Studies Of The Gene Chips And The Proteomics Of Hippocampus In The Rats With Disorders Of Cortical Development

PART ONE: RESEARCH OF THE MODELS WITH DCDCS INDUCED BY X-RAYObjective:To establish animal models with disorders of cortical development (DCDs) by x-ray and select the optimal dose of the established modle radiated by x-ray.Methods : Pregnant Sprague-Dawley rats were given x-ray with different radiation doses at embryonic day 17. There are five doses groups :the group1 125cGy, the group2 150cGy, the group3 175cGy, the group4 200cGy, the group5 225cGy, the control group 0 cGy .The following aspects were observed in all groups.①We calculated the survival rate of rat offspring in each group and observed their general state of behaviors.②The weight reflecting developmental condition of rat offsprings were measured at different time points.③We adopted Morris-water-maze experiment to test the learning and memory capacity of rat offsprings in each group on P28d.④After 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 on P56d..⑤We used kainic acid to induce seizure and compared their seizure threshold and mortality induced by seizure on P63d. The classical model induced by x-ray should be of hign survival rate, mental retardation, lower threshold of epileptic seizure and typically pathologic change of DCDs.Results:1) The survival rate of rat offsprings in the control group and the five model groups were 96.8%, 100%, 93.5%, 93.5%, 50%,20%. respectively. During the development of rats, No abnormality existed in the control, the model 1 and the model 2. The rat offsprings in model 3 were irritable, mild somnolence, fewer activities and dullness. The rat offsprings in model 4 were fewer activities, somnolence,obvious dullness and reduction of drinking water and feeding. No obvious seizure was observed in the control and five groups.2) Weight difference were not found among the rat offsprings of the control group and the model 1 and model 2 (P>0.05). The mean weight of rat offsprings in the model 3 reduced slightly at P0d and P14d(P<0.05), but the difference of weight disappeared at P28d and P56d(P>0.05). The weight of the rat offsprings in model 4 reduced heavily at different point(P<0.05).3) Morris-water-maze experiment showed that the rat offspring in model 1 did not show any disparity with the control group on the escape latency and times of crossing platform (P>0.05). The escape latency of rat offsprings in model 2 were longer than that of control group on first day and second day, but the difference disappeared on third day and fourth day(P>0.05). The escape latency and times of crossing platform of rat offsprings in the model 3 and the model 4 were always longer than those of the control group(P<0.05).4) The six rat were killed by P56d among the control and the model group. Brain weight of rat offspring in model 2 and model 3 obviously reduced(P<0.05). Reduced weight and abnormal construction of brain were displayed in the model 4(P<0.05). The incidence rate of DCDs of offsprings in the model 3 and model 4 reached 100%. Typical pathological features included a thin cortical plate, disorder of layer structure, 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 similar to cortical dysphasia and neuronal heterotopias in humans.5)The latent period of Kainic acid inducing seizure was shortened in the model 2 and the model 3(P<0.05), but the time of epileptic state and the mortality induced by seizure of rat offspring in the second group were the same as that of control group(P>0.05) in kainic acid inducing seizure experiment on P63d. The rat offspring in the model 4 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).Conclusion:1. X-ray by which the DCDs model were made was the good method. 2. The model induced by 175cGy was of hign survival rate, mental retardation, lower threshold of epileptic seizure and typically pathologic changes of DCDs.PART TWO: COMPARATIVE STUDY OF THE IMAGE OF BRAIN BETWEEN THR RATS WITH DCDS AND THE CHILDREN WITH HYDRANENCEPHALYObjective:Try to apply 1.5Tesla MRI on model rats with DCDs and to investigate brain image feature of the model rats and the children with hydranencephaly. To analyse imaging data and make comparative anlysis.Methods:Time-pregnant SD rats were irradiated on embryonic day E17 with 175 cGy or were left untreated. At P56d, the animals were scaned 1.5Tesla MRI, then were killed and their brains processed for histological studies. We collected the cases with hydranencephaly. All patients were scaned by MRI. At last we make comparative anlysis between the rats with DCDs and the patients. Results:1) Brain image feature of the model rat showed that small brain volume, enlargement of both lateral cerebral ventricle, the third ventricle and the four ventricle, thinning cortex, thinning corpus callosum and exposure of quadrigeminal bodies. 2) Brain image feature of the hydranencephaly showed that brain expansion, enlargement of both lateral cerebral ventricle , the third ventricle and the four ventricle, thinning cortex or absence of cortex, thinning corpus callosum or absence of callosum. 3) The results of comparison showed that the same aspects were enlargement of both lateral cerebral ventricle and the third ventricle, thinning cortex, thinning corpus callosum. The different aspects were hydranencephaly with more severe damage of cortex and hydrocephalus malformation.Conclusion:1. MRI provides a new approach for diagnosis of DCDs. 2.The characteristic of image of rats with DCDs don't simulate feature of hydranencephaly.PART THREE:RESEARCH OF THE GENE CHIP OF HIPPOCAMPUS IN LITTERS WITH DCDSObjective: :To obtain the differences expression gene among the hippocampus tissues between litters in the control group and litters with DCDs induced by x-ray and find the gene related intractable epilepsy in hippocampus in model litters. Methods: The animal model induced by 175cGy x-ray was established. The total RNAs were isolated from hippocampus tissues of newborn rats in Pod. cDNA were synthesized. Then,cRNAs which obtained by IVT hybrided with the rat affymetrix chip.The genechip were scaned by affymetrix scanner . The data were obtained and analyzed by software.Results: The different expression gene were shown : totally 149 differential expression gene including 45 upregulated genes and 104 downregulated gene. Based on the results of proteomics research, We found the gene related intractable epilepsy : GAP43 which may be involved in the sprouting of mossy fiber and reconstruction of nerve network.and SNAP 25 which may be involved in remolding of synapse. The expression of two gene was upregulated.Conclusions: 1. Expression of both GAP43 and SNAP25 gene were upregulated. 2. Both of them may be the key gene which were involved in the sprouting of mossy fiber and remolding of synapse to potentiate epileptic activities. Objective: To obtain the differences proteins of hippocampus between the animal model with DCDs induced by x-ray and the control and to find out the target and effective proteins related with intractable epilepsy.Methods : The animal model induced by 175cGy X-ray was established. 6 rat s were randomly selected in the model group and the control. We rapidly dissected the whole brain of each animal, extracted total protein of hippocampus and measure the protein concentration. Then the gel maps were obtained by 2D-E. The differential protein spots were analyzed by PDQUEST software. They will be identified by mass spectrometry. After consulting the NCBI, the different proteins were obtained.Results:①Well resolution and repetitiveness 2-DE gel images of rat brain proteome were obtained by 2D-E.②Eight different spots were obtained by consulting the NCBI, four upregulation proteins: GTP-binding protein,synapse-associated protein 90,synaptotagmin 8, calreticulin; four downregulation proteins: Cu-Zn superoxide dismutase ,dihydropyrimidinase-related protein , creatine kinase , glutathione S-transferase P.Conclusion: 1.The ideal brain 2D-E images could be obtained by 2D-E. 2.The obvious expression difference of hippocampus protein between the rats with DCDs and the control were found. The protein related with the sprouting of mossy fiber and remolding of synapse may play an important role to potentiate epileptic activities.