Optimization of condition in oxygen-glucose deprivation/re-oxygenation models of HT22 cells

Objective To optimize the HT22 cell oxygen-glucose deprivation/re-oxygenation models in hippocampal neurons of mice, discuss the basis of model establishment and test the related parameters. Methods HT22 cell growth curve was plotted by cell counting and cell doubling time was calculated, and then, the oxygen-glucose deprivation time and re-oxygenation time were determined by LDH assay. The CCK-8 assay, cell counting, cell morphology and flow cytometry were performed to further optimize the parameters of the models. Results The HT22 cell doubling time was 17 h 18 min. The oxygen-glucose deprivation time was 18 h and the re-oxygenation was 20 h. CCK-8 assay indicated that absorbance in cells of oxygen-glucose deprivation 18 h/re-oxygenation 20 h(OGD18 h/R20 h)group was 0.334 ± 0.138, which was significantly lower than that in the control group(0.756 ± 0.026, P<0.05); cells in the OGD18 h/R20 h group were shrank obviously. Flow cytometry indicated that the percentage of cells in terminal apoptosis or non-viable non-apoptotic cells was 24.56%±5.76% and that of viable apoptotic cells was 2.63% ± 0.55% in the OGD18 h/R20 h group, which were significantly higher than those in the control group, respectively(8.13%±0.87% and 0.80%±0.10%, P< 0.05). Conclusion The HT22 cell oxygen-glucose deprivation/re-oxygenation models are successfully established; both oxygen-glucose deprivation time and re-oxygenation time are related to the cell doubling time, and OGD18 h/R20 h can preferably help the establishment;. Key words: HT22 cell; Oxygen-glucose deprivation/re-oxygenation model; Cell doubling time; Cell death; Cell morphology