with the target antibodies listed in Table 2. After incubation at 4 °C overnight, the proteins were incubated with secondary antibodies. The images were captured using an ECL chemiluminescence imager.

Table 2 Antibody information