Abstract:

Abstract:Objective
 To explore time window of inducing neuronal apoptosis successfully via building and evaluating oxygen glucose deprivation/reperfusion (OGD/R) model in vitro to mimic ischemia and reperfusion inducing apoptosis of SH-SY5Y cells,and to build a basis for revealing the potential mechanisms of neuronal apoptosis. Methods  SH-SY5Y cells were employed and divided into control group and experimental group treated with   OGD/R  for different time. The cell viability was calculated by the method of MTT method.The OGD10h/R groups in which the cell viability was decreased approximately 50% 24 h after reperfusion and  control groups were selected for further assay. With light microscope,fluorescence microscope and transmission electron microscope the variation of structure and morphous in cells at different time were observed. The changes in  lactate dehydrogenase (LDH) levels, malondialdehyde (MDA) contents,superoxide dismutase (SOD)  activities  were detected at different time of reperfusion. Flow cytometry was used to analyze the cell cycle distribution and the apoptotic rates at different time of reperfusion. Results  The MTT results showed that the cell viabilities were decreased significantly with the prolongation of OGD time. At reperfusion 24 h,the cell viability was (44.91±1.21)% in OGD10h/R groups. The cell viabilities in other different OGD groups had significant differences (P<0.05 or P<0.01). In OGD10h/R groups the inverted microscope and HE staining results showed that the cell densities were decreased following varied shape and deep blue pycnotic nuclei.The AO/EB fluorescence staining results showed the apoptotic body emerged.Under electron microscope,the cells showed heterochromatin fringe agglutinated in nucleus which were viewed as the appearance of apoptosis and autophagia. The level of LDH in culture fluid and the contents of intracytoplasm MDA at different reperfusion time were increased with the prolonged time and at reperfusion 24 h respectively reached the peak and platform phase,compared with control groups the differences were significant (P<0.01). The intracytoplasm SOD activities at different reperfusion time were declined at first and recovered later with the prolonged reperfusion time,compared with control groups the differences were significant (P<0.01). By  flow cytometry analysis on cell cycle phase at different reperfusion time,in OGD 10h/R groups with the prolonged reperfusion time,the percentage of the cells in G0/G1 phase was increased at first and was decreased later with its peak of (74.09±2.62)% at reperfusion 24 h. The percentage of the cells in G2/M phase reached its peak of (26.85±1.35)% at R 0 h and was declined gradually later. The percentage of the cells in S phase was declined gradually and reached the lowest level of （19.2±1.58）% at reperfusion 24 h. Compared with control groups at the same time,the differences were significant (P<0.05 or P<0.01). Conclusion OGD10h and reperfusion (OGD10h/R) to mimic cerebral ischemia and reperfusion in vitro is a reliable model that induces SH-SY5Y cells apoptosis,which provide a basis for the futher study on neuronal apoptotic mechanisms.

Key words: oxygen glucose deprivation/reperfusion, SH-SY5Y cells, apoptosis