【作者】 刘勇；

【导师】 赵靖平；

【作者基本信息】 中南大学 ， 精神病与精神卫生学， 2009， 博士

【摘要】 第一部分MK-801重复处理围产期或成年期大鼠建立的精神分裂症模型的行为学评价目的:用Dizocilpine(MK-801)重复处理围产期或成年期大鼠建立精神分裂症(Schizophrenia,SZ)发育动物模型和慢性给药动物模型,通过对成年模型大鼠行为学改变的测评和比较,探讨两种SZ大鼠模型的表现效度及相关的病理机制。方法:新生雄性Sprague-Dawley(SD)大鼠于出生后(Postnatal,P)6天随机分为3组:SZ发育模型组、SZ慢性给药模型组和对照组。SZ发育模型组大鼠于P7-10天皮下注射MK-801(0.1mg/kg,2次/天);SZ慢性给药模型组大鼠于P47-60天腹腔注射MK-801(0.2 mg/kg,1次/天);对照组大鼠于相应时段注射0.9%的生理盐水(Normal Sodium,NS)。P63天各组大鼠进行自发活动、强迫游泳、社会交往和Morris水迷宫行为学测试。结果:(1)SZ慢性给药模型组自发活动总路程显著大于对照组(P＜0.01)。(2)强迫游泳测试,两模型组不动时间较对照组显著延长(P＜0.05)。(3)社会交往测试,SZ发育模型组社会行为较慢性给药模型组和对照组显著减少(P＜0.05);两模型组非攻击行为百分比显著低于对照组(P＜0.01)。(4)Morris水迷宫测试,训练的第3、4天两模型组的逃避潜伏期较对照组显著延长(P＜0.01或P＜0.05);第5天的记忆提取测试,两模型组在目标象限停留时间较对照组显著缩短(P＜0.05)。结论:(1)SZ慢性给药大鼠模型可部分模拟SZ的阳性症状;(2)两种SZ大鼠模型可部分模拟SZ的阴性症状;(3)两种SZ大鼠模型可部分模拟SZ的认知功能损害;(4)围产期MK-801重复处理建立的SZ发育动物模型是急性和慢性N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受体阻断剂处理诱导的SZ动物模型的有效补充,可用于SZ神经发育假说相关病理机制研究。第二部分MK-801重复处理建立的两种SZ模型大鼠脑组织PV、GAD67和KCC2表达的比较目的:通过对两种SZ模型大鼠脑组织γ-氨基丁酸(Gamma-aminobutyric acid,GABA)递质系统病理学改变的比较研究,探讨两种SZ大鼠模型的结构效度及相关的病理机制。方法:新生雄性SD大鼠于P6天随机分为2批,每批又随机分为3组:SZ发育模型组、SZ慢性给药模型组和对照组。造模方法同第一部分。P63天,第1批大鼠断头取脑,多聚甲醛固定后行组织切片,以免疫组化方法检测内侧前额叶皮质(medial prefrontal cortex,mPFC)和海马(hippocampus,HP)CA1区小清蛋白(Parvalbumin,PV)及谷氨酸脱羧酶67(glutamate decarboxylase 67,GAD67)的表达情况;第2批大鼠处死后取mPFC和海马组织匀浆,以免疫印记方法(Western blotting)检测mPFC和海马K~+-Cl~-协同转运蛋白2(K~+-Cl~- cotransporter 2,KCC2)的表达水平。结果:(1)两模型组mPFC和海马CA1区PV以及GAD67的表达显著低于对照组(P＜0.01或P＜0.05);(2)SZ发育模型组mPFC和海马KCC2的表达水平显著低于慢性给药模型组和对照组(P＜0.05)。结论:(1)两种SZ大鼠模型可部分模拟SZ的GABA系统病理学改变;(2)SZ发育模型成年大鼠部分脑区KCC2的表达减少。(3)两种SZ大鼠模型具有一定的结构效度,可用于SZ的相关病理机制研究。第三部分MK-801重复处理建立的两种SZ模型大鼠脑组织DA、Glu及其代谢产物浓度的比较目的:通过对两种SZ模型大鼠脑组织多巴胺(Dopamine,DA)和谷氨酸(Glutamate,Glu)及其代谢产物二羟苯乙酸(dihydroxy-phenylacetic acid,DOPAC)和γ-氨基丁酸(gamma-aminobutyric acid,GABA)浓度的检测和比较研究,探讨两种SZ大鼠模型的结构效度及相关的病理机制。方法:动物分组和造模方法同第一部分。P63天各组大鼠断头取脑,分离出mPFC和海马并匀浆,以高效液相色谱-库仑阵列电化学法检测组织匀浆中的DA、DOPAC、Glu和GABA,并计算分析DA和Glu的利用率。结果:(1)SZ发育模型组mPFC和海马DA和DOPAC浓度较对照组显著降低(P＜0.01);SZ慢性给药模型组mPFC区DOPAC浓度和DA利用率较对照组显著降低(P＜0.05),而海马DA利用率升高(P＜0.05)。(2)SZ发育模型组海马DOPAC浓度和DA利用率较慢性给药模型组显著降低(分别P＜0.01;P＜0.05)。(3)SZ发育模型组mPFC区Glu利用率较对照组显著降低(P＜0.05);SZ慢性给药模型组mPFC区GABA浓度和Glu利用率较对照组显著降低(分别P＜0.05;P＜0.01),海马Glu浓度也显著降低(P＜0.05)。结论:(1)两种SZ模型大鼠mPFC区DA系统功能减退;(2)SZ发育模型成年大鼠海马DA系统功能减退,而SZ慢性给药模型大鼠海马DA系统功能亢进。(3)两种SZ模型大鼠mPFC区Glu和GABA系统功能减退。(4)SZ慢性给药模型组大鼠海马Glu系统功能减退。第四部分SZ发育模型成年大鼠海马脑片LTP的表达改变及KCC2的作用机制研究目的:通过对SZ发育模型成年大鼠海马脑片长时程增强(long-term potentiation,LTP)的表达变化以及LTP诱导前后兴奋性突触后电位(excitatory postsynaptic potential,fEPSP)记录区域KCC2的表达改变研究,初步探讨KCC2在SZ认知功能损害中的作用。方法:新生雄性SD大鼠于P6天随机分为2组:SZ发育模型组(MK-801)和对照组(Control)。SZ发育模型造模方法同前。P61天各组大鼠断头取脑,制备急性海马脑片,根据脑片来源(组别)和灌流药物不同,脑片电生理实验分为4组:Control组、MK-801组、MK801+FURO组、MK-801+PTX组。实验采用海马CA1区场电位记录。以促强直刺激(Theta burst stimulation,TBS)诱导LTP表达,观察SZ发育模型大鼠海马脑片LTP的表达变化以及KCC2抑制剂呋塞米(Furosemide,Furo)和GABA_A受体阻断剂木防己苦毒素(Picrotoxin,PTX)对其表达的影响。以Western blotting方法检测LTP诱导前后海马脑片CA1区KCC2的表达改变。结果:TBS刺激诱导后40分钟,MK-801组标化后的fEPSP斜率(105±3%)显著低于Control组(149±2%)(P＜0.05);FURO灌流给药组即MK-801+FURO组fEPSP斜率(130±2%)较MK-801组显著增加(P＜0.05);PTX灌流给药组即MK-801+PTX组fEPSP斜率(146±2%)较MK-801组显著增加(P＜0.05),但与Control组比较无显著性差异(P＞0.05)。LTP诱导前,MK-801组CA1区KCC2的表达较对照组显著降低(P＜0.05);LTP诱导后,MK-801组CA1区KCC2的表达较诱导前无显著性差异(P＞0.05),而Control组CA1区KCC2的表达较诱导前显著降低(P＜0.05)。结论:(1)SZ发育模型成年大鼠海马突触可塑性受损;(2)SZ发育模型成年大鼠海马存在KCC2介导的GABA抑制功能紊乱;(3)KCC2可能参与了NMDA受体功能下调介导的认知功能损害。更多还原

【Abstract】 PartⅠBehavioral test in schizophrenia model rats repeatedly treated with MK-801 during perinatal or adult periodObjective:Rats repeatedly treated with N-methyl-Daspartate (NMDA) antagonists have been thought to be a developmental and chronically treated model of schizophrenia(SZ) during perinatal period or adulthood.In this study,we investigated the face validity and related pathomechanism in two SZ model by the test and comparison of behavioral changes in SZ model rats.Methods:Neonatal male Spragur-Dawley(SD) rats were randomly assigned at the postnatal day 6 to three groups:SZ developmental animal model group(subcutaneous injection with MK-801 at the postnatal day 7-10,0.1 mg/kg Bid),SZ chronically treated animal model group(intraperitoneal injection with MK- 801 at the postnatal day 47-60,0.2 mg/kg Qd) and normal control group(injection with saline during the corresponding periods). Behavioral test were measured in three groups at the postnatal day 63, including Locomotion test,Forced Swimming test,Social Interaction test and Morris Water Maze test.Results:(1) In Locomotion test,the total path was significantly increased in SZ chronically treated model group compared with both SZ developmental and control groups;(P<0.01).(2) In Forced Swimming test,the immobility time was significantly increased in two model groups as compared with control group(P<0.05).(3) In Social Interaction test,the social behavior was significantly decreased in SZ developmental model group compared with both SZ chronically treated and control groups(P<0.05);the non-aggressive behavior percentage was significantly decreased in two model groups compared with control group(P<0.01).(4) In Morris Water Maze test,the escape latent period was significantly longer in two model groups than that in control group at the training day 3-4(P<0.01 or P<0.05);in memory retrieval test,the detention time in the target quadrant was significantly shorter in two model groups than that in normal control group at the day 5 (P<0.05).Conclusions:(1) SZ chronically treated model may be used to simulate partly positive symptoms in SZ.(2) Both two models could be used to simulate partly negative symptoms in SZ.(3) Both two models could be used to simulate partly cognitive impairment in SZ.(4) SZ developmental model repeatedly treated with MK-801 in perinatal period is the efficient supplement for SZ animal model treated with acute and chronic NMDA receptor blocking pharmacon in adult rats,and may refer to study of pathomechanism related to neurodevelopmental hypothesis in SZ.PartⅡComparison of the expression of PV,GAD67 and KCC2 in brain tissue in both SZ modelsObjectives:Rats repeatedly treated with NMDA antagonists (MK-801) have been thought to be a developmental and chronically treated model of schizophrenia(SZ) during perinatal or adult period.In this study,we investigated the construction validity and related pathomechanism in two SZ model by the comparison of pathological changes of Gamma-aminobutyric acid(GABA) system in SZ model rats.Methods:At the postnatal day 6,neonatal male SD rats were randomly assigned to two batches(B1 and B2) with each batch being randomly divided into three groups:SZ developmental animal model group (subcutaneous injection with MK-801 at the postnatal day 7-10,0.1 mg/kg Bid),SZ chronically treated model group(intraperitoneal injection with MK-801 at the postnatal day 47-60,0.2 mg/kg Qd) and normal control group(injection with saline during the corresponding periods).At the day P63,the brain tissues of B1 rats were fixed with paraform for histological section after decapitation.And then the expression level of parvalbumin(PV) and glutamate decarboxylase 67(GAD67) in the tissue homogenate from medial prefrontal cortex(mPFC) and hippocampus CA1 region was examined by immunohisto-chemistry technique.The tissue homogenates of mPFC and hippocampus were obtained for measuring the expression levels of K~+-Cl~- cotransporter 2(KCC2) in both brain regions in B2 rats with Western blotting.Results:(1) The expression level of PV and GAD67 in the mPFC and hippocampus CA1 region in both SZ model groups was significantly decreased compared with control group(P<0.01;P<0.05,respectivelly).(2) The expression level of KCC2 in the mPFC and hippocampus CA1 region in SZ developmental model group was significantly decreased compared with both SZ chronically treated model and control groups(P<0.05). Conclusions:(1) Both SZ models may be used to simulate partly pathological changes of GABA system in SZ.(2) In SZ developmental model group,the KCC2 expression was downregulated in some brain regions.(3) Both SZ models show certain construction validity and could be used to pathomechanism study related to SZ.PartⅢComparison of concentration of DA,Glu and their metabolism production in brain tissue in both SZ models repeatedly treated with MK-801Objectives:We explored the construction validity and its related pathomechanism by the comparison of concentration of dopamine(DA), glutamate(Glu) and their metabolism production,dihydroxy-phenyl acetic acid(DOPAC) and GABA in brain tissue in both SZ model rats. Methods:The treated measures for SD rats was similar to that in PartⅠ. After decapitation at the day P63,DA,DOPAC,Glu and GABA of the tissue homogenate from the medial prefrontal cortex(mPFC) and hippocampus were examined with Coularray Electrochemic detection for high performance liquid chromatogram(HPLC) technique.Meanwhile, the utilization rate of DA and Glu was calculated.Results:(1) The concentration of DA and DOPAC in both mPFC and hippocampus regions was significantly decreased in SZ developmental model group compared with control group(P<0.01).The DOPAC concentration and DA utilization rate in the mPFC were significantly decreased in SZ chronically treated model and DA utilization rated was increased in the hippocampus region compared with control group(P<0.05).(2) The DOPAC concentration and DA utilization rate in the hippocampus region was significantly decreased in SZ developmental model group compared with SZ chronically treated model group(P<0.01;P<0.05,respectivelly). (3) Compared with control group,the Glu utilization rate in the mPFC was significantly decreased in SZ developmental model group,and the GABA concentration and Glu utilization rate in the mPFC and Glu concentration in the hippocampus were significantly decreased in SZ chronically treated model group(P<0.05).Conclusions:(1) The activity of DA system in mPFC was decreased in both SZ models.(2) The activity of hippocampus DA system was decreased in SZ developmental model rats,while the activity of hippocampus DA system was increased in SZ chronically treated model group.(3) The activity of Glu and GABA systems in the mPFC was decreased in both SZ model groups.(4) The activity of hippocampus Glu system was decreased in SZ chronically treated model group.PartⅣStudy of the impairment of LTP in hippocampal slice and mechanism of KCC2 down-regulation after maturation in SZ developmental model ratsObjective:Perinatal repeated MK-801-induced developmental animal models of schizophrenia were employed to detect LTP change and KCC2 expression on the same fEPSP record region,and discussed the role of KCC2 in cognitive impairment of Schizophrenia.Methods:Pups on postnatal day 6(P6) were randomly assigned to two groups:SZ developmental model group(MK-801) and control group.SZ developmental model was developed by the same method as that in Part Ⅰ.Hippocampus slice was obtained on P61 immediately after rats were sacrificed by decapitation.The slice for electrophysiological recording was divided into four groups according to differential slice(MK-801 or Control) and perfusion drugs(FURO or PTX):Control group,MK-801 group,MK-801+FURO group and MK-801+PTX group.The experimental results were recorded by employing the field potential in hippocampus CA1 region.Study the influence of KCC2 inhibitor Furosemide(FURO) and GABA_A receptor antagonist Picrotoxin(PTX) on theta burst stimulation(TBS) -induced LTP.Western Blotting was used to detect changes of KCC2 expression on hippocampus CA1 after LTP induction.Results:(1) 40 minutes after TBS stimulation,the normalized fEPSP slope in hippocampal slice(105±3%) was significantly lower in SZ developmental model group than that in control period(149±2%)(P<0.05).(2) The fEPSP slope(130±2%) was significantly increased after perfusion of FURO in MK-801+FURO group compared with MK-801 group(P<0.05).(3) The fEPSP slope was significantly increased after perfusion of PTX in MK-801+PTX group compared with MK-801 group(146±2%) but no significant difference compared with control group(P>0.05).(4) Before LTP induction,KCC2 expression in hippocampal CA1 region was significantly decreased in MK-801 group compared with control group;following LTP induction, no significant change of KCC2 expression in MK-801 group compared with that before LTP induction,while it was significantly decreased in control group compared with that before LTP induction.Conclusions:(1) Perinatal Repeated treatment of MKS01 will lead to impairment of hippocampal synaptic plasticity of matured rats;(2) Perinatal Repeated treatment of MKS01 will inhibit the down-regulation of KCC2 during LTP expression after maturation,which caused the disturbance of GABA inhibition.(3) KCC2 may be involved in cognitive impairment mediated by the function down-regulation of NMDA receptor.更多还原