Prelimary Investigation On Nruronal Damage And Change Of Neuralnetwork In The Cell Model Of Intractable Epilepsy

OBJECTIVE:To investigate the properties of primary cultured hippocampal neurons and observe the morphology of neurons in vitro process.And established the cell model of intractable epilepsy,using it to study neuronal damage and observe the morphologic change of neural network.METHODS:The hippocampal was isolated from the newborn rats and cultured in the serum-free neurobal medium adding B27 supplement.The growth characteristics of hippocampal neurons in vitro was observed througt phase-contrast microscope.The neurons were identified by immunochemistry with antiboby to neurofilament protein (NF).Primary hippocampal neurons cutured for 10th days randomly divided into 2 groups:(1) The cell model group of intractable epilepsy:after removing maintainable media,cultured hippocampal neurons were exposed to magnesium-free extracellular solution media for 3h,and then returned to maintainable media. (2) Control group:after removing maintainable media, cultured hippocampal neurons were exposed to regular extracellular solution media for 3h, and then returned to maintainable media.Lactic acid dehydrogenase(LDH) in supernatant of the two groups is measured in different time. And observed the morphologic change of neurons and network througt optical microscope and scanning electron microscop(SEM).RESULTS:The cultured hippocampal neurons can survived approximately 28 days in vitro by this method,hippocampal neurons were well-developed and network was most densely between 9th and 14th day. The purity of cultured neurons identified by NF reached 90% on the 9th day.Compared to the control group,the level of LDH in supermatant was significantly increased in the model group at different time(3h,6h,24h) after model preparation(P<0.05), with time prolonging, the level of LDH was increasing gradually. Unter phase-contrast microscope,neurons of model group migrated to each other closely after returned to maintainable media for 24h, network appeared to be "reticulated "gradually,then after returned to maintainable media for 3d,parts of neusons inosculated and the "reticulated "network become noticeable increasingly.Unter SEM, cell membranes of neurons were rough and the network piled up.Conclusions:(1) It is suggested that the serm-free neurobal medium adding B27 supplement could prevent glial cells growth,and the cultured neurons have fine viability and high purity.(2) The cell model of intractable epilepsy which established by cultured hippocampal neurons were exposed to magnesium-free extracellular solution media for 3h was Stable and sustained,it provided a good vitro model for studying the pathogenesis of refractory epilepsy.(3) The cell model of intractable epilepsy exists neuronal damage, with time prolonging, neuronal damage was more serious.(4) The cell model of intractable epilepsy exists morphologic change of neural network.