The Establishment Of Immature Epileptic Rat Model Induced By Pilocarpine And Expression Changes Of GFAP, SynapsinⅠ

Epilepsy is a common neurologic disorder that occurs more frequently in children.Despite some of the seizures can be controlled by reasonable administration of antiepileptic drugs(AEDs),there are still lots of them have no response to the AEDs and progressively become intractable epilepsy(IE),which has seriously detrimental effects on the developing brain.Meisal temporal lobe epilepsy(MTLE),originated from the hippocampus of temporal lobe,which represents about 10-30% of all childhood epilepsy cases,is the most common type of IE,and nearly 70%of them are pharmacoresistant.It is reported that lots of factors are related to the future refractoriness,such as status epilepticus (SE),less than 1 year of age at onset of seizures,neonatal seizures,febrile convulsion,trauma and infection of the developing brain,changes in type of epilepsy during the clinical course,and so on.But the mechanism of MTLE is still not clear,so children with epilepsy present unique challenges to the clinician,which means that it is necessary to establish an ideal model for the study of the mechanism contributing to epileptogenesis.MTLE is often the result of an early insult that induces changes and reorganization of the hippocampal formation.The morphological abnormalites in the hippocampus include neuron loss and mossy fiber sprouting(MFS),both of which are the representative pathologies of hippocampus in MTLE and play major roles in epileptogenesis.It is proposed that the initial neuron loss in the brain regions involves in seizure initiation,spread,and clinical expression;the zones of mesial temporal cell loss are linked to zones of epileptogenicity.The mossy fiber, also called the axonal of the granule cell,can sprout in pyramidal cell region of CA3 and supragranular of dentate gyrus,leading to the creation of new recurrent excitatory network and the increment of the seizure susceptibility.In addition to the neuron loss and mossy fiber sprouting,some proteins are also relevant to the development of epilepsy,such as glial fibrillary acidic protein(GFAP) and synapsinⅠ.GFAP is a cytoskeletal intermediate filament protein representing the activation of astrocytes. Astrocytes play an important role in the central nervous system,such as the regulation of ion homeostasis,neurotransmission and synapse formation.SynapsinⅠbelongs to one family of phosphoproteins,which is a critical factor in synaptic development,synaptogenesis,and neurotransmitter release.That is to say that synapsinⅠcould act as a molecular marker of synaptic growth and plasticity in neuronal networks that maintain high levels of activity,such as epileptogenesis.Most of the experiments on epilepsy in rats have been focusing on adulthood stage currently;there are few studies to mimic the clinical situation that patients have recurrent seizures in childhood.Besides,the immature brain differs considerably from the mature brain in the etiological factors,the development and propagation of seizures,the behavioral and EEG features of the seizures,the response to AEDs and their consequences.In our experiment,we employed pilocarpine on immature rats to induce status epilepticus,and investigated the morphological changes of hippocampal formation;by using immunohistochemistry and western blotting analysis,we examined the dynamic expression of GFAP,synapsinⅠin the subfield of hippocampus and evaluated whether they had correlation with the epileptogenesis.SECTION ONE The establishment of pilocarpine-induced animal model of mesial temporal lobe epilepsy and morphological changes of hippocampal formation in immature epileptic ratsObjective To establish an easy,reliable animal model depicting the human mesial temporal lobe epilepsy.Methods 165 3-week-old healthy male Sprague-Dawley rats were divided randomly into control group And LiCl-Pilocarpine experimental group.The experimental rats were intraperitoneally injected with LiCl(127mg/kg) and Pilocarpine (50mg/kg) to induce status epilepticus(SE).10%chloral hydrate was injected 90 minutes after SE onset.Survival rats were continuously observed for onset and recurrence of spontaneous seizures every day and were grouped and sacrificed at 1.5h(group A),3w(group B) and 8w (group C) after the last pilocarpine injection respectively.The control rats were mitted tales doses of 0.9%saline and grouped and sacrificed at 1.5h, 3w,8w after the saline injection.Nissl's staining(to evaluate neuron loss), Timm staining(to evaluate MFS) and immunohistochemistry were performed on half of each subgroup rats,and the rest were executed for the western-blotting analysis.Results 1.94.1%out of 135 rats were induced status epilepticus successfully with a high mortality of 68.8%.In group C,70%rats were observed for spontaneous recurrent seizures and EEG was featured by epileptic spike-wave when they grew up.In control rats,behavioral and EEG manifestations are normal.2.MFS and neuron loss were observed in group B and group C except for group A.MFS existed in dentate gyrus and CA3 subfield of the hippocampus and had a significant difference between group B and group C(P＜0.01).Neuron loss happened in dentate gyrus,CA1 and CA3 subfield,with the loss summit in group B,which had significant difference compared to the control group,group A and group C(P＜0.01).The neuron revived in group C,but in CA3 subfield and CA1 subfield it had significant difference compared to the control group(P＜0.05).Conclusions We employed LiCl and pilocarpine,and established an easy,ideal animal model depicting the human mesial temporal lobe epilepsy.Neuron loss and mossy fiber sprouting are involved in the epileptogenesis. SECTION TWO The study on dynamic expression of GFAP, synapsin I in hippocampus of the pilocarpine-induced immature epileptic ratsObjective To investigate the expression changes of GFAP, synapsin I in hippocampus of the immature epileptic rats during the development of epilepsy.Methods Based on the epileptic animal model set up in SECTION ONE,using western-blotting analysis and immunohistochemistry,we examined the dynamic expression of GFAP and synapsin I in the subfields of hippocampus.Results 1.Analyses of western blotting and immunohistochemistry indicated that expression of GFAP in CA3 subfield is up-regulated in both group B and group C,with significant difference between them(P＜0.01),either of which has significant difference compared to group A and the control group(P＜0.01).There is no significant difference between group A and the control group(P＞0.05).2.Analyses of western-blotting demonstrated that the expression synapsin I decreased dramatically in group A compared to the control group(P＜0.01),while showed higher expression in group B compared to the control group(P＜0.01).The expression in group C also has significant difference compared to control group,only this group demonstrated marked decrement(P＜0.01).Analyses of immunohistochemistry demonstrated that in dentate gyrus,CA1 and CA3 subfield of hippocampus,synapsin I immunoreactivity decreased highly in group A compared to control group(P＜0.01);it was enhanced greatly in group B compared to control group(P＜0.01),but only happened in CA3 subfield and dentate gyrus,with greater enhancement in CA3.The expression of synapsin I in group C is down-regulated again in the dentate gyrus and CA3 subfield,and has significant difference compared to control group(P＜0.01).There is no fluctuation of synapsin I expression in CA1 subfield in group B and group C,compared to the control group(P>0.05).Conclusions The increment of GFAP and expression changes of synapsinⅠin the subfields of hippocampus indicated their involvement in the development of MTLE in immature rats.CA3 subfield may be a more important region for the epileptogenesis according to the synapsinⅠdynamic expression in the hippocampal subfields.