Experimental animals
C57BL/6 mice were generated and kept in our laboratory according to a previous study. All mice used were male and 8–10 weeks old and weighed 22–25 g. The mice were maintained in individual cages under controlled conditions (22 ± 2 °C; 55% humidity) with free access to food and fresh water. All animal experiments were approved by the Ethics Committee of Chongqing Medical University.
Primary cell cultures
Cortical neuronal cultures were prepared from the whole cerebral cortices of C57BL/6 mouse embryos (E16). After removal of the meninges, the tissue was digested with 0.005% trypsin/0.002% EDTA (10 min, 37 °C), mechanically dissociated, and centrifuged at 1000 × g for 5 min. The cell pellet was resuspended in neurobasal medium (Gibco) containing B27 serum-free supplement (Gibco) and 500 N m L -glutamine. A total of 2 × 105 cells/well were seeded on sterile poly-L-lysine (Sigma-Aldrich)-coated glass cover slips in a 24-well plate and incubated at 37 °C and 5% CO2. After 1 h, the culture medium was changed completely. The purity of the neuronal cultures was > 95%, as confirmed by random staining with neuronal and glial markers. Five days after plating, the neurons had developed a dense network of extensions. To isolate primary macrophage cells, the cerebral hemispheres of 1-day-old postnatal mice were digested with 0.1% trypsin. The cells were seeded into a six-well plate coated with poly-L-lysine and cultured with Dulbecco's modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; HyClone, Logan, UT, USA). The culture medium was refreshed twice per week for 2 weeks. Macrophages were detached by gentle shaking and filtered through a nylon mesh to remove astrocytes. After centrifugation at 1000 × g for 10 min, the cells were resuspended in fresh DMEM supplemented with 10% FBS and plated at a final density of 5 × 105 cells/mL on a poly-L-lysine coated 6-well culture plate. The following day, the cells were subjected to experiments. The cell purity was determined by immunohistochemical staining using a macrophage-specific CD11b antibody. The macrophage cultures used were > 95% pure.
Lentivirus encoding TIPE2 construction
A TIPE2 expressing lentivirus gene transfer vector was constructed by Genechem Co., Ltd., Shanghai, China. The identity of the vector was confirmed by PCR and sequencing analysis. The recombinant TIPE2 expressing lentivirus (Lv-TIPE2) and the control lentivirus (Lv-CON) were prepared at a titer of 109 TU (transfection unit)/ml.
Lentivirus transduction and detection assay
Macrophage were transduced with lentiviral vectorsat a multiplicity
of infection (MOI) of 50 in complete medium containing polybrene
(5 µg/ml) at 37 °C and 5% CO2 for 18–24 h. The cells were then
washed and cultured in fresh medium containing 10% FBS. Cells
were harvested 3 days following transduction.
Preparation of erythrocyte lysates
The spleens were removed from C57BL/6 mice. Single-cell suspensions of splenocytes were prepared using stainless steel mesh screens. Then, 1 × 105 splenocytes were incubated with 1 ml of red blood cell lysis solution for 20 min and centrifuged at 2000 rpm for 10 min. The supernatants were utilized as erythrocyte lysates.
Cell treatment
Macrophages (1 × 10 5) were stimulated with 10 µL of erythrocyte lysate. After 3 days, the supernatants were removed and further analyzed for cytokine production by ELISA. Neurons were cultured in a 96-well plate at a density of 1 × 104 cells per well. For the toxicity experiments, neurons were serum-starved for 4 h and then treated with a mixture of macrophage-conditioned media. For the MTT and apoptosis assays, the cells were treated for 48 h.
ICH model
Briefly, mice were anesthetized by an intraperitoneal injection of 400 mg/kg chloral hydrate and fixed on a mouse stereotaxic frame (Stoelting). A 20-µl volume of autologous nonanticoagulated blood was collected from the tail vein of the mice and then injected into the caudate nucleus at 0.8 mm anterior to bregma, 2 mm lateral to bregma the left, and 3.5 mm deep under stereotactic guidance at a rate of 2 µl/min over a period of 10 min. The needle was held in place for 10 min after injection, and the microsyringe was pulled out after the blood had coagulated. The craniotomy was then sealed with bone wax, and the scalp was closed with sutures. Body temperature was maintained at 37 °C throughout the procedure, and the mice were given free access to food and water after they woke up. The mice that died due to anesthesia were excluded.
Administration of TIPE2 Lentivirus
To study the effects of TIPE2, mice received an intracerebral ventricular injection of TIPE2 Lentivirus or control lentivirus (1 × 10 9 plaque forming units [Pfu], 2 µl of a 10 mg/ml solution prepared in 0.9% NaCl) 10 min after ICH and sacrificed 48 h after ICH.
RT-qPCR
Total RNA was extracted from mouse brain tissue by using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Reverse transcription was performed with a First Strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania). Real-time PCR was performed on an ABI 7300 Sequence Detection System with a SYBR-Green PCR kit (both Applied Biosystems; Thermo Fisher Scientific, Inc.). All primers used in this study were purchased from Sangon Biotech Co., Ltd. (Shanghai, China), and the sequences were as follows: TIPE2, forward 5'-TGA AAC TCA GGT CCG CTT CT-3' and reverse 5'-TCC TAG TGC TGC CTC CAA CT-3'. For qPCR, 1 µl of cDNA, 1 nM primers and 12.5 µl of 2 × SYBR Green were mixed to obtain a final volume of 25 µl. Thermal cycling was performed as follows: 40 cycles at 95 °C for 30 sec, 56 °C for 30 sec and 72 °C for 30 sec. Relative gene expression levels were determined using the comparative Cq method with GAPDH as the control.
Western blot analysis
Total protein was isolated from ipsilateral lesional brain tissues using ice-cold RIPA buffer. The protein concentrations were measured with the BCA Protein Assay Kit (Forevergen Biosciences). The protein samples were separated using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. The proteins were detected by incubation with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. The immunoblots were visualized using ECL plus Western Blotting Detection Reagents (Pluslight, Forevergen Biosciences, China). GAPDH (1:1000, Cell Signaling Technology, USA) was employed as the loading control. Densitometry analysis was performed with the use of ImageJ software with normalization to GAPDH.
Enzyme-linked immunosorbent assay (ELISA)
Macrophages (1 × 105) were stimulated with 10 µL of erythrocyte lysate. After 3 days, the supernatants were removed and further analyzed for cytokine production by ELISA. ELISA was performed according to the manufacturer's instructions (Dakewe Biotech, Shenzhen, China) to assess the concentrations of TNF-α, IL-1β and IL-6 in the culture supernatant.
MTT assay
The viability of neurons was assessed using a 3-(4,5-dimethyl-2 -thiazolyl) − 2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma-Aldrich) assay. After 48 h, MTT reagent was added to the wells, and the cells were incubated for 4 h at 37 °C and 5% CO2. After centrifugation, the supernatant was removed from each well. The colored formazan crystal produced from MTT was dissolved with 0.15 mL of DMSO, then the A490 optical density (OD) value was measured with a multiscanner autoreader (Dynatech MR 5000; Dynatech Laboratories, Chantilly, VA, USA). The absorbance was measured at 570 nm. The mean of the readings of triplicate wells was taken as the value. The OD value for the control cultures was considered 100% viability, and the viability of the other samples is expressed as a percentage of the viability of the control cultures.
Flow cytometry analysis of apoptosis
An annexinV-fluorescein isothiocyanate (FITC) apoptosis detection kit (OncogeneResearch Products, Boston, MA) was used to detect apoptosis. The cells were seeded in 1-mL flasks and incubated in DMEM supplemented with 10% fetal bovine serum until they reached approximately 90% confluence. Then, the cells were harvested, washed with ice-cold PBS twice, and resuspended in binding buffer (10 mM Hepes, pH 7.4, 50 mM NaCl, 2.5 mM CaCl2, 1 mM MgCl2, and 4% bovine serum albumin). Annexin V-fluorescein isothiocyanate (0.5 mg/mL) and propidium iodide (0.6 mg/mL) were then added to a 250 mL aliquot (1 × 106 cells) of this cell suspension according to the manufacturer’s protocol. After a 15-minute incubation in the dark at room temperature, the stained cells were immediately analyzed with a flow cytometer (Beckman Coulter, USA). All of the samples were assayed in triplicate, and the cell apoptosis rate was calculated using the following formula: apoptosis rate = (apoptotic cell number / total cell number) × 100%.
Caspase assay
To detect caspase-3-like protease activities, the ApoAlert Caspase-3 Colorimetric Assay kit (Clontech, Palo Alto, USA) was used. Cytosolic lysates were prepared 48 h following transfection and incubated with 50 mm p-nitroanilide (pNA) conjugated to the caspase cleavage site Asp-Glu-Val-Asp (DEVD) for 1 h at 37 °C. Hydrolyzed pNA was detected using a Multiscan MS colorimeter (Thermo Labsystems, Vantaa, Finland) at 405 nm. For control experiments, the lysates were incubated with 10 mM caspase-3 inhibitor DEVD-fmk (Clontech) for 30 min before addition of the substrate.
Histochemical evaluation of macrophage activation
Three days after ICH, the animals were deeply anesthetized with pentobarbital and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4). After the mice were perfused and fixed, the perihematomal regions of the 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 perihematomal sections were 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 a 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 color reaction was conventionally developed with diaminobenzidine (DAB) and H2O2. Six representative sections from the brain of each animal were selected. IPP6.0 image processing software (Media Cybernetics, MD, USA) was utilized to count the number of positive cells.
Evans Blue Extravasation
Briefly, under anesthesia, Evans blue dye (2%, 5 mL/kg; Aladdin, Shanghai, China) was injected into the left femoral vein over a period of > 2 minutes and allowed to circulate for 60 minutes. Then, the mice were euthanized by intracardial perfusion of sterile saline. The brain samples were weighed, homogenized in sterile saline, and centrifuged at 15000 × g for 30 minutes. After that, an equal volume of trichloroacetic acid was added to the resultant supernatant. The samples were then incubated overnight at 4 °C and centrifuged again at 15000 × g for 30 minutes. The resultant supernatant was spectrophotometrically quantified for extravasated Evans blue dye at 620 nm.
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 of chloral hydrate (n = 5). Next, the cerebral tissues were removed, and the water on the surface of the cerebral tissues was blotted with filter paper. The brain samples were immediately weighed with 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%.
Evaluation of Neurological Scores
Neurological scores were determined by the Neurological Severity Scale, a composite of motor, sensory, reflex, and balance tests. Neurological function was graded on a scale of 1 to 18; a score of 1 point was awarded for the inability to perform a 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).
Statistical analysis
All experiments were repeated three times. The significance of differences between groups was determined by two-tailed Student’s t test and two-way ANOVA. P values of less than 0.05 were considered statistically significant.