An Experimental Study On Mechanisms Of Seizure Following Hypoxic Brain Injury

SECTION ONE AN STUDY ON ALTERNATIONS IN SEIZURE AND SEIZURE SUSCEPTIBILITY OF RATS WITH HYPOXIC BRAIN INJURYPARTⅠDEVELOPMENT AND EVALUATION OF HYPOXIC BRAIN INJURY MODEL IN RATSObjective:To establish and evaluate hypoxic brain injury model in rats,which offers experimental basis for research of pathophysiological mechanisms underlying hypoxic cerebral damage.Methods:Adult male Sprague-Dawley rats were exposed to 8% nitrogen oxygen atmosphere.After exposure to hypoxia, their behavior and electrophysiology were observed ,and pathological change in cortex and hippocampus was tested by light microscope and transmission electron microscope, respectively.Results:The rats subjected to hypoxia displayed apparent irritability and were not easy to be get hold of by experimenter and their electroencephalogram(EEG) presented various slow wave and epileptiform discharges by chance.Nissle staining demonstated neuronal cytoplasm condense and anachromasis,neurons array derangement and sparseness,and neurite reduction.TEM showed heterochromatin increase, perinuclear space enlargement,mitochondria swelling and vacuolar degeneration, mitochondrial cristae unclear, and rough endoplasmic reticulum and ribosome decrease.Conclusion: The model of hypoxic brain injury induced by nitrogen oxygen atmosphere could been successfully established in adult rats and this model was applicable for research of pathophysiological mechanisms underlying anoxic brain damage.PARTⅡMOSSY FIBER SPROUTING IN RATS HIPPOCAMPUS OF ONSET SUBCLINICAL SEIZURES FOLLOWING HYPOXIC BRAIN INJURYObjective: To explore the mechanisms of onset subclinical seizures following hypoxic brain injury.Methods:Adult male Sprague-Dawley rats were assigned randomly to control rats(n=12) and rats exposed to global hypoxia by 8% oxygen nitrogen atmosphere(n=91).Depending upon EEG recording epileptiform discharges, rats subjected to hypoxia were divided into subclinical seizures group and non- subclinical seizures group again, and then the changes in neuropathology, mossy fiber sprouting(MFS) in hippocampus and the expression of glial fibrillary acidic protein (GFAP) in cortex and hippocampus were studied by Nissle staining ,Timm staining,IHC staining and western-blot analysis.Results:A fraction of rats with exposure to hypoxia exhibited sharp wave,sharp and slow complex, and spike wave in their EEG tracing. Therefore,Subclinical seizures occurred in 23. 08% (21/91)rats following hypoxia. Compared with non-subclinical seizure group, neuronal loss of hippocampal CAl and CA3 region as well as temporal cortex were distinct in subclinical seizure group (P<0.05).In Subclinical seizure group, stained granule sedimentations appeared in stratum oriens of CA3 zone in 7 days following hypoxia ,the granule sedimentations were more stained and become bundle-like or patch-like in 14 days and in 28 days an obvious stained band is formed. Compared with non-subclinical seizure and control groups ,the scores of MFS in hippocampal CA3 region were higher in subclinical seizure group(P<0.05). But there was no significant difference of MFS within inner molecular layer(IML) of dentate gyrus(DG) in three groups above(P>0.05).Meanwhile,the expresssion of GFAP in subclinical seizure group was stronger than that of non- subclinical seizure group,especially in hippocampal area(P<0.05).Conclusion:Onset subclinical seizures occured following hypoxic brain injury in rats and rats with epileptiform discharges showed apparent neuronal loss,MFS in hippocampal CA3 subfield,and GFAP expression enhancement,which may be attributed to subclinical seizures following hypoxic cerebral damage.PARTⅢALTERATIONS OF SEIZURE SUSCEPTIBILITY AND PSD-95 ,VGLUT1,VGLUT 2 EXPRESSIONS IN RATS WITH HYPOXIC BRAIN INJURYObjective: To explore the pathophysiological mechanisms underlying change of seizure suscepitibility resulted from hypoxic brain injury in rats.Methods: Rats were assigned randomly to the two groups: control group(n=35)and exposed to global hypoxia group(n=35). To test the seizure threshold, rats from both groups(n=10) were treated with pentylenetetrazol (PTZ 10mg/kg.5min)intraperitoneally.To test the number of kindled rats and seizure scales,another rats from both groups(n=15) were injected intraperitoneally subconvulsive dosage of PTZ (35 mg/kg.2d) for 20 days, successively,and then rats kindled by PTZ in both groups were divided into simple kindling group and post-hypoxic kindling group, respectively.Furthermore,in order to manifest neuronal loss,vesicular glutamate transporter subtype1, subtype2(VGluT1,VGluT2),and postsynaptic density-95(PSD-95) expressions in the temporal cortex,midbrain,and hippocampal CA1 subfield,IHC and western blot techniques were used in the rest of both groups (n=10) and the rats kindled by PTZ in both groups.RESULTS:①As compared with the control group ,the dosage of PTZ-induction seizure was lower and latency to onset of PTZ-induction seizure was shortened in hypoxia group. Furthermore ,the number of rats kindled by PTZ was markedly enhanced and their seizure scales were more severe in hypoxia group (P<0.05).②The averaged numbers of neurons within temporal cortex ,midbrain and hippocampal CA1 subfield in hypoxia group were lower than those in control rats.As compared with hypoxia and simple kindling groups, both tempoxral cortex and hippocampal CA1 subfield had obvious neuronal loss in posthypoxic kindling group (P<0.05).③The number of PSD-95 immunoreactivity positive cells and optical density (OD) of PSD-95 immunoblotting were reduced in the both temporal cortex and hippocampus regions in hypoxia group(P<0.05).④OD of VGluT1 immunoreactivity positive cell within temporal cortex and hippocampus regions in hypoxic group were obviously higher than that in control group .Compared with hypoxia and simple kindling groups,OD of VGluT1 were markedly enhanced in posthypoxic kindling group (P<0.05).⑤OD of VGluT1 immunoblotting of posthypoxic kindling group was higher than that of the other three groups.Meanwhile,the VGluT1 immunoblotting OD of hypoxia group was higher than that of control group(P<0.05).⑥There was no significant difference in OD of VGluT2 IHC staining and immunoblotting in each groups(P>0.05).Conclusions: The enhancement of VGluT1 immunoreactivity,the reduction of PSD-95 expression,and apparent neuronal loss within associated regions may be lead to increased seizure susceptibility and hyperexcitability in brain,which may play an important role in the alterations of seizure susceptibility following hypoxic cerebral injury. SECTION TWO AN STUDY ON ALTERNATIONS OF EXCITABILITY AND EPILEPTIFORM DISCHARGES INDUCED BY HYPOXIA IN HIPPOCAMPAL NEURONSPARTⅠDEVELOPMENT OF PRIMARY RAT HIPPOCAMPAL NEURONS IN SERUM-FREE CULTUREObjective: To develop the method of cultivating of hippocampal pyramidal neurons in serum-free media and to detect electrophysiological properties of the neurons. Methods: Primary cultured hippocampal cells were carefully isolated from neonatal SD rat.After chopped into 1mm cubes,the cubes were mechanically dispersed with pipettes in the presence of trypsin (0.125%,g/ L).Hippocampal cells were cultured in DMEM/F12 enriched with 10 % fetal bovine serum and 10% horse serum for 24 hours and then they were cultured in DMEM/F12 with B27 supplement.The process of hippocampal neurons growth was observed.Neurofilament-200(NF-200) and Neuron- Specific Nuclear Protein(NeuN)were detected by ICC staining to identify neurons.Electrophysiological properties of pyramidal neurons in hippocampus were examined in whole-cell mode in the period of neurons mature (8th-14th day). Result: In this study the cultured hippocampal neurons could survive over 20 days and develop completely mature in the period from the 8th to 14th day.Morphology of hippocampal neurons with pyramidal or triangular phase–bright soma and its dendrities or axons were readily distinguished. Patch clamp recoding demonstreated that the sealing rate was over 80 %; resting membrane potential of hippocampal neurons were -57.7±9.2mV;the typical inward current (Na+) and outward current (K+) were elicited by command potential(-60mV～+ 60mV,step = l0mV,time=30ms);most neurons had action potentials when evoked by hyperpolarizing and depolarizing step currents(-60pA～+40pA,step=10pA,time=200ms),the threshold was 43.1371±5.3245 mV,the time of action potentials was 8.3571±3.1407ms and the range was 83.05±9.79Mv.Conclusion: The methods of primary cultivation of hippocampal pyramidal neurons with serum-free media improved the growth of neurons and cultured neurons can be applied for patch clamp recording. PARTⅡALTERATIONS OF EXCITABILITY FROM RAT HIPPOCAMPAL NEURONS INDUCED BY VARIOUS HYPOXIC DURATIONObjective:To explore alterations of excitability from hippocampal neurons subjected to hypoxia in various duration.Methods:Hypoxia episodes( 5,10,15,20,25,30,35,40 min duration) of primary cultured hippocampal neurons were produced by switching from ACSF equilibrated with 95% O2/ 5% CO2 to ACSF equilibrated with 95% N2 / 5% CO2.Membrane potentials of neurons in the different hypoxia duration were measured by whole-cell recording,and then threshold intensity,that is minimum current intensity inducing action potential in neuron,were tested by step currents(-60pA～+40pA, step=10pA,time=200ms) in neurons exposed to various hypoxia duration. Neurons in control group were perfused with ACSF equilibrated with 95% O2/5%CO2.Results:Membrane potential values of hippocampal neurons with exposure to hypoxia for 10 and 15min were higher than those in control group(P<0.05);and as compared with control group, membrane potential values of hippocampal neurons with exposure to hypoxia for 25,30,35, 40min were decreased(P<0.05).Threshold intensities of neurons subjected to hypoxia for 10,15min were higher than those in control group (P<0.05),however,,threshold intensities of neurons with exposure to hypoxia for 25,30,35,40min were lower than those in control group(P <0.05).Conclusion:Hippocampal neurons take on hyperpolarization of membrane potential,enhancement of threshold intensity,and thereby decrease of excitability in early hypoxic stage. Hippocampal neurons present depolarization of membrane potential,reduction of threshold intensity,and so increase of excitability in late hypoxic stage.PARTⅢEFFECT OF ACUTE HYPOXIA ON EPILEPTIFORM DISCHARGES EVOKED BY GLUTAMATE IN RAT HIPPOCAMAPL NEURONSObjective:To explore effect of acute hypoxia on epileptiform discharges induced by glutamate in primary cultured hippocampal neurons.Methods : Hypoxia episode( 30min duration) of hippocampal neurons were established by switching from ACSF equilibrated with 95% O2/5%CO2 to ACSF equilibrated with 95%N2/5%CO2,and then recovering ACSF equilibrated with 95%O2/5%CO2.Membrane potential,membrane capacity,and membrane input resistance of hippocampal neurons were measured in pre-hypoxia,hypoxia,post-hypoxic 10,20,30,40min.Under gap-free sampling mode,spontaneous action potentials of hippocampal neurons were recorded in control, hypoxia,glutamate and hypoxia+ glutamate groups and then spike frequency was calculated.Whole-cell recording showed whether there was action potentials of hippocampal neurons elicited by glutamate(2.5μmol/L ) in hypoxia and control groups. Neurons in control group were perfused with ACSF equilibrated with 95% O2/5%CO2.Results : Membrane potential values of neurons in hypoxia, post-hypoxic10, 20,30,40min were lower than those in pre-hypoxia (P<0.05) and there were no significant difference among membrane potentials of neurons in post-hypoxic 10,20 ,30 ,40min(P>0.05). Tendency of membrane capacity change in neurons were similar to that of membrane potential. However, in contrast to membrane potential and membrane capacity, membrane input resistance of neurons were lower in hypoxia, post-hypoxic 10,20,30,40min (P<0.05). There was strikingly significant difference in distribution of action potentials with more than 3Hz in each group (P<0.05). Percent of spike with more than 3Hz in hypoxia group was higher than that in control group,and also percent of spike with more than 3Hz in hypoxia+glutamate group was higher than that in the other groups.Both reversible depolarization and successive action potential were induced by glutamate (2.5μmol/L) in hypoxia group,but reversible depolarization was only elicited in control group.Conclusion:Hippocampal neurons with exposure to hypoxia may present alterations of electrophysiological characteristics,augmentation of spontaneous discharge frequency,which may be result to reduction of the threshold for epileptiform activity.Furthermore,hypoxic damage leaded to enhancement of susceptibility of epileptiform discharges in hippocampal neurons.