| Epilepsy is one kind of chronic brain disease caused by various etiological factors. It is characterized by a series of clinical syndrome constituted of abrupt,temporal,paroxysmal,recurrent attacking clinical and breadboard manifestation induced by excessive discharge of neurons within cerebrum. Epilepsy is a quite high-incidence-rate and rather harmful disease, whose incidence is about 5‰in general population. Temporal lobe epilepsy (TLE), a representative case of partial epilepsy, is a kind of gyrus uncinatus seizure induced by interior basilar epileptic focus of temporal lobe, accounting for 60～70% of the whole disease. The correlation between incidence rate of epilepsy and age reflects the fact that different age stages may have different etiological factors. TLE mainly occurs among youth (10-20 age), 62% of which occurs before 15 age firstly. Generally, sickness rate is the highest before one year old, lower during 1-10 age., In a word, puerile epilepsy accounts for most part of the whole epilepsy disease. Therefore, exploring the pathogenesis of puerile epilepsy is of important significance. The pathogenesis of TLE is quite complicate and vague. Sclerosis of hippocampus is the most universal pathological change, and degeneration,necrosis,lost of neurons and gliosis are mainly features during the sclerosis of hippocampus. It has been demonstrated that neurons lost,gliosis and neurogenesis of hippocampus reside in the pathological path of epilepsy. At present, the establishment of epileptic model in animal is the most predominant method to explore the pathogenesis of human epilepsy and to obtain relative information. So far, the kindling model which is much more close to natural physiological state and human epilepsy is the most ideal model of chronic epilepsy in human. Therefore, by establishing the kindling model, the present study is to observe and explore the pathogenesis of TLE at various levels including entirety,organ and molecule in order to provide new methods in the clinical therapy of puerile epilepsy.It has been reported that neuron stem cells and neurogenesis exist in brain tissue during childhood and even adult age. During the process of epilepsy lost of neurons,gliosis and neurogenesis also exist in hippocampus. As the totipotent cell of nervous system, neural stem cells (NSCs) have strong capability of splitting and proliferation, and can differentiate directionally to neurons and glial cells under the effect of many factors. Much more documents have indicated that many factors participate in the regulation of neurogenesis in microenvironment of NSCs. The expression of NSE and nestin during embryo,infant and adult hint that they are key microenvironment factors of NSCs in the process of normal differentiation and proliferation. In addition, as an important embryonic developmental protein, the mammalian noggin is similar in its sequence to that of Xenopus. In humans, the noggin gene has been localized to chromosome 17q22, and in the mouse, it is localized to a region of chromosome 11. Noggin cDNA contains a single reading frame encoding a 26 kD protein, which has been identified to effect neural induction through antagonizing bone morphogenetic protein-4 (BMP4). In addition, the spread expression of chordin as another antagonist of BMP4 during postgastrulation embryogenesis and in infant brain, has been demonstrated, indicating it is an important neural inducer that keeps working in the adult nervous system .Up to now, there has been no report regarding noggin and BMP4 genes expression and their functions in the normal childhood rat and ones after PTZ kindling.In this project, the expression of noggin and BMP4 in the neural development of hippocampus of young rats after PTZ kindling was detected using immunohistochemistry,in situ hybridization histochemistray (ISHI} and reverse transcription-polymerase chain reaction(RT-PCR). Next, by using 5- bromodeoxyuridine (BrdU)-labeling and immunohistochemistry (IHC) methods, the effect of Noggin and BMP4 on the neural development of hippocampus on epileptic childhood rats was explored.The main results were as follows:After the establishment of epileptic childhood rats model kindled by improved PTZ, the expression of NSE and nestin in hippocampus at different levels and phases were observed, and Timm's coloring of DG was also observed. The results indicated that expression of NSE increased obviously in hippocampus of experimental groups, in which CA3 was visible, then the DG and CA1, while CA2 was weaker. The expression of nestin begin to appear in DG,CA3 and CA1 after 3 days of PTZ kindling, reaching to maximum in 7th day, then beginning to decrease gradually, recovering to the control level after 60 days. The expression of nestin is different in different areas of hippocampus, in which DG is visible, then the CA3 and CA1, CA2 is weaker. Both the expression of NSE and nestin in hippocampus of experimental group are much larger than the control group (P<0.01). Timm's colouring reveals superficial granular layer and dental molecular layer of DG begin to appear MF germination, and increase gradually during the stationary phase after PTZ kindling, and there are moderate degenerative and necrotic neurons in hippocampus and DG.. But, MF germination of DG appears progressive increase during chronic phase. Slight degenerative and necrotic neurons also exist in hippocampus and DG accompanying gliosis,formation of glial scar and atrophy of hippocampus. In the next, the variation of noggin mRNA and BMP4 mRNA were observed dynamically based on the injury of neurons and gliosis in hippocampus: PTZ kindling induces an increasing expression of noggin, reaching to maximum in 7th day, then beginning to decrease gradually, recovering to the control level after 60 days; the expression of BMP4 also increases after PTZ kindling reaching to maximum in 5th day, then beginning to decrease gradually, still higher than the control level after 90 days. However, ISHI reveals that the expression of BMP4 begin to increase in 7th day after PTZ kindling, reaching to maximum in 5th day, then beginning to decrease gradually, still higher than the control level after 60 days, in which DG is visible, then the CA3, CA1 and CA2 are weaker. Then, using the technique of RT-PCR, we observed that: the expression of noggin mRNA increase obviously after PTZ kindling in hippocampus, reaching to maximum in 7th day, then beginning to decrease gradually, recovering to the control level after 60 days; the expression of BMP4 mRNA also increase obviously after PTZ kindling in hippocampus, reaching to maximum in 5th day, then beginning to decrease gradually, recovering to the control level after 90 days. Finally, the effect of noggin on hippocampus neurogenesis of epileptic childhood rats was studied using anti-sense noggin oligodeoxynucleotide, combined with BrdU labeled and ICC methods. It was shown that the number of noggin mRNA positive cells in the hippocampus of adult rats decreased significantly when treated with antisense of noggin, whereas there is no obvious change in the number of BMP4 mRNA positive cells. In addition, the number of BrdU labeling cells decreased significantly in the hippocampus of adult rats after giving antisense noggin, indicating inhibitory effect on the proliferation of neural progenetior cells in the hippocampus by antisense of noggin.Conclusion: 1. The chronic epileptic animal model resembling TLE in human is established successfully, which provides an ideal platform to study the pathogenesis and electrophysiological feature of TLE. 2. Injury of neurons,gliosis and germination of mossy fiber exist in the process of PTZ kindling. 3. The expression of NSE and Nestin increase in hippocampus of epileptic childhood rats. 4. Noggin and BMP4 participate in the neural development of Noggin may play a promoting role on transforming from hippocampus precursor cell to genuine neurons,plasticity alteration of newly born neurons and formation of abnormal excitability loop. 5. BMP4 may have a role in gummosis of hippocampus,recurrent and spontaneous attack of chronic epilepsy and its long-term maintenance.In the present study, the influence of Noggin and BMP4 on neural development of hippocampus was preliminarily explored in chronic epileptic rats, which have some effect on the genesis and development of epilepsy. Further study on the action mechanism of noggin and BMP4 has some sense on the regulation of neurogenesis and neural plasticity in epilepsy,prevention of epileptic formation and brain tissue injury as well as endogenous repair and reconstruction of higher brain function, providing new perspectives of inducing endogenous proliferation,differentiation and repair independently.
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