| Epilepsy is a brain dysfunctional syndrome which characteristics issudden, recurrent and transient neurological disorder caused by abnormaldischarge of neurons with various reasons. There are about700Chinese withepilepsy currently, and this condition afflicts at least800000Americans also.Over the past30years, there are about30000drugs were used to carry outepilepsy treatment in animal experiments all over the world, it has achievedgood effect, many epilepsy patients can get relief through the drug therapy,unfortunation, pharmacoyherapy usually fails to achieve long-term remission,it can not inhibit the seizure or recurrent. Currently, the surgery is mainlytaken to treat the refractory epilepsy to resect the epileptic zone, it can notchange the neuronal hyperexcitability and abnormal function of ion channelcompletely, most patients still rely on long-term standard pharmacoyherapy.with the pathogenesis and molecular biology technology developing, the genetherapy as a new method to control and treat epilepsy effectively will bringinnovative and promising alternative.In the pathogenesis research of epilepsy, it is considered that thehippocampal synaptic reorganization associated with temporal lobe epilepsyclosely, and in hippocampal synaptic reorganization process, moss fibersprouting (MFS) is an important symbol of synaptic reconstruction, and at thesame time with synaptophysin (P38) and synaptic growth-associated protein43(GAP-43) abnormal expressing, this kind of synapse formation reverse toaggratate epilepsy seizures.The synaptic ultrastructural study found that the morphology, quantity,structure, and function of new synapses in status epilepticus, are different from normal brain neural, these new synapse formation is excitatory circuitry orinexcitatory circuitry, also have aroused wide public attention. The study ofsynaptic ultrastructural shows that most of these recurrent synaptic isasymmetry synapses, and this is a typical characteristics of excitabilitysynaptic. So moss fiber sprouting and granular cell formation of ectopicsynapse is considered to be the important basic structure induced to seizures.The study of pharmacoyherapy in epilepsy over many years,neuropeptide Y (NPY) as a neurotransmitter and neuromodulator wide spreadover the central nervous system and peripheral nervous system, and it has thehighest concentration in the hippocampus of central nervous system. NPY andits receptors have the relationship with the epileptogenesis and moss fibersprouting, and exogenous NPY has antiepileptic effect. But the neural functionof NPY is very complex in epilepsy model, it appear inconsistent and evenconflicting experimental results with different animal model or differentmethod of administration.In this study, NPY gene carried by the recombinant adeno-associatedvirus as a vector, is transfered to specific target area of brain, the influence ofNPY gene to epileptic seizure rats which induced by Kainic acid (KA) wasobserved about the MFS, synaptic element expression, synaptic growth-associated protein (GAP-43) in hippocampal CA3using Timm dyeing, PCR,western blot method, and the influence of rAAV2/1-NPY-EGFP onhippocampal synaptic ultrastructural using transmission electron microscope.Part1The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on seizure and mossy fiber sprouting inhippocampus in rat model induced by kainic acidObjective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on seizure and mossy fibersprouting in hippocampus in rat.Methods:30clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=12), KA group (n=12) and controlgroup(n=6). NPY group in which10μl of rAAV2/1-NPY-EGFP (titer5× 1011v.g./ml) were injected to ventricle, KA group, in which amount saline wasinjected to ventricle. Ten minutes later, KA were injected respectively tohippocampal CA3area of two groups, The control group respectively wereinjected in ventricle and CA3area of same amount of saline for two times.Theseizure situation, EEG wave frequency and amplitude were observed afterinjection of2and4week. Mossy fiber sprouting(MFS) in the hippocampuswere detected by Timm’s staining after injection of4w.Result:1The seizure degree in rats of NPY group gradually reduced withobservation time. At4W post-injection, in rats of NPY group, the onset ofsymptoms and EEG epileptic discharge frequency and amplitude decreased(P<0.05), compared to rats of positive control group.2The scores of mossy fiber sprouting in the hippocampal CA3area werehigher in KA group compareed to NPY group.3The control group had noseizure and no significant difference of mossy fiber sprouting.Conclusion: rAAV2/1-NPY-EGFP gene transfection can inhibit seizuresand reduce the hippocampal MFS, which provide strong support to the NPYgene therapy for clinical refractory epilepsy.Part2The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on expression of synaptophysin inhippocampus in rat model induced by kainic acidObjective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on expression of synaptophysin inhippocampus in rat.Methods: Clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=24), KA group (n=24) and controlgroup(n=24). NPY group in which10μl of rAAV2/1-NPY-EGFP(titer5×1011v.g./ml) were injected to ventricle, KA group, in which amountsaline was injected to ventricle. Ten minutes later, KA were injectedrespectively to hippocampal CA3area of two groups, The control grouprespectively were injected in ventricle and CA3area of same amount of saline for two times. Three groups were taken the hippocampal tissue, to observedthe changes of expression of synaptophysin after injection of2and4weekwhich experimentation was performed by fluorescent quantitative PCR andwesten-blot.Result:1The seizure degree in rats of NPY groups decreased significantly after4W injection compared with KA groups (P<0.05).2The results of PCR and Westen-blot on synaptophysin (P38)expression showed that there are obvious difference between NPY groups andKA groups after4W(P <0.05), and there are no difference after2W.Conclusion: rAAV2/1-NPY-EGFP gene transfection can inhibit seizuresand reduce the synaptophysin (P38) expression, thus to inhibit epilepsy.Part3The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on expression of synaptic growth-associatedprotein43(GAP-43) in hippocampus in rat model induced by kainic acidObjective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on expression of synapticgrowth-associated protein43(GAP-43) in hippocampus in rat.Methods: Clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=48), KA group (n=48) and controlgroup(n=48). NPY group in which10μl of rAAV2/1-NPY-EGFP(titer5×1011v.g./ml) were injected to ventricle, KA group, in which amountsaline was injected to ventricle. Ten minutes later, KA were injectedrespectively to hippocampal CA3area of two groups, The control grouprespectively were injected in ventricle and CA3area of same amount of salinefor two times. Three groups were taken the hippocampal tissue, to observedthe changes of expression of GAP-43after injection of2and4week whichexperimentation was performed by immunohistochemical and fluorescentquantitative PCR.Result: The results of immunohistochemical and fluorescent quantitativePCR on GAP-43expression showed that there are obvious difference between NPY groups and KA groups after4W(P<0.05), and there are no differenceafter2W.Conclusion: rAAV2/1-NPY-EGFP gene transfection can inhibit seizuresand reduce the GAP-43expression, thus to inhibit the synaptic reorganization.Part4The effects of neuropeptide Y gene transfection using recombinantadeno-associated virus vector on ultrastructure features of MFS inhippocampus in rat model induced by kainic acidObjective: To study the effects of neuropeptide Y gene transfection usingrecombinant adeno-associated virus vector on ultrastructure features of MFSin hippocampus in rat.Methods: Clean level Wistar rats which weight are250~270g, wererandomly divided into NPY group (n=24), KA group (n=24) and controlgroup(n=24). NPY group in which10μl of rAAV2/1-NPY-EGFP(titer5×1011v.g./ml) were injected to ventricle, KA group, in which amountsaline was injected to ventricle. Ten minutes later, KA were injectedrespectively to hippocampal CA3area of two groups, The control grouprespectively were injected in ventricle and CA3area of same amount of salinefor two times. Three groups were taken the hippocampal tissue, mossy fibersprouting(MFS) in the hippocampus were detected by Timmhistochemistry,to observed the changes of the synaptic quantity, synapticvesicles density, and the synaptic cleft under the electron microscope.Result:1Most of the mossy fiber sprouting synaptic terminals were asym-metric synapses, the quantity of asymmetric synapses increased in KA groupand NPY group after2W, the quantity of asymmetric synapses decreased inNPY group after4W, there are obvious difference between NPY groups andKA groups after4W(P <0.05).2The synaptic cleft increased in KA group and NPY group after2W, anddecreased in NPY group after4W, there are obvious difference between twogroups after4W(P <0.05).3It is showed that the quantity of synaptic vesicles reducing, shape of synaptic vesicles being irregular, and the density of synaptic vesicles beingreorganization in KA group and NPY group after2W. The synaptic vesiclesdensity(SVD) is normal after4W, there are obvious difference between NPYgroups and KA groups after4W(P <0.05).4It is showed that the nucleus swelling, entoblast disappearing, mitoch-ondrial vacuolar degeneration, mitochondrial ridge and membrane dissolved,and the rough endoplasmic reticulum degranulation under the electronmicroscope in KA group and NPY group after2W, comparing to NPY groupafter4W, there are significant difference(P <0.05).Conclusion: Most of the mossy fiber sprouting synaptics terminals areasymmetric synapses, this kind of new synaptic is irritability, and can beinhibited by NPY. The apoptosis of neuron in hippocampal increased in rat ofepilepsy, and this apoptosis of neuron can be inhibited by NPY. |
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