The Effects Of Neuropeptide Y Gene Transfection Using Recombinant Adeno-associated Virus Vector On Seizure In Rat Model Induced By Kainic Acid And Its Mechanism

Epilepsy, a brain dysfunctional syndrome caused by abnormal dischargeof neurons with many reasons, is characterized by temporary and recurrentdisorder of brain functions. At present, there are at least7million patients withepilepsy in China. Among this population, approximately20%of epilepticpatients do not respond adequately to the anti-epileptic drugs, which remain aknotty problem for modern clinical medicine. Recurrent and long-termseizures not only seriously affected the patients′life quality, but also broughtimmense burden and pressure to their family and society.Currently, the surgery is mainly taken to treat the refractory epilepsy, butonly the onset of epileptic focus is removed by the surgical excision, with nocomplete change to the neuronal hyperexcitability and abnormal function ofion channel, while surgery itself have certain risks, such as anesthesiaaccidents, bleeding, infection, limb paralysis and so on. With this betterunderstanding of the pathogenesis of epilepsy and the development ofmolecular biology techniques, gene therapy to completely cure epilepsy bringsnew hope to the patient with epilepsy.The data shows that neuropeptide Y mainly located in the cerebralcortex、hippocampus、thalamus、corpus striatum in the central nerve system;NPY and its receptors had closely connection with the origination of epilepsy.Because the complicated mechanism in different epileptic model, theexperimental results were always inconsistent or contradictory with each otherdue to the different animal models and methods of administration.In this study, neuropeptide Y was considered as the treated gene, andrecombinant adeno-associated viral was acted as vectors. After the NPY genewas transferred to the specific target in the brain tissue, the effects of NPY overexpression on seizures in chronic epileptic rat induced by kainic acid wereobserved and further PCR, Western blotting, micro-dialysis technique, patchclamp techniques were used to analyze the biological mechanisms of NPYregulation to seizures.Part1The expression of recombinant adeno-associated virusneuropeptide Y gene in rat models of chronic epilepsyObjective: To compare the approach of intraventricular injection with thehippocampal injection in the expression of rAAV2/1-NPY-EGFP in epilepticrat brain.Methods:36rats with successful model were randomly divided into twogroups: the hippocampal and the ventricular injected group with18rats ineach group. rAAV2/1-NPY-EGFP gene has been transferred into the brainunder stereotactic guidance. After frozen sections of brain tissue were made,the expressions of rAAV2/1-NPY-EGFP gene were observed by thefluorescent microscopy; at the same time, the expressive volume and greyvalue of rAAV2/1-NPY-EGFP-positive cells were also detected.Results: The expressions of rAAV2/1-NPY-EGFP had been found bothintraventricular injected group and hippocampal injected group at2wpost-injection. At4w post-injection, the expression of rAAV2/1-NPY-EGFPpositive cells in volume and gray value in ventricular injected group werehigher than that in the hippocampal injected group, and the difference wasstatistically significant (P <0.05).Conclusion: rAAV2/1-NPY-EGFP in epileptic brain tissue underpathological conditions can be expressed effectively. The injected approach toventricle may be better than the hippocampus.Part2Effects of neuropeptide Y gene transfection using recombinantadeno-associated virus on behavior, EEG, and pathologicmorphology in rats with chronic epilepsy.Objective: To explore the effects of gene transfection ofrAAV2/1-NPY-EGFP on rat seizures, EEG, and pathological morphology. Methods: After successful model experimental animals were randomlydivided into4groups: control group, in which saline was injected tohippocampus CA3area; KA group, in which KA was injected to hippocampalCA3area with no virus injection; rAAV2/1-empty-EGFP group, in which10μlof rAAV2/1-empty-EGFP(titer5×1011v.g./ml) were injected to ventricle insuccessful rats chronic model; rAAV2/1-NPY-EGFP group, in which10μl ofrAAV2/1-NPY-EGFP(titer5×1011v.g./ml) were injected to ventricle insuccessful rats chronic model. The seizure situation﹑the onset latency andEEG wave frequency and amplitude were observed. Number of injuredneurons in the hippocampus CA3area HE staining. The ultrastructuralchanges of hippocampal CA3area were observed by the transmission electronmicroscopy (TEM).Results:1The seizure degree in rats of rAAV2/1-NPY-EGFP groupgradually reduced with observation time; at4W post-injection, in rats ofrAAV2/1-NPY-EGFP group, the onset of symptoms and onset latencysignificantly reduced (P<0.05), EEG epileptic discharge frequency andamplitude also decreased (P<0.05), compared to rats of KA group andrAAV2/1-empty-EGFP group.2HE results showed: at2-4w after genetransfection, hippocampal CA3neurons in the control group generally normalmorphology while number of normal neurons in KA group and therAAV2/1-empty-EGFP significantly reduced; The neurons showed neuralsparseness of arrangement, cell swelling and degeneration, unclear contours,nucleoli fuzzy, lightly stained cytoplasm companied with proliferation of glialcells in different degrees. At2,3w rats in rAAV2/1-NPY-EGFP group showeddifferent degrees of reduction in the number of neurons; at the4w, HE stainingshowed the lighter degree of cell damage, mild swelling and degeneration ofcells, cells loosely arranged and mild gliosis; the number of harm neuronsdecreased compared to KA group and the rAAV2/1-empty-EGFP group withsignificant difference (P<0.05).3TEM results suggest that hippocampal CA3area in control rats was normal; number of hippocampal neurons in KA groupand the rAAV2/1-empty-EGFP group has reduced, showing cytoplasmicconcentration, nuclear enrichment, increased marginalization of hetero- chromatin, expansion of perinuclear gap, significant degeneration of remainedneurons, microglia proliferation around the necrotic cell, mitochondrialswelling and cristae derangement or broken, missing mitochondrial cristae.While rAAV2/1-NPY-EGFP group showed mild degeneration of neuronsedema, minor deformation of the cell body, mitochondrial edema andcomplete crest.Conclusion: rAAV2/1-NPY-EGFP gene transfection can inhibit seizuresand reduce the hippocampal pathological changes, which provide strongsupport to the NPY gene therapy for clinical refractory epilepsy.Part3The dynamical research of Recombinant neuropeptide Y genetransfection using recombinant adeno-associated virus onneurotransmitters in rats with chronic epilepsyObjective: To investigate effects of rAAV2/1-NPY-EGFP genetransfection on neurotransmitters in hippocampal extracellular fluid in centralnervous system.Methods:30μL of microdialysis dialysate were collected in rats of fourgroups at different time points and the concentrations of Glu and γ-GABA ofrat hippocampal dialysate were examined by high performance liquidchromatography.Results: At2w and3w after gene transfection the Glu concentration inmicrodialysis in rAAV2/1-NPY-EGFP group showed no significant differencecompared to KA and rAAV2/1-empty-EGFP group (P>0.05); but at3w, theGlu concentration in microdialysis has been reduced; at4w post-injection, Gluconcentration in rAAV2/1-NPY-EGF group(28.36±1.68μmol/L) wasdecreased by about50%compared to that in KA group(63.00±2.85μmol/L)and the rAAV2/1-empty-EGFP (59.05±8.35μmol/L) with significantdifferences (P <0.05).Conclusion:rAAV2/1-NPY-EGFP gene transfection inhibits the releaseof excitatory amino acids in the epileptic rats, NPY regulation of neurotran-smitters may be involved in the mechanism of inhibition to epileptic seizures. Part4Effects of neuropeptide Y gene transfection using recombinantadeno-associated virus on the expression of NMDA receptorsubunits in rats with chronic epilepsy.Objective: To investigate the effects of rAAV2/1-NPY-EGFP geneexpression on the expression of hippocampal NMDA receptors.Methods: After rats in4groups were killed2,3,4w after vector injectionrespectively, hippocampal NMDA receptor subunits NR1, NR2A and NR2BmRNA were detected with quantitative PCR and the expression of NMDAreceptors protein were also done by Western blotting analysis.Results:1There was significantly increased of NPY expression at2,3,4w post-injection in the KA group, rAAV2/1-empty-EGFP group and therAAV2/1-NPY-EGFP rats compared with the control group (P <0.05). At4wafter vector injection, expression of NPY mRNA and protein inrAAV2/1-NPY-EGFP group were higher than the KA group and therAAV2/1-empty-EGFP group.2At2,3,4w post-injection, hippocampalNMDA receptor subunits NR1, NR2A and NR2B mRNA and proteinexpression in KA group and the rAAV2/1-empty-EGFP were significantlyincreased compared with the control group with the significant difference(P<0.05).The expression of mRNA and protein of hippocampal NMDAreceptor subunits in rAAV2/1-NPY-EGFP group decreased significantly at4wpost-injection, compared to the KA group and the rAAV2/1-empty-EGFPgroup (P <0.05).Conclusion: rAAV2/1-NPY-EGFP gene transfection inhibits theexpression of NMDA receptors in epileptic rat hippocampus. NPY may playanti-epileptic and neuroprotective effects through downregulation to theexpression of hippocampal NMDA receptor subunits.Part5The inhibitory action of neuropeptide Y on epileptiform dischargesin hippocampal neuronsObjective: To investigate the effect of NPY on epileptic discharge ofprimary cultured neurons.Methods: The model of epileptic discharges of hippocampal neuronswas formed by12d primary cultured hippocampal neurons treated with magnesium-free solution for3h, and action potentials of epileptichippocampal neurons were detected by whole cell current clamp mode.Effects were observed of0.1μM and1μM NPY application for10s on thefrequency and amplitude of neuronal action potential.Results:1A stable model of epileptiform discharges can be formed byhippocampal neurons treated with3h magnesium-free extracellular fluid withfrequency16~23/s and amplitude75~96mV.2Compared with the previousadministration, two concentration of NPY can reduce action potentialfrequency and amplitude (P<0.05); there was significant difference betweenthe two NPY concentration (P <0.05).1μM NPY significantly inhibited thefrequency and amplitude of action potential.Conclusion: NPY can inhibit neuronal epileptiform activity induced bymagnesium-free extracellular solution, which provides evidence of cellelectrophysiology to apply NPY to suppress seizures.Part6Effects of neuropeptide Y on neurons in hippocampal epilepsyinduced currents in NMDAObjective: To investigate NPY on NMDA induced inward currents ofprimarily cultured rat hippocampal neurons in vitro.Methods: After the model of epileptic discharge of hippocampalneurons was established, effects of various concentrations(0.1μM and1μM) ofNPY on100μM NMDA induced inward current by patch-clamp voltage mode.Results: NPY can suppress the NMDA-induced inward current inhippocampal epileptic neurons. Its effect has in a concentration dependentmanner.Conclusion: NPY may decrease neuronal excitability and play ananti-epileptic role by regulating the activity of the NMDA receptors inepileptic hippocampal neurons.