IkB signaling riluzole was pretreated in order to observe its protection to inhibit Mn cytotoxicity, cell cycle progression, and apoptosis of astrocytes caused by Mn exposure. Materials and Methods Astrocytes Culture The procedure for astrocytes isolation from the cerebral cortex of newborn Sprague Dawley rats, which were approved by institutional guidelines for the care and use of laboratory animals in China Medical University, Shenyang, China, and conformed to the National Institutes of Health Guide for Care and Use of Laboratory Animals. The subsequent culture was carried out according as previously described by Hertz et al. The cells were used if more than 95% of them stained positively for the astrocyte marker glial fibrillary acidic protein using immunocytochemistry. Methyl Thiazolyl Tetrazolium Assay The cultured astrocytes were exposed to 0, 125, 250, and 500 M MnCl2 for 24 h. In addition, the astrocytes were pretreated with 100 M riluzole for 6 h before no MnCl2 bcr-abl exposure or 500 M MnCl2 exposurefor 24 h. The viability of the astrocytes was determined using the MTT assay as described by Marks et al.
LDH Leakage Assay According to the p38 MAPK signaling pathway procedures described above, astrocytes were exposed after seeding into 96 well plates. LDH leakage assay was determined as described by Wang et al. Morphological Observation According to the procedures described above, astrocytes were pretreated with riluzole and/ or exposed with Mn. Morphological observation was carried out using an Olympus IX70 inverted phase contrast microscope fitted with a digital camera system to capture images. Analysis of Apoptosis by Flow Cytometry Cells apoptosis was determined by FCM assay according to the method reported by Brumatti et al. using Annexin V/PI apoptosis detection kit. Cell Cycle Progression Analysis by Flow Cytometry According to the method of Riccardi et al. DNA content and cell cycle distribution were analyzed using Flow cytometry. Cytofluorometric analysis was performed on a minimum of 10,000 cells per sample, using the Cell Quest software. Statistical Analysis The data were presented as mean standard deviation. All statistical analysis was performed using SPSS software, version 11.5. Data were analyzed using one way analysis of variance followed by the post hoc Duncan multiple range celestone test when F was significant. Statistical significance was set at P0.05. Results Effects of MnCl2 Exposure and Riluzole Pretreatment on Cell Viability Cell viability was assessed by the MTT method.
Our results indicated that cell viability declined in a concentration dependent manner after Mn exposure. Exposure to 500 M MnCl2 for 24 h resulted in loss of almost half of astrocytes. Comparing with the control group, there was no obvious changes of cell viability in the riluzole control group. When compared to 500 M MnCl2 group, the cell viability increased to 88.2413.33% in the riluzole pretreatment group. Effects of MnCl2 Exposure and Riluzole Pretreatment on LDH Leakage LDH leakage was measured to indicate cell membrane integrity. The results had shown that LDH leakage increased in the concentration dependent manner after Mn exposure. After 125 500 M MnCl2 incubation, astrocytes displayed obvious cell shrinkage, nuclear condensation, and loss gradually.