The Effects Of Valproate On Epileptic Mossy Fiber Sprouting Induced By The ERK1/2

Objective: To observe the effects of valproate sodium on the expression of phosphorylated ERK1/2, growth associated prorein and synaptophysin after epileptiform discharge of hippocampal neurons, and investigate the anti-epileptic mechanism of valproate sodium from cell molecular organism point of view.Methods: Neonatal Wistar( < 24 hours) rats were adopted. Hippocampal neurons were cultured in neurobasal medium supplemented with 10% fetal bovine serum, and placed into a 37oC, 5% CO2 cell culture incubator. The neurons were devided into (1) Normal control group: On day 9 after beginning the incubation, we replaced the maintenance medium with normal extracellular fluid for three hours, then recorded the discharge using whole-cell patch clamp techniques. (2) Model group: On day 9 after beginning the incubation, we replaced the maintenance medium with magnesium-free extracellular fluid for three hours, then recorded the epileptiform activity using whole-cell patch clamp techniques. (3) valproate sodium group: In concentration-response relationship experiment, 30 min before epileptiform discharge, neurons were incubated with different concentrations of valproate sodium (50mg/L,75mg/L and 100mg/L). Expression of p-ERK1/2 in different concentrations were detcteded by immunofluorescence technique. In time-response experiment, neurons were incubated with valproate sodium (50mg/L) at different time points (30 min before epileptiform discharge and 0min, 30min, 2h and 6h after epileptiform discharge), western blot were used to observe the changes of pERK1/2, GAP-43 and SYP.Results: (1) Hippocampal neurons epileptiform discharge was recorded by EPC-10 system in whole-cell patch clamp. Action potential could be recorded in neurons, who had been treated with normal extracellular fluid for 3 hours. Epileptiform discharges of neurons recorded from cultured neurons that were exposed to magnesium-free extracellular fluid for 3 hours manifested larger, longer duration synaptic potentials and multiple action potentials.These evolved into continuous tonic high-frequency burst discharges that resolved into recurrent epileptiform discharges after return to normal medium.(2) Immunofluorescence labeling, In the normal control group, pERK1/2 were detected in the cytoplasm predominantly; pERK1/2 could be observed bouth in the cytoplasm and nucleus in the model group; In valproate sodium group, the expression of pERK1/2 was inhibited significantly (p <0.01) with different concentration valproate sodium. However, there was no significant difference in different concentrations of valproate sodium(p >0.05).(3) We measured the levels of pERK1/2, SYP and GAP-43 by western blotting. In the time-response experiment, GAP-43 and SYP expressed all the time, and peaked at 30min, whose tendency were the same as pERK1/2 in the model group.Compared with model group, there were significant differences expression of these proteins in the valproate sodium group at each time point. Moreover, it was most obviously at 30min before epileptiform discharge.Conclusion: (1) Neurons cultured in Neurobasal medium with fetal bovine serum was fit to be treated with magnesium-free extracellular fluid for three hours. The neuronal epileptiform discharge model was stabile.(2) ERK1/2 was activated after neuronal epileptiform discharge, and accompanied by increased levels of GAP-43 and SYP, which were considered to be markers of MFS. In addition, the expression of these proteins could be decreased by valproate sodium.(3) We observed the effects of valproate sodium on the MFS after epilepsy, and investigated the anti-epileptic mechanism of valproate sodium from cell molecular organism point of view.