Animals
A total of 36 adult male C57BL/6J mice between ten to twelve weeks old were used in this study. These mice were purchased from the experimental animal center of our institute and weighed between 22 and 25 g. All experiments were carried out under NIH’s Guide for the Care and Use of Laboratory Animals and were approved by the Ethics Committee of Yantai Ludong Hospital (Code: 160688M001).
SAH Model
An endovascular perforation method was used to establish the animal model of SAH as previously described [21, 22]. In brief, 50 mg/kg pentobarbital sodium was injected to carry out anesthetization. A heat blanket was then used to maintain the rectal temperature of mice at 37 ± 0.5 °C. The internal carotid artery, external carotid artery and left common carotid artery were then exposed through a midline incision at the neck, followed by ligation and dissection at the left external carotid to leave a 3-mm stump. A 5-0 nylon suture was then inserted into the left internal carotid artery to perforate the artery at the bifurcation of the anterior and middle cerebral arteries. The identical procedures were performed on sham-operated mice except for the artery perforation.
Experimental Protocol
The mice were assigned into the groups listed below: (1) SAH group (n= 12), (2) sham group (n= 12), (3) SAH + MT group (n= 12). For the MT group, MT was dissolved in 1% ethanol in 1 mL sterile saline, then intraperitoneally injected at a dose of 150 mg/kg and 12 h after SAH [23].
Neurological Score
Neurological deficits were evaluated at 96h after the onset of SAH using an 18-point system containing six tests per previous published protocol [22, 24]: forelimbs outstretching (0–3), spontaneous activity (0–3), climbing (1–3), symmetry in the movements of all limbs (0–3), response to vibrissae touch (1–3) and body proprioception (1–3). A higher score indicates an elevated function.
Brain Water Content
The standard wet-dry method was used to measure the brain water content in each mouse. In brief, the mice were sacrificed 96h post-operation to have their brains immediately isolated and separated into the right and left cerebellum, cerebral hemispheres and brain stem. These brain parts were weighed respectively to obtain their wet weight. Subsequently, the brain sections were dried for 24 hours at 105°C and weighed to obtain the dry weight. Finally, the brain water content was calculated as (wet weight - dry weight)/wet weight× 100%.
RNA isolation and real-time PCR
Tissue and cell samples were treated with TRIZOL (Invitrogen, Carlsbad, CA) to extract total RNA, which was then converted to cDNA on a PE9600 PCR instrument (Perkin-Elmer, Foster City, CA). The mRNA expression of H19, miR-675, HIF1A, and TLR4 was then measured using real-time PCR using an SYBR Green kit following the manufacturer’s instructions. Accordingly, U6 and GAPDH were used as internal control. The thermocycle conditions were: 95℃ for 5 min, 45 cycles of 95℃ for 10s and 60℃ for 50s, 90℃ for 1 min, 55℃ for 1 min, 95℃ for 30s. All experiments were repeated in triplicated and the data was analyzed by the 2−ΔΔCt method [25]. The primers pairs used were: H19-forward: 5'-TGCTGCACTTTA CAACCACTG-3’; H19-reverse: 5’- ATGGTGTCTTTGATGTTGGGC-3'; miR-675-forward: 5’-TGGTGCGGAGAGGGCCCACAGTG-3’; miR-675-reverse: 5’-GCGAGCACAGAATTAATACGAC-3’; HIF1A-forward: 5’-CGTGTTATCTGTCGCTTTGAGTC-3’; HIF1A-reverse: 5’-GTCTGGCTGCTGTAATAATGTTCC-3’; TLR4-forward: 5’-GGCATCATCTTCATTGTCCTTG-3’; TLR4-reverse: 5’-AGCATTGTCCTCCCACTCG-3’; U6-forward: 5’-CGCTTCGGCAGCACATATACTA-3’; U6-reverse: 5’-CGCTTCACGAATTTGCGTGTCA-3’; GAPDH-forward: 5’- CTTTGTCAAGCTCATTTCCTGG-3’, GAPDH-reverse: 5’-TCTTGCTCAGTGTCCTTGC-3’.
Cell culture and transfection
U251 and SH-SY5Y cells were cultured at 37℃ and in 95% humidity and 5% CO2 with a Dulbecco's modified eagle medium (DMEM) (Invitrogen, Carlsbad, CA) containing 10% fetal calf serum (FCS) (Invitrogen, Carlsbad, CA). When the cultured cells grew to 90% adherence, they were passaged. Subsequently, the cell transfection was accomplished using Lipofectamine 2000 following the instruction of the manufacturer. For MT treatment, the cells were treated with 1 µm and 5 µm of MT for 48 h before the cells were collected for subsequent analyses [23].
Luciferase assay
Full length 3' UTR of HIF1A was amplified by PCR and cloned into a pGL3 vector (Promega, Madison, WI) downstream of the firefly luciferase reporter gene. Subsequently, a Quick-change site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to generate mutated 3' UTR of HIF1A, which was also inserted to a pGL3 vector and used for subsequent transfection experiments. Similarly, miR-675 mimics were designed and synthesized before they were co-transfected to U251 and SH-SY5Y cells with either wild type (WT) or mutated 3' UTR of HIF1A. At 48 h after transfection, the cells were harvested and the luciferase activity in transfected cells was measured using a dual-luciferase reporter assay (Promega, Madison, WI). In order to investigate the effect of HIF1A on the promoter of TLR4, the full length of TLR4 promoter was amplified by PCR and cloned into the pGL3 vector. Subsequently, HIF1A and TLR4 promoter were co-transfected to U251 and SH-SY5Y cells. At 48 h after transfection, the cells were harvested and the luciferase activity in transfected cells was measured using the dual-luciferase reporter assay.
Western blot analysis
Tissue samples (basal cortical samples) were pooled and cell samples were lysed and resolved using 12% SDS-PAGE. Subsequently, the resolved proteins were blotted onto a membrane, which was then blocked at room temperature for 1 h with Tris-Buffered Saline and Tween 20 (TBST) containing 5% bovine serum albumin (BSA) and incubated at 4℃ overnight with primary antibodies against HIF1A (ab92498, Abcam, Cambridge, MA) and TLR4 (ab8378, Abcam, Cambridge, MA). After TBST washing, the membrane was further incubated for 1h at 37℃ with HRP-labeled second antibodies (ab6728, Abcam, Cambridge, MA). Finally, after being developed using chemiluminescence reagents (Santa Cruz Biotech, Santa Cruz, CA), the optical density values of target proteins were measured.
Apoptosis analysis
Treated U251 and SH-SY5Y cells were harvested by centrifugation. Subsequently, the cells were suspended in PBS to a concentration of 5-10 × 104 cells/mL and incubated with Annexin V-FITC and Propidium iodide. In the next step, the cell apoptosis profiles in different groups were analyzed using a FACSCanto II flow cytometer (BD, Franklin Lakes, NJ). The final apoptosis rate = early apoptosis rate + late apoptosis rate.
Immunohistochemistry assay
Histochemical immunostaining assay was done using a streptavidin-peroxidase (SP) three-step method. All samples were first fixed in 4% formalin and embedded in paraffin. After antigen repair, the slides were incubated overnight at 4℃ with primary antibodies against HIF1A (ab92498, Abcam, Cambridge, MA) and TLR4 (ab8378, Abcam, Cambridge, MA). After being washed repeatedly with PBS, the slides were incubated for 10 min at room temperature with the secondary antibody. Subsequently, the reaction was terminated by an anti-biotin-labeled peroxidase solution, and the slides were colorized with DAB. After re-staining with hematoxylin, the slides were dehydrated with anhydrous ethanol and dried before they were mounted in neutral gum and observed under a microscope.
TUNEL assay
The frozen tissue samples were recovered at room temperature for 5-10s, followed by the addition of a resuscitation solution. After resuscitation, the samples were stained using a TUNEL kit (Beyotime, Shanghai, China). Subsequently, the samples were immersed in a 3% H2O2 solution at room temperature for 10 min and then rinsed with PBS for 5 min. After the addition of 50 uL of proteinase K solution (20 µg/mL), the samples were hydrolyzed for 20 min at room temperature to remove tissue proteins. On the next, the samples were washed with PBS and the antigen retrieval was done using a citrate buffer (0.01 M). Subsequently, the sections were cooled to room temperature and washed with PBS. Then, 50 μL of a TdT enzyme solution was added to the slices and incubated at 37 ℃ for 1 h, whereas the reaction solution containing no TdT enzyme was used as a negative control. After the slices were washed with PBS, 50 μL of peroxidase-labeled anti-digoxigenin were added on the slides and incubated at 37 ℃ for 30 min away from the light. Finally, a diamidino-2-phenylindole (DAPI) solution was added dropwisely onto the slides at room temperature and incubated for 10 min before the slides were observed under a fluorescence microscope. The nuclei showing a green color were considered as apoptotic cells, while the nuclei showing a blue color were considered as normal cells. A total of 10 visual fields were selected randomly to calculate the apoptotic index.
Statistical analysis
Statistical analysis was performed using SPSS16.0 statistical software. The measurement data were presented by mean values and standard deviation, while the average values between two groups were compared using t-tests. The comparison between multiple groups were conducted using one way ANOVA followed by the Scheffe’s method as the post hoc text. A P value of < 0.05 was considered statistically significant.