Neuroprotective effects of citicoline on 6-hydroxydopamine-treated mesencephalic dopaminergic neurons in primary culture

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 Ca~(2+),[Ca~(2+)]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(P0.05).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[Ca~(2+)]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(P0.01).The densities of [Ca~(2+)]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 [Ca~(2+)]i in 6-OHDA group was(49.30±7.62)%.MMP significantly increased(P0.01).(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 [Ca~(2+)]i,maintain MMP at a relatively higher level.