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• W2129015955 abstract "Physiological levels of H2S exert neuroprotective effects, whereas high concentrations of H2S may cause neurotoxicity in part via activation of NMDAR. To characterize the neuroprotective effects of combination of exogenous H2S and NMDAR antagonism, we synthesized a novel H2S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamide (S-memantine) and examined its effects in vitro and in vivo. S-memantine was synthesized by chemically combining a slow releasing H2S donor 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48) with a NMDAR antagonist memantine. S-memantine increased intracellular sulfide levels in human neuroblastoma cells (SH-SY5Y) 10-fold as high as that was achieved by ACS48. Incubation with S-memantine after reoxygenation following oxygen and glucose deprivation (OGD) protected SH-SY5Y cells and murine primary cortical neurons more markedly than did ACS48 or memantine. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by sodium sulfide (Na2S) or ACS48, but suppressed by memantine and S-memantine. S-memantine prevented glutamate-induced glutathione depletion in SH-SY5Y cells more markedly than did Na2S or ACS48. Administration of S-memantine after global cerebral ischemia and reperfusion more robustly decreased cerebral infarct volume and improved survival and neurological function of mice than did ACS48 or memantine. These results suggest that an H2S-releasing NMDAR antagonist derivative S-memantine prevents ischemic neuronal death, providing a novel therapeutic strategy for ischemic brain injury. Physiological levels of H2S exert neuroprotective effects, whereas high concentrations of H2S may cause neurotoxicity in part via activation of NMDAR. To characterize the neuroprotective effects of combination of exogenous H2S and NMDAR antagonism, we synthesized a novel H2S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamide (S-memantine) and examined its effects in vitro and in vivo. S-memantine was synthesized by chemically combining a slow releasing H2S donor 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48) with a NMDAR antagonist memantine. S-memantine increased intracellular sulfide levels in human neuroblastoma cells (SH-SY5Y) 10-fold as high as that was achieved by ACS48. Incubation with S-memantine after reoxygenation following oxygen and glucose deprivation (OGD) protected SH-SY5Y cells and murine primary cortical neurons more markedly than did ACS48 or memantine. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by sodium sulfide (Na2S) or ACS48, but suppressed by memantine and S-memantine. S-memantine prevented glutamate-induced glutathione depletion in SH-SY5Y cells more markedly than did Na2S or ACS48. Administration of S-memantine after global cerebral ischemia and reperfusion more robustly decreased cerebral infarct volume and improved survival and neurological function of mice than did ACS48 or memantine. These results suggest that an H2S-releasing NMDAR antagonist derivative S-memantine prevents ischemic neuronal death, providing a novel therapeutic strategy for ischemic brain injury. Hydrogen sulfide has been proposed as a gaseous signaling molecule along with nitric oxide and carbon monoxide (1Olson K.R. The therapeutic potential of hydrogen sulfide: separating hype from hope.Am. J. Physiol. 2011; 301: R297-R312Crossref PubMed Scopus (157) Google Scholar). A number of studies suggested therapeutic potential of H2S-donating compounds and H2S gas itself for a number of animal models of human disease including ischemic brain injury (2Caliendo G. Cirino G. Santagada V. Wallace J.L. Synthesis and biological effects of hydrogen sulfide (H2S): development of H2S-releasing drugs as pharmaceuticals.J. Med. Chem. 2010; 53: 6275-6286Crossref PubMed Scopus (213) Google Scholar, 3Predmore B.L. Lefer D.J. Development of hydrogen sulfide-based therapeutics for cardiovascular disease.J. Cardiovasc. Translat. Res. 2010; 3: 487-498Crossref PubMed Scopus (59) Google Scholar). Gaseous H2S, however, may be difficult to be used clinically because of its characteristic odor and toxicity at high concentrations (1Olson K.R. The therapeutic potential of hydrogen sulfide: separating hype from hope.Am. J. Physiol. 2011; 301: R297-R312Crossref PubMed Scopus (157) Google Scholar, 4Reiffenstein R.J. Hulbert W.C. Roth S.H. Toxicology of hydrogen sulfide.Annu. Rev. Pharmacol. Toxicol. 1992; 32: 109-134Crossref PubMed Scopus (766) Google Scholar). Na2S and sodium hydrosulfide (NaHS) have been used as H2S donor compounds in the majority of experimental studies (2Caliendo G. Cirino G. Santagada V. Wallace J.L. Synthesis and biological effects of hydrogen sulfide (H2S): development of H2S-releasing drugs as pharmaceuticals.J. Med. Chem. 2010; 53: 6275-6286Crossref PubMed Scopus (213) Google Scholar, 3Predmore B.L. Lefer D.J. Development of hydrogen sulfide-based therapeutics for cardiovascular disease.J. Cardiovasc. Translat. Res. 2010; 3: 487-498Crossref PubMed Scopus (59) Google Scholar). However, because the half-lives of these sulfide salts are very short in biological fluid, plasma sulfide levels rapidly increase after bolus administration of Na2S or NaHS and then return to baseline instantaneously (5DeLeon E.R. Stoy G.F. Olson K.R. Passive loss of hydrogen sulfide in biological experiments.Anal. Biochem. 2012; 421: 203-207Crossref PubMed Scopus (127) Google Scholar). To sustain “physiological” levels of sulfide in circulation after bolus administration, many slow-releasing H2S donor compounds, including ACS48, have been developed (2Caliendo G. Cirino G. Santagada V. Wallace J.L. Synthesis and biological effects of hydrogen sulfide (H2S): development of H2S-releasing drugs as pharmaceuticals.J. Med. Chem. 2010; 53: 6275-6286Crossref PubMed Scopus (213) Google Scholar, 6Lee M. Tazzari V. Giustarini D. Rossi R. Sparatore A. Del Soldato P. McGeer E. McGeer P.L. Effects of hydrogen sulfide-releasing L-DOPA derivatives on glial activation: potential for treating Parkinson disease.J. Biol. Chem. 2010; 285: 17318-17328Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). While it has been reported that low and physiological levels of H2S protect neurons, H2S also exhibits neurotoxicity especially at high concentrations (4Reiffenstein R.J. Hulbert W.C. Roth S.H. Toxicology of hydrogen sulfide.Annu. Rev. Pharmacol. Toxicol. 1992; 32: 109-134Crossref PubMed Scopus (766) Google Scholar). Some investigators have suggested that H2S-induced neurotoxicity may be mediated via enhancement of N-methyl-d-aspartate receptor (NMDAR) 2The abbreviations used are: NMDARN-methyl-d-aspartate receptorS-memantineN-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamideOGDoxygen and glucose deprivationDMFN,N-dimethylformamideHATUO-(7-azabenzotriazol-1-yl)-N,N,N‘,N‘-tetramethyluronium hexafluorophosphateLDHlactose dehydrogenaseSSA5-sulfosalicylic acidMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideCVcrystal violetTTC2,3,5-triphenyltetrazolium chlorideBCAObilateral carotid artery occlusion. activity (7Chen M.J. Peng Z.F. Manikandan J. Melendez A.J. Tan G.S. Chung C.M. Li Q.T. Tan T.M. Deng L.W. Whiteman M. Beart P.M. Moore P.K. Cheung N.S. Gene profiling reveals hydrogen sulphide recruits death signaling via the N-methyl-d-aspartate receptor identifying commonalities with excitotoxicity.J. Cell. Physiol. 2011; 226: 1308-1322Crossref PubMed Scopus (25) Google Scholar, 8Qu K. Chen C.P. Halliwell B. Moore P.K. Wong P.T. Hydrogen sulfide is a mediator of cerebral ischemic damage.Stroke. 2006; 37: 889-893Crossref PubMed Scopus (242) Google Scholar, 9Cheung N.S. Peng Z.F. Chen M.J. Moore P.K. Whiteman M. Hydrogen sulfide induced neuronal death occurs via glutamate receptor and is associated with calpain activation and lysosomal rupture in mouse primary cortical neurons.Neuropharmacology. 2007; 53: 505-514Crossref PubMed Scopus (103) Google Scholar), because toxicity of H2S was abolished by NMDAR antagonist in vitro and in vivo (8Qu K. Chen C.P. Halliwell B. Moore P.K. Wong P.T. Hydrogen sulfide is a mediator of cerebral ischemic damage.Stroke. 2006; 37: 889-893Crossref PubMed Scopus (242) Google Scholar, 9Cheung N.S. Peng Z.F. Chen M.J. Moore P.K. Whiteman M. Hydrogen sulfide induced neuronal death occurs via glutamate receptor and is associated with calpain activation and lysosomal rupture in mouse primary cortical neurons.Neuropharmacology. 2007; 53: 505-514Crossref PubMed Scopus (103) Google Scholar). Based on these observations, we hypothesized that a hybrid NMDAR antagonist that is capable of slowly releasing H2S in circulation is more effective in protecting neurons than H2S donor compounds alone. N-methyl-d-aspartate receptor N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamide oxygen and glucose deprivation N,N-dimethylformamide O-(7-azabenzotriazol-1-yl)-N,N,N‘,N‘-tetramethyluronium hexafluorophosphate lactose dehydrogenase 5-sulfosalicylic acid 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide crystal violet 2,3,5-triphenyltetrazolium chloride bilateral carotid artery occlusion. In the current study, we sought to characterize the efficacy of a novel H2S-releasing NMDAR antagonist derivative, S-memantine, in cerebral ischemia and reperfusion injury using in vitro and in vivo approaches. We also compared neurotoxicity of Na2S, ACS48, and S-memantine in a human neuroblastoma cell line and murine primary cortical neurons. Here, we report that S-memantine exhibits high therapeutic potential with low toxicity against ischemic neuronal death. All reagents were purchased from Sigma-Aldrich unless otherwise specified. 4-(3-thioxo-3H-1,2-dithiol-4-yl)-Benzoic acid (ACS48) was synthesized as described previously (Fig. 1A) (6Lee M. Tazzari V. Giustarini D. Rossi R. Sparatore A. Del Soldato P. McGeer E. McGeer P.L. Effects of hydrogen sulfide-releasing L-DOPA derivatives on glial activation: potential for treating Parkinson disease.J. Biol. Chem. 2010; 285: 17318-17328Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). N-((1r,3R,5S,7r)-3,5-Dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzamide (S-memantine) was synthesized using the following two steps as shown in Fig. 1B: First, under nitrogen atmosphere, at room temperature, 4-(propan-2-yl)-benzoic acid (0.5 g; 3.05 mmol) and memantine (0.82 g; 4.57 mmol) were mixed in 5 ml of anhydrous N,N-dimethylformamide (DMF), and N,N-diisopropylethylamine (2.12 ml; 12.19 mmol) was added. After cooling to room temperature, O-(7-azabenzotriazol-1-yl)-N,N,N‘,N‘-tetramethyluronium hexafluorophosphate (HATU) (1.045 g; 2.75 mmol), dissolved in 5 ml of DMF was gradually added, followed by an overnight stirring at room temperature. After evaporation of DMF, extraction and purification, N-((1r,3R,5S,7r)-3,5-dimethyl-adamantan-1-yl)-4-isopropylbenzamide (Amide 1) was obtained. In the second step, under nitrogen atmosphere, sulfur (5.05 g, 158 mmol) was melted at 140 °C and amide-1 (2.16 g, 13.2 mmol) was added. The reaction mixture was stirred for 24 h at 190 °C to form a layer of reddish brown solution. After cooling the reaction mixture back to 140 °C, 100 ml toluene was added followed by further cooling to room temperature. Acetone was added to form a suspension that was filtered and the filtrate was concentrated to dryness. The dry residue was purified by flash chromatography. Fractions containing pure S-memantine were dried under vacuum to give 0.678 g (19% yield) of reddish brown crystals. The purity of the final product was greater than 98%. The structure of the final product was confirmed by mass spectrometry (Finnigan LCQ Advantage, ESI+) and 1H NMR spectroscopy. C22H25NOS3: m/z calculated: [M+H]+ 415.11; found: 415.64. 1H NMR (400 MHz, DMSO-d6) 9.21 (s, 1H), 7.82 (d, J = 6.8Hz, 2H), 7.68 (s, 1H), 7.63 (d, J = 8.4Hz, 2H), 2.12–2.14 (m, 1H), 1.92–1.93 (m, 2H), 1.71–1.78 (m, 4H), 1.16–1.39 (m, 6H), 0.86 (s, 6H). Human neuroblastoma SH-SY5Y cells were cultured in Eagle's medium/Ham's F-12 50/50 Mix (DMEM/F12, Cellgro by Mediatech, Inc.) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were seeded into 96 well plates (2 × 104 cells per well) for OGD and measurements of toxicity, 6 cm dishes (5 × 105 cells per dish) for measurements of sulfide and reduced glutathione (GSH), or 10 cm dishes (1 × 106 cells per dish) for measurements of intracellular calcium. Cell culture medium was replaced every 2 days, and the cultures were maintained at 37 °C in 95% air/5% CO2 in a humidified incubator. Cells were used after reaching 80% confluent. Primary neuronal cultures were prepared from the cortex of embryonic day 15 C57BL6J mice. In brief, brains were harvested and the hemispheres were dissected under a microscope. The cortical neurons were dissociated in Neurobasal medium (Invitrogen) with B27 supplement (antioxidant plus, Invitrogen). The cells were seeded into 24 well plates coated with poly-d-lysine (Becton Dickinson Labware, 2 × 105 cells per well), followed by the medium-change with fresh one on the next day. The half of culture medium was replaced with Neurobasal medium with B27 supplement (antioxidant minus) every other day, and the cultures were maintained at 37 °C in 95% air/5% CO2 in a humidified incubator. Cells were used for experiments 11 days after seeding. ACS48 and S-memantine were dissolved in dimethyl sulfoxide (DMSO), then, diluted to desired concentration with culture medium. The final concentration of DMSO was adjusted to 1%. We confirmed that 1% DMSO did not affect cell viabilities of SH-SY5Y and primary cortical neurons using lactose dehydrogenase (LDH) method (data not shown). Concentration of free sulfide in SH-SY5Y cells was measured using high performance liquid chromatography (HPLC) (10Tokuda K. Kida K. Marutani E. Crimi E. Bougaki M. Khatri A. Kimura H. Ichinose F. Inhaled hydrogen sulfide prevents endotoxin-induced systemic inflammation and improves survival by altering sulfide metabolism in mice.Antioxidants Redox Signal. 2012; 17: 11-21Crossref PubMed Scopus (90) Google Scholar). Briefly, SH-SY5Y cells were seeded into 6 cm dishes (5 × 105 cells per dish). After being 80% confluent, 20 μm H2S donor was added to the dish and incubated at 37 °C. Cells were washed with ice-cold Tris-HCl (100 mm, pH 9.5, DTPA 0.1 mm) buffer, scraped, transfered to Eppendorf tubes, and centrifuged. MBB (10 mm in acetonitrile, 50 μl) was added to 100 μl of supernatant. After 30 min of incubation at room temperature in dark, 50 μl of 200 mm 5-sulfosalicylic acid (SSA) was added. After centrifugation, supernatant was analyzed by HPLC. For sulfide levels in medium, after centrifugation, supernatant was used for MBB reaction as above. Plasma and brain sulfide levels were measured 90 min after intraperitoneal administration of Na2S, ACS48, or S-memantine. Blood was drawn from left ventricle and centrifuged to collect the plasma. After perfusion with Tris-HCl buffer via left ventricle, brain was harvested, homogenized in Tris-HCl, and centrifuged. Plasma and supernatant of brain homogenate were derivatized with MBB and analyzed by HPLC. OGD for SH-SY5Y was performed by placing cells in a hypoxia chamber (STEMCELL Technologies Inc.) for 15 h, followed by 24 h of reoxygenation as previously reported (Fig. 2A) (11Serra-Pérez A. Verdaguer E. Planas A.M. Santalucía T. Glucose promotes caspase-dependent delayed cell death after a transient episode of oxygen and glucose deprivation in SH-SY5Y cells.J. Neurochem. 2008; 106: 1237-1247Crossref PubMed Scopus (17) Google Scholar). Briefly, medium was replaced with glucose-free RPMI 1640 with l-glutamine (Cellgro by Mediatech, Inc), deoxygenated with anaerobic gas mixture (93% N2-5% CO2-2% H2) for 30 min before using. Cells were then placed in a hypoxia chamber, flushed with anaerobic gas mixture (93% N2-5% CO2-2% H2) and incubated at 37 °C. After 15 h of hypoxia, medium was replaced with DMEM/F12 and incubated for 24 h at 37 °C in 95% air/5% CO2 in a humidified incubator. OGD for primary cortical neurons was performed with the similar protocol as above (Fig. 2B). Briefly, the culture medium was replaced with deoxygenated Neurobasal medium without glucose, and then placed in the hypoxic chamber for 2.5 h. After the OGD, the medium was replaced with Neurobasal medium with glucose and incubated for 21 h at 37 °C in 95% air/5% CO2 in a humidified incubator. Control cells without OGD and reoxygenation were incubated in the fresh Neurobasal medium with glucose and incubated for 21 h at 37 °C in 95% air/5% CO2, then, used for viability experiment. Microtiter plate containing cells was centrifuged at 250 × g for 10 min, and the supernatant was used for LDH measurement with LDH Cytotoxicity Detection Kit (Roche). After aspirating medium, remaining cells were washed with PBS, then, 100 μl of 1% Triton-X was added to each well, followed by incubation at 37 °C for 30 min. Medium and lysates were used for LDH measurement at wavelength 492 nm. Percentage of released LDH was calculated with following formula {LDH (medium)/LDH (medium + cell) × 100}. The average value of control (cells without OGD) was deducted as background. 10 μl of thiazolyl blue tetrazolium bromide solution (5 mg/ml in pH 7.4, PBS) was added to each well containing 100 μl of medium and cells, followed by incubation at 37 °C for 4 h in the dark. Isopropanol (100 μl, 0.04 n HCl) was added to dissolve the blue dye. After dissolving completely, absorbance was measured with a plate-reader (Synergy 2, BioTek Instrument) at test wavelength 570 nm and reference wavelength 670 nm. Cell viability was determined by absorbance at 570 nm and reported as ratio to control cells (without OGD). After aspirating culture medium, cells were fixed and stained by 0.5% CV in 95% (v/v) ethanol for 5 min, then washed by tap water several times. After taking photographs, 1% sodium dodecyl sulfate solution was added to each well to elute blue dye. Absorbance was measured with a plate reader at 595 nm of wavelength. Values were shown as ratio to control (cells without OGD). Intracellular calcium level was measured by a previously described method using Fura-2/AM with some modifications (12Gao M. Zhang W.C. Liu Q.S. Hu J.J. Liu G.T. Du G.H. Pinocembrin prevents glutamate-induced apoptosis in SH-SY5Y neuronal cells via decrease of bax/bcl-2 ratio.Eur. J. Pharmacol. 2008; 591: 73-79Crossref PubMed Scopus (92) Google Scholar). Briefly, cells were trypsinized, pelleted, resuspended in the medium, and incubated with 5 μm Fura-2/AM (Invitrogen) in HEPES buffer (pH 7.4, NaCl 110 mm, KCl 2.6 mm, MgSO4 1 mm, CaCl2 (Fisher Scientific) 1 mm, HEPES 25 mm, and glucose 11 mm) at 37 °C for 40 min, and then washed twice. Cells were resuspended in HEPES buffer and transferred to a cuvette. Na2S, ACS48, memantine or S-memantine at 20 μm was added to the cuvette with or without glutamate (100 μm), respectively. Final cell concentration was 1 × 105 cells/ml. We were not able to examine the effects of higher concentration of S-memantine than 20 μm because of its poor solubility to HEPES buffer. The fluorescence intensity ratio was measured with Spectra Max M5 (Molecular Devices, CA) at a wavelength of λex = 340/380 nm and λem = 510 nm. Intracellular GSH level of SH-SY5Y was measured using HPLC method as previously reported (13Kimura Y. Goto Y. Kimura H. Hydrogen sulfide increases glutathione production and suppresses oxidative stress in mitochondria.Antioxidants Redox Signaling. 2009; 12: 1-13Crossref Scopus (508) Google Scholar). Briefly, cells were seeded into 6-cm dishes (5 × 105 cells per dish) and treated with 50 μm H2S donors or memantine w/wo 2 mm glutamate for 8 h, followed by washing with ice-cold PBS. Cells were scraped and transferred to an Eppendorf tube and sonicated. Some fraction of lysate was used for protein assay. After centrifugation, 75 μl of supernatant, 26 μl of 2-(cyclohexylamino) ethanesulfonic acid (CHES, 0.5 m, pH 8.4) and 4 μl of 50 μm MBB were mixed and incubated at room temperature in dark for 30 min. Acetic acid (100 μl, 30% v/v) was added, followed by centrifugation at 15,000 × g for 10 min after 5 min incubation of the tube on ice. The supernatant was analyzed using HPLC at wavelength of λex = 370 nm and λem = 486 nm. Protein levels in SH-SY5Y were determined by standard immunoblot techniques using primary antibodies (1:1,000, Cell Signaling Technology Inc., Danvers, MA) against cleaved caspase-3, caspase-3, phosphorylated Akt at threonine 308, Akt, phosphorylated ERK1/2 at threonine 202, and tyrosine 204, Erk1/2, and β-tubulin. Bound antibody was detected with a horseradish peroxidase-linked antibody directed against rabbit IgG (1:10,000∼1:25,000; Cell Signaling Technology Inc.) and was visualized using chemiluminescence with ECL Advance kit (GE Healthcare). After approval by the Massachusetts General Hospital Subcommittee on Research Animal Care, all animal experiments were performed in accordance with the guidelines of the National Institutes of Health. Male mice (C57BL/6J, 8–9 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME) and given access to food and water ad libitum in our animal facility until the time of experiments. Mice were anesthetized with ketamine (80 mg/kg, intraperitoneal) and xylazine (12 mg/kg, intraperitoneal). Body temperature was kept at 37 ± 0.5 °C during whole procedure. Cerebral ischemia was induced by 40 min of bilateral common carotid artery occlusion (BCAO) with microsurgical clips. Na2S, ACS48, S-memantine, or memantine at 25 μmol/kg or vehicle was intraperitoneally administered 1 min after the initiation of reperfusion. After reperfusion and recovery from anesthesia, mice were intraperitoneally given 1 ml of 5% dextrose-enriched lactated Ringer's solution daily for 1 week. Neurological score was evaluated as described previously (14Thal S.C. Thal S.E. Plesnila N. Characterization of a 3-vessel occlusion model for the induction of complete global cerebral ischemia in mice.J. Neurosci. Methods. 2010; 192: 219-227Crossref PubMed Scopus (25) Google Scholar). The next eight items were checked and scored to evaluate neurological function: 1) Grasping movement reflex (inducing the catching reflex by running a little rod over the plantar surface of the paw): 0–4 points, 2) stop at the edge of a table: 0 or 1 point, 3) turning the head (turning the head when touching the ear from behind with a little rod): 0–2 points, 4) falling reflex (lifting the mouse at the tail and lowering with the front legs toward the ground): 0 or 1 point, 5) spontaneous motility (moving behavior on a flat surface): 0–2 points, 6) circling behavior (moving behavior on a flat surface): 0 or 2 points, 7) pelt property (appearance of the coat): 0 or 1 point, 8) flight reaction (spontaneous behavior on a flat surface): 0 or 1 point. Total 14 points. The higher score means worse neurological function. ACS48 and S-memantine were dissolved in the corn oil/DMSO (v/v, 95/5) suspension. Na2S was dissolved in saline 5 min before administration. Mice were intraperitoneally given 4 μl/g of these solutions 1 min after reperfusion following 40 min of BCAO. Mice were decapitated and brains were harvested 24 h after BCAO and reperfusion. Coronal sections (2 mm thickness) of the cerebrum were then soaked into 1% 2,3,5-triphenyltetrazolium chloride (TTC) solution in PBS at 37 °C for 30 min. After taking photographs under the same condition, infarct volume was calculated with Image J software ver.1.44. Photographs were gray-scaled, then, brighter area than threshold determined using image J software was calculated as infarct area. Values were shown as ratio of cerebral infarct volume to total volume. The average value of the brighter region volume in control mice was deducted from calculated area as background. All data are presented as means ± S.E. Data were analyzed by ANOVA using Sigmastat 3.01a (Systat Software Inc., Chicago, IL) and Prism 5 software package (GraphPad Software, La Jolla, CA). Newman-Keuls multiple comparison post hoc test or Bonferroni post hoc test were respectively performed for One-way Anova or Two-way Anova test as required. Smaller p values than 0.05 were considered significant. To determine the timing and levels of sulfide release by different sulfide donors, sulfide concentrations after addition of Na2S, ACS48 or S-memantine to the Dulbecco's modification of DMEM/F12 with 10% FBS (without cells) was measured using HPLC as reported (10Tokuda K. Kida K. Marutani E. Crimi E. Bougaki M. Khatri A. Kimura H. Ichinose F. Inhaled hydrogen sulfide prevents endotoxin-induced systemic inflammation and improves survival by altering sulfide metabolism in mice.Antioxidants Redox Signal. 2012; 17: 11-21Crossref PubMed Scopus (90) Google Scholar). Fig. 3A shows time-dependent changes of sulfide concentrations in the medium after addition of 20 μm of each compounds at time 0 at pH 7.4. Although Na2S raised sulfide levels immediately, sulfide levels induced by Na2S decreased rapidly and became lower than sulfide levels induced by ACS48 and S-memantine at 1.5 h and 8 h after addition to the medium, respectively (p < 0.01 by two-way ANOVA with Bonferroni post-test). ACS48 and S-memantine increased sulfide levels to 3.6 μm and 5.1 μm after 24 h, respectively. Sulfide levels in the medium were higher after addition of S-memantine than ACS48 at all time points examined (∼ 2.1-fold, p < 0.01 by two-way ANOVA with Bonferroni post-test). Interestingly, both ACS48 and S-memantine released very little sulfide in PBS whereas ACS48 released more sulfide than did S-memantine in Tris-HCl (pH 9.5) and in DMEM/F12 without FBS (supplemental Fig. S1). Incubation of SH-SY5Y cells with Na2S, ACS48, and S-memantine increased intracellular sulfide levels with different magnitude and time course. Intracellular sulfide levels peaked around 1.5 h after addition of Na2S and ACS48 to the medium that disappeared by 8 h (Fig. 3B). In contrast, incubation of SH-SY5Y cells with S-memantine increased intracellular sulfide level more markedly than incubation with ACS48 at all time points after addition (∼10-fold at 4 h, p < 0.001 by two-way ANOVA with Bonferroni post-test). In a separate experiment, we examined whether or not incubation with memantine itself or incubation with ACS48 and memantine would affect intracellular sulfide levels in SH-SY5Y cells. We found that memantine itself did not affect intracellular sulfide levels in SH-SY5Y cells incubated with or without ACS48 (Fig. 3C). Hence, it was indicated that chemical bonding between ACS48 and memantine would be important for the high intracellular sulfide levels achieved after addition of S-memantine. We examined the effect of Na2S since it has been widely used as a therapeutic compound against neuronal ischemia in vitro. Fifteen hours of OGD followed by 24 h of reoxygenation induced cell death in SH-SY5Y cells as indicated by increased LDH release into the medium (Fig. 4). Addition of Na2S to the culture medium at various time points (pre-OGD or 0.5, 2, 5, and 8 h after the end of OGD) and concentrations (10 and 50 μm) failed to improve cell viability (Fig. 4). We examined whether or not ACS48, memantine, and S-memantine improves viability of SH-SY5Y cells subjected to 15 h of OGD followed by 24 h of reoxygenation. Based on our time- and dose-ranging studies, we determined that 50 μm and at 8 h after the end of OGD were the most effective dose and time point to add ACS48 or S-memantine to improve viability of SH-SY5Y cells after OGD (Fig. 5, A and B). Addition of S-memantine to the medium at 50 μm at 8 h after the end of OGD improved the viability of SH-SY5Y cells more markedly than did addition of ACS48 or memantine at the same dose and time point, as indicated in LDH release, MTT, and CV assays (Fig. 6, A–D).FIGURE 6Effects of H2S donors and memantine on cell viability after OGD. A–D, ACS48, memantine, S-memantine at 50 μm or vehicle was added 8 h after the end of OGD. LDH released in the culture medium was measured 24 h after the end of OGD. A, LDH release from SH-SY5Y after OGD, n = 5 or 6 each. *, p < 0.001 versus vehicle; #, p < 0.05. B, MTT assay, n = 5 or 6 each. No-OGD control (control) differs significantly from all other groups (p < 0.001). *, p < 0.01 versus vehicle; #, p < 0.05. C, CV assay and D, photographs of wells containing SH-SY5Y stained with CV after OGD, n = 5 each No OGD control (control) differs significantly from all other groups (p < 0.001). *, p < 0.001 versus vehicle, #, p < 0.05. E, LDH released from murine primary cortical neurons measured 2.5 h after the end of OGD. ACS48, memantine, S-memantine at 50 μm, or vehicle was added 0.5 h after the end of OGD. LDH released in the culture medium was measured 21 h after the end of OGD, n = 5 or 6 each. *, p < 0.001 versus vehicle, #, p < 0.001 versus ACS48 and memantine.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also examined whether or not ACS48, memantine, and S-memantine improves survival of murine primary cortical neurons after 2.5 h of OGD followed by 21 h of reoxygenation. Based on our dose- and timing-ranging studies (Fig. 5, A and B), we added ACS48 and S-memantine at 50 μm at 30 min after the end of OGD. S-memantine exhibited more robust neuroprotective effects compared with ACS48 or memantine, as assessed by LDH release assay (Fig. 6E). To define the role of NMDAR in cytotoxicity of H2S, we examined whether or not memantine suppresses toxicity of Na2S and ACS" @default.
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