【作者】 杨鹏；

【导师】 曾水林；

【作者基本信息】 东南大学 ， 人体解剖与组织胚胎学， 2004， 硕士

【摘要】 研究目的:应用微电极核团毁损术毁损帕金森病(Parkinson’s Disease,PD)大鼠的腹侧苍白球(ventral pallidum, VP),观察其旋转行为的变化,检测术后脑内相关神经递质的改变及相应脑区的形态变化,以探讨“细胞刀”治疗 PD 疗效和可能机制。研究方法:成年 SD 大鼠 120 只,雌雄不拘,体重 200~300g。借助脑立体定位仪,用微量注射器向左侧黑质致密部(Substantia Nigra pars Compact,SNc)和中脑腹侧被盖区(Ventral Tegmental Area,VTA)注射 6-OHDA 溶液,术后向大鼠腹腔内注射阿朴吗啡(apomorphine,APO)诱发动物向右侧旋转,制备 PD 模型。筛选出合格的模型,行微电极直流电 VP 核毁损术,毁损后每周进行一次阿朴吗啡腹腔注射诱导并记录其旋转行为,对比其 VP 毁损前后旋转行为变化。并分别在术后 1 周、2周、4 周、8 周处死,用 HE 和 Caspase-3 免疫组织化学染色,观察毁损前后 VP区细胞坏死及凋亡情况;TH 和 NADPH-d 染色观察 TH 阳性神经元和 NOS 神经元的变化;通过高效液相色谱(High Performance Liquid Chromatography ,HPLC)观察毁损后不同时间点相关脑区氨基酸类和单胺类神经递质的变化情况。结果:1. VP 毁损后,动物的旋转次数与自身毁损前及与模型对照组、假手术组相比明显减少(P<0.05);2. HE 染色显示 VP 毁损区中心有组织脱落,周围呈现均质红染,无细胞结构,外周有大量炎性细胞浸润;3.NADPH-d 结果表明:与正常大鼠相比,模型对照组纹状体区 NADPH 阳性神经元数量明显增加(P<0.05);而在 VP 毁损术后,毁损侧纹状体内 NADPH 阳性神经大幅度增加,与其它各组相比较均有显著差异(P<0.01);4.TH 染色显示:与正常大鼠相比 PD 大鼠黑质内 TH阳性神经元数量明显减少(P<0.01);VP 毁损后,与模型对照组及假手术组相比无差异(P>.05);5. Caspase-3 免疫组织化学显示:正常大鼠黑质及纹状体内没有观察到 Caspase-3 阳性神经元,PD 鼠黑质内有较多的 Caspase-3 阳性细胞,VP 毁损术后,其黑质内 Caspase-3 阳性神经元数量上没有变化(P>0.05),在 VP 毁损组毁损区周围 Caspase-3 阳性细胞显著增加,与模型对照组及假手术组相比有统计学意义(P<0.05);6.HPLC 检测各组大鼠纹状体内多巴胺及其代谢产物和谷氨酸、γ-氨基丁酸,显示:①与正常组相比,PD 鼠 DA 含量显著下降(P<0.01);VP 毁损术后,DA 亦大幅下降,与模型对照组及假手术组相比有显著性差异(P<0.01)。 I<WP=5>中文摘要②PD 大鼠纹状体内谷氨酸比正常大鼠减少(P<0.05);与模型对照组相比,VP毁损术后谷氨酸量明显增加,在第二周时达高峰,以后逐渐下降,但仍高于模型对照组。③模型对照组 GABA 含量比正常组减少(P<0.05),VP 毁损术后第一周没有变化(P>.05),第二周开始升高(P<0.05),以后又呈逐渐下降,但也高于模型对照组。结论:1.VP 毁损术对 PD 大鼠的旋转行为有明显的改善作用。2.VP 毁损后对 PD大鼠纹状体内 DA 含量显著下降,而氨基酸在毁损后第二周时达高峰,以后逐渐下降,到两个月时仍旧高于未毁损组。3.微电极核团毁损术对 PD 症状有明显改善作用,其作用机理可能与苍白球受损后中断了其异常兴奋的传导,同时与产生大量的 NO 对纹状体产生进一步的长时间的损害以及兴奋性氨基酸的毒性作用、毁损区细胞的凋亡等有关。更多还原

【Abstract】 Objective: To oberserve the effect of Parkinson’s disease (PD) rats by microelectrodelesion neucli of ventral pallidum(VP),detect the change of neural transmitter andmorphology in the brain, to explore the mechanism of cell knife therapy PD.Methods: A total of 120 adult Sprague-Dawlay(SD), weighting 250-300g ,byinjecting 6-OHDA into left substantia nigra pars compact(SNc) and ventral tegmentalarea(VTA) of rat and inducing with apomorphine, we escablished Parkinson’sDisease(PD) modle. After lesioning procedure was determined by measuring thenumber of full turns over 30 min following intraperitoneal injection ofapomorphine(APO). Only those had mean net rotation of seven turns/min wereused.Then those rats were received surgery with microelectrode lesioning, and therotational behavior of rats were observed by APO induced. Those rats were sacrificedat 1,2,4 and 8 weeks after the surgery procedure, and neural necrosis and apoptosis wasoberserved with Hematoxylin and eosin(HE) staining and Caspase-3immunohistochemistry staining. The change of TH positive cell and NOS positive cellwere observed with TH immunohistochemistry and NADPH-d histochemistrystaining.Then content of neuro transmitter about amino acid and monamine wasdetected with High Performance Liquid Chromatography(HLPC).Results: 1.After VP lesion, compared with the number of rotation of lesion rats wassignificantly decreased(P<0.05).2. HE staining show:area of VP zone neural necrosiswas observed in central area of VP lesion zone.There was some tissue falled in the VPlesion region, little eosinophilic cytoplasm no cell structure, histological signs of aninflammatory response were present,with perivascular coffing and infiltration of cellsaround VP lesion region.3.Compared with normal rats, NADPH-d positive cells in thestriatum increased(P<0.05), and those positive cells in the ipsilateral striatum of VPlesion group were also increased. Compared with PD and sham group,there wasmarkedly difference(P<0.05).4.Results of TH immunohistochemistry staining revealed Ⅲ<WP=7>英文摘要TH positive cells in SNC of PD were significantly lower than normalrats(P<0.05).There was no differdnce among VP lesion PD rats and shamgroups(P<0.05).5.there were many Caspase-3 positive cells in the SNc of PD rats,andno change of number of Caspase-3 positive cells in VP lesion and sham surgry groupswas ovserved(P>0.05),but around VP lesion region,Caspase-3 positive cells werestrikingly increased,compared with PD group and sham groups(P<0.05).6.Result ofHPLC indicated: ① Content of DA was significantly less than normalgroup(P<0.01).After lesion of VP content of DA was also strikinglydecreased,compared with PD rats and sham groups.②Content of Glu was lower thannormal rats(P<0.05).After VP lesion ,content of Glu was remarkablely increased,andreached the peak at 2 weeks, then subsiding by 14 days but higer than control groups.③Content of GABA in PD rats was less than that of normal rats(P<0.05).After VPlesion, content of GABA was no change at one week, and obvious increased at 2weeks(P<0.05),then decreased gradually but much high than PD group in 2 months.Conclusions: 1.The rotational behavior of PD rats was significantly improved by VPlesion.2.Production of DA was remarkablely influenced by VP lesion.Contents ofamino acid reach the Peak at 2 weeks after VP lesion,then decrease gradually.3.VPlesion can obviously improved symptom of PD,it maybe result from lesion of VPinhibited abnormal discharge,and the increased NO content in the striatum canlonger reinforce the injury to VP,and the toxicity of exciting amino acid,and theapoptosis in lesion region.更多还原