Neuroprotective effect of melatonin against ischemia/reperfusion-induced neuronal apoptosis in mouse cerebellum

Abstract

Background Some experiments have demonstrated that melatonin (N-aceyl-5-methoxytryptamine, Mel) has antioxidation. However, whether it has neuroprotective effect in the ischemia/reperfusion injury of central nervous system is unclear. Objective To observe the protective effect of Mel on ischemia/reperfusion-induced cerebellar neuronal apoptosis of rats, and the action mechanism. Design Controlled observation experiment. Setting Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology. Materials Eight Sprague-Dawley rats aged 7–8 days and weighing 10–12 g were provided by Medical Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technology. Anti-cytochrome C monoclonal antibody was purchased from R & D Company; 7-dichlorodihydrofluorescein diacetate(DCFH-DA), rhodamine 123 and Mel were purchased from Sigma Company (USA). Lactate dehydrogenase (LDH) kit was purchased from Nanjing Jiancheng Bioengineering Institute. Methods This experiment was carried out in the laboratory for Department of Biochemistry and Molecule Biology, Tongji Medical College between October 2002 and March 2004. Cerebellar neurons of rats were cultured in vitro. After oxygen-glucose deprivation (OGD) for 90 minutes, 1×10 −4,1×10 −6, 1×10 −9 mol/L Mel was added, respectively, namely high-, middle-, and low-concentration Mel groups. Cells, which were cultured by OGD, served as model group, and control group, in which OGD intervention was omitted, was set. ▪Cytochrome C level of mitochondrial cells in each group was detected by ELISA method. ▪LDH activity in the cell culture fluid was measured, and cell membrane permeability change was analyzed. The cells in the Mel group with the lowest LDH activity served as Mel treatment group, i.e. cells were cultured with OGD, and then Mel was added; Meanwhile, Mel prevention group was set, i.e. Mel was added before OGD. Intervention was not changed in the model group and control group. ▪ DNA level was analyzed and cell apoptosis was observed by agarose gel electrophoresis(AGE). ▪Mitochondrial transmembrane potential of cells, and apoptotic way in each group were analyzed by confocal laser scanning microscopy. Main Outcome Measures ▪Mitochondrial cytochrome C level of cerebellar nerve cells. ▪LDH activity of cerebellar nerve cells. ▪ DNA AGE results. ▪Mitochondrial transmembrane potential change. Results ▪Mitochondrial cytochrome C level of cerebellar nerve cells: cytochrome C was obviously released at 6 hours of OGD-reperfusion. Mel inhibited the release of cytochrome C in dose-dependent manner. ▪LDH activity of cerebellar nerve cells: LDH activity ( A value) was significantly lower in the high- and middle-concentration Mel groups than in the model group ( P < 0.05). LDH activity ( A value) in the low-concentration Mel group was 0.415 0±0.012 9, indicating that Mel could decrease LDH activity of OGD-treated cell supernatant and promote membrane stablization in dose-dependent manner. ▪AGE results of DNA: 1×10 −9 mol/L was considered as the best concentration of melatonin. Cell DNA was extracted for AGE. Results presented typical ladder shape, indicating apoptosis appeared, while apoptosis was lessened in the Mel treatment group and Mel prevention group. ▪Mitochondrial transmembrane potential change: Experimental results showed that green fluorescein was evenly distributed in cerebellar granule cells cultured normally, and the axons of neurons were very clear. The body of neurons was condensed and the axons disappeared after cerebellar granule cells undergoing OGD injury. Mel could completely reverse the effect of OGD. Conclusion Mel can enhance cerebellar neuronal membrane stabilization of rats in dose-dependent manner, and suppress OGD-induced apoptosis of cerebellar granule cells by preventing against mitochondrial apoptosis.