Abstract:

Objective     To explore the optimal condition of primary culture for anterior cingulate cortex(ACC) neurons in neonatal rats and identify their purity and viability. Methods     Neurons were isolated from the ACC of neonatal rats, and then digested with a low concentration of tripsin for a long time. When the cells were cultured till day 4, Ara-c was added to purify them. The cultured ACC neurons were identified by the microtubule-associated protein2(MAP2) immunocytochemical method. Viability of ACC neurons in the cell cycle(about 14 d) was investigated by the measurement of optical density(OD) values at 570 nm using the MTT method.  Results     ACC neurons from neonatal rats were successfully cultured under the present experimental condition. On day 5 of culture, most cells appeared multipolar and had more than two ecptoms and their interlacing looked like a piece of a net. On day 7, many kinds of connections presented among these cultured cells and even aggregation of cells appeared. The cultured neurons were stained by immunocytochemistry(ICC) and the green immunoreactive product was seen in neurons under a fluorescence microscope, indicating that the cultured neurons were positive. The MTT experiment shows that viability of ACC neurons cultured from day 7 to day 9 was the best. Conclusion     ACC neurons in neonatal rats can be cultured in vitro, and primary cultured ones may serve as a cell model in vitro for  research into ACC, indicated by the result of purity and viability in ACC neurons.

Key words: Gyrus cinguli; Neurons; Immunohistochemistry; Primary culture; Viability assessment