Organotypic Hippocampal Slice Cultures (OHSC)
All animals were cared for following procedures approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco. OHSC were prepared from 7-day-old Sprague-Dawley rats (Charles River, Wilmington, MA). Rat pups were anesthetized with 2-3% isofluorane, decapitated and their hippocampi dissected out and placed in 4°C Gey's Balanced Salt Solution (UCSF Cell Culture Facility, San Francisco, CA). Next, the hippocampi were transversely sliced (400 μm) with a tissue slicer (Siskiyou Design Instruments, Grants Pass, OR), transferred onto 30 mm diameter membrane inserts (Millicell-CM; Millipore, Bedford, MA), placing 6 slices / insert. The inserts were placed into six-well culture trays adding 1.2 ml of culture medium per well. The slice culture medium consisted of 50% Minimal Essential Medium (Eagles with Earle's Balanced Salt Solution; UCSF Cell Culture Facility), 25% Earle's balanced salt solution (UCSF Cell Culture Facility), 25% heat-inactivated horse serum (UCSF Cell Culture Facility), Penicillin-Streptomycin 50 μg/ml, and the glucose 1.8 mg/ml. The OHSC were grown in an incubator in 5% CO2 / air at 37°C for 9 days, with the media changed every second day. The slices were selected with propidium iodide (PI, 2 μg/ml; Invitrogen) before further analysis to exclude those damaged during processing.
In Vitro Oxygen and Glucose Deprivation (OGD)
Before OGD, hippocampal slices were assigned randomly to different treatment groups: four groups received OGD and one of the treatments: vehicle (5 μl of 0.1M PBS), recombinant human erythropoietin (EPO; R&D Systems; 1 IU/ml of medium; the dose was established by a dose response curve with greatest effect at the selected dose, data not shown); anti-EPOR antibody (M-20, Santa Cruz; 2.5 μg/ml of medium); or wortmannin (Sigma-Aldrich; 100 nM). An additional group was not exposed to OGD and served as the control group. Anti-EPOR was used with the intention to block the EPO-EPOR signaling pathway at the receptor site. Wortmannin was chosen to block downstream EPO signaling since it is a specific PI3K inhibitor, blocking one of the signaling pathways upstream of AKT (Kilic et al., 2005
Hippocampal slices were exposed to OGD by transferring the inserts to glass vials containing 20 ml of glucose-free artificial rat spinal fluid (aCSF), which was bubbled for at least 30 minutes prior and throughout the experiment with 95% N2 / 5% CO2. The partial pressure of oxygen of this solution was less than 2 mmHg (measured with a Clark oxygen electrode; Cameron Instrument Co., Port Aransas, TX). The vials were immersed in a water bath so that aCSF temperature was 37° ±0.2°C, monitored with a thermocouple thermometer. After 10min of OGD, slices were removed and recovered in fresh culturing medium with the added treatment (if applicable), prewarmed to 37°C. An additional group of OHSC slices was used initially as OGD-sham, where instead of aCSF normal culturing medium was used and was bubbled with 95% air / 5% CO2; we have not found any significant difference from the non-OGD control and therefore omitted this data. The aCSF was composed of NaCl (124 mM), NaHCO3 (25.7 mM), KCl (4.0 mM), MgCl2 (1.0 mM), CaCl2 (2.5 mM), KHPO (1.0 mM), with the pH adjusted to 7.30.
Cell viability after OGD
Cell viability after OGD was assessed in a separate set of experiments using PI fluorescence. At the beginning of the experiment, PI was added to the OHSC medium (5 μg/ml) for 30 min, after which the medium containing PI was replaced with Earl's basic salt solution and washed with it 4 more times. The fluorescent digital images of OHSC were taken using a Nikon Diaphot 200 inverted microscope (Nikon Corporation, Tokyo, Japan) and SPOT Jr. Digital Camera (Diagnostic Instruments Inc., Sterling Heights, MI). Excitation light wavelength was 490 nm and emission was 590 nm. All OHSC except those in the control group were then exposed to OGD, placed back in normal culture medium and treated according to the treatment groups they belonged to (described under OGD). Twenty-four hours later they were re-imaged the same way. Cells were then exposed for 1 hour to an Earle's solution containing 100 uM NaNC (sodium cyanide) and 1 mM iodoacetic acid to kill all living cells, and re-imaged for the third time.
The images were analyzed with ImageJ software (version 1.42k; U. S. National Institutes of Health, Bethesda, MD). Mean PI fluorescence was measured for the CA1, CA3 and DG regions as well as for the whole slice. Cell death was expressed as the percentage of total death using the following calculation: (PI fluorescence after treatment – PI fluorescence before tretament) / PI fluorescence after total death * 100.
Hippocampal precursor cell isolation and culture
Hippocampal precursor cells were isolated as described previously (Sall et al., 2009
). Sprague-Dawley rats were separated from the dam on postnatal day 2 and decapitated. Hippocampi were immediately dissected out and placed in 10mL ice cold Hanks Basic Salt Solution without calcium. Whole hippocampi pooled from 10 animals were collected by centrifugation for 2 minutes at 300rcf (relative centrifugal force). Supernatant was removed and hippocampi were minced with a razor blade then triturated 5 times using a 200 μl pipette. Cells were then re-suspended in 10mL Hanks Basic Salt Solution for 2 minutes. After settling for 2 minutes the supernatant was transferred to a new tube. Cells were then collected by centrifugation at 300rcf for 5 minutes. Supernatant was discarded and cells were re-suspended in proliferation medium consisting of 3:1 Dubelco's Modified Eagles Medium: Ham's F12 (UCSF cell culture facility), 1% penicillin and streptomycin, 1× B-27 supplement (Invitrogen, Grand Isle, NY), 20ng/mL basic fibroblast growth factor (Chemicon, Temecula, CA), 0.75units heparin/ml (Abraxis, Schaumburg, IL). HPC were then grown in an incubator in 5% CO2
/ air, at 37°C with media changed 3× per week. Non-adherent proliferating neural precursor cells were transferred to a new cell culture flask every 5 to 7 days to purify multipotent HPCs from differentiated progeny. HPC had been grown in culture for 2 to 3 weeks at the time of use.
Before plating the cells into 8 well chamber slides (Thermo Fisher Scientific, Rochester, NY), the chambers were coated with poly-L-ornithine and laminin, washed with PBS and than filled with 250 μl of culturing medium. Depending on the treatment groups, EPO was added to some wells (1 IU/ml).
In vitro hypoxia of HPC and differentiation
In vitro hypoxia was induced by placing the 8 well chamber slides carrying the HPC in a modular incubator chamber (Billups-Rothenberg, Del Mar, CA) which was flushed with 95% N2 / 5% CO2 for 15 min, decreasing the PO2 to 0 mmHg, as measured with the MiniOX I oxygen analyzer (MSA Medical Products, Pittsburgh, PA), and then sealed. The slides were left in the chamber at 37°C for 4 hours and afterwards placed back into the standard incubator conditions of 95% air / 5% CO2.
To differentiate HPC, basic fibroblast growth factor containing medium was replaced with differentiation medium consisting of Neurobasal-A (Invitrogen, Grand Isle NY), B27 supplement, 1% penicillin-streptomycin (UCSF cell culture facility, San Francisco, CA), L-glutamine (Invitrogen, Carlsbad CA), and 5% fetal bovine serum (UCSF cell culture facility, San Francisco, CA). After 24 hours, the medium was replaced with a serum free differentiation medium.
Western blot analysis (WB)
Groups of OHSC were used for Western blot analysis 72h after OGD, during which time the medium was not changed. The inserts carrying OHSC were flushed briefly with ice cold 0.1 M phosphate-buffered saline (PBS) before the slices were transferred into glass tubes containing ice cold lysis buffer composed of HEPES pH 7.9 (10mM), KCl (10mM), EDTA (0.1 mM), and EGTA (0.1 mM). Protease inhibitors and reducing agents were also used: pepstatin A (2 μg/ml), phenylmethylsulfonyl fluoride (PMSF; 0.5 mM), a protease inhibitor cocktail tablet (Roche diagnostics GmbH, Mannheim, Germany), and dithiothreitol (DTT; 1 mM). Protein concentration was determined by the Bradford method (BioRad, Richmond CA) using bovine serum albumin (BSA) as the standard. Equal amounts cytoplasmic protein (40 μg) were separated on 4 – 12% SDS polyacrylamide gels (Invitrogen Life Technologies, CA). Rainbow recombinant protein molecular weight marker (Amersham) was used as a size reference. Proteins were transferred to PVDF membranes (BioRad Laboratories, CA). The membranes were incubated in blocking buffer, 5% nonfat dry milk in TBST (20 mM Tris – HCl, pH 7.6, 137 mM NaCl, 0.1% Tween-20), at room temperature for 2 h with rotation. The membranes were then incubated with rabbit antibody against doublecortin (DCX; Cell Signaling, 1:1000) overnight at 4°C. Mouse anti-β-actin monoclonal antibody (Santa Cruz Biotechnology, 1:2000) was used as a loading control for protein quantization. Following rinses with TBST, the membranes were incubated with peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology, 1:2000) in blocking solution for 1 h. After rinses with TBST, signal of bound antibodies was developed by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL). ImageJ was used to measure the optical densities of the protein signals on scans of X-ray films. The relative optical density was calculated using the optical density of DCX protein signals divided by the optical density of loading control (β-actin) for each experiment.
To evaluate the EpoR expression in HPC after OGD, the cells were kept in proliferation medium, exposed to 4h of hypoxia, and collected in lysis buffer 24 hours after start of hypoxia. Collected samples were then analyzed as above.
At 9 DIV the OHSC were exposed to OGD, treated according to the treatment group they belonged to, and incubated under standard conditions for 5 additional days, changing the medium on the third and fourth day. On the fourth day, 20 μM 5-bromo-2-deoxyuridine (BrdU, Sigma) per well was also added to culturing medium. After five days, cultures were washed briefly in 0.1 M ice-cold PBS and fixed in ice-cold 4% paraformaldehyde in PBS for 1 hour. After 24 h incubation with 30% sucrose, OHSC were cryosectioned (Leica CM 1850, Leica Instruments, Nussloch, Germany) and 20 μm coronal sectioned slices were used for immunodetection. DNA denaturation was achieved by treatment with 2 M HCl at 37 °C for 10 min and 500 mM boric acid pH 8.4 at room temperature for 10 minutes. Thereafter OHSC were incubated for 2 hours in blocking solution (10% goat serum, 0.3% Triton X-100 in PBS) and overnight with mouse anti-BrdU antibody (1:500; BD Biosciences) or rat anti-BrdU antibody (1:200; Abcam) and rabbit anti-β-III tubulin antibody (Tuj1; 1:2000; Covance), mouse anti-GFAP antibody (1:500; Millipore), mouse anti-ED1 antibody (1:50; Millipore) or rabbit anti-NG2 antibody (1:1000; Millipore) in blocking solution containing 2.5% goat serum at 4 °C. After rinsing with PBS, the slices were incubated for 2 h at room temperature with secondary Alexa Fluor 488 and/or 568 (Invitrogen, 1:500). After rinsing, the slices were exposed to DAPI for 5 minutes and then coverslipped with ProLong Gold (Invitrogen). Controls included omission of primary antibodies. The images were taken using a Nikon TE2000 C1 spectral confocal microscope (Nikon, Tokyo, Japan) at the Nikon Imaging Center, UCSF. To estimate the number of proliferating cells in the DG 4-5 days after OGD, the OHSC images were analyzed using ImageJ software. The mean BrdU fluorescence in the region was measured for the control, vehicle and EPO-treated groups of OHSC.
The undifferentiated HPC were placed in 8 well chamber slides, fixed and then processed using a similar protocol as for OHSC to stain for the markers Sox2 (BD Biosciences, 1:500) and Nestin (BD Biosciences, 1:500). The differentiated HPC were fixed 5 days after hypoxia and stained using anti-Tuj1 and anti-GFAP antibodies. To count the number of cells that differentiated either into Tuj1 positive or GFAP positive cells, the images were analyzed with the Metamorph Offline software (Molecular Devices, Sunnyvale, CA; version 7.0r3).
A statistical analysis was performed by using SPSS 16 for Mac (SPSS Inc, Chicago, IL). Numerical data were collected from at least three different experiments and are presented as mean ± standard error of the mean or standard deviation (for EPOR expression on NPC). Data from immunoblots were evaluated using one way ANOVA with Bonferroni post-hoc testing. Students t test was used for comparison of the ratio of GFAP/Tuj1 positive cells between different groups of HPC. Differences were considered significant at P < 0.05.