Identification of OPN, TNC and E-selectin as potential recognition proteins in cerebral vasospasm after subarachnoid hemorrhage
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-?m 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.
HE 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-?m 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 ?g)
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.