Effect of intermittent high glucose on oxygen-glucose deprivation/refurnish neuronal survival / 中华危重病急救医学

ABSTRACT

Objective To investigate the effect of intermittent high glucose on oxygen-glucose deprivation/refurnish (OGD/R) neuronal survival.Methods The primary cultured hippocampal neurons of mice were sub-cultured when the cell fusion reached about 80％.Cells in logarithmic growth phase were placed in a hypoxic incubator (37 ℃,5％ CO2,95％ N2) to simulate cell hypoxia.The culture medium was replaced by glucose-free Hank equilibrium salt solution (HBSS) to simulate cell hypoglycemia.The normal glucose and oxygen control group was set up.Cell morphology was observed under inverted phase contrast microscope after 6 hours of hypoxia and hypoglycemia treatment,and cell viability was detected by CCK-8 cell proliferation assay kit,and then grouping experiment was carried out.The cells were randomly divided into four groups.The cells were cultured in different concentration glucose medium under normal oxygen,5％ CO2 and 37 ℃ for 72 hours to prepare OGD/R model of cell ischemia/reperfusion.The low-glucose control group was cultured in medium containing 5.5 mmoFL glucose.The constant high-glucose group was cultured in medium containing 33.0 mmol/L glucose.The intermittent high-glucose group was cultured in medium containing 33.0 mmol/L glucose for 3 hours then in medium containing 5.5 mmol/L glucose for 2 hours alternately for 3 times during the day,and overnight in medium containing 33.0 mmol/L glucose at night.The hyperosmotic control group was made up of 5.5 mmol/L glucose medium and mannitol.The osmotic pressure was the same as that of the constant high-glucose group,and the effective glucose concentration was the same as that of the normal glucose and oxygen group,so as to eliminate the effect of osmotic pressure changes caused by the high-glucose medium on the results.Cell morphology was observed under inverted phase contrast microscope after 72 hours of cell culture in each group.Cell viability was measured by CCK-8 kit,and apoptotic rate was measured by flow cytometry.Results The inverted phase contrast microscope showed that the cells in the normal glucose and oxygen control group were plump and refractive,and had obvious nucleus,clear processes and high cell activity.After 6 hours of hypoxia and hypoglycemia treatment,the cells were shrunk,refractive index was poor,the nucleus was unclear,the processes were not clear,and the cell activity was significantly lower than that of normal glucose and oxygen control group (A value0.34±0.06 vs.1.09±0.06,P ＜ 0.01),which indicated that the model of oxygen-glucose deprivation (OGD) was successfully prepared.After 72 hours of culture with different concentrations of glucose,the cells in the low-glucose control group were shrunk,the cell membrane was incomplete,the nucleus was unclear,and number of necrotic cells were more.In the constant high-glucose group,the refractive index of cells was poor,a large number of cells floated,and the nucleus was not obvious.In the intermittent high-glucose group,the cell morphology was normal,the refractive rate of cells was decreased slightly,and the necrotic cells were less.In the hypertonic control group,the cell status was close to that in the constant high-glucose group.Compared with the low-glucose control group or constant high-glucose group,the cell viability in the intermittent high-glucose group was significantly increased (A value2.04±0.15 vs.0.64±0.18,1.16±0.16,both P ＜ 0.01),the apoptotic rate was significantly decreased [(59.60 ± 2.55)％ vs.(78.15 ± 15.77)％,(95.60± 0.14)％,both P ＜ 0.05].There was no significant difference in cell activity or apoptotic rate between the hypertonic control group and the constant high-glucose group [cell activity (A value)1.07 ± 0.07 vs.1.16 ± 0.16,apoptotic rate(87.80 ± 4.53)％ vs.(95.60 ± 0.14)％,both P ＞ 0.05].Conclusion Intermittent high glucose within a certain range had protective effect on OGD/R neuronal survival.