Pathological Study And Stereology Of Brain About X Ray Lesion-Induced Diffuse Cortical Dysplasias In Rats

OBJECTIVE:This study was to observe the routine pathological characteristics and precise pathological characteristics(cerebral hemisphere and hippocampal volume, medullated nerve fibers,synapse and neuronal quantity and so on) of animal models of diffuse cortical dysplasias(DCD) in cortex by the method of histiocyte pathology and stereological methods, and to observe behavioral character, electroencephalography,seizure susceptibility, and discuss the mechamism of pathologic changes and epileprogenesis in this model.METHODS: To establish a animal model of DCD in one way.The way was that pregnant SD rats were exposed to X-ray using a single dose of 150 cGy on embryonic day 17(E17) for 84 seconds.The brain of their offsprings might receive lesions through this method.The experimental rats were divided into normal control groups,irradiated adult rats and model groups.To observe behavioral character, EEG,seizure susceptibility.The volume density,length density,total volume and total length of the myelinated fibers in white matter were estimated with the stereological techniques and transmission electron microscope technique..We observed the following aspects to confirm the success ratio, the credibility, histiocyte pathology and immunohistochemical studies in the animal models of DCD: Postnatal P0, P1, P2, P3, P7, P14, P21, P28, P56, P84 and 31 weeks,the offsprings of irrated pregnant rats were killed to be used to histomorphology and histopathologic studies.The expressions of NeuN,GFAP,GAP-43,Spy in cortex and hippocampus were observed by immunohisochemistry.Slice followed by quantitative analysis of neuron density,cell,and diameter of nuclear and the distribution DCD and white matter neurons using two-dimensional cell counting methods-Gundersen.To use statistics analysis by mean standard deviation(?x±s),P<0.05 represent significant, student test analysis or nonparametric Kruskal-Wallis H test and Nemenyi test in SPSS10.0.RESULTS: (1)Animal model groups have activities than the normal control groups. Most of DCD rats showed increased activities, excitation and restlessness,scratching and frequent " watching face-like activities". About 60% DCD rats showed frequent spontaneous interictal epileptiform spikes and spontaneous seizures arising independently from the hippocampal or the frontal neocortical structures.We observed about 10% spontaneous seizures;however,clinical stages of seizures and EEG were significantly increased in about 90% DCD rats by an administration of kainic acid. typical spikes wave, sharp wave, sharp and slow wave were discharged in EEG.(2) We can succeed in developing the animal models of DCD, the method was that pregnant rats were exposed to X-ray on embryonic day 17. All of the irradiated pregnant rats'offsprings were caused brain lesions resulting in neuronal migration disorders and quadrigeminal bodies expose ,callosum absence, cerebral ventricle expansion, volume of cerebra shrink.The DCD rats cortex shows laminar disorganization and 60% thinning and four,five-layered lamination.(3)In addition, Weight of DCD rats and brain decrease about 19% and 57%. volume of brain, cortex, hippocampus and CA1 decrease about 36%, 19%,39% and 32%.cortex/brain,hippocampus/brain,CA1/hippocampus decrease about 20%,21%and 27% in DCD rats.HE stains and Nissl stains showed there are clusters of large neurons in both superficial and deep layers in the model rats cortex. Neuron density, cell density and neuron percentage increase 99 double,121 double,64 double in DCD rats. A modest enhancement of excitability may occur in CA1 pyramidal neurons of irradiated rats due to diminished network inhibition and possibly aslo to increased synaptic excitability in the region. DCD could result in mossy fibers sprouting in hippocampi CA3 area. We demonstrate that there are mossy fiber sprouting in hippocampal CA3 region(P<0.05)in DCD rats, but no mossy fiber sprouting in normal control groups and irradiated adult rat.(4)We found several morphological changes in these specimens: Swelling and expanding of interior membrane system. Dissociation of plasma membrane and myelinolysis.These might resulting in an increased susceptibility to seizures,leading to further seizures. There was significant decrease 64% of the total length of the myelinated fibers in white matter between normal and DCD rats.However there was significant decrease 80% of the total length of the myelinated fibers in white matter, between normal and DCD rats. The total volume of the myelinated fibers in white matter of DCD rats was decreased by 52%.(5)The expressions of NeuN,GFAP,GAP-43 and Spy in cortex and hippocampi CA1,CA3 area were observed by immunohisochemistry. The increased 30 doubles, 16 doubles, 10 doubles and 10 doubles expression of NeuN ,GFAP ,GAP-43 and Spy around CD in white matter of DCD rats may have relationship with the mechanisms of the intractable epilepsy (P<0.05). In the present study, we had found out hippocampal mossy fiber sprouting in adult rats of DCD induced by X-ray injury. Because the increasing expression of GAP-43 and Spy in the DCD rats'hippocampi CA3 area of DCDs may have relationship with the mechanisms of the intractable epilepsy (P<0.05). CONCLUSIONS: 1. Neonatal X-ray irradiated injury on embryonic day 17 could result in an ideal animal model simulating human gray matter heterotopia, tuberosa sclerosis, hippocampal dysplasia, periventricular nodular heterotopia and corpus callosum absence.It is important that study mechanisms of epileptogenicity of the intractable epilepsy correlated with diffuse cortical dysplasias.2. We conclude that prenatal X-ray exposure disrupts critical developmental processes and prenatal neocortical structures, ultimately resulting in neocortical disorganization and hippocampal malformations. However, these studies provide important mechanisms of epileptogenicity because of mossy fiber sprouting in hippocampi CA3 area,DG area and hyperexcitability of the cortical synapse network.3. There was significant decrease of the myelinated fibers with large and small diameters. The present study for the new stereological techniques and TEM technique to quantitatively study the age-related changes of the myelinated fibers in white matter of DCD rats.Biological stereology method is a good approach to study and research biology because it can detect precise pathological characteristics.4. Application of the stereological methods to study expression of NeuN and GFAP exhibits internal attribution of ectopia nodus,and ectopia nodus is grey matter, mainly be made up of neuron and horizontal cell. It provide important mechanisms of epileptogenicity because of increasing Spy and GAP-43 than control animals in hippocampi CA3. It is a good antibody that detection mossy fiber sprouting in Spy antibody with SP immunohistochemistry is sensitive more than Timm's histochemistry.