| Objective:The dissertation is to investigate the optimal dose and methods of the established animal model of disorder of cortical developments (DCDs) induced by Carmustine, to explore the mechanism of apoptosis in hippocampus of the rats with DCDs and correlated apoptosis, to estimate the application effects of BDA as a nerve tracer in rat cortical and hippocampus neuron for the study of abnormal neural network of DCDs, and to study the clinical effective of topiramate for intractable epilepsy by DCDs.Methods:1 The establishment of rat model of DCDs induced by CarmustinePregnant Sprague-Dawley rats were given intraperitoneal injection of carmustine with different doses at embryonic day 17. The rats in model one were given injection of carmustine with 5mg/kg, the rats in model two 10mg/kg, the rats in model three 15mg/kg, and the rats in model four 20mg/kg. The histologic alterations are suggestive of DCDs in rat offspring with the control group treated shamly.The following aspects were observed in all groups. We calculated the survival rate of rat offspring in each group and observed their general state on birth and the postnatal daily behaviors including consciousness, viability and epileptic seizure. The weight reflecting developmental condition of rat offspring were measured at P0,P7,P14,P21,P56 and P84. We adopted Morris-water-maze experiment to test the study and memory capacity of rat offspring in each group and used Kainic acid inducing seizure to compare their seizure threshold and mortality induced by seizure. After measuring the wet weight of brains and observing their appearance, we examined the changes of brain pathology in cerebral cortex and hippocampus to confirm the types of DCDs at P21 and P84. We assessed whether we could establish the animal model of DCDs induced by Carmustine successfully by above aspects or not. We also judged the best drug dose of successful model and evaluated the credibility of methodology in the animal model of DCDs.2 The expression of bax and bcl-2 in hippocampus of the rats with disorder of cortical developmentsRats with DCDs were established by BUCN. AT P0, P15, 30, P45, and P60, the number of apoptosis cell in hippocampus were detected by TUNE,the protein expression of Bax and Bcl-2 were detected by immunohistochemistry method, the mRNA of bax and bcl-2 were detected by RT-PCR method in the BCNU-exposed groups and the control groups at the same age, then whose outcomes was analyzed by computer micrograph analysis system.3. Application of BDA as a nerve tracer in rat cortical neuronAdult SD rats were used in this experiment and 10%BDA was injected slowly into the bilateral sensory and motor cortex and hippocampus with a mini syringe through four round-shaped bone windows with 3mm diameter, formed by a dental dril1, with the total volume 4u1.After survival for 14 days, brains were removed and stored in a freezer. Sections were made in a cryostat,and stained by the free-floating method for BDA before the morphous of these neurons were observed.4 Clinical effects of Topiramate for the Intractable Epilepsy by Disorders of Cortical DevelopmentA group of 100 patients with intractable epilepsy by DCDs were diagnosed by MRI and physician of neurology, and treated with topiramate(100mg.d-1～200mg.d-1). We observed their change of seizures frequency compared with the basic-line before treatment.Results:1. The assessment of rat model of DCDs induced by Carmustine1) The survival rate of rat offspring in models group and control groupThe survival rates of rat offspring in control group , model one, model two, model three and model four were 100%, 100%, 93.1%, 83.3%and 0% respectively. All the rat offsprings in model four died at P0.2) The results of general ethology observationThe observation results of rat offspring in model one were similar with those of control group. No abnormality existed in model two at the process of growth and development. The rat offspring in model three were poorer than control group on birth, followed by fewer activities, dullness and disturbance of intelligence in daily life. No obvious seizure was observed in all models group.3) Condition of growth and development in rat offspringThe rat offsprings in control group and model one were found to have no difference in weight (P>0.05). The mean weight of rat offspring in model two reduced slightly at P0(P<0.05), but the difference of weight disappeared when they became adult. The rat offspring in model three indicated an overall reduction in mean weight at every time point, so the phenomenon was significantly with age(P<0.05).4) Morris-water-maze experimentThe rat offspring in model one did not show any disparity with control group on the escape latency and times of crossing platform (P>0.05.The escape latency of rat offspring in model two was longer than that of control group on first day and second day, but the difference disappeared on third day and fourth day. The times of crossing platform of rat offspring in model two was equal with that of control group(P>0.05. In the training period escape latency of rat offspring in model three was always longer than that of control group(P<0.05). After removing platform on fifth day, the times of crossing platform of rat offspring in model three was not so many as that of control group(P<0.05).5) Kainic acid inducing seizure experimentThere was no difference between model one and control group on latent period and time duration of Kainic acid inducing seizure and mortality induced by seizure of rat offspring(P>0.05). The latent period of Kainic acid inducing seizure was shortened in model two(P<0.05), but the time of epileptic state and the mortality induced by seizure of rat offspring in model two were the same as that of control group(P>0.05). The rat offspring in model three had a significantly lower seizure threshold and longer time of epileptic state than those of control group, the mortality induced by seizure of rat offspring was obviously higher than that of control group(P<0.05).6) Comparison of cerebral appearance and brain weight and pathological examination of brainThe disparation of cerebral appearance and brain weight didn't exist between model one and control group. There was no pathologic change in model one. Rat offspring in model two have no significant change in weight and construction of brain, the incidence rate of DCDs was 41.67%. Reduced weight and abnormal construction of brain were displayed in model three, the difference increased with age. The incidence rate of DCDs reached 100%. Typical pathological features included that model rats showed a thin cortical plate and disorder of layer structure, nerve cells in cerebral cortex became thinner and dysphasia, and distinct clusters of neuronal elements that represented heterotopias emerged in cerebral cortex and hippocampus. These morphologic features of DCDs found in this model were shared with cortical dysphasia and neuronal heterotopias in humans.2. Expression of Bax and Bcl-2 in hippocampus of the rats with DCDs at distinct time pointAt P15, P30, P45, and P60, the number of apoptosis and Bax/ bcl-2 masculine neuron, and the mRNA of bax/ bcl-2 in hippocampus in the model group are higher than that in the control group. The ratio of Bax/Bcl-2 in model group was significantly higher than those in the control groups (P<0.05), but at P0 there were not differences.3. Application of BDA as a nerve tracer in rat cortical neuronThe BDA injected into the bilateral sensory and motor cortex and hippocampus is taken by the axon and soma well,and the soma, the axon and dendrite of neuron are observed clearly with a 200×magnification.4. Clinical effects of Topiramate for the Intractable Epilepsy by Disorders of Cortical DevelopmentThere are 37 patients seizure free, 33 patients the frequency reduced more than 75%, and 20 patients more than 50% with the total effective reached 90%. All subjects didn't have obvious side effects.Conclusions:1.The method that pregnant SD rats are given intraperitoneal injection of carmustine with 15mg/kg on embryonic day 17 can establish an ideal model of DCDs in offspring. But carmustine treatment with 5mg/kg or 10mg/kg or 20mg/kg of pregnant rats can not successfully establish the model of DCDs in offspring. Rat offsprings in the model show poor growth and development, abnormal behaviors, disorders in study and memory , lower threshold of epileptic seizure and have typically pathologic change of DCDs. They commendably reveal the features of ethology and pathology in humans. The neuronic abnormality of neuropeptide Y and calretinin in the brain of model rat offspring provide theoretical clue for the mechanism of epileptic seizure induced by DCDs. DCDs of rat offspring in the model can simulate cortical dysplasia and neuronal heterotopia that is similar in humans. The rat offspring in our model have higher survival rate than that of rat offspring in mode that carmustine treatment with 20mg/kg of pregnant rats on embryonic day 15 in abroad. Our model has better repeatability and being a new , pragmatic animal model of DCDs.2.At P15, P30, P45 and P60, Apoptosis exists notably in hippocampus of rat of disorder of cortical developments.The protein and the mRNA expression of bax and bcl-2 are higher in model group than those in the control group, but at P0, there were no differences. So we can conclude that bax and bcl-2 take part in apoptosis of hippocampus neuron, which may be associated with the clinical spectrum of DCDs, such as epilepsy and IE.3. BDA can demonstrate the soma, the axon and dendrite of neuron in cortex and hippocampus. It is biologically stable and has good availability , and can be transported for a long distance. The experiment process is simple and provides a reliable method for the morphological study of cortical and hippocampus neuron and for the study of abnormal neural network of DCDs correlated to epilepsy and IE.4.As a new antiepilepsy drug, topiramate to IE by DCDs, has a good control rate, excellence rate and effective rate, and has no severe side effect. So we can conclude that topiramate can be a choice drug for IE by DCDs.
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