Biochemical experiments involving animals were performed on brains removed from 8–10-week-old male wild-type, SR−/−
(Basu et al., 2009
) and nNOS−/−
animals. Animals were maintained on a 12 h light/dark cycle at a room temperature of 23°C, with free access to food and water. All animal-use procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals
and approved by the Johns Hopkins University Animal Care and Use
Antibodies to SR and nNOS were from BD Biosciences (San Jose, CA), to NMDA receptor NR1, β-tubulin and actin from Millipore (Billerica, MA), and to GAPDH from Calbiochem (San Diego, CA).
Oxygen-glucose deprivation (OGD)
The OGD experiments were done as described previously (Eliasson et al., 1997
). Briefly, cortical cultures grown for 12–14 days in Neurobasal media supplemented with horse serum at 37°C were treated with 10 mM L-serine for 5 h and then exposed to a gas mixture of 5% CO2
in an airtight chamber with OGD buffer containing 125 mM NaCl, 3 mM KCl, 1.6 mM CaCl2
, 0.2 mM arginine, 25 mM HEPES pH 7.4, 1 mM D-glucose and 1.25 mM Na2
for 1 h at 37°C. The OGD solution was then replaced with regular neuronal culture media and the cells grown for 24 h at 37°C. Toxicity was assayed by microscopic examination with computer-assisted cell counting following staining of all nuclei with 1 μg/ml Hoechst 33258 stain and dead cells with 7 μM propidium iodide. Total and dead cells were counted, and percent cell death determined. Experimenters were blinded.
Middle cerebral artery occlusion (MCAO)
All mice were 8–10-week-old male SR−/−
and matched wild-type littermates weighing between 20–25 g. Transient focal ischemia was induced by a 90 min occlusion of the middle cerebral artery (MCA) in wild-type and SR−/−
mice, as described previously (Zeynalov and Doré, 2009
). Successful occlusion was confirmed by an 87–90% reduction in cerebral blood flow (CBF), as measured by laser-Doppler flowmetry. After reperfusion was begun and isoflurane anesthesia was discontinued, the animals were kept in a humidified thermo-regulated chamber until they became completely awake (usually within 20–30 min). At 24 h of reperfusion, the mice were deeply anesthetized and the brains processed for analysis of infarct volume. Brains were harvested, sliced into 2 mm thick sections and stained with 1% 2,3,5-triphenyltetrazolium chloride. Infarct volume was calculated as a percent of the contralateral hemisphere and corrected for swelling.
NMDA excitotoxicity assays
Weight and rectal temperature of each mouse was recorded before the surgical procedure. Anesthesia was induced with 3.0% halothane and thereafter maintained at 1.0% halothane. Each mouse was mounted on a stereotaxic frame, and 0.3 μL of NMDA (67 mM), prepared in phosphate-buffered saline, was injected into the right striatum over a 2 min period; the needle was left in situ for an additional 5 min to prevent back flow. After injections, mice were placed in a humidified, thermoregulated chamber maintained at 31°C and were returned to their cages after full recovery from anesthesia. Throughout the experimental procedure, mouse rectal temperature was monitored and maintained at 37.0 ± 0.5°C. Forty-eight hours after injection, brains were harvested and immediately frozen in 2-methylbutane (pre-cooled over dry ice); 20 μm sections were cut on a cryostat and stained with cresyl violet to measure lesion volume. Brain sections were photographed and analyzed with SigmaScan Pro 5.0.
NO formation was assessed in cortical neurons cultured for 12–14 days at 37°C or from 6–8 week-old mouse brains. Cultures, treated with 10 mM L-serine for 5 h, were incubated 5 min with 2 μM 4-amino-5-methylamino-2′7′-difluorofluorescein diacetate (DAF-FM DA) (Invitrogen), a specific dye that emits fluorescence intracellularly only upon interaction with NO. The cells then were gently washed with fresh media and subjected to immunofluorescence microscopy with excitation wavelength at 495 nm and emission wavelength at 515 nm for 15 min with continuous signal recording. For measurements from mouse tissue, brains were sliced into 300 μm sections using a Mcllwain Tissue Chopper and equilibrated with 95% oxygen/5% CO2 at 37°C for 30 min in pre-oxygenated artificial cerebrospinal fluid (ACSF) buffer containing 125 mM NaCl, 3 mM KCl, 1.6 mM CaCl2, 0.2 mM arginine, 25 mM HEPES pH 7.4, 11 mM D-glucose and 1.25 mM Na2HPO4. The slices were then incubated with 0.2 mM DAF-FM DA at 37°C for 1 h, following which they were mechanically lysed, centrifuged at 14,000 rpm for 10 min and the protein concentration measured with the Biorad protein assay solution. Lysate (0.25 mg protein), reconstituted in 1 ml 20 mM ACSF buffer at pH 7.4, was then subjected to fluorescence measurements to detect NO generation as above.
S-nitrosylation Biotin Switch Assay
The assay was carried-out as described previously (Jaffrey and Snyder, 2001
) but with minor modifications. Briefly, brain tissue from wild-type, SR−/−
mice was homogenized in HEN buffer (250 mM Hepes-NaOH, pH 7.7, 1 mM EDTA, 0.1 mM Neocuproine) supplemented with 100 μM deferoxamine (DFO) and centrifuged at 13,000 × g
for 20 min at 4°C. Lysate (0.24 mg protein) was added to blocking buffer (HEN buffer plus 25% SDS and 20 mM methymethanethiosulfonate (MMTS)) at 50°C for 20 min with frequent vortexing. The MMTS was then removed by acetone and the proteins precipitated at −20°C for 20 min. After acetone removal, the proteins were resuspended in HENS buffer, which is HEN + 1% SDS. To the suspension was added 1 mM biotin-HPDP in DMSO with 1 mM ascorbic acid. After incubation for 2 h at 25°C, biotinylated proteins were precipitated by streptavidin-agarose beads, which were then washed with HENS buffer. The biotinylated proteins were eluted by SDS-PAGE sample buffer and subjected to Western blot analysis. For quantitation of protein S
-nitrosylation the signals were densitometrically analyzed using the softwares EagleSight 3.2 (Stratagene) and Odyssey 2.1 (Li-Cor).
D-serine from wild-type and SR−/−
cortical neuronal cultures, treated with 10 mM L-serine for 5 h, was measured as described earlier (Kartvelishvily et al., 2006
All data are expressed as means ± SEM of three independent experiments each performed in triplicate unless otherwise indicated. Data were analyzed by unpaired Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).