Effect And Mechanism Of Plic-1on Epileptic-seizure

PART ONE: EXPRESSION OF PLIC-1IN EPILEPTIC PATIENTS ANDKINDLED ANIMAL MODELObjective: To investigate the expression of Plic-1both in cortex of TLEpatients and normal patients.To investigate the expression of Plic-1in both hippocampus andcortex between spontaneously group and non-spontaneously group inlithium chloride-pilocarpine kindled epileptic rat and normal rat.Co-localized Plic-1with MAP2,GFAP,GAD67,GABAβ2/3by doubleimmunofluorescence on frozen sections from the hippocampus or cortex ofeither TLE patients or rat models.Method: Neocortical tissues adjacent to epileptic foci at temporal region30samples from TLE patients and10samples were randomly collected.Control neocortical tissue samples were randomly obtained from10non-TLE patients who need therapeutic surgical tissue resection from headtrauma but excluded any obvious histological changes and no seizuremanifestations or usage of AEDs. Hippocampus and cortex between spontaneously group andnon-spontaneously group in lithium chloride-pilocarpine kindled epilepticrat (220g-280, male) were obtained2month after kindled.All the tissues above were used in Western-blot and double-stainningfor investigation expression difference of Plic-1between epileptic groupsand control groups.Result:1. Western blot: In human neocortical tissue, Plic-1expression wassignificantly lower in TLE group than that in controls (*P＜0.01).2. In the cortex of animal model (2month after seizure onset): Plic-1expression in neocortical tissue was significantly decreased inspontaneous seizure group compare to non-sponstanous seizure groupin pilocarpine model when spontaneously seizure demonstrated;(*P＜0.05).In hippocampus of epileptic animal model (2month after seizure onset):Plic-1expression in spontaneous seizures group was significantlydecreased than in non-spontaneous seizures group or control group (*P＜0.01).3. Immunofluence staining revealed that Plic-1was positive in membraneand cytoplasm of neurons and sometimes showed punctuate distributionin nuclei. Double labeling revealed that Plic-1was co-localized withMAP2, GAD67and GABAAβ2/3but not with GFAP in the cortical region of TLE patients or in the cortex and hippocampus of epilepticrats model.Conclusion: Plic-1was showed decreased in TLE patient and epileptic ratmodels compared to control group. Plic-1was enriched in these neuronsand co-localized with GABAA receptors at inhibitory synapses.Theseindicated that Plic-1involved in epileptic-seizure and epilepsy. PART TWO: ADMINISTRATION OF PePαPEPTIDE INCREASESEIZURE SEVERITY IN TWO EPILEPTIC RAT MODELSObjective: To investigate interruption Plic-1connection with GABAARcould affect behaviors seizure and GABAARβ2/3expression.Method:We constructed the PePα peptide which could interrupt connectionbetween Plic-1and GABAAR or scrambled peptide (Control group).Microinjected the constructed PePα peptide or scrambled peptide into CA1region of hippocampus in both pilocarpine epileptic model andpentylenetetrazole (PTZ) epileptic model. We evaluated behavior scores intwo epileptic rat models and GABAARβ2/3expression after kindled.1.48male SD rats (220-280g) were randomly divided into two groups, andeach group divides into normal control group（n=8）, Scrambled peptidegroup（n=8）and PePα peptide group（n=8）. Before intraperitoneal injectionof pilocarpine or pentylenetetrazole, rats were microinjected withsaline(10μl), PePα peptide [10μl（1mg/kg）] or scrambled peptide [10μl（1mg/kg）] separately.1. Intraperitoneally injected pilocarpine or pentylenetetrazole (PTZ), thelatency to first seizure and Racine score in1hours after first seizurewere scored in pilocarpine model. The latency to first seizure andseizure severity and percentage of GTCs were scored in PTZ model.2. The intensity of fluorescence of Plic-1and GABAARβ2/3were examined in CA1region of Hippocampus, Doubled-staining cells werecounted in CA1region of Hippocampus.3. Western-blot were used to evaluated the Plic-1and GABAARβ2/3expression in hippocampus24hs after kindled.Results:1. Behavior Scores:Pilocarpine model:Racine score in seizure severity: PePα peptide significantly enhancedseizure severity in PePα peptide group compared to scramble peptide groupand control group (*P<0.05, after10min,20min and50min).Seizure onset latency: PePα peptide group shorten in latency than twocontrol groups (*P<0.01).PTZ model:PePα peptide significantly enhanced seizure severity in PePα peptidegroup compared to scramble peptide group and control group: PTZ scalescore:(*P<0.05);Seizure onset latency: PePα peptide group shorten in latency thanscramble peptide group and control group (*P<0.01);Percentage of GTCs: PePα peptide group increase percentage of GTCsthan two control groups (*P<0.05).2. The intensity of fluorescence of Plic-1and GABAARβ2/3:Immunofluorescence intensity of GABAARβ2/3staining in PePα peptide group was weaker compared with other two control groups(*P<0.01). No significance in fluorescence intensity of Plic-1(P>0.05).Double labeling cells counting: No significance in GABAergic neuronswith Plic-1labeling in PePα peptide group in CA1region compared to twocontrol groups:(P>0.05).3. Western-blot：Total GABAARβ2/3expression hippocampus: Significant reduction inPePα peptide group compared to two control groups (*P<0.05).Total Plic-1expression hippocampus: No significant differencebetween three groups (P＞0.05).Conclusion： The effective dose of PePα peptide significantly shorted thelatency to first seizure onset and aggravated the seizures severity.Interrupted Plic-1connection with GABAAR caused decreased ofGABAARβ2/3expression. PART THREE: UP-REGULATION OF PLIC-1BY RECOMBINANTLENTIVIRUS-PLIC-1INCREASES GABAAR EXPRESSION ANDDECREASE SEIZURE SEVERITY IN EPILEPTIC RAT MODELObjective: To investigate if Plic-1over expressed could have oppositeeffect of PePα in behaviors seizure and GABAARβ2/3expression in PTZmodel.Method:We constructed recombinant over expressed lentvirus-Plic-1geneand injected into CA1region of hippocampus in PTZ model. Evaluatebehavior scores in PTZ rat models and GABAARβ2/3expression.1.24male SD rats (220-280g) were randomly divided into normal controlgroup（n=8）, LV-GFP group（n=8）and LV-Plic-1group（n=8）. Beforeintraperitoneal injection of pentylenetetrazole, rats were microinjected withsaline (20μl), LV-GFP (20μl) or LV-Plic-1(20μl) separately.2. Intraperitoneally injected pentylenetetrazole (PTZ), the latency to firstseizure and seizure severity and percentage of GTCs were scored.3. The intensity of fluorescence of GABAARβ2/3were examined in CA1region of Hippocampus.4. Western-blot were used to evaluated the Plic-1and GABAARβ2/3expression in hippocampus24hs after PTZ kindled.Results:1. Behavior Scores: PTZ scale score in seizure severity: LV-Plic-1group showedsignificantly alleviation in seizure severity compared to LV-GFP group andcontrol group (*P<0.05).Seizure onset latency: LV-Plic-1group showed longer in seizure onsetlatency compared to two control groups but without significance (P>0.05).Percentage of GTCs: No significant difference between three groups(P>0.05).2. The intensity of fluorescence of GABAARβ2/3: Immunofluorescenceintensity of GABAARβ2/3staining in LV-Plic-1group was strongercompared with other two control groups (*P<0.01).3. Western-blot：Total Plic-1expression hippocampus: Plic-1expression in LV-Plic-1group was higher than LV-GFP and control groups (*P <0.05).Total GABAARβ2/3expression hippocampus: GABAβ2/3expressionin LV-Plic-1group significantly elevated than LV-GFP group and controlgroup (*P <0.05).Conclusion： By over expressed Plic-1in CA1region in hippocampus inPTZ model significantly alleviated seizures severity after kindled. Overexpressed Plic-1caused increase of both Plic-1and GABAARβ2/3expression. PART FOUR: PLIC-1AFFECT SYNAPTIC INHIBITION OFPYRAMIDAL NEURONS IN CA1Objective: To investigate interruption Plic-1connection with GABAAR orover expression Plic-1could affect synaptic inhibition of pyramidalneurons in CA1region in Mg-free rat brain slices or epileptic rathippocampus brain slices in vitro.Method: We used16normal rats (P2-4) to prepared Mg-free rats brainslices. Miniature IPSC changes after perfused with PePαpeptide orscrambled peptide in Mg-free hippocampal slices were collected andcalculated as amplitude, frequency and decay time.We used25normal male rats (220-280g).Injected saline or LV-GFP orLV-Plic-1before PTZ induced. Epileptic rats to prepared epileptic ratsbrain slices. Each randomly selected hippocampal slices were bathed withMg-free condition. Miniature IPSC changes in hippocampal slices fromdifferent groups were collected and calculated as amplitude, frequency anddecay time.Result:1. Mg-free rat brain slices:Mean amplitude of mIPSCs: After adding PePαpeptide: A significantreduction was recorded in the pyramidal neurons in CA1region comparedwith Mg-free control condition(14.016±0.735pA in PePα peptide,21.566±1.541pA in Mg-free,*P＜0.01). mIPSC variation after scramble peptide application showed no significance difference as compared withMg-free control(P>0.05).Frequency of mIPSCs: PePα peptide decreased in current frequencythan in Mg-free control condition (*P＜0.01). Scrambled peptide had nosignificant change in frequency current than in Mg-free control condition(P>0.05).Decay time of mIPSC: PePα peptide significantly accelerated meanmIPSC decay time (5.831±0.546ms in Mg-free+PePα peptide,9.629±0.977ms in Mg-free*P <0.05). Scrambled application did not affect thedecay time (Mean mIPSC decay time:15.997±3.00ms in Mg-free+scrambled peptide,10.950±2.228ms in Mg-free, P>0.05).1. Epileptic rats brain slices:Mean amplitude of mIPSCs: mIPSC in LV-Plic-1group displayedsignificant higher amplitude compared with LV-GFP group and controlgroup (Mean mIPSC amplitude:27.490±2.942pA in LV-Plic-1,17.894±1.620pA in LV-GFP,15.203±1.509pA in control,*P<0.01). mIPSC inLV-GFP group had no significant change in amplitude current than controlgroup (P>0.05).Mean mIPSC frequency: mIPSC in LV-Plic-1group displayedsignificant higher frequency compared with LV-GFP group and controlgroup:*P=0.033compare to LV-GFP group;*P=0.040compare to controlgroup). mIPSC in LV-GFP group had no significant change in frequency current than control group (P>0.05).Decay time of mIPSC: The decay time of mIPSC was significantlyprolonged in LV-Plic-1group than other two control groups (7.445±0.775ms in LV-Plic-1,2.968±0.423ms in LV-GFP,3.233±0.673ms in controlgroup,*P<0.01compare to LV-GFP;*P<0.01compare to control). mIPSCin LV-GFP group had no significant change in decay time than controlgroup (P>0.05).Conclusion: These results suggested that PePα peptide interruptedconnection between Plic-1and GABAAR could decrease GABAARmedicated mIPSC in amplitude, frequency and decay time at postsynapticsite. Over expressed Plic-1could enhance GABA-mediated mIPSC inamplitude, frequency and decay time at postsynaptic in seizure. Thus,Plic-1could regulate GABA-mediated mIPSC in seizure.