Animals
The animal use and care protocols, including all operation procedures, were approved by the Animal Care and Use Committee of Taishan Medical University and conformed to the guide for the care and Use of Laboratory Animals by the National Institute of Health.
Rat SAH Model
The SAH model was established as described previously. The animals were anesthetized with intraperitoneal (IP) injection of 10% chloral hydrate (400 mg/kg). Then, the animals were placed in a stereotactic frame with the head tilted down at ~30. With the help of an operating microscope, a small suboccipital incision was made to expose the atlas arch, occipital bone, and atlas occipital membrane. Then, the membrane was punctured with a 27-gage needle, and 0.3 ml autologous femoral arterial blood was injected into the cisterna magna within 10 min with an infusion pump. Animals in the sham group were injected with 0.3 ml sterile saline. After blood injection, instantly sealed the hole with absorbable sponge to avoid the fistula and closed the wound. To preserve the blood in the basal pool, rats were placed in a head-down prone position at a 30-degree angle for 30 minutes.The animals with SAH were separated into 6 subgroups randomly and sacrificed by decapitation prior to anesthesia with 10% chloral hydrate (400 mg/kg; IP) at 12 h, day 1, day 3, day 5 and day 7 post-SAH (n=6). Sham animals (n=6) experienced the same surgery but without injection of blood into the cisterna magna. Consequently, the sham animals were sacrificed 24 h following the sham surgery. No signs of peritonitis such as decreased food intake and abdominal swelling were observed following the administration of 10% chloral hydrate. Death was verified using the following criteria: Confirmation of the absence of visible breathing and measurable heartbeat, confirmation of pupil dilation and absence of the tail pinch reflex.
Western blot analysis
Dissolve the frozenbrain tissue in a solvent which is 20 mM Tris, pH 7.6 and contains 0.2 % sodium dodecyl sulfate, 1 % Triton X-100, 1 % deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.11 IU/ml aprotinin (all bought from Sigma–Aldrich, Inc., St. Luis, MO, USA). Centrifugate the lysate at 4 ℃at 12,0009g for 20 minutes . The samples (60ug per lane) were electrophoresis separated with 8 % sodium dodecyl sulfate polyacrylamide gel and electrically transferred to PVDF membrane (Bio-Rad Laboratory, Hercules, California, USA).
At room temperature, the membrane was closed with 5 % skimmed milk for 2 h , hatched for one night at 4 ℃ using primary antibodies directed against MCM7 (from Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:200 dilution ,Ki67 (from Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:100 dilution ,re-caspase-3 (from Santa Cruz Biotechnology, Santa Cruz, CA, USA) at1:200 dilution . The GAPDH (at the dilution of 1:6000, Sigma–Aldrich, Inc., St. Luis, MO, USA) served as loading control. Firstly the membrane was washed six times in PBS-Tween 20 (PBST)for 10 min each ,and then it was hatched in the suitable horseradish peroxidase-bound secondary antibody( at the dilution of 1:400 in PBST) for 2 h. The imprinted protein bands were developed by enhanced chemiluminescence (ECL) western blot detection reagents (Amersham, Arlington Heights, IL, USA) and then were exposed to X-ray film. Developed films were digitized employing an Epson Perfection 2480 scanner (Seiko Corp, Nagano, Japan). Optical densities were gained by means of Glyko Bandscan software (Glyko, Novato, CA, USA).
RNA isolation and quantitative real-time PCR
Rat Brain tissues were isolated using TRIzol Reagent(TAKARA Biotechnology) according to the manufacturer's specification. The concentration of RNA was confirmed by spectro photometric analysis (OD260/280).The quantity of RNA was gauged by OD260.The isolated RNA was in store at −80 ℃ before being analyzed. RNA was reverse-transcribed to cDNA employing Reverse Transcriptase Reagent(TAKARA Biotechnology) and oligo-dt primers. Quantitative real-time PCR analysis was performed employing the Agilent Technologies Stratagene Mx3000P real-time PCR system(Genetimes Technology, Inc.)and real-time SYBR Green PCR technology. The volume of the reaction mixtures was 25 μl,containing1 μl cDNA, 12.5 μl SYBR Green (TAKARA Biotechnology),1 μl forward primer,1μl reverse primer (10 μM) and nuclease-free water . The primers were synthesized by Life Technologies (Invitrogen, Shanghai,China)and the sequences used were from a database at NCBI for rat MCM7 and GAPDH. MCM7 forward and reverse primers were 5′-GTAGAGGGAGAACCGAGGGT -3′ and 5′-CGTACTTCCCGGATCACTCG-3′; GAPDH forward and reverse primers were 5′-AAGAAGGTGGTGAAGCAGGC-3′ and 5′-TCCACCACCCTGTTGCTGTATAGTG-3′. After 95 ℃ for 30s, 40 PCR cycles were performed, each consisted of a denaturation step(95 ℃, 5s)and an annealing step(60 ℃, 30s).Total RNA concentrations from each sample were normalized by quantity of GAPDH messenger RNA(mRNA),and the expression levels of target genes were evaluated by using the 2−ΔΔCq method. All samples were analyzed in triplicate.
Immunofluorescence staining
Immunofluorescence staining was performed according to our previous study in our laboratory. Brain tissue was fixed with 4% paraformaldehyde overnight and dipped in 20% saccharose PBS for 2 days and then in 30% saccharose PBS for another 2 days to dehydrate the tissue. The slices were cut into 6 micron thickness and sealed with PBS solution containing 5% FBS and 0.1% Triton X-100 at normal room temperature for 2 hours. After rinsing, they were treated with anti-glial fibrillary acidic protein (GFAP)(astrocyte labeled, 1:100; Sigma), mouse monoclonal primary antibodies against neuronal nucleus (NeuN) (neuronal marker, 1:500; Santa Cruz), anti Ki67(proliferation indicators,1:50; Santa Cruz), anti-re-caspase-3 (apoptosis indicators,1:50; Santa Cruz) were hatched overnight in a 4°C refrigerator. Secondly antibodies bound to FITC and TRAITC were hatched at 25°C for 2 h. Finally, the sections were observed with a Leica fluorescence microscope made in Germany.
Cell culture .
Primary astrocytes were gained from the cerebral cortex of 10 neonatal (2 day-old) male Sprague-Dawley rat pups obtained from Shandong Experiment Animal Center. The rats were beheaded and their brains were rapidly acquired. Cerebral cortex was digested by trypsin and separated by grinding with a grinder at 0°C . The mixture was inoculated in Dulbeco modified eagle medium (DMEM) at a density of 5×107 cells per 75 cm2 flask, which contained F12 nutrients (1∶1; Gibco Thermoelectric Fisher Scientific Inc.). Cells were cultured in this medium for 7-8 days. Before the next experiment, the astrocyte culture was subcultured twice and the medium was changed to serum-free DMEM/F12. The transfection experiment was started 24 hours after the culture.
siRNAs and transfection
The synthetic MCM7 siRNA and negative control siRNA were purchased from Shanghai Gene Pharma Co. Ltd. The astrocytes cell were transiently transfected according to the manufacturer's protocol. The sequence of negative control siRNA is 5'-UCUCCGAACGUGUCACGUT-3'.The sequences of MCM7 siRNA are 5'-CTAGTAAGGATGCCACCTA-3', 5'-GAUGUCCUGGACGUUUACA-3' and 5'-GCTCATGAGGCGTTACATA-3'.
Statistical methods
All experimental datas were analyzed by STATA 7.0 statistical software. All values are expressed as the mean standard error of the mean, and each experiment is repeated at least three times. Statistical evaluation was processed by two-tailed Student’s t-test between two groups. Statistical evaluation of multiple groups was performed using a one-way analysis of variance followed by Dunnett’s post-hoc test. P value less than 0.05 was considered statistically significant.