Changes Of NF-κB Expression In Hippocampal Neurons Epilepsy Model

Objective1、Temporal lobe epilepsy is a refractory epilepsy and common clinical syndrome in neurology,howhile its pathogenesis has not been fully elucidated.At present,the role of NF-κB in diseases of the nervous system is one of the hotspots of neurology.2、To investigate the pathogenesis of hippocampal neurons epilepsy in vitro.3、To explore the NF-κB expression and activity of hippocampal neurons discharge induced by magnesium-free extracellular fluid, and further verify the credibility of NF-κB results in rat epilepsy model,which provide some theoretical basis for clinical antiepileptic drugs.MethodsIn this study, primary hippocampal neurons prepared from suckling mice (within24hours of birth) were cultured in vitro, and7days later, the experimental agents were administered.1、Morphological observation:To obtain morphology of cells in different periods and groups when observing them with inverted phase contrast microscope.2、Immunofluorescent staining of MAP2was administered to analyse the purity of neurons:the neurons labeled by MAP2antibody,DAPI nuclear staining,observed and acquised images under a fluorescence microscope.3、Using the patch clamp technique detects epileptiform discharges of neurons.4、Western blotting was chosen to observe the expression levels of NF-κB between the control group and experimental group.5、Immunofluorescent staining was administered to test the NF-κB expression and subcellular localization.Results1.Morphological observation and purity identification of neurons:The primary hippocampal neurons, when cultured after48h, were adhered to the dishes with an elliptical or triangular body and neuritis; when cultured after7days, the neurons were with plump size and plenty of neuritis interweaving a net. The purity of neurons was50to60%on the second day, and the purity of neurons was up to95% when cultured after7days by immunofluorescent staining. The hippocampal neurons by magnesium-free extracellular fluid grew well,part of the neurons aggregation,woven into the neural network.2、Patch clamp test results show that the mg-free extracellular solution for3hours and then returned to normal extracellular fluid,occurs neuron last outbreak epileptic spontaneous discharge,the voltage amplitude of60-80mV,substantial increase in the frequency.3、Western blotting:Western blot analysis showed that the NF-κB protein expression relative gray value difference was statistically significant between the hippocampal neurons treated by magnesium-free (experiment group) and normal control group (p<0.05).4、Immunofluorescent staining:The NF-κB of hippocampal neurons in normal control group expressed mainly in the cytoplasm,however the experimental group treated by magnesium-free mainly in the nucleus,which prompted neurons were activated,consistent with the western blot results.Conclusion1、In primary cultured hippocampal neurons, magnesium-free extracellular fluid can induce hippocampal neurons to produce stable neurons status epilepticus model in vitro.2、Western blot experiment results show that:increased expression of NF-κB in total protein was detected in magnesium-free groups vs normal control groups by western blotting(P<0.05).3、In primary hippocampal neurons, the immunofluorescence results show that:the NF-κB is mainly cytoplasmic expression in normal control group;while in the magnesium-free treatment group,NF-κB expression mainly in the nucleus.NF-κB nuclear translocation prompts neuron activation.4、In summary,the above results suggest that NF-κB is one of the important mechanisms involved in epileptic activity.