| Background and objective:Epilepsy is one of the common seen neurological disorders in children, with prevalence and incidence of 3.5‰-6.6‰. Mesial temporal lobe epilepsy (MTLE) is the most common epilepsy syndrome with pharmacologically intractable partial-onset seizures. The pathological substrate is hippocampal sclerosis (HS). To date, most of the patients with MTLE do poorly responds to anti-epileptic drugs (AEDs) and surgically interruption, which is a headachy problem in the world. So, the studies about the pathogenesis of MTLE associated with HS, the responds to AEDs and mechanisms of drug-resisitence, are the focus and key fields about epilepsy in the recent decades.The epilepsy in children is quite different with the epilepsy in adults. In early development of the central nervous system, the structure and function, the neurotransmitters and receptors related to epilepsy had enormous room for growth. The influence of seizures to the immature brain and the effect of the AEDs are various in the different stages of the brain's development. Quite a lot of research data shown that, a fairly high ratio of MTLE adult patients with HS can be pursued long history of epileptic seizures, particularly febrile convulsion in childhood. The brain plasticity change after long epileptic seizures in early childhood can produce long-term effects in the individual. MTLE with HS is closely related to the abnormal brain plasticity formation. Under go developmental changes from infancy to adult after the influence of seizures forms, plasticity and learning also contributes to the pathophysiology of epilepsy.The current molecular researches related to MTLE have been focused on regeneration and synaptic plasticity, neurons damage regulation, cellular metabolism and protein synthesis, neurotransmission and signal transduction, cytoskeleton changes and ion channels changes and ect. But a great part of these studies were limited to a certain aspect of a certain time, lacking a systematic and consecutive research. So, considered epileptogenesis's unceasing development and changes, this study aimed to explore the pathogenesis and development of MTLE from a general and consecutive perspective, by using proteomics method to separate and identify the differentially expressed proteins in the different stages of immature rat models with MTLE, combined with bio-informatics technology to perform a global comparative analysis and function classification with the differentially expressed proteins. Then, the wide-broad AEDs valproate (VPA) was administrated in early stage of MTLE development to observe its therapeutic effects and to explore the related mechanisms. Furthermore, to approach the mechanisms and impact of VPA's administration in different aspects, the expression levels of partial significant proteins in the postoperative hippocampus from patients with drug-resistant MTLE were validated, to explore new strategies and targets for clinical intervention in the future.Methods:The research includes three parts:1. For seizure induction, the 3-week-old Sprague-Dawley rats were intraperitoneally injected pilocarpine with different doses of 10 mg/kg,30 mg/kg,50 mg/kg and 100mg/kg, to obtain the most suitable dosage of pilocarpine. The surviving animals were continuously monitored for 8 weeks to observe the spontaneous seizures. After the chronic spontaneous MTLE induced, EEG was recorded to observe the epileptic discharges, nissl and Timm staining were performed to evaluate neurons loss and mossy fiber sprouting, so as to validate the chronic MTLE models.2. The MTLE rats were divided into 6 groups based on different stages (acute stage, latent stage, chronic stage):acute control group, acute seizure group, latent control group, latent seizure group, chronic spontaneous seizure group, chronic no spontaneous seizure group. Proteins in the hippocampus were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) technology. PDQuest software was used to analyze 2-DE images, and MALDI-TOF-MS was used to identify the differentially expressed proteins. Then the differentially expressed proteins were analyzed and classified globally and then the protein interaction network was construction by applying bio-informatics software. Western blot techniques were used to determine the differential expression levels of the partial proteins.3. VPA was administrated at or before the onset of spontaneous seizures for 2 weeks to observe the MTLE rats'responds to AEDs. Nissl and Timm staining were performed to evaluate changes of the hippocampal morphology, so as to evaluate VPA's therapeutic effect. Proteins in hippocampus of VPA-treated and un-treated rats were separated and identified. The differentially expressed proteins were analyzed and classified globally by bio-informatics software to explore the interest proteins related to the VPA's treatment. Furthermore, the expression levels of partial proteins in the postoperative hippocampus from patients with drug-resistant MTLE were detected, to validate the results clinically.Results:1.The seizure rate in 10mg/kg pilocarpine group was 0%, in 30mg/kg group was 30%, in 50mg/kg group was 60%, in 100mg/kg group was 10%. The most suitable dosage of pilocarpine was 50 mg/kg. The chronic MTLE rate was 63.3%. In chronic MTLE rats, there were spontaneous focal motor seizures observed; the EEG shown typical epileptiform discharge; a widespread cell loss and degeneration, reactive gliosis and plasticity of neurons were noted in the hippocampus by nissl staining, and Timm staining found abnormal mossy fiber terminal staining increased in inner molecular layer of dentate gyrus.2. The 2-DE patterns of different stage's hippocampus with chronic MTLE of immature rats were established.110 differentially expressed proteins were identified, which could be divided into several main groups based on their functions:cytoskeleton modification, cell junction, energy metabolism, synaptic plasticity and neurotransmitter release, heat shock proteins, signal pathway regulatory molecules, and etc. The expression levels of several differentially expressed proteins were verified by western blot technique, and the results were coincidence with the proteome analysis.3. The administration of VPA in the early stage could control the spontaneous seizures in the immature MTLE rats. The results of nissl and Timm staining shown that VPA could prevent the neurons loss and the abnormal mossy fiber sprouting which conformed the neuroprotective effects of VPA. The synapse-related proteins synapsin-1, dynamin-1 and neurogranin were differentially expressed between the VPA-treated group and un-treated group. The western blot results both in the MTLE rats and human hippocampus were similar to the changes noted in 2-DE.Conclusion:1. This study successfully established a chronic MTLE model of immature rat, which was well coincidence with human chronic temporal epilepsy in seizure pattern, EEG recording and hippocampus pathology. This may conduce to further studies involving etiology or possible intervention of early stage's hippocampus damage induced adult chronic MTLE2. The consecutive and dynamic 2-DE patterns of hippocampus in immature rats with MTLE were established firstly in this study. The 110 differentially expressed proteins identified by 2-DE in combination with MALDI-TOF-MS may be closely related to the mechanism of temporal lobe epilepsy, which provided important clues to elucidate the pathophysiological mechanisms of chronic MTLE and to identify an optimum medication intervention for the development of pharmacological therapies targeted at MTLE.3. The administration of VPA in the early stage could obtain seizure free based on the immature MTLE rat models, what provided an optimum medication intervention time of anti-epileptic drugs. The synapse-related proteins synapsin-1,dynamin-1 and neurogranin involved in the pathogenesis of chronic MTLE and the pharmacological effects of VPA, which probable developed into the focus of the study related to intractable epilepsy in the future. |
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