Animals
Adult male Sprague-Dawley rats (12-week old; 366–390 g) purchased from BioLASCO, Taiwan, and postnatal day 1 (P1) Sprague-Dawley rat pups from our breeding colony were used. Animals were housed in an AAALAC International-accredited Center for Laboratory Animals, with maintained room temperature (24 ± 1 °C) and 12 h light/12 h dark cycle. Standard laboratory rat chow and tap water were available ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Kaohsiung Chang Gung Memorial Hospital (IACUC Number: 2015100501), and were conducted in accordance with the Animal Protection Law set forth by the Council of Agriculture, Taiwan and AAALAC-International Guide for the Care and Use of Laboratory Animals.
Induction of focal cerebral ischemia
We used the middle cerebral artery occlusion (MCAO) method to induce acute focal cerebral ischemia in adult male Sprague-Dawley rats, following published procedures [25,26,27,28] with modifications. In brief, under 2% isoflurane anesthesia, a punctate incision with a 25G needle was made in the ventral wall of the left common carotid artery (CCA). A nylon monofilament suture (RWD Life Science; Shenzhen, China), 5 cm in length with a silicone-rubber coated tip (0.43 ± 0.02 mm diameter), was advanced into the CCA lumen towards the MCA via the internal carotid artery. The distance travelled was typically 20 mm. After 90 min of MCAO, the suture was withdrawn, the incision covered using cyanoacrylate glue, and patency of perfusion in the CCA verified. The wound was then closed after recording the end of occlusion time (beginning of reperfusion time). Animals were given 3 ml of normal saline subcutaneously to prevent dehydration, and were continuously monitored for full recovery from anesthesia. Sodium penicillin (10,000 IU; YF Chemical, Taiwan) was given intramuscularly to prevent postoperative infection. As a routine, the effects of 90-min transient MCAO, and 6 h or 24 h after reperfusion (MCAO/R) were evaluated in this study. Animals that received the same surgical procedures under isoflurane anesthesia but without MCAO served as the sham controls. The selection of animals to receive MCAO, MCAO/R or as the sham-controls was completely randomized. Detailed procedures for induction of focal cerebral ischemia are described in Additional file 1: Supplementary materials and methods.
Magnetic resonance imaging (MRI)
We carried out sequential MRI acquisition using a 9.4 T horizontal-bore animal MR scanning system (Biospec 94/20; Bruker, Ettingen, Germany) in rats under 1.5% isoflurane anesthesia to measure brain infarction and edema. T2-weighted coronal imaging (T2WI) was carried out using multislice turbo rapid acquisition with refocusing echoes (Turbo-RARE) sequence; ImageJ version 1.48v was used to quantify areas of hyperintensity in the acquired images (Additional file 2: Figure S1a). Diffusion weighted imaging (DWI) was performed using DtiEpi sequence on the same spatial brain dimension as in T2WI. ParaVision 5.1 software (Bruker) and MIstar (ver. 3.2.63; Apollo Medical Imaging Technology, Melbourne, Australia) were applied to process the DWI and apparent diffusion coefficient (ADC) maps respectively. The parameters for MRI acquisition were provided in Additional file 1: Supplementary materials and methods.
Triphenyltetrazolium chloride staining
2,3,5-Triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS) at 0.05% (w/v) concentration and used immediately for staining. The brain was isolated, sliced into serial 2-mm-thick coronal sections, and incubated in 0.05% TTC solution at 37 °C for 45 min [29]. After staining, the slices were washed in PBS and fixed in 4% paraformaldehyde (Sigma-Aldrich) at 4 °C for 24 h. Quantification of infarct volume was based on the ratio between areas stained in red indicating normal tissue and areas stained in white indicating infarct lesion (Additional file 2: Figure S1b).
Immunofluorescence staining
As reported previously [30], immunofluorescence staining was carried out using a mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (Thermo Fisher Scientific, Rockford, IL, USA) antibody or rabbit polyclonal anti-Pnn antibody (Sigma-Aldrich). The secondary antibodies used included a goat anti-rabbit IgG conjugated with Alexa Fluor 488 and a goat anti-mouse IgG conjugated with Alexa Fluor 568 (Thermo Fisher Scientific). Viewed under a Fluoview FV1000 laser scanning confocal microscope (Olympus; Tokyo, Japan), immunoreactivity for GFAP exhibited red fluorescence, and Pnn exhibited green fluorescence. ImageJ (version 1.48) was used to quantify GFAP immunoreactivity in the cortical and striatal areas.
Culture of rat primary astrocytes
Culture of rat primary astrocytes was performed as described previously [31] with modifications. Briefly, the cerebral cortex was aseptically dissected from postnatal day 1 (P1) Sprague-Dawley rat pups and placed in Gibco Minimum Essential Media (MEM; Thermo Fisher Scientific) containing 2 mg/ml trypsin (Sigma-Aldrich). After incubation at 37 °C in the CO2 incubator for 30 min, the cortical tissues were mechanically triturated with pipette until they dissociated into single cells. Fetal bovine serum (FBS; 10% final concentration) was then added to stop the activity of trypsin. Cells were seeded into polyethylenimine-coated T75 culture flask at a density of 2 × 106 cells and incubated at 37 °C in the CO2 incubator for 3–4 h until the cells attached. On replacement with Dulbecco’s Modified Eagle Medium (DMEM; Thermo Fisher Scientific) that contains high glucose supplemented with N-2 supplement, 10% heat-inactivated FBS and 1% penicillin/streptomycin, cells were incubated at 37 °C in a humidified 5% CO2 incubator. After 7–8 days, the confluent cultures were shaken for 30 min to minimize microglia contamination. The remaining primary astrocytes were trypsinized and re-seeded for further experiments.
Oxygen-glucose deprivation and re-oxygenation
Oxygen-glucose deprivation (OGD) and re-oxygenation (OGD/R) were employed to mimic the hypoxic-ischemic condition of stroke in in vitro experiments. Hypoxic exposure was provided by a Heracell 150i CO2 incubator (Thermo Fisher Scientific, Waltham, MA, USA). Glucose-free DMEM was prepared by gassing in the hypoxic chamber with 5% CO2, 1% O2, and 94% N2 for at least 12 h. For the OGD experiment, the culture medium was removed and astrocytes were washed once with pre-warmed (37 °C) PBS. OGD was induced by incubating the primary astrocytes in the pre-gassed glucose-free DMEM medium in a hypoxic chamber with 5% CO2, 1% O2, and 94% N2 for 24 h. The normoxic control cells were incubated at 37 °C in a humidified atmosphere of 95% air/5% CO2. Following OGD exposure, the incubation medium was replaced with the conditioned medium and the cultured cells were returned to a CO2 incubator at 37 °C. Cells were collected after 24 h of OGD exposure or after 24 h of OGD/R for subsequent experiments. The selection of astrocytes to receive normoxia, OGD or OGD/R was completely randomized.
siRNA transfection
ON-TARGET plus SMART pool for rat Pnn siRNA (Cat# L-100331-02-0050) was obtained from Dharmacon (GE Healthcare, Lafayette, CO, USA). The siRNA was resuspended in 1x siRNA buffer (60 mM KCl, 6 mM HEPES-pH 7.5, and 0.2 mM MgCl2) to attend a 20 μM stock concentration. Rat primary astrocytes cultured until reaching 40–50% confluent were transfected with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The RNAi duplex-Lipofectamine RNAiMAX complexes were prepared in Gibco Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific) and incubated for 5 min at room temperature. The Lipofectamine RNAiMAX was used to transfected the primary astrocytes in 100-mm culture dishes at a final concentration of 10 nM siRNA. After 48 h of incubation at 37 °C in a CO2 incubator, siRNA-transfected primary astrocytes were used for OGD and OGD/R studies. For non-specific siRNA control, the ON-TARGET plus Non-Targeting Pool (Cat# D-001810-10-20) obtained from Dharmacon was used. The effective concentrations and transfection time for siRNA treatment were determined according to the results presented in Additional file 3: Figure S2.
WST-1 cell viability assay
Cell viability was evaluated using Cell Proliferation Reagent WST-1 (Roche, Basel, Switzerland) according to the manufacturer’s protocol. Primary cultured astrocytes were seeded into 96-well plates at a density of 5 × 104 cells per well. After replacing the incubation medium with the WST-1 Reagent diluted in a fresh growth medium (1:10), cells were incubated at 37 °C in a CO2 incubator for 4 h. Absorbance was then measured at 450 nm with the reference wavelength at 650 nm using a Thermo Scientific Multiskan Spectrum microplate spectrophotometer.
Lactate dehydrogenase leakage assay
Lactate dehydrogenase (LDH) released from damaged primary astrocytes into the culture medium was determined using a Cytotoxicity Detection KitPLUS (LDH) kit (Roche) according to the manufacturer’s instructions. The LDH reaction mixture (100 μl) was added to 100 μl of culture medium and incubated for 30 min at room temperature. Stop solution was added and the absorbance was measured at a wavelength of 490 nm using a Thermo Scientific Multiskan Spectrum microplate spectrophotometer.
Determination of ATP levels
Changes in cellular ATP levels were determined using an ATP Detection Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). Primary astrocytes were lysed with RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS), followed by homogenization through sonication and centrifugation at 12,000 x g for 15 min at 4 °C. The supernatants were collected and used for ATP determination. ATP reaction mixture containing luciferase-luciferin buffer (100 μl) was added to 10 μl of cell lysate. Luminescence was measured using a Centro LB 960 Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany). ATP values were determined from a standard curve and normalized to the protein content of each sample.
Flow cytometry
To determine cell death status, primary astrocytes were harvested and washed, and stained with the eBioscience Annexin V-FITC Apoptosis Detection Kit (Invitrogen, Carlsbad, CA, USA). For each experiment, cells harvested from OGD or OGD/R group pooled from two dishes were used. The stained cells were analyzed by the Gallios Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA). Annexin V- and propidium iodide (PI)-double negative cells were classified as viable, annexin V-positive and PI-negative as early apoptotic, annexin V- and PI-positive cells as late apoptotic, and annexin V-negative and PI-positive cells as necrotic. Data from the experiments were analyzed by the Kaluza software (Beckman Coulter). Detailed procedures were described in Additional file 1: Supplementary materials and methods and Additional file 4: Figure S3.
Protein extraction and Western blot analysis
Cortical or striatal tissues from animals, as reported previously [32], were homogenized on ice in a T-PER tissue protein extraction buffer (Thermo Fisher Scientific) that contains protease and phosphatase inhibitors, and centrifuged at 10,000 x g at 4 °C for 10 min. In some experiments, proteins from the cytosolic fraction of the samples were extracted by a commercial kit (Active Motif, Carlsbad, CA, USA). Primary astrocytes, as described previously [33], were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS and 1% protease and phosphatase inhibitor), followed by homogenization through sonication and centrifugation at 12,000 x g for 15 min at 4 °C. In all cases, the supernatant was collected, and the concentration of total proteins was determined by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
Protein samples (50 μg) were electrophoresed on a 7.5% or 12.5% SDS-PAGE gels and then transferred onto a polyvinylidenedifluoride (PVDF) membrane. The membrane was incubated overnight at 4 °C with primary antibodies: anti-Pnn (Abcam, Cambridge, MA, USA), anti-ERK (Cell Signaling Technology, Danvers, MA, USA), anti-phospho-ERK (Cell Signaling Technology), anti-Bcl2 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-cleaved caspase-3 (Cell Signaling Technology), or anti-β-actin (Merck Millipore, Darmstadt, Germany). The membrane was subsequently incubated with a specific horseradish peroxidase (HRP)-conjugated secondary antibody for 90 min, visualized through enhanced chemiluminescence using the UVP BioSpectrum 600 Imaging System (Analytik Jena, Jena, Germany). The immunoblot bands were quantified using VisionWorks LS Image Acquisition and Analysis Software (Analytik Jena), and were expressed as the ratio relative to β-actin protein.
Statistical analysis
All values are expressed as mean ± SEM. One-way or two-way analysis of variance with repeated measures was used to assess group means, followed by the Scheffé multiple range test for post hoc assessment of individual means. P < 0.05 was considered statistically significant. It should be mentioned that analysis of the experimental data was carried out in a single-blind manner.