Neuronal Loss And Caspase3 Expression In Sombati's Epileptic Model

Neuronal loss and Caspase3 expression in Sombati's epileptic model Epilepsy is one of common neurological diseases, affecting over 1% of the adult population and over 2% of children. Temporal epilepsy (TE) is one of the most common subtype in partial epilepsy, which accounts for 20- 30% of all epileptic population. Most of TE will progress to intractable and refractory to medical treatment, so it is harmful to the community. It is belived that TE is associated to hippocampal neuron loss, but the particular mechanism is unknown. Sombati developed a recurrent spontaneous epileptic model in primary cultured hippocampal neuron after transient Mg free culture in 1995. This model is similar to TE in clinic. Sombati found hippocampal neuron loss after recurrent epileptic discharge in the model, but the particular way by which the neuron loss isn's investigated. This research investigated the particular way by which the neuron loss and caspase3 expression in hippocampal neuron after recurrent epileptic discharge and aimed to give some clues hippocampal sclerosis in TE patients. This research consists of two parts. 1. Neuron loss in Sombati's epileptic discharge model Hippocampal neuronal cultures were prepared from hippocampal tissue isolated from newborn SD rats and processed according to Sombati's procedures and testified by whole cell recording, then DNA ladder electrophoresis, Tune! stain and fluorescence labelling were applied to detect apoptotic cells. Dead neurons at different time were calculated with flow cytometry finally. This research found that the model neurons menifested recurrent spontaneous spike discharge from 4-16Hz and paroxysmal depolarizing shifts(PDSs). Model cells show apparent 5 morphological changes, some cells exhibit slight swelling cell body andthinning and beeding neurites, others lost their normal shaPe ethibitingWhole cell blebbing. There scattered positive staining cell in the culture inTunel stain. The purple stained nuclei look like kidney or blebbing formand lost normal shaPe. There were no positive staining in cells withnormal outword aPPearance. Both controlled and model cultures havethree tyPes of positive lablling in fluorescence labelling. Cells onlylabelled with PI were necrotic neurons, whereas with Annexin-V wereeary apoptotic ones or aPoptotic body. Cells labelled with PI andAnnexin-V necrotic or lately aPoptotic neurons. Both groups show DNAladdering in electrophoresis. Flow cytometry find necrOtic cells haveincreased within 3 hours after Mg free culture in model group, thenaPproximate to identical in both groups. The aPoptotic cells have beenincreasing significantly after 6 hours Mg free cultUre in model group,since then there have been more aPoptotic cells in model grouP thancontrol in unit time.This research document that neurons die in two ways after recurrentepileptic discharge: becausing of enormous energy consumPing, theneurons die in necrosis mainly in the early phase, whereas die in aPoptosisin late phase.2. Caspase3 cDNA cloning and it's exPressing in Sombati modelAPoptosis means cells suicide themselves and it is induced byapoptotic triggers. The pathway can be divided into four stages and thestages are presented as a sequential series of events including initiation,propagation, conunitriellt, and execution and each stage is programed bycertain genes. Caspase3 is the key member of Caspase family. Aiming toexPlore the molecular mechanism of neuronal aPoptosis in Sombati modeland afford basis for control neuronal aPoptosis, this researc...