Animals
All experimental protocols for this study were approved by the Animal Ethics Committee of Chongqing Medical University. The study complied with the National Institutes of Health guide for the care and use of Laboratory Animals and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. C57BL/6 mice (male, weight about 25 g) were purchased from and bred at the Animal Center of Chongqing Medical University. All mice were housed in a light and temperature controlled room with free access to food and water.
ICH mouse model induction
Mice were anesthetized with 10% chloral hydrate (350mg/kg)and were placed in a stereotaxic frame (Alcott Biotech, Shanghai, China). Through a hole drilled in the skull, a 32-gauge needle was implanted into the striatum, 2.0 mm lateral to the midline, 1.0 mm anterior to the coronal suture, and at a depth of 4.0 mm from the surface of the brain. Each mouse was microinjected with 25 μl of autologous whole blood (right striatum) taken from the tail vein over 10 min using a microinfusion pump (ALC-IP600, Alcott Biotech). Then, the needle was pulled out without blood reflux after 5 min of dwelling, and the wound was sutured. Only the mouse observed to have a neurological deficit was regarded as a successful model. The mice in the sham operation group had the same operation, but no blood was injected.
Intracerebroventricular injection
The in vivo transfection was performed according to the method described as follows: the stereotaxic coordinates were 0.5mm posterior and 1.0mm lateral to bregma and 2.5-3.0mm ventral to the surface of the skull. The Ad-Nedd4L siRNA , Ad-TRAF3 RNAi or control (1×109 plaque forming units [Pfu], Sangon Biotech, Shanghai, China) were added to 2 μl of a 10 mg/ml solution prepared in 0.9% NaCl. In the inhibitor treatment experiments, mice were received intracerebroventricular injection of 0.9% NaCl (2 μl) in the presence of the P38 inhibitor EO1428 (50 μM, Sigma-Aldrich), or the NF-κB p65 inhibitor SC75741 (50 μM, Sigma-Aldrich), respectively. The solution was mixed gently, left for 15 min and then injected intracerebroventricularly (i.c.v.) using a micro syringe (Hamilton, NV, USA) under the guidance of the stereotaxic instrument (RWD Life Science).
BBB permeability
To evaluate BBB permeability, Evans blue (Aladdin, China) was injected intraperitoneally (100 μl of 4% solution in saline) . After 3 h circulation, mice were transcardially perfused with cold phosphate-buffered saline (0.1M, PBS, pH7.4) under deep anesthesia. Afterwards, the brain was removed and divided into left and right hemispheres and stored at -80 °Cimmediately. The right part of the brain was homogenized in 1100 μl PBS, sonicated, and centrifuged (12,000 g, 4 °C, 30 min). The supernatant was collected and added an equal amount of trichloroacetic acid (TCA) to incubate overnight by 4 °C. After centrifugation (12,000 g, 4 °C, 30 min), Evans blue stain was measured by spectrophotometer (Thermo Fisher Scientific, USA) at 610 nm.
Immunofluorescence staining
The mice were deeply anesthetized and were transcardially perfused with 20 ml icecold PBS followed by 20 ml of 4% paraformaldehyde at 24 h post-ICH. The whole brain was collected and then fixed in 4% paraformaldehyde for another 24 h. Afterwards, the brain was fixed in 20% sucrose solution until the tissue sink to the bottom followed by 30% sucrose solution for another 24 h. After being frozen at -25 °C, the brain was cut into 10-μm-thick coronal sections using a cryostat (CM1860; Leica Microsystems, Germany). To conduct double immunohistochemistry staining, the brain sections were incubated with primary antibody of CD11b or Nedd4L (1:100, Santa cruz) overnight at 4 °C. After being incubated with the appropriate secondary antibody (1:200, Bioss) at 37 °C for 1 h, the sections were visualized and photographed with a fluorescence microscope (UHGLGPS, OLYMPUS, Japan). Microphotographs were analyzed with cellsens Standard software. The numbers of positive cells were identified and counted in three different fields in peri-hematoma area from five random coronal sections per brain, and data were expressed as cells/field.
Real-time polymerase chain reaction (PCR)
The ipsilateral hemisphere was homogenized using RNAiso Plus (Takara) and ceramic beads for 1 min in a speedmill according to the manufacturer’s instructions (Alytik Jena). RNA was isolated according to the manufacturer’s instructions and reverse transcribed to obtain cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara). Real-time PCR was performed using cDNA samples with SYBR@Premix ExTaq™II (Takara, Tli RNaseH Plus) by the One-step Plus analyzer (ABI). We normalized the results for each individual gene using the housekeeping gene beta-actin. The 2−ΔΔCT method was used to calculate relative gene expression levels.
Western blotting
Briefly, the mice were perfused with 0.01 mol/l phosphatebuffered
saline after ICH, and the cerebral tissues from the perihematomal region were isolated (n=5). The perihematomal tissues were lysed in 1 ml of radioimmunoprecipitation assay lysis buffer, and then the protein was extracted, electrophoresed, and transferred onto polyvinylidene fluoride membranes (Amersham Pharmacia). The polyvinylidene fluoride membranes were incubated with primary antibodies overnight, followed by incubation with peroxidase-conjugated secondary antibodies for 6 h. The same membranes were probed with an antibody for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Protein signals were detected with an enhanced chemiluminescence system. The signals were quantified by scanning densitometry and computer-assisted image analysis. Protein levels were expressed as the ratio of the values of the detected protein band to the GAPDH band.
Evaluation of neurological scores
The neurological scores were assessed by Neurological Severity Scores, according to the motor, sensory, reflex, and balance tests. Neurological function was assessed on a scale of 1-18; a score of 1 point is regarded as the inability to perform the test or for the lack of a tested reflex. The higher the score, the more severe the injury (normal score: 2-3; maximal deficit score: 18).
Measurement of brain edema
Brain hemisphere were quickly separated and weighted to assess the wet weight (wW) using an electronic analytic balance. After drying the brain hemisphere in an oven at 100 ◦C for 24 h, dry tissue weight (dW) was assessed. The percentage of water was calculated according to the following formula: brain water content (%) = (wW-dW)/wW×100%.
Statistics analysis
The data are expressed as the mean ± SD of at least 3 separate experiments performed in triplicate. The differences between groups were determined with the Student’s t-tests and a one-way analysis of variance (ANOVA) using SPSS 13.0 software. Differences were declared significant at *P < 0.05.