Study On The Correlated Molecular Mechanism Of Epilepsy With Calcium Overloading

Part IStudy on the association of calcium overloading in hippocampal CA3 region of epileptic rats with the pathogenesy of epilepsyObjective To clarify the role of calcium overloading in hippocampal CA3 region on the epileptogenesis induced by pentylenetrazol (PTZ) and the blockage of nimodiping . Methods 26 adult male rats were randomly divided into two groups, one group containing 19 rats was used to measure calcium overloading, the other containing 7 rats was used to observe the histopathological changes. (1)The epileptic model was induced by PTZ(55mg/kg). Hippocampal slices were made when the epileptic seizures attacked, and then were incubated with ACSF. Calcium overloading in hippocampal CA1, CA3 region of epileptic rats was measured by a fluorescent inverted microscope, and the inhibition by the nimodipine on the calcium overloading was observed, too.(2)When the epileptic seizures attacked, the hippocampal slices were made. Histopathological changes were observed, and the neuronal loss in hippocampi CA1, CA3 and CA4. Results Calcium overloading in hippocampi CA1 , CA3 region was more pronounced than in that of the normal rats, and calcium overloading in hippocampal CA3 region was more pronounced than in that of CA1 region. Calcium overloading was significantly decreased by the inhibition of nimodipine. Histopathological changes also showed that neuronal denaturation , putrescence in CA3 region was most obvious of whole hippocampi region. The degree of cortical neurons in hippocampi was CA3>CA4>CA1>CA1 from serious to light. The number of lost neurons in CA3 was also the most and was significantly more than that of in CA1. Which was coincidence with the study of calcium overloading. Conclusion Calcium overloading is involved in the epilepogenesis, calcium overloading in hippocampi CA3 region plays the main role in the epilepogenesis, and nimodiping can adjust the calcium influx, and can be the assistant medicine of treating epilepsy. Part IIResearch on the relationship between expression of mGLuR1α and neuronal apoptosis in rat brain with PTZ-induced seizuresObjective To study on the relationship between expression of mGLuR1α and neuronal apoptosis in rat brain with PTZ- induced seizures and identify the role of mGLuR1α on PTZ- induced seizures. Methods 36 adult male SD rats were randomly divided into control group and epileptic groups, and epileptic groups were again divided into 6h, 12 h, 24 h, 48 h, 72 h group after PTZ-induced seizures. Each group contained 6 rats. Immunohistochemistry method and RT-PCR technique were used to measure the expression of mGLuR1α in neurons at different point of time after PTZ-induced seizures, TUNEL technique and flow cytometry were used to measure the change trends of neuronal apoptosis. Results (1) TUNEL positive neurons began to express at 6h following epilepsy, apoptotic neurons increased significantly at 12h, began to decrease at 24h, and were still in the state of downtrend at 72h. The apoptotic ratio of control group and PTZ group at different point of time also showed it reached the highest at 12h after PTZ-induced seizures, and failed -off following time lasting. (2) mGLuR1α mRNA transcription achieved the highest level at 6h, and falled-off following time lasting, it was still higher than control group at 72h. But the expression of mGLuR1α protein was slightly late, it reached the peak at 12h, and began to decrease at 24h. (3) The earliest and peak time of mGLuR1α expression were basally coincidence with the reaction time of apoptotic neurons. Conclusion PTZ-induced seizures result in the temporarily high expression of mGLuR1α, and its mechanism may be related to the neuronal apoptosis induced by epileptic seizures.. Part IIIEffects of (s)-4C3HPG (antagonist of mGLuR1) on the PTZ-induced seizuresObjective To observe the effect of (s)-4C3HPG on the PTZ-induced seizures and identify the role of mGLuR1α on the epileptic seizures. Methods 14 adult male SD rats were divided into saline+PTZ and (s)-4C3HPG+ PTZ group, each group contained 7 rats. They were preconditioned with the injection of saline and (s)-4C3HPG through the lateral cerebral ventricle respectively, and then their behavioral changes were observed. Brain tissue slices were prepared for the detection of histology, the expression of mGLuR1α and neuronal apoptosis at 12h after injection of PTZ. Results (1) The latency of PTZ-induced seizures in the preconditioned rats was prolonged, and (s)-4C3HPG lessened the severity of epileptic seizures. (2) The histopathologic changes of two group all showed main of hydropic degeneration, cortex and hippocampi showed obvious, and hippocampal CA3 showed the most severity in hippocampi. Neurons of control group showed obvious necrosis and defluxion, but few neurons of (s)-4C3HPG group showed degeneration and necrosis. There was significant difference between the two groups of survived neurons(P<0.01). The numbers of apoptotic neurons of (s)-4C3HPG group were obviously less than that of control group. (3) The mGLuR1α positive cells of (s)-4C3HPG group were obviously less than that of control group, mGLuR1α mRNA was also obviously down regulation. Conclusion The active changes of mGLuR1α following epilepsy are associated with the neuronal damadge and apoptosis, (s)-4C3HPG can lessen the exitotoxicity generated by PTZ-induced seizures and has protection on the rat brain of epileptic seizures. Part IVProtective Effect of Ginkgo Biloba Extract and mild hypothermia on Brain Injury By PTZ-induced seizures in Rat一 Protective Effect of Ginkgo Biloba Extract on Brain Injury ByPTZ-induced seizures in RatsObjective To explore the effect of Ginkgo Biloba Extract (GBE) on the PTZ-induced seizures and the protection of GBE on the brain injury by epileptic seizures. Methods 42 adult male SD rats were divided into two groups. Each group contained 21 rats. One group was used to measure the effect of bilobalide B (GB) on the calcium overloading in hippocampal CA3, the other was used to observe the effect of GBE on the epileptic rats. (1) The hippocampal slices were made when epileptic seizures attacked, then hippocampal slices were incubated with ACSF. The effect of bilobalide B (GB) on the calcium overloading in hippocampus CA3 was observed by using a fluorescent inverted microscope. (2) The behavioral changes were observed in vivo after the preconditioned rats with different dose of GBE were injected with PTZ .The hippocampal slices were made at the time of 12 h after epileptic seizures attacked, then the behavioral, histological and apoptotic changes were observed in vivo and compared with the control group. Results GB (150μmol) lessened the calcium overloading in hippocampal CA3, the effect of GB (50μmol) was not obvious. The experiment in vivo showed that GBE (50mg/kg) could slightly lessen the latency and seventy of epileptic seizures, the effect of GBE (100mg/kg) was significant. The numbers of neuronal loss and apoptosis were obviously decreased under the effect of two doses of GBE, and there was significant variability compared with control group. But the effect of different doses of GBE was dissimilarity, the larger the doses were, the more obvious the effect was. Conclusion GBE can lighten calcium overloading, palliate the epileptic seizures , lessen the neuronal loss and apoptosis, thus it has protection on the brain injury by PTZ-induced seizures. 二, The effect of mild hypothermia on the pentylenetrazol (PTZ)-inducedepileptic seizures in ratsObjective To observe behavioral seizures in rats with scalp temperature of 41.0, 37.0, 32.0 and 26.0 degrees c, also observe the histopathological changes in hippocampus of epileptic rats and count the number of the survived neurons. Methods Male SD rats were divided into four groups with scalp of 41.0, 37.0, 32.0 and 26.0 degrees c. Scalp temperatures were controlled with ice bags to debase temperature and incandescent bulbs to elevate temperature. Behavioral seizures in rats with above four different temperature were observed at the same time. Hippocampal slices were prepared from epileptic rats and dyed with hematoxylin and eosin (HE). The histopathological changes in hippocampus were examined by Olympus optics microscope and survived neurons were counted in the region with the most obvious histological demage. Results The degree of behavioral seizures appeared lighted, the duration of seizures were shortest and the lost neurons were least among the four groups when scalp temperature was 32.0 degree c, but of the other there groups, following elevation of scalp temperatures, seizures appeared more and more serious and lost neurons were more and more. Conclusion mild hypothermia has protective effect on brain. Part VThe study of manganese ion enhanced functional MRI (ME-fMRI) on the encephalic region correlated with epileptic seizuresinduced by PTZObjective To determine the value of the encephalic region correlated with epilepsy by Mn²⁺-fMRI and further to determine the correlation of epilepsy and calcium overloading. Accordingly, the doubling study on the pathogenesy and allocation is carried out . Methods 42 male adult cats were divided into two groups: (1) Group of PTZ-induced seizures (epileptic group containing 30 cats) was again divided into behavioral observed group, epileptic group, 24 h group after epileptic seizures attacked. (2) Saline group (control group containing 12 cats). The epileptic cat model was made to by intramuscular injected with PTZ, the behavioral and EEG changes were observed, and the ME-fMRI was undertaked at the time of acute epileptic seizure and at the time of 24 hours after the epileptic seizure; the pathological examination was made on the TI-Weighted MRI. Signal enhanced encephalic regions and compared to the control group. Results Epileptic seizures can be successfully induced by PTZ (60mg/kg). The EEG of the epileptic cats by PTZ showed paroxysmal spine-slow waves with high amplitude. The ME-fMRI showed diffuse signal enhancement in the cerebral cortex of cats with generalized tonic-clonic convulsive seizures compared with control animals. The enhancement rate of frontal-parietal-occipital lobe was 34.6%, and 22.9% in temporal lobe compared with the control group. Signal enhancement on frontal-parietal lobe persisted 24 hours after epilepic seizures were induced. The neurons of enhanced encephalic regions showed obvious degeneration necrosis. Conclusion Frontal-parietal lobe was the correlated encephalic regions of epilepy, Mn²⁺-fMRI could play an important role on the allocation and revealing pathogenesy of epileptic seizures.