Neuron Plasticity Play A Role In Epileptogenous Mechanism

Epilepsy is a common and chronic clinical syndrome characterized by repeatedly paroxysmal discharge of abnormal electrical rhythms in some part of the brain and transient functional disturbance of central nervous system. The study of epileptogenous mechanism has already been focus of epilepsy research such as ion channel, neurotransmitter, neuronal plasticity and glia. Especially, the relation between neuronal plasticity and epileptogenesis has drawn attentions to many researchers. Progresses have made great in the last few years in this point. After siezure, in the neuvous system neural plasticitical changes have take place, which including structural plasticity and functional plasticity. Structural plasticity, for group, has new synapse forming (increasing density or number) and synapsy reorganizing ; for single synapse, Structural plasticity has structural altering or changes of structural parameter such as the changes of postsynaptic density, synaptic interface curvature and synaptic perforation. Functional plasticity namely synaptic transmission efficiacy includes enhancing (excitability)or decreasing(depress) of synaptic transmission efficiacy,which long-term potentiation(LTP) is a typical marker. Study has confirmed that there exist neural plasticitical changes after seizure in the neuvous system such as abnormal synaptic connection forming among neurons, pathological neural circle pathway forming and excitability of brain increasing in patients and animal models. Immediately early genes(IEG) play key role in the previous process. IEG (including c—fox., C—jun., krox) and their coding genetic acting protein(AP) P38s a third messager, which target genes of AP named late reaction gene(LRG). LRG's expression products include neurotransmitter, neurotrophin, neuromodulator, receptors, synapse binding protein. They change excitivity of neural system net and neural system complement ,for example, decreasing of GABA and GLU releasing , decreasing of GABA and GLU receptor s sensitivity, altering of synaptic protein forming, change of releasing process of neurotransmitter, nervous sprouting and new synapse forming. But, all these change take place after seizure . After seizure Patient has bad effect such as cognitive and limbic function damage differently from few seconds to few hours, even days. Whether the previous pathological feature of neuronal plasticty is primary or secondary do not know. To avoid the interference of seizure, the epileptogenesis animal model, i.e animal model before seizure was usd to study epileptogenous mechanism so that the authentic epileptic pathological features could directly been observed. Pentylenetetrazol is a central nervous system drug that induce chronic and acute epilepsy model to study epileptogenous mechanism .Kindling is a natural epileptogenous phenomenon. Pentylenetetrazol-kindling animal model is ideal epilepsy model to study epileptogenous mechanism. Previous studies have confirmed that changes of neuron plasticity have taken place such as mossy sprouting,new synapse forming and reorganizing by the methods of electronic microscope and Timm stain in epileptogenesis of pentylenetetrazol-kindling rat. But these is not all, problems still need being resolved such as molecularobiological base , neuronal apoptosis and regulating mechanism.Apoptosis is a kind form of neural cell death and key reason of neural cell abnormal excitability and synaptic circle forming. After seizure, the number of neural cell apoptosis and apoptosis related genes(P53, bax) increases obviously. Especially increasing of p53 expression makes neuron died broadly and leads to change of neuron plasticity of synapse connection reorganizingNuclear factor κappa B (NF—κB) is an important nuclear transcript factor and regulates many genes expression and affects many cellular biological functions. Studies show that NF—κB is related to signal transduction of synaptic plasticity and neuron plasticity forming. Growth-associated protein-43(GAP-43) is a nervous system characteristic protein. Two kinds changes of neuron plasticity are confirmed being related to GAP-43:(1)synaptic structural change makes GAP-43 increase ; (2) changes of synaptic transduction efficacy that long term potential and long term depression can alter GAP-43 phosphatize state and mRNA expression.As special presynaptic terminal membrane protein, P38 stand for synaptic density and location. Examining its expression level is a way that exams synapsy forming and synaptic functional state. Glia do not express P38, therefore it stand for neuron plasticity. Neural cell adhesion molecule (NCAM) is a membrane glucoprotein . Studies show that Neural cell adhesion molecule is closely related to synaptic plasticity and neuron plasticitical formation.To explore whether apoptosis and neuronal plasticity exist, neuronal plasticity-associated Proteins and Genes NF—κB, GAP-43, NCAM and P38 plays an important role in neuronal plasticity and whether NF—κB was activated and the mechanism of the NF—κB being activated in epileptogenesis. In this study, pentylenetetrazol was used to kindle rats and the methods of Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL), Flowocyctometer, Reverse transcription polymerase chain reaction (RT-PCR) and Western Blot was used to examine the apoptosis and the expression of NF—κB, GAP-43, P38, NCAM and NF—κB activation and the mechanism of the NF—κB being activated respectively to explore the epileptogenous mechanism.Objective:1. To explore whether apoptosis play an role in epileptogenesis by observing neural cell apoptosis in epileptogenesis of pentylenetetrazol- kindling rat;2. To confirm that neuronal plasticity play an important role in epileptogenesis,neuronal plasticity-associted Proteins GAP-43 and NF—κB, P38 plays an important role in neuronal plasticity in this process by observing the expressions of NF —κB, GAP-43 and P38 in epileptogenesis of pentylenetetrazol-kindling rat3. To investigate the expression of neural cell adhesion molecule 140(NCAM-140) in epileptogensis of penlylenetrazol-kindling rat and its significance.4. To investigate how NF—κB play an important role in neuronal plasticity by observing effects of pentylenetetrazol on intracellular Ca²⁺ and NF—κB in neuron-like PC12 cells. Methods1. Pentylenetetrazol (PTZ, 40mg/kg, intraperitoneal injection, daily) was used to kindle rats and the methods of TUNEL and Flowocyctometer was used to examine the apoptosis. The difference of apoptosis between control group, no-drug group and drug group(pentylenetetrazol,30mg/kg, ip,daily) during kindling were measured by image analysis system.2. PTZ was used to kindle rats and the methods of RT-PCR and Western Blot was used to examine and the expression of NF—κB mRNA, GAP-43and P38 protein in hippocampus respectively. The difference of expression of NF—κB, GAP-43and P38 in hippocampus between between control group, no-drug group and drug group during kindling were measured by image analysis system.3. Pentylenetetrazol was used to kindle rats and the Reverse transcription polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of NCAM in hippocampus. The difference of mRNA expression of NCAM in hippocampus between control group, no-drug group and drug group at different time point during kindling were measured by image analysis system;4. The subcultured neuron-like PC12 cells were pretreated with PTZ. The changes of the concentration of intracellular Ca²⁺ and the expression of NF—κB in nuclear under different conditions were measured by confocal laser scanning microscope(CLSM)。Results1. no-drug group group were kindled at 17～23d after PTZ, ip . drug group were kindled at 30～38d after PTZ add Phenobarbital(30mg/kg,daily), ip .2. The expression of NF—κB mRNA, growth-associated protein-43 and P38 in hippocampus in no-drug groups increased compared with that in control group(P<0.01); The expression of NF—κB mRNA, growth-associated protein-43 and P38 in hippocampus in drug group decreased compared with that in no-drug group (P<0.05) .3. All rats in no-drug group were kindled at 17-23d of PT Z,ip,while rates in drug group were kindled at 30-38d of PTZ ip .During PTZ kindling, before the occurrence of spontanerus seizure, rats were confirmed by behavivo and EEG at 4d, 10d ,16d .The mRNA expression of NCAM in hippocampus in no-drug group continuously increased compared with that in control without using PTZ. The mRNA expression of NCAM in drug group decreased compared with that in no-drug group (P<0.01) .4. Both the concentration of intracellular Ca²⁺ and the expression of NF—κB in nuclear in the PTZ-treated group were higher than those in the control group.Conclusions1. During kindling apoptosis has no existence; It is suggested that apoptosis does not plays an important role in the process of the epileptogenesis;2.The expression of NF—κB, GAP-43 and P38 increased before PTZ-kindling. It is suggested that changes of neuronal plasticity have taken place in epileptogenesis , GAP-43, NF—κB and P38 play an important role in neuronal plasticity in epileptogenesis. Neuronal plasticity plays an important role in neuronal plasticity in epileptogenesis;3. The expression of NCAM mRNA is positively correlated with the process of PTZ-kindling. It is suggested that NCAM plays an important role in the process of the epileptogenesis.4. PTZ could activate NF—κB and increase intracellular Ca²⁺.Ca²⁺ might modulate the activation of NF—κB. It is suggested that NF—κB, Ca²⁺ and the pathway which Ca²⁺ modulate the activation of NF—κB play an important role in neuronal plasticity.