Fluoro-Jade B法显示神经毒物红藻氨酸和MPTP致小鼠基底神经核损伤引起神经元的变性死亡

目的探讨应用Fluoro-JadeB(FJB)染色法检测基底神经核神经元变性死亡的方法。方法制备纹状体定位注射红藻氨酸(KA)损伤大鼠模型、腹腔注射MPTP损伤小鼠模型、以及培养纹状体神经元KA损伤细胞模型,用FJB荧光染色显示和分析变性死亡的神经元。结果FJB染色可清晰地显示KA和MPTP致大、小鼠损伤引起的变性神经元胞体和部分突起。在脑组织切片上,KA模型纹状体内、MPTP模型黑质致密部内分布许多FJB染色阳性的变性神经元,而对照组动物未见变性神经元。在培养纹状体神经元体系内,FJB也能清楚地显示KA损伤后变性的神经元。单位面积内计数FJB阳性神经元占培养纹状体神经元总数的比率(均值±标准差),KA损伤组为(8·42±1·09)%,较对照组(3·42±0·45)%明显增多(P<0·01)。结论FJB方法快速、简便、经济、可靠。可以用于脑组织切片和培养细胞显示变性神经元,能成功检测出KA和MPTP损伤后基底神经核的变性神经元。     

阻断Notch信号通路降低1-甲基-4苯基-1,2,3,6-四氢吡啶诱导的多巴胺能神经元损伤

目的探讨Notch信号通路的缺失对1-甲基-4苯基-1,2,3,6-四氢吡啶(MPTP)诱导的中脑多巴胺能神经元损伤的影响。方法选用5月龄TH-Cre Rbpj基因敲除小鼠及野生型小鼠共48只,腹腔注射MPTP建立帕金森病(PD)模型,通过行为学、免疫组织化学和Western blotting等方法研究阻断Notch信号通路对MPTP诱导的中脑黑质多巴胺能神经元损伤的影响。结果 MPTP处理的Rbpj CKO小鼠运动能力强于MPTP处理的野生型小鼠;Rbpj CKO小鼠黑质致密部神经元数目较野生型小鼠减少;MPTP处理后,野生型小鼠多巴胺能神经元数目明显下降,而Rbpj CKO小鼠多巴胺能神经元数目无明显改变;Western blotting结果显示,MPTP处理后Rbpj CKO小鼠和野生型小鼠Notch-1胞内结构域(NICD-1)的表达均明显增加,且Rbpj CKO小鼠表达量增加更为显著。结论Notch信号通路缺失导致多巴胺能神经元数量减少,阻断Notch信号通路可以降低MPTP诱导的多巴胺能神经元损伤。     

脑内炎症对黑质多巴胺能神经元的选择性损伤作用

目的 探讨脑内炎症反应对中脑黑质多巴胺能神经元的选择性变性作用.方法 健康SD雄性大鼠10只,随机分为实验组和对照组,实验组行脂多糖(LPS)右侧脑室定位注射,对照组注射生理盐水.注射后48周用免疫组织化学或组织化学法观察黑质多巴胺能、中缝核5-羟色胺能及基底核胆碱能3种不同类型神经元的变性及上述不同脑区小胶质细胞的激活情况.结果 免疫组织化学染色显示,LPS组在黑质、海马、纹状体、中缝核部位均可见到OX6阳性小胶质细胞,说明不同脑区均出现炎症反应.不同类型神经元染色结果显示,LPS组黑质多巴胺能神经元胞体变小、染色变浅、突起减少甚至消失,神经元数量比对照组减少40.1%(P<0.01);5-羟色胺能神经元及胆碱能神经元形态及数量均无明显改变.结论脑室注射LPS导致的脑内炎症反应可选择性引起黑质多巴胺能神经元变性损伤.     

G-CSF对帕金森病小鼠运动能力和黑质纹状体神经元的影响

目的:探讨粒细胞集落刺激因子(G-CSF)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)所致小鼠帕金森病(PD)模型中脑黑质致密部(SNpc)及纹状体(STR)多巴胺(DA)能神经元的影响。方法:正常健康雄性C57BL/6J小鼠随机分为对照组、MPTP组及MPTP+G-CSF组,采用腹腔注射MPTP法制作小鼠PD模型,MPTP+G-CSF组于最后一次MPTP注射后再腹腔注射G-CSF。爬杆实验观察小鼠的行为学改变;尼氏染色技术观察各组小鼠黑质致密部神经元数量及形态学变化;免疫组织化学法检测酪氨酸羟化酶(TH)在各组小鼠中脑黑质致密部及纹状体的表达; Western Blot法检测各组小鼠中脑TH蛋白的表达量。结果:与对照组相比,MPTP组小鼠较对照组爬杆用时显著延长(P 0. 05),尼氏染色显示黑质致密部神经元数量显著减少(P 0. 05),黑质致密部、纹状体TH表达量显著减少(P 0. 05); MPTP+G-CSF组较MPTP组爬杆用时显著缩短(P 0. 05),神经元丢失减少(P 0. 05),神经元形态较完整;与MPTP组比较,MPTP+G-CSF组黑质致密部、纹状体TH表达水平显著增高(P 0. 05)。结论:G-CSF能够改善PD小鼠运动功能,减少黑质致密部多巴胺能神经元丢失,增加TH表达水平。     

Lactacystin诱导黑质神经元变性死亡的帕金森病大鼠模型方法

目的:探讨脑内定位注射蛋白酶体抑制剂Lactacystin诱导大鼠黑质神经元变性及运动症状的帕金森病大鼠模型方法。方法:成年雄性SD大鼠16只随机分为Lactacystin组和对照组,将Lactacystin 8μg(溶于0.8μl生理盐水)定位注入左侧内侧前脑束,对照组注射等量生理盐水。于1、3、5周时间点观察其运动行为,处死动物后进行中脑酪氨酸羟化酶和Fluoro-Jade C染色,显示分析黑质多巴胺神经元生存和变性死亡。结果:Lactacys-tin组黑质致密部酪氨酸羟化酶阳性神经元急剧减少,伴有大量Fluoro-Jade C阳性的变性神经元出现。Lactacystin组动物出现自发活动减少、运动缓慢、震颤和自发旋转,并呈逐渐加重的运动行为改变。结论:Lactacystin能够高效诱导黑质多巴胺神经元变性和运动障碍的大鼠模型,是研究帕金森病发病机制和神经保护的优良方法。     

尼莫地平对帕金森病模型小鼠黑质多巴胺神经元的保护作用

目的:研究尼莫地平在1-甲基-4-苯基-1,2,3,6四氢吡啶(MPTP)所致帕金森病(PD)模型小鼠中对黑质多巴胺能神经元的保护作用。方法:雄性健康C57BL/6N小鼠随机分为对照组,腹腔注射生理盐水;模型组,腹腔注射MPTP(25 mg/kg);尼莫地平治疗组,每次注射MPTP前3 h灌胃给予尼莫地平(15 mg/kg)。观察各组小鼠行为学变化,免疫组织化学和免疫蛋白印迹法观察中脑黑质酪氨酸羟化酶(TH)、前列腺素E2(PGE2)和肿瘤坏死因子α(TNF-α)的表达变化。结果:与对照组小鼠比较,MPTP模型组小鼠出现典型的PD症状,中脑黑质TH阳性神经元大量丢失,PGE2和TNF-α阳性细胞显著增多,蛋白水平明显升高;经尼莫地平治疗后,上述症状得到明显改善。结论:在MPTP所致PD小鼠模型中,尼莫地平通过抑制炎症因子PGE2和TNF-α的表达从而对多巴胺能神经元具有一定的保护作用。     

MPTP对小鼠中脑α-突触核蛋白线粒体表达和线粒体形态改变的影响

目的:观察MPTP对中脑黑质神经元线粒体表达α-突触核蛋白及线粒体形态改变的影响。方法:运用腹腔注射MPTP诱导C57/BL小鼠中脑黑质神经元损伤,以生理盐水腹腔注射小鼠作为对照组。运用线粒体纯化结合Western Blot分析线粒体α-突触核蛋白的水平,运用免疫组织化学标记结合激光共聚焦方法观察线粒体结构的改变。运用MPP+干预原代培养的多巴胺能神经元,观察线粒体形态的改变。结果:MPTP可以诱导小鼠中脑线粒体α-突触核蛋白的水平升高,并导致线粒体出现碎片化,碎片化的线粒体结构与α-突触核蛋白共定位。运用MPP+处理原代培养的中脑腹侧神经元,发现MPP+可以选择性地诱导多巴胺能神经元的线粒体碎片化。定量研究显示:多巴胺能神经元经MPP+处理后,线粒体的长度显著降低(P0.01)。结论:MPTP可以在体诱导多巴胺能神经元的线粒体碎片化,线粒体α-突触核蛋白水平的升高可能与其碎片化密切相关。     

JNK信号通路对亚急性帕金森病MPTP模型小鼠黑质iNOS表达的影响

目的:研究JNK在1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)所致小鼠帕金森病(PD)模型中对诱导型一氧化氮合酶(iNOS)表达的调控作用,探讨PD黑质多巴胺(DA)能神经元变性失活的可能机制.方法:采用神经毒素MPTP制备亚急性PD小鼠模型,健康雄性C57BL/6N小鼠随机分为模型组、JNK抑制剂组和对照组,采用行为学观察、免疫组织化学和免疫印迹法,观察模型小鼠行为学变化及中脑黑质酪氨酸羟化酶(TH)、 iNOS和磷酸化c-Jun(p-c-Jun)的表达变化;观察给予JNK通路特异性抑制剂SP600125对上述变化的影响.结果:与对照组相比,模型小鼠出现典型PD样症状.中脑黑质区TH阳性神经元下降约65%,中脑黑质TH表达水平显著降低约75%;黑质区iNOS和p-c-Jun阳性细胞明显增加,且p-c-Jun特异性表达于细胞核,中脑黑质iNOS与p-c-Jun表达水平明显升高.经SP600125处理后,模型小鼠PD样症状减轻,与对照组比较,TH阳性细胞数和TH表达水平仅下降约15%和25%,与模型组比较,黑质区iNOS与p-c-Jun阳性细胞减少,且p-c-Jun仅表达于胞质,中脑黑质iNOS与p-c-Jun表达水平明显下降.结论:JNK通路在MPTP诱导的亚急性PD小鼠黑质iNOS表达中可能起重要的调控作用;JNK通路特异性抑制剂SP600125可能对帕金森病小鼠具有一定的神经保护作用.     

巴曲酶抑制黑质纹状体损伤后的细胞凋亡并改善行为学功能

目的研究巴曲酶对黑质纹状体通路损伤的保护作用及可能机制。方法制备小鼠黑质纹状体通路损伤模型并给予巴曲酶腹腔注射,采用行为学方法观察运动和神经功能的变化;FJC染色观察神经元凋亡变化;免疫组化染色观察络氨酸羟化酶的变化。结果巴曲酶具有改善脑损伤后神经功能损伤的效果,急性期减少纹状体神经元凋亡,增加黑质中络氨酸羟化酶阳性细胞数目。结论巴曲酶在黑质纹状体通路损伤中具有神经保护作用,并具有临床应用潜在价值。     

色钉菇乙酸乙酯提取物对帕金森病模型小鼠黑质多巴胺神经元的保护作用

目的:探讨色钉菇乙酸乙酯提取物能否改善帕金森病(PD)小鼠的行为症状以及对黑质多巴胺(DA)能神经元保护作用。方法:以成年C57BL/6小鼠为对象,设置空白对照组、MPTP模型组和100 mg/kg、200 mg/kg、400 mg/kg三个剂量的色钉菇乙酸乙酯提取物的干预组。通过站立次数和爬杆得分等指标评价行为症状,以TH的免疫荧光方法检测存活的黑质DA能神经元,FJC染色法显示黑质致密部变性的DA能神经元。结果:在行为上,实验处理之前各组之间无显著性差异(P0.05);而实验处理后,于站立次数指标上,400 mg/kg剂量干预组显著地高于实验对照组(P0.05),与空白对照组无显著性差异(P0.05);在爬杆得分指标上,200 mg/kg组和400 mg/kg组均显著地高于实验对照组(P0.05),但与空白对照组无显著性差异(P0.05)。在TH标记的黑质多巴胺能神经元数量上,只有400 mg/kg组显著地高于实验对照组(P0.05),与空白对照组无显著性差异(P0.05)。而FJC标记的变性细胞只在MPTP组、100 mg/kg和200 mg/kg组表达,空白对照组和400 mg/kg组未见FJC阳性细胞。结论:色钉菇乙酸乙酯提取物能显著地改善PD的行为症状和保护黑质DA能神经元,且呈剂量依赖性。     

Fluoro-Jade: novel fluorochromes for detecting toxicant-induced neuronal degeneration

Two anionic fluorescein derivatives can be used for the simple and definitive localization of neuronal degeneration in brain tissue sections. Initial work on the first generation fluorochrome, Fluoro-Jade, demonstrated the utility of this compound for the detection of neuronal degeneration induced by a variety of well-characterized neurotoxicants, including kainic acid, 3-nitropropionic acid, isoniazid, ibogaine, domoic acid, and dizocilpine maleate (MK-801). After validation, the tracer was used to reveal previously unreported sites of neuronal degeneration associated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), methamphetamine, and d-fenfluramine. Preliminary findings with a second generation fluorescein derivative, Fluoro-Jade B, suggest that this tracer results in staining of optimal contrast and resolution in animals dosed with kainic acid. These 2 tracers can be combined with other histologic methods, including immunofluoresence and fluorescent Nissl stains. Recent preliminary findings on a number of specialized applications of Fluoro-Jade include the detection of apoptosis, amyloid plaques, astrocytes, and dead cells in tissue culture.     

低剂量MPTP致雌鼠黑质听觉P50诱发电位动态变化的实验研究

目的:功能学角度了解低剂量MPTP对雌鼠黑质听觉P50诱发电位的动态影响及帕金森病动物模型旋转行为的出现与黑质听觉P50诱发电位的关系。方法:45只大鼠随机分为三组:(1)正常对照组;(2)MPTP致帕金森病模型组;(3)生理盐水对照组。动态监测术后第7至14天MPTP型帕金森病动物模型黑质听觉P50诱发电位。结果:MPTP组的P50T/C值在第11~14天均较正常组及生理盐水溶剂对照组(I NT-Saline组)降低,且有统计学意义(P<0.01)。结论:低剂量MPTP可渐进性引起正常雌鼠黑质听觉P50诱发电位降低,且在帕金森病动物模型黑质听觉P50诱发电位降低的同时出现旋转行为。     

人参皂苷Rg1对MPTP所致亚急性帕金森病模型小鼠中脑黒质p-c-Jun与COX-2表达的影响

本文研究了人参皂苷Rg1(Ginsenoside Rg1,Rg1)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine,MPTP)所致亚急性Parkinson病(PD)模型小鼠中脑黒质磷酸化c-Jun(p-c-Jun)与环氧合酶-2(cyclooxygenase-2,COX-2)表达的影响,以期探讨Rg1保护PD中脑黑质多巴胺(DA)能神经元可能的分子机制。实验采用MPTP制备亚急性PD小鼠模型。通过行为学观察,免疫组织化学SP法和免疫蛋白印迹法,观察PD模型小鼠行为学变化,中脑黑质酪氨酸羟化酶(TH)、COX-2和p-c-Jun表达水平的变化,并观察给予人参皂苷Rg1对上述变化的影响。结果显示,模型小鼠出现典型PD样症状。与对照组小鼠相比,黑质区TH阳性神经元数下降约65%(P<0.001),TH表达水平显著降低约75%;黑质区COX-2阳性细胞明显增多,p-c-Jun特异性表达于黑质区细胞核内,且COX-2与p-c-Jun表达水平均明显升高。经Rg1(10mg/kg)处理后,模型小鼠PD样症状减轻,与对照组比较,在MPTP第5次注射后7d,TH阳性细胞数和TH表达水平仅下降约15%和20%;与模型组比较,黑质区COX-2阳性细胞明显减少,p-c-Jun主要表达于细胞浆,部分表达于细胞核,表达水平也明显降低。以上实验结果表明,人参皂苷Rg1对MPTP诱导的亚急性Parkinson病小鼠中脑黑质细胞的神经保护作用可能是通过阻抑p-c-Jun核内表达,从而减弱COX-2表达及其介导的黒质区炎症反应。     

氯胺酮对帕金森病小鼠模型α-突触核蛋白表达的影响

目的:观察氯胺酮(ketamine,Ket)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的帕金森病(Parkinson's disease,PD)小鼠模型黑质-纹状体区α-突触核蛋白(α-synuclein,α-Syn)异常表达的影响。方法:选取48只健康雄性小鼠随机分为3组:对照组(NaCl组)、模型组(MPTP组)和治疗组(MPTP+Ket组)。采用rotarod实验和gait analysis system评价3组小鼠行为学差异;免疫组化染色观察黑质区酪氨酸羟化酶阳性神经元数目变化;免疫荧光染色和Western blot检测小鼠脑黑质-纹状体区α-Syn的表达。结果:(1)Rotarod实验及gait analysis system结果显示,MPTP组较NaCl组转棒仪圈数减少,步距减小(P0.05),MPTP+Ket组较MPTP组转棒仪圈数增加,步距增宽(P0.05);(2)免疫组化及免疫荧光实验结果表明,MPTP组较NaCl组多巴胺能神经元数目减少,黑质-纹状体区荧光强度增加(P0.05),MPTP+Ket组较MPTP组多巴胺能神经元数目增多,荧光强度减少(P0.05);(3)Western blot实验结果显示,MPTP组较NaCl组黑质-纹状体区的α-Syn表达增多(P0.05),而MPTP+Ket组较MPTP组的α-Syn表达量减少(P0.05)。结论:氯胺酮对PD小鼠黑质-纹状体区α-Syn异常表达具有抑制作用。     

Experimental Modeling of Functional Deficiency of the Nigrostriatal Dopaminergic System in Mice

The dopaminergic nigrostriatal system of the brain is a key component in controlling motor behavior. The bodies of dopaminergic neurons are located in the compact zone of the substantia nigra and their axons, forming the nigrostriatal tract, project to the striatum. The aim of the present work was to develop an experimental model of functional deficiency of the dopaminergic neurons of the nigrostriatal system with no impairment to motor behavior, i.e., the presymptomatic stage of parkinsonism. The model was created in mice by administration of single subcutaneous injections of low doses (12 mg/kg) of 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is converted to MPP⁺ in the brain, this being a neurotoxin for dopaminergic neurons. These experiments showed that 14 days after administration of MPTP the animals a) lacked any impairment to motor behavior; b) had no decrease in the dopamine level or any dopamine neuron degeneration in the substantia nigra; c) showed significant reductions in dopamine levels in the striatum due to degeneration of half of its dopaminergic fibers. The absence of changes in motor behavior in the presence of significant changes in dopamine metabolism and structural damage to dopaminergic axons in the striatum provides evidence for the activation of compensatory mechanisms in the brain. Thus, we have developed an experimental model of the presymptomatic stage of parkinsonism, which is characterized by degeneration of the axons of dopaminergic neurons in the striatum with no changes in the bodies of these neurons in the substantia nigra; this model will be used for further studies of compensatory mechanisms.     

环加氧酶-2对帕金森病模型小鼠黑质多巴胺能神经元的影响

目的　观察COX 2对帕金森病小鼠黑质多巴胺能神经元的影响。方法　选用C5 7BL种系环加氧酶 2 (Cyclooxygenase 2 ,COX 2 )缺陷小鼠 ,腹腔注射 1 甲基 4 苯基 1,2 ,3,6 四氢吡啶 (MPTP)制备帕金森病小鼠模型 ,用免疫组织化学方法。结果　行为学及免疫组织化学观察显示 ,野生型帕金森病小鼠的死亡率明显高于COX 2缺陷杂合子帕金森病小鼠 (P <0 0 1) ;野生型帕金森病小鼠黑质致密部酪氨酸羟化酶 (Ty rosineHydroxylase ,TH)免疫反应阳性神经元数目较杂合子帕金森病小鼠明显减少 (P <0 0 1)。结论　COX 2很可能与帕金森病时黑质多巴胺能神经元的损伤有关     

Effects of locus coeruleus lesions on parkinsonian signs, striatal dopamine and substantia nigra cell loss after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in monkeys: a possible role for the locus coeruleus in the progression of Parkinson's disease

Six pairs of female squirrel monkeys were given a daily intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 9-14 days, beginning the same day on which they received either a bilateral 6-hydroxydopamine lesion or a sham lesion of the locus coeruleus. Sham animals developed typical parkinsonian signs (i.e. tremor, bradykinesia, hypokinesia and reduced blink rate) which largely recovered by six to nine weeks after the start of MPTP treatment. At nine weeks, post mortem levels of striatal dopamine in these same animals were partially reduced (by 45%), and this only in the putamen, compared to values obtained from three non-operated, normal control animals. Additionally, histological examination revealed a moderate loss of neuronal cell bodies in the substantia nigra, pars compacta. In marked contrast, the locus coeruleus-lesioned monkeys exhibited little or no recovery from the parkinsonian signs induced by MPTP. Post mortem examination of these animals revealed profound decreases in caudate (by 84%) and putamen (by 91%) dopamine content, and severe neuronal cell loss in the substantia nigra pars compacta of all animals. These neurological, biochemical and histological assessments indicate that lesioning of the locus coeruleus impairs the recovery which usually occurs from the parkinsonian manifestations induced by MPTP in squirrel monkeys. The results support the hypothesis that deficient locus coeruleus noradrenergic mechanisms underlie the progression of Parkinson's disease.     

人参皂甙Rg1对帕金森病小鼠黑质JNK细胞凋亡通路的影响

目的 探讨人参皂甙Rg1(ginsenoside Rg1)抗帕金森病(PD)小鼠黑质神经元凋亡的可能机制。方法 采用MPTP制备帕金森病(PD)小鼠模型，经人参皂甙Rgl预处理后，用尼氏染色、TH和活化型caspase-3组织化学染色及TUNEL染色法观察黑质神经元的损害情况，并计算神经元的凋亡率，同时应用蛋白免疫印迹法检测黑质神经元磷酸化．JNK(c—Jun氨基末端激酶)及磷酸化c—Jun蛋白表达水平。结果 人参皂甙Rg1预处理可减轻PD小鼠黑质致密带Niss1阳性神经元和TH阳性神经元的丢失现象，同时减少磷酸化．INK及磷酸化C-Jun蛋白表达水平，活化型caspase-3表达减少，降低黑质神经元的凋亡率。结论人参皂甙Rg1能减少MPTP诱导的小鼠黑质神经元凋亡，其机制与阻断．JNK细胞凋亡通路激活有关。     

Might deep brain stimulation of the subthalamic nucleus be neuroprotective in patients with Parkinson’s disease?

Parkinson’s disease (PD) is characterized by nigral degeneration of dopaminergic neurons in the pars compacta of the substantia nigra. Rather than treating only the symptomatic aspects of Parkinson’s disease, one may also consider treatments designed to retard, arrest, or even reverse this degenerative process. Such strategies could include preventive or restorative treatments instead of purely palliative treatments. A recent hypothesis states that glutamate output from the subthalamic nucleus (STN) to the substantia nigra contributes to the neurotoxic process underlying dopaminergic cell death in Parkinson’s disease. Furthermore, high-frequency stimulation (HFS) of the STN inhibits neurons resulting in the suppression of their glutamate output. Experiments in both rats and monkeys provide preliminary data supporting this hypothesis. Kainic acid (KA) lesions of the STN prevent the loss of dopaminergic neurons in the substantia nigra after intrastriatal injection of 6-hydroxydopamine (6-OHDA) in rats, and after systemic administration of MPTP in monkeys. In PD patients, the background level of their disease is evaluated in the off medication/off stimulation state (UPDRS III score), over a period of 5 years. Thirty percent of the patients are stabilized and 18% have persistent improvement of their disease-related impairment. Further experiments are needed, including controlled clinical trials utilizing functional imaging of the dopamine transporters and post-synaptic receptors.