Establishment of the model of oxygen-glucose deprivation and reperfusion in hippocampus neurons from neonatal rat in vitro

Abstract

Objective To culture primary hippocampus neurons and establish a better oxygen-glucose deprivation and reperfusion (OGD/R) model for further research on neurons in vitro. Methods L-poly lysine and rat tail collagen were used as the growth medium in vitro culture of neurons, then hippocampal tissues were isolated from the newborn SD rats (within 24 hours of the birth), digested by single trypsin and bath shock, and made into cell suspension. The suspension was cultured with nutrient solution. 7 days later, to replace the medium with sugar free blood, and expose neurons to hypoxia in a three gas incubator containing 95% nitrogen (N2) for 2 hours, then cells were returned to former incubator under normoxic conditions for 24 hours to build a OGD/R model. Evaluating the OGD/R model with observing the morphological changes of neurons and the cell viability with anti-microtubule-associated protein-fluorescein-5-isothiocyanate (anti-MAP2-FITC) immunofluorescent staining and cell activity assay kit cell counting kit-8 (CCK-8) before and after the establishment of OGD/R model. Results With a fluorescence microscope, neuronal dendrites and axons stretched and overlapped into a network under normal circumstances were observed. After the establishment of OGD/R model, neuronal synapses retracted, cell disintegrated, reticular structure was destroyed and a decrease in neuronal viability was found by CCK8 test. Conclusions The neurons obtained by the aboved method were in good growth state, and the purity was higher than 95%. The OGD/R model of hippocampus neurons was successfully established by the way of replacing the normal medium with the sugar free medium combined with high purity N2 for 2 hours from neonatal rat in vitro. Key words: Hippocampus neurons; Primary cultivation; Hypoxia-ischemia, brain; Oxygen-glucose deprivation and reperfusion; Three gas incubator; In vitro; Rats