BACKGROUND:Chloride channels participate in non-neuronal apoptosis.However,it remains unclear whether chloride channels are involved in ischemic neuronal apoptosis.OBJECTIVE:To explore the effects of 4-acetamido-4’-isothiocyanatostilbene-2,2’-disulfonic acid (SITS) and 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS),two chloride channel blockers,on the hippocampal neuronal apoptosis induced by 3-morpholinosydnonimine (SIN-1) based on the nitric oxide toxicity theory of neuronal apoptosis following ischemic brain injury.DESIGN,TIME AND SETTING:Comparative observation and in vitro experiments were performed at the laboratory of Zhuhai Campus of Zunyi Medical College from January to May 2009.MATERIALS:SIN-1,SITS,and DIDS were purchased from Sigma,USA.METHODS:Hippocampal neurons from Sprague-Dawley rats,aged 1 day,were cultured in vitro for 12 days and randomly assigned to control,SIN-1,or chloride channel blocker groups.SIN-1 group neurons were induced by SIN-1 for 18 hours to establish a model of ischemic neuronal apoptosis.Neurons in chloride channel blocker groups were treated with SITS or DIDS plus SIN-1 for 18 hours.The controls were cultured in DMEM/Ham’s F12 complete medium alone.MAIN OUTCOME MEASURES:The apoptotic neurons and nuclear appearance were detected by Hoechst 33258 fluorescence staining;neuronal viability was quantitatively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis.Caspase-3 activity was analyzed by Western blot.RESULTS:SIN-1 (1 mmol/L) dramatically induced apoptosis (50%-60%).SITS and DIDS inhibited nitric oxide-induced neuronal injury in a dose-dependent manner,suppressed caspase-3 activation,reduced neuronal apoptosis,and improved neuronal survival.CONCLUSION:Chloride channel blockers can protect against neuronal injury induced by NO.Chloride channels might be involved in neuronal apoptosis following cerebral ischemia. BACKGROUND: Chloride channels participate in non-neuronal apoptosis. Although it remains unclear whether chloride channels are involved in ischemic neuronal apoptosis. OBJECTIVE: To explore the effects of 4-acetamido-4’-isothiocyanatostilbene-2,2’-disulfonic acid SITS) and 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS), two chloride channel blockers, on the hippocampal neuronal apoptosis induced by 3-morpholinosydnonimine (SIN- 1) based on the nitric oxide toxicity theory of neuronal apoptosis following ischemic brain injury. DESIGN, TIME AND SETTING: Comparative observation and in vitro experiments were conducted at the laboratory of Zhuhai Campus of Zunyi Medical College from January to May 2009. MODIALS: SIN-1, SITS, and DIDS were purchased from Sigma , USA.METHODS: Hippocampal neurons from Sprague-Dawley rats, aged 1 day, were cultured in vitro for 12 days and randomly assigned to control, SIN-1, or chloride channel blocker groups. SN-1 group neurons were induced by SIN- 1 for 18 hours to est ablish a model of ischemic neuronal apoptosis. Neurons in chloride channel blocker groups were treated with SITS or DIDS plus SIN-1 for 18 hours. The controls were cultured in DMEM / Ham’s F12 complete medium alone. MAIN OUTCOME MEASURES: The apoptotic neurons and nuclear Appearance were detected by Hoechst 33258 fluorescence staining; neuronal viability was quantitatively determined by 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide analysis. Caspase-3 activity was analyzed by Western blot.RESULTS: SIN SITS and DIDS inhibited nitric oxide-induced neuronal injury in a dose-dependent manner, suppressed caspase-3 activation, reduced neuronal apoptosis, and improved neuronal survival .CONCLUSION: Chloride channel blockers can protect against neuronal injury induced by NO. Chloride channels might be involved in neuronal apoptosis following cerebral ischemia.