Animals
8 week-old male specific pathogen-free (SPF) C57BL/6 mice were purchased from Chongqing Medical University and were housed in standard polypropylene cages at the animal facility until the day of the experiment. All procedures were performed in accordance with guidelines established by the Animal Care and Use Committee of Chongqing Medical University.
Macrophage culture
BMDMs (bone marrow derived macrophages) were isolated from the marrow of the femurs and tibias of C57BL/6 mice. The legs of the animals were sprayed with 70% EtOH, and the skin and muscle tissue were removed from the bones. The bones were sprayed with 70% EtOH, transferred to a sterile-flow hood and cut at both ends. The marrow was flushed out into a sterile falcon tube in Dulbecco’s modified Eagle’s medium supplemented with heat-inactivated fetal bovine serum (FBS; 50 ml; 10%) and penicillin-streptomycin (5 ml; 1%; Gibco). The cell suspension was triturated using a sterile Pasteur pipette, filtered through a nylon mesh filter into a sterile tube and centrifuged (400 × g, 5 min). The supernatant was removed, and the pellet was resuspended in red blood cell lysis buffer (Sigma-Aldrich, Gillingham). The suspension was centrifuged (400 × g, 5 min), the supernatant was discarded and the cells were washed using DMEM and centrifuged once more (400 × g, 5 min). The pellet was resuspended in 20 ml of DMEM supplemented with L929-conditioned media (20%). Cells were seeded in sterile cell culture flasks (T175 cm2 flasks). On day 2, non-adherent cells were removed from the flask, the media was replaced and the remaining adherent cells were maintained in culture for a further 6 days.
Neuronal cultures
Cortical neuronal were prepared from embryonic day 16–18 C57BL/6 mice. Briefly, the cerebral cortices and hippocampus of fetus mice were dissected out and the meninges were carefully removed. Cells (1 × 106 cells/mL) were maintained in poly-d-lysine (Sigma, St. Louis, MO) coated plates in Dulbecco’s modified eagle medium (DMEM) medium (Life Technologies) with 10% fetal bovine serum (FBS) (Life Technologies). After 4–6 h of culture, the cultures were replenished with Neurobasal medium (Life Technologies) containing 100 U/mL penicillin, 100 µg/mL streptomycin, 2% B27, and 0.5 mM glutamine (Life Technologies) at 37 ◦C with 5% CO2. The medium was changed every three days.
Preparation of erythrocyte lysates
Whole blood collected from 30 to 50 mice was pooled and leuko reduced using a Neonatal High-Efficiency Leukocyte Reduction Filter (Purecell Neo; Pall Corporation). Blood was centrifuged at 400 × g for 15 minutes, and the volume reduced to a final hemoglobin level ranging from 17.0 to 17.5 g/dL, as determined by a modified Drabkin hemoglobin assay at a 1:251 dilution of stored RBCs to Drabkin reagent (Ricca Chemical Company). Washed stored RBCs were prepared with 3 washes using 10 volumes of phosphate-buffered saline (PBS) and centrifugation at 400 g. After the final wash, the washed stored RBCs were resuspended in PBS to a final hemoglobin concentration of 17.0 to 17.5 g/dL for transfusion. Supernatant was obtained using a 400gspin of stored RBCs and 400 µL of this solution were transfused undiluted. RBC ghosts were obtained by hypotonic lysis of twice the volume of stored RBCs (ie, for 400 µL of ghosts, 800 µL of stored RBCs were hemolyzed) with PBS to distilled water (1:15), followed by multiple washes with the same buffer and centrifugation at 30 000 g until a white pellet was obtained. The white pellet of RBC ghosts was resuspended in PBS. Stroma-free RBC lysate was prepared by freeze-thaw of washed stored RBCs followed by centrifugation at 16 000 × g to pellet and remove the stroma.
Cell treatment
Macrophages (1 × 105) were stimulated with propofol (0 ~ 100 µM) plus 10 µl PBS or erythrocyte lysates for 24 hs. After then, the supernatants were removed and further analyzed for cytokine production with ELISA.
Real-time PCR
The ipsilateral hemisphere was homogenized using RNAiso Plus (Takara) and ceramic beads for 1 min in a speedmill plus according to the instructions of the manufacturer (Alytik Jena). RNA was isolated according to the instructions of the manufacturer and reverse transcripted 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 analysis
Proteins from cultured macrophages were resolved using SDS-PAGE and transferred onto polyvinylidene fluoride membranes using electroblotting. The membranes were incubated with primary antibodies, all diluted to 1:1000 (Cell Signaling Technology), at 4˚C overnight. GAPDH (1:200; Santa Cruz Biotechnology, Dallas, TX) was used as the loading control. The membranes were incubated with HRP-conjugated goat anti-rabbit secondary Abs (1:2500; Sigma-Aldrich, St. Louis, MO) at 25˚C for 1 h. Bound Abs were visualized using a chemiluminescence detection system. Protein levels were calculated as the ratio of the target protein value to the GAPDH value.
Enzyme-linked immunosorbent assay
The supernatants or brain tissue extracts were harvested, and TNF-α, IL-1β and IL-6 productions were determined by ELISA. The specimens were assayed using respective Enzyme Linked Immunosorbent Assay (ELISA) kits (Minneapolis, MN, USA) according to the instruction manuals.
Cytotoxicity assay
The cytotoxic activity of macrophages was measured by a 6 h lactate dehydrogenase release assay using CytoTox96 Non-radioactive Cytotoxicity Assay kit (Promega, Charbonnie’res-les-Bains, France) on 5 × 103 neuron/well. Neuron was then added to the wells with 2 × 103 macrophage. Experiments were performed in quadruplet and the percentage of lysis was determined by OD490 measurement as described in the manufacturer’s instructions. The percentage of cell mediated cytotoxicity was calculated according to the equation: % Specific Lysis = (effector/target cell mix LDH release-spontaneous effector LDH release)/(maximum target LDH release - spontaneous target LDH release) × 100%:
Oligonucleotide transfection
All of the transient transfections were performed with Lipofectamine 2000 Reagent (Invitrogen). MiRNA oligonucleotide transfections were performed according to an established protocol. Briefly, macrophages were seeded in 6-well plates at a density of 2 × 105 cells per well and were grown overnight to 60%-80% confluency. Next, miRNA mimic (Pre-miR ™ miRNA precursor) or miRNA inhibitor (Anti-miR ™ miRNA inhibitor) (Ambion) was added to the culture media at a final concentration of 100 nM according to the manufacturer’s recommendations. The sequences of miR-494 used were shown as following: miR-494 mimic, 5′-UGAAACAUACACGGGAAACCUC-3′, miR-494 inhibitor, 5′-UUCUCCGAACGUGUCACGUUU-3′. Transfection efficiency (> 90%) was measured by qRT-PCR. For small interfering RNA (siRNA) inhibition were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and were introduced into the macrophage at final concentrations of 100 nM according to the siRNA transfection protocol. Control siRNA-transfected macrophages were used as the negative control. Transfection efficiency (> 80%) was measured by qRT-PCR. After 6 h of transfection, the medium was replaced with normal glucose or low-serum (2% FBS) medium and cells were incubated for 48hs.
Vector construction and luciferase reporter assays
Luciferase reporter constructs were used, and luciferase assays were performed as described previously. Briefly, the mouse Nrdp1 3′-UTR sequence was amplified by PCR from mouse genomic DNA, and ligated into the pMIR-REPORT luciferase vector multiple cloning site (Ambion, Austin, TX) to yield pMIR- Nrdp1 3′-UTR (NRDP1 3′-UTR). Another pMIR-REPORT luciferase construct containing the Nrdp1 mRNA 3′-UTR with a mutation by site-directed mutagenesis was generated as a negative control and named Mut- Nrdp1 3′-UTR. Macrophage were plated in 6-well plates and allowed to reach 60%-80% confluence overnight. Cells were then co-transfected with a reporter construct (pMIR-null REPORT plasmid, pMIR- Nrdp1 3′-UTR, pMIR-Nrdp1 3′-UTR-Mut). After 24 h, cells were harvested, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s recommendations. Luciferase activity was normalized to control TK Renilla construct expression (pRL-TK, Promega).
ICH model
After anesthetizing mice with 1–3% isoflurane inhalation and ventilating them with oxygenenriched air (20%:80%), we injected a total of 0.5 µL containing 0.075 units of collagenase VII-S (No. C9572, Sigma, St. Louis, MO) at 0.1 µL/min into the left basal ganglion at the following coordinates relative to bregma: 0.8 mm anterior, 2 mm lateral, and 2.8 mm deep. The craniotomy was sealed with bone wax, and the scalp was closed with 4 − 0 silk sutures. Rectal temperature was maintained at 37.0 ± 0.5 °C throughout the experimental and recovery periods (DC Temperature Controller 40-90-8D; FHC Inc., ME). Sham-operated mice received the same treatment, including needle insertion, but collagenase was not injected. The mortality rate in untreated animals is 4.6%.
Intracerebroventricular injection
To investigate the anti-inflammatory effects of propofol, propofol (2 µg/2 µl) or PBS (2 µl) was pretreated with a single intracerebroventricular (i.c.v.) injection in the ipsilateral ventricle 15 min before ICH. For the injection into the ipsilateral ventricle, a small burr hole was made in the parietal region (1.0 mm posterior and 1.0 mm lateral to the bregma) under the guidance of the stereotaxic instrument (RWD Life Science).
Histochemical evaluation of microphage activation
3 days after ICH, the animals were deeply anaesthetized with pentobarbital and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB,pH7.4). After the mice were perfused and fixed, the perihaematomal region of cerebral tissues were collected, fixed in 4% paraformaldehyde for 24 h, dehydrated in 30% sucrose solution for 48 h, embedded, frozen, and cut into 25-µm sections using a Leica CM1900 cryostat. The perihaematomal region was treated with 3% H2O2 in 0.01 M phosphate-buffered saline (PBS) and preincubated in 5% normal goat serum. The samples were then incubated in a primary antibody solution containing rat anti- Iba antibody (Serotec, Fullerton, CA, USA, 1:200) overnight at 4 ◦ C. After washing, the samples were incubated in a secondary IgG antibody (1:200) for 1 h at room temperature (RT). Finally, the sections were incubated in horseradish peroxidase (HRP)-Streptavidin (1:200) for 1 h at RT, and the colour reaction was conventionally developed with diaminobenzidine (DAB) and H2O2. For each animal, six representative sections of each brain were selected. IPP6.0 image processing software (Media Cybernetics, MD, USA) was utilized to count the number of positive cells.
Evans Blue Extravasation
Briefly, the Evans blue dye (2%, 5 mL/kg; Aladdin, Shanghai, China) was injected and administered > 2 minutes into the left femoral vein under anesthesia, where it was allowed to circulate for 60 minutes. Then mice were euthanized by an intracardial perfusion with sterile saline. Then brain samples were weighed, homogenized in sterile saline, and centrifuged at 15000 g for 30 minutes. After that, equal volume of trichloroaceticacid was added to the resultant supernatant. Those samples were then incubated overnight at 4 °C and centrifuged again at 15000 g for 30 minutes. The resultant supernatant was spectrophotometrically quantified for the extravasated Evans blue dye at 620 nm.
Evaluation of neurological scores
A standardized battery of behavioral tests was used to quantify neurological function at 3ds post-ICH. The neurological scores were determined by Neurological Severity Scores, a composite of motor, sensory, reflex, and balance tests. Neurological function was graded on a scale of 1–18; a score of 1 point is awarded for 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). Tests were conducted by an observer blinded to the treatment group.
Brain water content measurement
Brain water content was measured in mouse cerebral tissues after ICH. Briefly, mice were randomly sampled from each group and anesthetized by intraperitoneal injection with chloral hydrate (n = 5). Next, the cerebral tissues were removed, and the surface water on the cerebral tissues was blotted with filter paper. Brain samples were immediately weighed on an electric analytic balance to obtain the wet weight and then dried at 100 °C for 24 h to obtain the dry weight. Brain water content was calculated using the following formula: brain water content (%) =(wet weight - dry weight) / wet weight × 100%.
Statistical Analysis
All experiments were independently performed three times. The differences between groups were determined with the one-way analysis of variance (ANOVA) using SPSS 13.0 software. P values of less than 0.05 were considered to be statistically significant.