The Research About Relevant Mechanisms And Expression Of N-Shc, Egr-4and KCC2in The Immature Neurons Of Focal Cortical Dysplasias Ⅱa Relative Intractable Epilepsy

The expression and effect of Egr-4、N-Shc and KCC2in thefocal cortical dysplasias IIa relative intractableepilepsyObjectiveTo investigate the expression and effect of Egr-4、 N-Shc and KCC2in the focalcortical dysplasiasIIa(FCDIIa) relative intractable epilepsy.Methods1. The expression of N-Shc and KCC2were checked by super SP staining ofimmunohistochemistry in ten cases of FCDIIa relative intractable epilepsy and tencases of normal brain tissue which surgical removal of acute intracranial hypertensionafter traumatic brain injury were taken as a control group.2. The contents of N-Shc protein were measured by Western blotting in six cases ofFCDIIa and normal brain tissue respectively.3. The expression of N-Shc, Egr-4and KCC2mRNA were measured by the methodsof RT-PCR in ten cases of FCDIIa and normal brain tissue respectively.Results1. Immunohistochemistry：compared with the control group, the expressions of N-Shcand KCC2are significantly positive in dysmorphic neurons(DNs) of FCDIIa.2. Western blotting： the expression of N-Shc pretein in FCDIIa are significantlyincreased compared with the control group.3. Real-time qPCR: the transcription and protein expression of N-Shc, Egr-4andKCC2were higher than that in control group. Conclusion1. The increasing of N-Shc and KCC2in the DNs may have a close relationship withdevelopment of epilepsy in the FCDIIa.2. In the development of epilepsy of the FCDIIa, the BDNF may activate twopathways BDNF-SHC-KCC2and BDNF-EGR-KCC2expressed in the DNs,whichcausing increased expression of KCC2and induced neuronal chloride ionconcentration. Ultimately causing inhibitory effects of GABA-mediated andinhibition of epilepsy. The second partThe expression and effect of N-Shc and KCC2in the primaryculture of isolated embryonic rat cerebral immature cortexneuronsObjectiveTo investigate the expression and effect of N-Shc and KCC2in the primary culture ofisolated embryonic rat cerebral immature cortex neurons after BDNF intervention.Methods1、The rats with18pregrant days were anesthetized by hydral and were disinfectedwith alcohol, then took out cortical tissue of the embryonic rats,removed theleptomeninges under an anatomical microscope, dissociated cortical tissue with0.125%trypsin and then dissociated mechanically. The cerebral cortex neurons wascultured in vitro and divided into Group A: immature groupⅠ; Group B: immaturegroup Ⅱ; Group C: mature group. Group A1: BDNF intervention immature group Ⅰ;Group B1: BDNF intervention immature groupⅡ; Group C1: BDNF interventionmature group. Group A:Primary neurons at days in vitro （Div）1were cultured byserum-free medium for12hours. Group B: Primary neurons at days in vitro （Div）4were cultured by serum-free medium for12hours; Group C: Primary neurons at daysin vitro （Div）7were cultured by serum-free medium for12hours; Group A1:Primary neurons at days in vitro （Div）1were treated with BDNF (50ng/ml) andcultured by serum-free medium for12hours; Group B1: Primary neurons at days invitro（Div）4were treated with BDNF (50ng/ml) and cultured by serum-free mediumfor12hours; Group C1: Primary neurons at days in vitro （Div）7were treated withBDNF (50ng/ml) and cultured by serum-free medium for12hours; The expression ofNSE in cerebral cortex neurons were measured with immunohistochmistry in eachgroups．The growth and purity of cerebral cortex neurons were observed under themicroscope.The expression of N-Shc and KCC2mRNA and pretien were measuredby the methods of RT-PCR. 2、 The cerebral cortex neurons cultured under hypoxia condition at days in vitro（Div）4and divided into Group D: control group; Group E: BDNF group; Thecerebral cortex neurons cultured under normal condition at days in vitro （Div）4anddivided into Group D1: control group; Group E1: BDNF group; Group D: Primaryneurons at days in vitro （Div）4were treated with CoCl2（100μmol/ml） andcultured for30minutes; Group E: Primary neurons at days in vitro （Div）4weretreated with CoCl2（100μmol/ml） and cultured for30minutes and then treated withBDNF (50ng/ml); Group D1: Primary neurons at days in vitro （Div）4were culturedby serum-free medium for30minutes; Group E1: Primary neurons at days in vitro（Div）4were cultured by serum-free medium for30minutes and then treated withBDNF (50ng/ml). Cell morphology were observed under the microscope,andintracellular chloride fluorescence intensity were measured via laser scanningconfocal microscope and chloride fluorescence probe in each groups．Results1、 The groups of serum-free medium (DIV1and DIV4): Morphologicalcharacteristics of cortical neurons were immature. The group of serum-free medium(DIV7): the body of cerebral cortex neurons showed cone-shape,appeared shine andhad halation aroundly.They also had three or four processes which were strong andits’ terminal branch growed into neural networks and supported for each other. Thecells (DIV7) were confirmed to be cerebral cortex neurons by NSE staining. Thepurity of cerebral cortex neurons was more than90%in all groups.2、 The transcription and protein expression of N-Shc and KCC2were higher afterBDNF treated. That were the highest in the BDNF intervention immature groupⅡ.3、 Cultured under hypoxia conditions, compare with the BDNF treated group, thechloride ion fluorescence intensity of cerebral cortex neurons cultured after30mintures were stronger than that in control group (P＜0.01). Cultured under normalconditions, compare with the BDNF treated group, the chloride ion fluorescenceintensity of cerebral cortex neurons after cultured30mintures were stronger than thatin control group (P＜0.01). Conclusion1、 The groups of serum-free medium (DIV1and DIV4): Morphologicalcharacteristics of cortical neurons were immature.However, The groups of serum-freemedium (DIV7) was negative.2、The purity of cortical neurons in all groups were more than90%,and which weresuitable for cytological experiments.3、 In the immature fetal rat cortical neurons, the BDNF may activateBDNF-N-Shc-KCC2pathway expressed in cells and induced the increasingof KCC2.4、 Through the intervention of immature fetal rat cortical neurons in vitro,andsimulated hypoxia pathological state during clinical epileptic seizures. We believe thatthe BDNF may induced the increasing of KCC2in the immature fetal rat corticalneurons under hypoxic conditions.