Male Wistar rats (240–260 g) were obtained from the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University, and were housed with free access to food and water in a temperature- and humidity-controlled facility with a 12 h light/dark cycle. Prior to experimental utilization, all animals were given a 1 week acclimatization period. All animal studies were approved by the School of Medical Science of Harbin Medical University, with all efforts having been made to reduce animal use and suffering when possible.
Reagents and antibodies
Rabbit anti-rat MAP1LC3 were purchased from Cell Signaling Technology (USA). Rabbit anti-rat Sphk1 was obtained from NOVUS. Rabbit anti-rat SQSTM1, rabbit anti-rat TRAF2, mouse anti-rat Ibal, and mouse anti-rat Neun were purchased from Abcam (USA). Fluorescently labeled anti-rabbit and anti-mouse IgG were from Invitrogen (USA). A TUNEL staining kit was obtained from Roche (Switzerland). S1P, Rapamycin (RAP), and 3-MA were obtained from Sigma. An siRNA specific for rat TRAF2 was purchased from GenePharma Corporation (Shanghai, China).
Focal cerebral ischemia-reperfusion model
In order to study CIRI in vivo, we employed a slightly modified version of the previously described middle cerebral artery occlusion (MCAO) model (Lv et al., 2011). Briefly, 10% chloral hydrate (350 mg/kg) was intraperitoneally injected into rats in order to achieve deep anesthesia, after which the neck was opened such that the common carotid artery (CCA) was visible. The proximal ends of the CCA and the internal CA (ICA) were then clamped, and a small V-shaped incision was made in the external carotid artery (ECA). A bolt of thread was then inserted through this incision for MCAO embolization, with a knot being created at the site of thread entry. The clamp on the CCA was then loosened, with the CCA being adjusted until positioned 45 degrees to the right lateral side. The thread was then inserted further until resistance was detected. Blood flow was disrupted for 2 h, after which the embolic thread was removed to the ECA stump as a means of simulating IRI. Control animals underwent the same surgical manipulation but without thrombus insertion. After completion of all surgical procedures, rats were warmed using heating pads and were individually housed with free access to food and water. Animals were subjected to downstream analyses at 12, 24, and 48-h post-surgery.
Immunofluorescent staining was used to visualize the microglial expression of Sphk1 (NOVUS Biologicals, JA31-14), MAPLC3 II (Cell Signaling Technology, D11) , SQSTM1/p62 (Abcam, ab155686), and TRAF2 (Abcam, ab126758) in our experimental system. Briefly, we initially used xylene to deparaffinize ischemic penumbra tissue sections from model animals. Sections were then treated with citric acid to facilitate antigen retrieval, followed by three washes in PBS. Samples were next blocked for 1 h using serum, followed by probing overnight at 4°C with appropriate primary antibodies, further washing, and probing for 1 h at room temperature using fluorescently conjugated secondary antibodies. Samples were then imaged via Olympus FV300 confocal microscope (200×). To ensure accuracy, 4-5 fields of view in different cortical regions were selected at random, with positive areas being averaged across these regions.
The expression of Sphk1 (NOVUS Biologicals, JA31-14), MAPLC3 II (Cell Signaling Technology, D11), SQSTM1/p62 (Abcam, ab155686) and TRAF2 (Abcam, ab126758) in OGDR and MCAO model systems was measured via Western blotting. Briefly, protein levels in ischemic penumbra tissue samples were quantified via BCA assay. Next, 10-12% SDS-PAGE was used to separate the proteins, and they were transferred onto cellulose acetate membranes. Blots were next blocked for 2 h using 5% non-fat milk followed by overnight probing with primary antibodies at 4°C. Blots were then washed prior to probing for 1 h with appropriate fluorescent secondary antibodies. Blots were then imaged with an infrared Odyssey 3.0 instrument, with β-actin being used as a means of normalizing protein expression across samples.
Lentiviral knockdown of SphK1
SphK1-knockdown rats were generated via administering 3.5% chloral hydrate (350 mg/kg) to anesthetize rats and placing them within a stereotactic device. A lentivirus targeting SphK1 (GenePharma Corporation Shanghai, China) was then directly administered (2 μg/μl) to the right lateral ventricle at a position 1.0 mm behind the anterior iliac crest, 2.0 mm to the side of the stereotactic instrument using the midline for guidance, and 3.5 mm ventral of the surface of the skull. At 2 weeks post-injection, rats were subjected to MCAO procedures.
Injection of inhibitors
Selected inhibitory compounds (RAP and 3-MA, Sigma) were administered at doses selected based upon prior studies (23-25). RAP and 3-MA were both delivered via intracerebroventricular injection (i.c.v.). Dosing with RAP (35 pmol; dissolved in ethanol and diluted to < 2% ethanol using normal saline) was conducted at both 2 h prior to MCAO and 24 h post-perfusion, while 3-MA was administered at the onset of reperfusion.
Culture of primary cells
Neonatal rats (1-2 days old) were used as a source for primary microglia. Briefly, animals were euthanized and the meninges and cortical tissue were separated from one another. The cortices were then dissociated and passed through a 200 μm filter. The filtered cells were resuspended in high glucose (4.5 g/L) DMEM containing 5% fetal calf serum and penicillin/streptomycin (all from Hyclone, USA). Cells were then cultured at 37 °C. On day 10 of this culture process, flasks were shaken overnight (300 rpm) at 37 °C, and purified microglia were collected and cultured for 2-3 days in media prepared as above (complete media) with the addition of 1% microglia growth supplement (ScienCell, USA). Immunocytochemical staining for the microglial marker Iba1 was used to confirm that these cultures were at least 98% pure.
To culture primary neurons, SD rats (embryonic day 17) were utilized as in previous reports (26). First, meninges were removed and cortical tissue was dissected, with the resulting tissue fragments being used to prepare a single cell suspension via passage through a 200 μm filter. The resultant cells were then cultured for 7-9 days using Neurobasal ™ medium supplemented with 2% B27 supplement (both from Invitrogen) and penicillin/streptomycin, after which cells were used for downstream experiments. Cultures were confirmed to be at least 96% pure based on staining for NeuN.
Lentiviruses carrying an SphK1-specific shRNA (Lv-SphK1-shRNA) were prepared using the AdEasy lentivirus Vector System (GenePharma, Shanghai, China) via cloning the appropriate sequence into the pShuttle-CMV vector, followed by subsequent recombination into the pAdeasy-1 lentiviral vector. The resultant lentivirus was used to infect microglia for 24 h at a high titer (MOI = 100). Media was then exchanged for fresh complete media with appropriate supplements and the cells were cultured for 24 h, after which they were again infected as above.
Cells were used for in vitro OGDR modeling experiments. OGDR conditions were achieved by removing normal cellular media and instead culturing cells for 2 h in Earle’s balanced salt solution (with no added glucose) at 37°C in a low-oxygen (95% N2/3% CO2/2%O2) incubator. In parallel, control cells were cultured in Earle’s balanced salt solution containing 10 mM glucose in a normoxic incubator. After the 2 h low-oxygen incubation, cells were then returned to a normoxic incubator as a means of initiating reperfusion.
Phosphate-buffered glutaraldehyde (2.5%) and osmium tetroxide (1%) were used to fix samples, after which they were dehydrated using a series of graded acetone solutions. Epoxy resin was then used to embed tissues, after which semi-thin (1 mm) tissue sections were prepared and toluidine blue-stained. Sections that were 600-Å thick were then prepared from these stained sections, with the resultant sections being lead citrate and uranyl acetate stained. A transmission electron microscope (JEM-1200X, SHIMADZU, Japan) was then used to visualize 5-8 random fields of view per brain section (13,500 ×; n=6 rats/group).
In order to knock down TRAF2 expression, microglia cultured as above were transfected with either a negative control siRNA or a rat TRAF2-specific siRNA at 70 nM with the XtremeGENE siRNA transfection reagent (Roche) based on provided directions. Briefly, cells were plated in 12-well plates at 1.4×105/well, and were allowed to rest for 48 h post-transfection prior to downstream use. A pEGFP expression vector (BD Biosciences Clontech, CA, USA) was used in parallel wells as a means of monitoring the efficiency of transfection, which was usually > 70%.
Measurement of apoptotic cell death
The survival of OGDR-treated cells was analyzed using a TUNEL staining kit (Roche, Switzerland). Briefly, neurons that had been cultured on coverslips were fixed using 4% paraformaldehyde, after which they were treated using 0.3% hydrogen peroxide to quench any endogenous peroxidase activity. Samples were then incubated for 1 h with the TUNEL reaction solution, followed by a 30 minute counterstain with DAPI at 37 °C. After staining, cell samples were assessed via fluorescent microscopy (Olympus), with the TUNEL staining frequency being determined by normalizing TUNEL-positive areas in individuals to total DAPI-positive area.
OGDR-induced production of IL-1β, IL-6, and TNF-α by microglia was quantified in cell culture supernatants at the indicated time points via ELISA based on provided directions. Briefly, 96-well plates were initially coated overnight with capture antibodies specific for these cytokines of interest (2–4 μg/ml). Next, 1% BSA was used to block these plates, after which 50 μl of appropriate standards or samples were added to each well. Plates were then washed and probed with a biotinylated secondary antibody, followed by further washing and streptavidin-mediated color development. Absorbance at 450 nm was then measured to quantify protein levels.
Detection of apoptosis by flow cytometry
Primary neurons were seeded in 6-well plates at a density of 5 × 105 cells/well. After 7 days, they were transduced with an appropriate lentivirus, and were then subjected to hypoxic conditions. Cells were then collected, washed with PBS, and 1x105 cells were resuspended in 500 μL of binding buffer. Cells were then stained using an Annexin-V-Fluos kit based on provided directions, with 100 μL of reaction solution being added per sample containing HEPES, Annexin-V, and PI. After staining for 15 minutes, 1 mL of PBS was added per sample, and cells were then analyzed via flow cytometry.
Data are presented as means ± standard deviation from triplicate experiments. Data were compared via Student’s t-tests and ANOVAs as appropriate using SPSS v22.0. P < 0.05 was the significance threshold for these analyses.