Experimental animals
Male C57BL/6J mice (aged 10-12 weeks with an initial body weight of 20 ± 2 g) were purchased from the Third Military Medical University Animal Center. The mice were housed at a temperature of 25 ± 2 °C and a relative humidity of 70% ± 5% under natural light/dark conditions and allowed free access to food and water. The animal experiments were performed in strict accordance with the ethical guidelines of the Third Military Medical University Animal Studies Committee and the National Institutes of Health Guide (NRC 2011) for the Care and Use of Laboratory Animals.
MCAO model
To establish the middle cerebral artery occlusion (MCAO) model[9], the mice were anesthetized with isoflurane (3% for induction and 1.5% for maintenance). After a ventral neck incision was made, the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were carefully exposed. Microvascular aneurysm clips were applied to the right CCA and the ICA. A-coated 6-0 filament (RWD, Shenzhen, China) was introduced into the arteriotomy hole, fed distally into the ICA, and advanced to a predetermined distance 8 mm from the carotid bifurcation toward the MCA. After 60 min of MCAO, the filament was withdrawn, the collar suture was tightened, and the skin incision was closed. Sham mice underwent neck dissection and coagulation of the ECA but not MCA occlusion. Laser Doppler flowmetry (PeriFlux system 5000; Perimed, Stockholm, Sweden) was used as a quality control; success was determined based on an 80% decrease in regional CBF in the mice after MCAO. Dead animals and failed MCAO model mice were excluded from the experiments.
Western blot assay
Total protein samples from the brain tissues of mice were lysed using cold RIPA buffer containing 1 mM phenylmethylsulfonylfluoride according to the manufacturer’s protocol (Beyotime Technologic Inc., China). The protein content was determined using a bicinchoninic acid protein assay kit (Beyotime Technologic Inc., China). Equal amounts of protein lysates (30 μg in each lane) were loaded onto sodium dodecyl sulfate (SDS)-polyacrylamide gels (8-12%), electrophoretically separated and transferred onto PVDF membranes (Millipore, USA). After blocking nonspecific binding sites with 5% dry milk for 1 h in TTBS at 37 °C, the membranes were individually incubated with primary antibodies against TRAF6 (Bioss Technologic Inc., China) and β-actin at 4 °C overnight. Then, the membranes were further incubated with a corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at a 1:500 dilution for 2 h at room temperature. Protein bands were visualized with enhanced chemiluminescence (ECL) reagents (Pierce; Thermo Fisher Scientific Inc., Waltham, MA, USA). β-Actin was used as an internal reference for relative quantification. The densitometric values were analyzed with ImageJ 1.43u (National Institutes of Health, USA) and normalized to β-actin as an internal control (IOD of target protein versus IOD of GAPDH).
Quantitative real-time PCR assay
Total RNA samples were obtained from brain tissues using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. cDNA was synthesized using a SuperScript cDNA Synthesis Reagent Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Quantitative real-time PCR was performed using Maxima SYBR Green/ROX qPCR Master Mix according to the manufacturer’s instructions (Thermo Fisher Scientific) on an ABI-StepOnePlus Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The cycle threshold (CT) values for each sample were normalized to those of GAPDH, and the fold differences were determined using the 2-ΔΔCT method.
Immunohistochemistry
Immunostaining was performed as described previously[23]. Paraffin-embedded sections of brain tissues were dewaxed and rehydrated with xylene and graded alcohols. Endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol for 5 min, and then the sections were microwaved in EDTA buffer solution (pH 8.0) for 20 min and cooled. After the sections were incubated with 5% BSA and 0.1% Triton X 100, they were incubated with a rabbit anti-TRAF6 antibody (Bioss Technologic Inc., China) at 4 °C overnight. Normal nonimmune rabbit serum was used as a negative control. After incubation with a secondary antibody at room temperature for 60 min, the slides were incubated with goat anti-rabbit immunoglobulin conjugated to peroxidase-labeled dextran polymer (EnVision+System-HRP; Boster, China) for 20 min. Then, the sections were stained with 3,3-diaminobenzidine (DAB) and counterstained with hematoxylin. After dehydration and drying, the sections were mounted with neutral gum, covered with coverslips and observed under a microscope.
Immunofluorescence staining
The sections were treated with 5% goat serum and 0.1% Triton X-100 to block nonspecific binding. Then, the sections were incubated overnight at 4 °C with a rabbit anti-TRAF6 antibody (Bioss Technologic Inc., China) and an anti-GFAP antibody (mouse monoclonal, 1:600; CST, USA). After washing with PBS, the sections were incubated with a mixture of Alexa Fluor 647-conjugated donkey anti-rabbit IgG and Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:500; Life Technologies) for 30 min at 37 °C. To identify the nuclei of the cells, the sections were incubated with 4',6-diamidino-2-phenylindole (DAPI) for 5 min. After staining, the sections were mounted with Ultramount (DAKO) and photographed with a confocal fluorescence microscope (TCS-TIV; Leica, Nussloch, Germany).
Neurological score
Animals were scored as previously described[24, 25]. The behavior of MCAO mice was assessed 24 h after cerebral ischemia. Animal behavior was recorded and subsequently analyzed by two observers who were blinded to the animal group designations. Neurological deficits were scored using the following scale: 0 points, escapes to supports; 1 point, hangs onto string with forepaws, hindpaw(s) and tail; 2 points, hangs onto string with forepaws and hindpaw(s); 3 points, hangs on with forepaws and moves laterally on string; 4 points, hangs on with forepaw(s); 5 points, falls off within 2 s. The mean score on the scale was used as the final score for each animal.
TTC staining and assessment of cerebral infarct volume
The cerebral infarct volume was determined as previously described and assessed using the TTC method[9, 26].After MCAO, animals were sacrificed, and the brains were removed and frozen at -80 °C for 5 min. The brains were then quickly removed and sectioned coronally to obtain 7 sections. The slices were incubated for 20 min at 37 °C in darkness with 2% triphenyl tetrazolium chloride (TTC) solution (Sigma-Aldrich, St. Louis, MO). After rinsing with PBS (0.1 M), the slices were fixed overnight in 4% PFA (4 °C) and photographed with a camera. Red areas indicated normal brain tissue, whereas pale white areas indicated the infarcted area. The infarct area was measured by using the following formula: percentage of infarct volume = 100 × (unstained volume (mm3)/edema index)/total volume (mm3).
Nissl staining
Nissl staining was performed as previously described[27].Paraffin-embedded sections of brain tissues were dewaxed and rehydrated with xylene and graded alcohols. The sections were incubated in 0.5% cresyl violet solution (Beyotime Technologic Inc., China) for 20 min. After washing with distilled water, the sections were hydrated in serial alcohol solutions (70%, 80%, 95%, 100%) and xylene. The sections were mounted with neutral gum, covered with coverslips and observed under a microscope. Quantitative analysis of the ratios of viable neurons was performed using Image-Pro Plus 5.0 image processing software (Media Cybernetics, Rockville, MD).
TUNEL staining
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed using a TUNEL kit (Roche, Mannheim, Germany) according to the manufacturer’s protocol as previously described.[27] After the sections were deparaffinized and rehydrated, antigen retrieval was performed, and endogenous peroxidase activity was blocked with methanol solution containing 0.3% H2O2 for 5 min. Then, the brain slides were incubated with proteinase K for 30 min at 37 °C and TUNEL reaction mixture for 1 h at 37 °C. After three washes with PBS, the sections were treated with 50 ml converter-POD for 30 min at 37 °C. Three or more random fields were used to assess cell death and calculate the apoptotic index. The apoptosis level was expressed as the percentage of TUNEL-positive cells relative to total cells.
Water maze test
To assess spatial learning and memory, the mice were subjected to the Morris water maze test starting from day 14 after MCAO/ reperfusion[28]. Briefly, the Morris water maze apparatus consisted of a circular tank containing water in a room with salient visual cues. The mice were trained to find a hidden platform in the water maze (0.8 m in diameter) for 5 consecutive days. Each mouse underwent five trials (with randomly assigned starting positions) per day to locate the platform, and the intertrial interval was 10 s. For each trial, the mouse started from one of the four quadrants facing the wall of the pool; the trial ended when the animal climbed onto the platform, which was submerged 2 cm underneath the water in the middle of one of the quadrants of the water tank. If a mouse did not locate the platform within 60 s, it was guided to the platform. The swimming path and the percentage of time spent in the target quadrant were recorded by a video camera. The mean latency was recorded for each, and the spatial test was performed on the fifth day to evaluate the memory retention of each animal.
Lentiviral vector transfection
In vivo lentiviral vector transfection was performed as described previously. Mice were injected with a TRAF6-overexpressing lentivirus vector or a TRAF6-negative lentivirus vector. The mice were anesthetized with isoflurane (3% for induction and 1.5% for maintenance), their heads were shaved, and they were placed in a stereotaxic frame. A midline incision was made, and burr holes were made using a high-speed dental drill. Next, 1-2 μl of the TRAF6-overexpressing lentivirus vector or TRAF6-negative lentivirus vector was injected into the brain over a 5-10 min period. Virus administration was performed with a 10-µl Hamilton syringe (Hamilton Company, USA), which was controlled by a microinfusion pump (RWD, Shenzhen, China). The location of the lentivirus infusion was determined by immunohistochemistry.
Statistical analysis
All assays were performed in triplicate. The data are shown as the mean ± SD. To compare mean values between two groups, independent Student’s t-test was used. To compare mean values among more than two groups, two-way ANOVA was used. Fisher’s exact test was used to compare the tumor metastasis rate between two groups.