An Easy and Effective Way to Produce Experimental oxygen-glucose Deprivation in Cultured Neurons
摘要
BACKGROUND: Oxygen-glucose deprivation (OGD) in cultured neurons simulates stroke to a certain degree and plays an important role in studying processing and pathophysiological mechanism of ischemic neuronal injury. OBJECTIVE: To produce experimental OGD models in cultured neurons. DESIGN, TIME AND SETTING: A grouping controlled study was performed at the Center Laboratory of Third Hospital, Peking University from January 2007 to March 2008. MATERIALS: Fetal Wistar rats with gestational age of 17-19 days were collected in this study. METHODS: Primary cultures of cortical neurons that were derived from fetal Wistar rats with gestational age of 17-19 days were performed to remove pollutional non-neuronal cells. OGD was produced by incubation with non-glucose balanced salt solution and 2% Oxyrase in 7-day cultured cortical neuron cultures. Cell cultures were kept in a humidified 37 ℃ incubator. In the control group, cell culture medium was replaced with balanced salt solution containing 20 mmol/L glucose. In the sham operation group, balanced salt solution containing 20 mmol/L glucose and Oxyrasewere used to replace the medium. MAIN OUTCOME MEASURES: Oxygen concentration in the culture medium was measured with blood gas analysis; neuronal death in the experimental group was observed under phase contrast microscope; lactate dehydrogenase activity was detected with lactate dehydrogenase assay; effect of oxygen-glucose deprivation on neuronal viability was observed with trypan blue staining. RESULTS: Measurement of oxygen concentration showed that hypoxia could be quickly achieved shortly after the addition of Oxyrase; lactate dehydrogenase assay revealed that after treatments of neuron cultures with Oxyrase and non-glucose balanced salt solution, lactate dehydrogenase release increased significantly with the treatment time; trypan blue staining and phase contrast microscope showed that cell viability decreased after treatments of Oxyrase and non-glucose balanced sal