All procedures were approved by the Animal Ethics Review Committee of Nanchang University and were in accordance with the institution’s Guidelines for Animal Experiments.
Animals and experimental groups
Adult male SD rats (weighing 300–350 g) were provided by the Laboratory Animal Center of Medical College of Nanchang University and housed in an air-filtered unit with free access to food, water is under a 12-h light/dark cycle. The temperature in the feeding room and the operation room was between 22 °C and 25 °C. The animals were randomly assigned to two groups: (1) sham group (n = 12), (2) SAH group (n = 12), fresh autologous caudal artery blood (0.3 ml) was injected into the cisterna magna of SD rats.
Induction of experimental SAH
Rats were anesthetized with an intraperitoneal injection of 10 % chloral hydrate (300 mg/kg). In the prone position of the rat, the atlanto-occipital membrane was exposed. After the animal was turned into a supine position, fresh autologous non-heparinized blood (0.3 ml) withdrawned from caudal artery. The fixed animal which in a stereotactic frame and a 25-gauge needle was inserted through the atlanto-occipital membrane into the cisterna magna. Then, the 0.1 ml of cerebrospinal fluid (CSF) was slowly withdrew and the autologous blood was injected into the cisterna magna at a speed of 0.15 ml/min. After the injection, the animal was placed at an angle of 30° in head-down position for about 30 min in order to facilitate the blood settle around the BA. The same procedure was repeated 24 h later with a 0.3 ml fresh autologous blood injection. Shan-operated rats underwent the same procedure without the blood injection.
India ink angiography
Gelatin-India ink solution was made by dissolving gelatin powder (7 g) in 100 ml PBS mixed with 100 ml India ink. The ascending aorta was cannulated with a blunted 20-gauge needle attached to flexible plastic tubing, which was connected to a syringe on a micro pump. After an incision made in the right atrium which allowed the outflow of perfusion solutions, 100 ml of PBS, 15 min of 10 % formalin, and 10 min of 3.5 % gelatin-India ink solution were infused through the closed circuit. All perfusates were passed through a 0.2-μm pore size filter. The rat was refrigerated at 4 °C for 24 h to allow gelatin solidification. The brains were harvested and high-resolution pictures of the circle of Willis and basilar arteries (BAs) were taken with a scale before and after the removal of a subarachnoid clot. The brain was stored in 10 % neutral buffered formalin. An experienced person who was unaware of the treatment group measured the smallest lumen diameter of BA, used Image J software for three times and determined a mean value per segment.
H&E staining
After five days, the second hemorrhage rat was perfused with PBS. The brainstem containing BA was immediately removed and post-fixed in the 4 % paraformaldehyde (PFA) 100 ml for 24 h. The entire length of BA was divided into proximal, middle, and distal. After it dissected, the middle section was deparaffinized, hydrated, washed, and stained with Hematoxy-lin-eosin (H & E) staining.
Immunohistochemical staining
Transections of rats organs were processed into paraffin blocks, and were cut into 10-μm slices. After deparaffinized, the slices were heated and boiled for 15 min in citrate buffer solution (0.01 M, PH = 6.0) for retrieval antigen. Each section was treated with 3 % hydrogen peroxide for 20 min at room temperature and was used to diminish nonspecific staining. After rinsing with PBS, the slices were blocked with 5 % normal goat serum in PBS (0.01 M, PH = 7.4) for 20 min at room temperature. The slices were incubated overnight at 4 °C with the Rabbit Anti-osteopontin antibody (bs-0019R, Bioss Biotechnology corporation, Beijing, China), Rabbit Anti-Tenascin C antibody (bs-1039R, Bioss Biotechnology corporation, Beijing, China), and Rabbit Anti-E-Selectin antibody (bs-1273R, Bioss Biotechnology corporation, Beijing, China). After rinsing with PBS, the specimens were incubated with biotinylated secondary antibody (ZSGB-BIO, Beijing, China) at room temperature for 20 min and then re-incubated with horseradish peroxidase-labeled streptavidin for 20 min. The immunoreactivity was revealed by 3′diaminobenzidine (DAB) solution and counterstained with hematoxylin. The primary antibody was omitted for the negative control.
Western blotting
Rats were killed under the deep anesthesia after five days in the second hemorrhage. The BA, brain tissue, heart, liver, kidney, lung, spleen, pancreas, spinal cord, thoracic aorta, abdominal aorta, pulmonary artery and mesenteric artery were isolated. The specimens were homogenized in ice-cold extract buffer (PH = 7.4) and centrifuged at 20,000 rpm for 20 min at 4 °C. The protein concentration was determined by using a BCA kit (Thermo Fisher Scientific, USA). Equal amounts of protein samples (10 μg) were loaded on a tris glycine gel, separated by 12 % SDS-PAGE, electrophoresed, and transferred to a polyvinylidencedifluoride (PVDF) membrane. Then, the membrane was blocked with 5 % skimmed milk for 2 h at room temperature, incubated overnight at 4 °C with the Rabbit Anti-osteopontin antibody (bs-0019R, 1:200, Bioss Biotechnology corporation, Beijing, China), Rabbit Anti-Tenascin C antibody (bs-1039R, 1:200, Bioss Biotechnology corporation, Beijing, China), and Rabbit Anti-E-selectin antibody (bs-1273R, 1:200, Bioss Biotechnology corporation, Beijing, China). GAPDH (1:1000, Cell Signaling Technology, USA) was blotted on the same membranes as a loading control. The membrane was then incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:4000, ZSGB-BIO, Beijing, China) for 2 h at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence method and quantified with Image J software (NIH). Results were expressed as a relative density to GAPDH.
Statistical analysis
The SPSS software package v 19.0 was used for all analyses. Data expressed as mean ± standard deviation (SD). The death rates were used in the chi-square test for contrast. Differences between these means were evaluated by the one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls (SNK) test for multiple comparisons. *P <0.05 were considered statistically significant.
