Abstract: Objective To study the neuroprotective effects of citicoline (CC) on the toxicity induced by 6-OHDA towards dopaminergic mesencephalic neurons in primary culture-Parkinson′s disease (PD) model in vitro and its mechanism. Methods Mesencephalic neurons in culture were prepared from embryonic 15-day Wistar rats. Cultures were treated for 6, 8, 10 d with various concentrations of CC (2,1,0.1,0.01 and 0.001 mmol·L^-1). At 11th day, the cultures were co-treated with the toxin 6-OHDA (50 μmol·L^-1) for 0.5 h, the cells were collected. Seven groups were categorized as follows:CC (2,1, 0.1, 0.01 and 0.001 mmol·L^-1) +6-OHDA, control and 6-OHDA group. The cell viability was evaluated with MTT assay. Intracellular free Ca²⁺，Ca²⁺i， was labeled by using the fluorescent dye Fluo3-AM and detected by flow cytometer. By measuring the intracellular Rhodamine 123 fluorescence density with flow cytometer, mitochondrial membrane potential (MMP) was evaluated. Results Cultures were treated with 2, 1 and 0.1 mmol·L^-1 CC, the viability of cell was increased (P<0.05). 1, 0.1, 0.01 and 0.001 mmol·L^-1 CC significantly attenuated 6-OHDA-induced neurotoxic effects. As compared with 6-OHDA, the viability of neurons increased, the mean Ca²⁺i was significantly lower in cells treated with CC(1, 0.1, 0.01 and 0.001 mmol·L^-1 ) plus 6-OHDA (49.30±7.62)% than that in 6-OHDA group without CC treatment (P<0.01). The densities of Ca²⁺i in CC (1, 0.1, 0.01 and 0.001 mmol·L^-1) plus 6-OHDA groups were (32.23±1.87)%,(17.09±7.45)%,(21.71±8.89)%,(29.18±4.71)%, respectively，and the density of mean Ca²⁺i in 6-OHDA group was (49.30±7.62)%. MMP significantly increased（P<0.01）. Conclusion CC has an important effect on dopaminergic cell survival in vitro in a validated model of PD. The neurotoxic effect of 6-OHDA can be reduced by CC and CC can increase the viability of neurons, decrease Ca²⁺i, maintain MMP at a relatively higher level.

Key words: neurons, cells, cultured, dopamine, Parkinson′s disease, neuroprotective remedy