[Effect of intracellular-free Ca2+ concentration on transient magnesium-free treatment induced epileptic injury in developing cortical neurons of rats].
OBJECTIVE
To investigate the effect of intracellular-free Ca2+ concentration ([Ca2+]i) on injury following transient Mg(2+)-free treatment in vitro in developing cortical neurons.


METHODS
Embryo cortical neurons of rats cultured for 6 d and 17 d were directly exposed to Mg(2+)-free media, or pretreated with NMDA receptor antagonists or calcium channel antagonist before being exposed to Mg(2+)-free media. MTT assay was used to study the injury of neurons. [Ca2+]i were measured using fluo-3, a fluorescent calcium-sensitive dye and laser-scanning confocal microscope, and calculated by the fluorescent intensity.


RESULTS
Compared with control, MTT conversion rates decreased after transient (3 h) Mg(2+)-free treatment in neurons cultured for 6 d and 17 d in vitro, (59.1 +/- 6.87)% and (51.2 +/- 5.90)%, respectively. In neurons pre- and co-treated with 10 mumol.L-1 MK-801, 50 mumol.L-1 AP-5 and 10 mumol.L-1 nifedipine, MTT conversion rates were higher than those of neurons with only Mg(2+)-free treatment. Peak values of [Ca2+]i in neurons cultured for 6 d and 17 d were 2.4 +/- 0.23 and 3.2 +/- 0.32, respectively. Peak value of neurons 17 d in vitro was significantly higher than that of neurons 6 d in vitro (P < 0.05). In neurons pre- and co-treated with MK-801, AP-5 and nifedipine, [Ca2+]i were lower than those of neurons with only Mg(2+)-free treatment.


CONCLUSIONS
Neuronal injury and [Ca2+]i changes following Mg(2+)-free-treatment-induced seizure were different between neurons 6 d and 17 d in vitro. It suggested that the age-dependent [Ca2+]i changes might play a role in an age-dependent manner of injury following Mg(2+)-free-treatment-induced seizure. NMDA receptor-Ca2+ pathway activation was crucial in the [Ca2+]i change and the cellular injury induced by Mg(2+)-free treatment.
Department of Pediatrics, Peking University First Hospital, Beijing 100034, China.
Haiyan
H
Cao
Yuwu
Y
Jiang
Qihua
Q
He
Yue
Y
Chen
Lan
L
Yuan
Xiru
X
Wu
D000814:Aniline Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002478:Cells, Cultured; D003470:Culture Media; D016291:Dizocilpine Maleate; D004827:Epilepsy / Q000139:chemically induced / Q000150:complications / Q000378:metabolism*; D008274:Magnesium; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate; D014966:Xanthenes
D000814:Aniline Compounds; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D003470:Culture Media; D016194:Receptors, N-Methyl-D-Aspartate; D014966:Xanthenes; C059715:Fluo-3; D016291:Dizocilpine Maleate; D008274:Magnesium; D002118:Calcium