Effects Of The Excitation Mechanism By GKS On KA Rat

PART ONE ELECTROPHYSIOLOGICAL CHARACTERISTICS OF NERVE CELLS IN RIPE RATS HIPPOCAMCAL SLICES AND THE INTERVENTION OF MK801 ON EPILEPTIC DISCHARGEObjectiveTo explore the excitation injury mechanism of Glu and KA jet douche on rats hippocamcal slices and the intervention of MK801 on epileptic discharge.MethodsWe used ripe rats about 7-8 weeks old and 180±10g weight. First, the brain tissues were brought out after the rats were decapitated, which were then cut into slices of 300μm thickness. All the slices were put into a container with an air mixture of 95% O2 and5% CO2 as well as saturated ACSF, followed by incubation for 1h at 37°C in 35~37°C thermostatic water bath, then they were moved into 24~25°C thermostatic water bath for incubation for about 3~4 h before use. Observation the following issues with whole cell patch clamp recording technique:①the basic electrophysiological characteristics of nerve cells in CA3 area of hippocampus and their reaction to Glu and GABA;②the whole cell Na+,K+,Ca2+ current;③Characteristics and influential factors of epileptic discharge in the nerve cells in CA3 area of hippocampus induced by Glu and KA; the influence of MK801 on the epileptic discharge in the nerve cells of hippocamcal slices.Result1.Survival rate of incubated hippocamcal slice cells was more than 80%, cells in CA3 area had a big size, and localized with great number. The cell surfaces of neurons were very smooth with uniform glisten and obvious neurites;2.There were some kinds of difficults for seal between nerve cells in ripe hippocampal slices with about 60~70% of successful rate(n=400).The resting potential of cells was 61.2±6.8mv(n=200).According to the reading of C-slow and G-series, membrane capacity Cm and series resistance Rs were 16.36±2.7pF and 10.03±1.7G?respectively. Time constant t(=Cm×Rs)was 159.4±41.23μs(n=57). Stimulation with constant current could produce multiple action potentials, while hippocamcal nerve cells induced by Glu displayed a reversible depolarization as well as paroxysmal action potential, meanwhile, GATA could inhibit the release of action potential induced by current stimulation; 3.The rapid voltage-dependent activated and deactivated inward Na+ and outward K+ current could be recorded under the whole cell mode; Ca2+ current, an inward current , which was activated at 40mV and reached the peak at 0Mv,its activation and deactivation was very slow with a characteristic of internal current. When the common extracellular fluid was changed to be non-calcium one, this current disappeared;4.All the treatments including constant current stimulation, Glu perfusion or KA showed a spontaneous, paroxysmal or cluster epileptic discharge. MK801 sprouting could inhibit the epileptic discharge of hippocamcal nerve cells, inhibition rate of Glu group was higher than KA group.Conclusion1.Stripping of hippocampus in ripe rats was easier with little injury; Hippocampus had ordered neurons and fiber arrangement, and it was more easier for us to know the exact sites of fiber and synapse for carrying out further Electrophysiological work;2.There were intact Na+,K+,Ca2+ ion channels in the hippocamcal slices of ripe rats after incubation;3.All the treatments including constant current stimulation, Glu perfusion and KA could induce the excitation and the recurrent of spontaneous epileptic discharge in hippocamcal cells;4.MK801 could not only block the action potential, but also inhibit the epileptic discharge induced by Glu and KA, which facilitated the nerve cells maintaining normal function and reducing adverse reactions. PART TWO THE EFFECT AND REGULATION MECHANISM OF NMDAR ON THE EPILEPSY RATS AFTER GAMMA KNIFE RADIOSURGERYObjectiveTo explore the occurrence and development of NMDAR excitability injury after gamma knife radiosurgery(GKS) on the rats with kainic acid-induced seizure.MethodsWistar rats were divided into the following three groups:①A group, the control group(normal rats);②B group: epileptic rats model induced by kainic acid(KA);③C group: the epileptic rats induced by kainic acid with GKS. Observed the following index on the 7th d, 14th d, 21st d, 35th d, 49th d after irradiation irrespectively, including observation of morphology and ultramicrostructure changes with the light microscope, electron microscope and Timm's staining; an overview of learning and memory functions in rats with water maze; observation of histomorphological changes in rats after GKS with immunochemistry; detection of NMDA protein and mRNA expression levels by Western blot and RT-PCR respectively, detection of Glu intracellular concentration with high efficiency liquid chromatography, A fluorometric method for estimating the changes of the intracellular calcium content with single wavelength dye Fura-2/AM. Results1.There was a significant decrease of attacking times of rats after GKS (P<0.05);2.Neuronal injury was the main morphological change in rats after GKS, which displayed as follows: condensation and degeneration of neurons, increase and congregation of heterochromatin along the edge near the nuclear membrane, chromatin gathering, nuclear shrinkage, as well as the irregular emboly of nuclear membrane;3.There was an obvious decrease of mossy fiber sprouting 21~49 days after GKS (p<0.05);4.The memories of epileptic rats after GKS was decreased, which was in significantly positive correlation with the NMDA receptors level(P=0.005,r=0.816);5.mRNA level of NMDA was down-regulated obviously, which was in significantly positive correlation with the NMDA protein level(P=0.004,r=0.912);Meanwhile, this change was consistent with the concentration alteration of intracelluar Glu and free calcium.ConclusionsStable irradiation model could be constructed by exposing kainic acid-induced epileptic rats to GKS (40Gy, 4mm collimating apparatus) within 49 days. Seizure control by exposure of the rats to GKS wouldn't aggravate brain disorder. GKS could directly or indirectly affect on the intracellular Glu concentration in brain cells, which further regulated the mRNA level of NMDA and led to the down-regulation of calcium. Might the down-regulation of Glu-NMDA-Ca2+ was the basis for stopping the epileptic attacks.