ABSTRACT
Objective:To develop an in vitro neuroinflammation model by establishing a microglia-neuron co-culture system. Methods:Mouse microglia (BV-2), motor neurons (NSC34) and hippocampal neurons (HT-22) were selected.This experiment was performed in two parts.Experiment Ⅰ BV-2 microglia were stimulated with different concentrations of lipopolysaccharide (LPS, 10, 100, 500 and 1 000 ng/ml). Microglia culture supernatant(Conditioned Medium) was extracted and two types of neurons were cultured separately.The concentration of LPS that resulted in a significant 50% decrease in neuronal viability was selected using the CCK-8 method for establishment of the Transwell co-culture system.Experiment Ⅱ Microglia were cultured in the upper chamber of Transwell, and neurons were seeded in the lower chamber.Microglia were divided into 2 groups ( n=12 each) using the random number table method: control group and LPS group.In control group and LPS group, microglia were cultured for 6 h with cell culture medium and LPS, respectively, then the medium was replaced with fresh medium, microglia were continuously incubated for 12 h, and then the cells in the upper and lower chambers were combined.The cells were incubated using the BV-2-NSC34 Transwell co-culture system for 12 h and using the BV-2-HT-22 Transwell co-culture system for 24 h. The concentrations of interleukin-1beta (IL-1β) and IL-18 in neuronal culture supernatant were measured by enzyme-linked immunosorbent assay, the apoptotic rate of neurons was determined by flow cytometry, the expression of Bcl-2 and Bax mRNA in neurons was detected by quantitative real-time polymerase chain reaction, and the expression of cleaved caspase-3, Bcl-2 and Bax in neurons was detected by Western blot. Results:Experiment Ⅰ LPS concentration for stimulation was 10 ng/ml in BV-2-NSC34 Transwell co-culture system and 1, 000 ng/ml in BV-2-HT-22 Transwell co-culture system.Experiment Ⅱ Compared with control group, the concentrations of IL-1β and IL-18 and apoptotic rate of neurons were significantly increased, Bax protein and mRNA expression was up-regulated, Bcl-2 protein and mRNA expression was down-regulated, and cleaved caspase-3 expression was up-regulated in LPS group ( P<0.05 or 0.01). Conclusions:The microglia-neuron co-culture system is successfully established by the conditioned medium technique and Transwell co-culture system, which provides an experimental protocol for establishment of neuroinflammation models associated with postoperative cognitive dysfunction.