Protection Of Intravenous Anesthetic On Cultured PC12 Cells Impaired By NMDA

Experiment I Protection of propofol on cultured PC12cells impaired by NMDAObjective: To evaluate the protective action of propofol on cultured pheochromocytoma(PC12) cells impaired by N-methyl-D-aspartate (NMDA), and explore their possible protective action mechanisms. Method: 1. Cellular culture: The differentiated PC 12 cells were inoculated in the 96 well cellular culture plate with DMEM complete nutrient solution, used to detect the concentration of lactic dehydrogenase(LDH) and MTT(values of OD57onm)- To measure the concentration of incellular calcium ions ([Ca2+]j), we used 6 well cellular culture plate to inoculate the PC 12 cells. While, 100mm diameter cellular culture utensil was used to inoculate cells for measuring the activity of nitric oxide synthase(NOS). The culturing PC 12 cells were put into the CC>2 cell incubator and cultured at 37癈, 5% CO2 for 3 or 4 days and then above respective experiments were performed. 2. LDH and MTT(OD570nm): The PC 12 cells were exposed to 300 u molL"1 NMDA in the absence or presence of propofol 1.56-40014 mol'L^for 4h. The degree of cellular impairment and the cellular viability were detected using LDH and MTT assays, to evaluate the protective efficiency of propofol on the NMDA-induced lesion in PC 12 cells. 3. The measurement of [Ca2+]i: After cells exposing to 300 u mol.L"1 NMDA in the absence or presence of propofol 12.5 or 125 U mol'L"1 for 4h,the change of [Ca2+]i were detected by Fura-2/AM fluorescencelabelling assay, to observe the effects of propofol on incellular calcium ion overload induced by NMDA. 4. The measurement of NOS: The NOS activity of the PC 12 cells treated with NMDA in the absence or presence of propofol was measured with ultraviolet spectrophotometer assay. NOS activity at each unit of tissue was calculated using an admitted formula. Result: 1. After being exposed to NMDA 300 mol'L-1 for 4h, the PC 12 cells were impaired obviously, the release of LDH increased and values of OD570nm decreased significantly(P<0.01), compared with control group. While at the presence of propofol 6.25, 25, 100, 400 U molL"1 for 4 h, the LDH release was decreased and the values of ODsvonm increased in a concentration dependent manner. 2. The PC 12 cells were exposed to NMDA 300 P molL"1 for 4 h, the [Ca2+]f raised significantly. However, after treated with propofol 12.5, 125 u mol.L"1 simultaneously for 4 h, the [Ca2+]j showed less increasement (P<0.01), compared with NMDA group. 3. The NOS activity was increased obviously(P<0.01) when the PC 12 cells were treated with NMDA 300 u molL"1 for 4 h. While propofol 12.5 or 125 u mol.L"1 were used together with NMDA 300 u mol'L"1, the activity of NOS was depressed evidently (P<0.01). Conclusion: 1. The PC 12 cells were obviously impaired when exposed to NMDA 300 U mol'L"1 for 4h. The intravenous anesthetic, propofol 6.25-100 u mol'L"1 can extenuate the impairment when added to the PC12 cells together with NMDA 300 u mol'L"1. It showed that propofol is an effective neuronal protective drug. 2. After the PC 12 cells were treated with NMDA for 4h, the [Ca2+]j and the NOS activity increased significantly. Propofol 12.5 and 125 u mol.L"1 can antagonize these action of NMDA evidently. These results indacate that the protective action of propofol on PC 12 cells impaired by NMDA may bethrough depressing the [Ca2+]J5 inhibiting the activity of NOS , decreasing the production of nitric oxide, and then inhibiting the oxidant stress. In one word, the inhibition of propofol on the NMD A receptor-Ca2+-NOS pathway may one of its cytoprotection mechanisms. Key words: N-methyl-D-aspartate; intravenous anesthetic, propofol; lactic dehydrogenase; calcium ion; nitric oxide synthase; cell, PC 12; protective action, mechanisms.Experiment II Protection of midazolam on cultured PC 12cells impaired by NMDAObjective: To evaluate the protective action of midazolam on cultured PC 12 cells impaired by NMDA, and explore their possible protective mechanisms. Method: 1. Cell culture: The same as the experiment I. 2...