Research on the improved culture of hippocampus neurons from rat in vitro

Abstract

Objective To establish an improved culture method of rat hippocampus neurons for further research on the hippocampus neuron related diseases such as hypoxia-ischemic brain damage model. Methods The culture of neonatal rat hippocampus neurons was performed by using improved method of this study. The criteria for judging the success of primary cultured hippocampus neurons in vitro was that the expression of microtubule-associated protein (MAP) 2 in neuronal cell bodies and dendrites could be seen under fluorescence microscope. The improved method was as follows: ① primarily L-polylysine and rat tail collagen were used as the growth medium in vitro culture of neurons, then hippocampal tissues were isolated from the newborn rats (within 24 h of the birth), digested by single trypsin and bath shock, and then made into cell suspension. The suspension was collected and inoculated in the culture of DMEM medium with 10% fetal bovine serum by suitable planting density, and then the neurons were moved into nutrient solution after 4 h. The half of medium was replaced every 3 d. The morphological changes of neurons in different stages were observed under phase-contrast microscope. ②The derivation of cells was identified by immunofluorescent staining with anti-MAP2-fluorescein-5-isothiocyanate (FITC) immunofluorescence assay. Results ①The results in vitro culture of hippocampus neurons showed a large number of hippocampal neurons began to adhere to the glass slides within 30 min.After 2 h, the adherent wall was obvious, and a few cells began to develop small neuritis. Up to the 5th day, many neurites extended to form dense network. The neuron differentiation was more mature, and the synapses between neurons were more closely linked on the 7th day.Up to the 21st day, hippocampus neurons began to degrade and denature, cell bodies were in a state of shrinkage, and residual traces of cell bodies could be found, halo around cell bodies disappeared, refraction weakened, dendrites were fused and extended into long and thick state, and networks of neurons were thick and aging. ②Anti-MAP2-FITC immunofluorescence assay demonstrated that the rate of positive cells of MAP2 was 96.3%. Conclusions The neurons obtained by the above improved method are in good growth state, and have higher purity, which provide the conditions for further study of hippocampus neuron related diseases, such as hypoxia-ischemic brain damage model. Key words: Hippocampal neurons; Primary cultivation; Cells, cultured; Morphological characteristics; Fluoroimmunoassay; Hypoxia-ischemia, brain; Rats