银杏叶提取物对帕金森病模型小鼠多巴胺能神经元保护作用研究

目的研究银杏叶提取物（GBE）对帕金森病模型小鼠黑质（SN）多巴胺（DA）能神经元的保护作用。方法36只C5,Bk小鼠随机3组,每组12只。其中,帕金森病（PD）模型组的小鼠采用1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）腹腔注射（30mg／kg×5d）诱导PD;GBE预处理组的小鼠于注射MPTP前2h腹腔注射GBE（60mg／kg×8d）;正常对照组的小鼠只注射等体积生理氯化钠溶液。应用免疫组织化学染色观察小鼠黑质致密带（SNzc）酪氨酸羟化酶（TH）免疫反应阳性细胞数量的变化,用高效液相色谱法（HPLC-ECD）检测小鼠纹状体（Str）内DA及其代谢物二羟基苯乙酸（DOPAC）和高香草酸（HVA）含量。结果PD模型组小鼠SN内酪氨酸羟化酶（TH）阳性细胞数量、Str内DA及其代谢产物DOPAC和HVA的含量明显减少（P〈0．01）,GBE预处理组小鼠SN内TH阳性细胞数量、Str内DA及其代谢产物DOPAC和HVA的含量明显增多（P〈0.05）,但仍低于正常对照组（P〈0．01）。结论GBE对MPTP致帕金森病小鼠SNDA能神经元具有明显的保护作用。     

帕金森病小鼠黑质多巴胺能神经元凋亡诱导因子核移位的研究

目的探讨帕金森病(PD)小鼠模型黑质凋亡诱导因子(AIF)的核移位情况及其与多巴胺(DA)能神经元损伤之间的关系。方法采用1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)制备PD小鼠模型,2h、24h、72h后取中脑组织进行酪氨酸羟化酶免疫染色观察黑质DA能神经元的损害情况,AIF免疫组化染色和Western blot方法检测AIF的核移位情况。结果与对照组比较,PD组小鼠黑质DA能神经元数量随时间呈递减趋势,并出现AIF核移位,2h时达高峰,之后随时间呈下降趋势。结论AIF核移位是PD小鼠黑质DA能神经元损伤的早期指标,在PD的发病过程中发挥重要作用。     

p38MAPK抑制剂对MPTP模型小鼠黑质多巴胺能神经元的保护作用

目的:研究SB239063在1-甲基-4-苯基-1,2,3,6四氢吡啶(MPTP)所致帕金森病(PD)模型小鼠中抑制p38丝裂原激活蛋白激酶(mitogen-activated protein kinase,MAPK)活化对多巴胺(DA)能神经元的保护作用。方法:雄性C57BL/6N小鼠随机分为MPTP(30 mg/kg)模型组;SB239063(5 mg/kg)抑制剂组;SB239063(15 mg/kg)抑制剂组;SB239063(25 mg/kg)抑制剂组。抑制剂组于每次注射MPTP前3 h腹腔注射SB239063;对照组注射与模型组和抑制剂组等量生理盐水和DMSO。采用免疫组织化学和免疫蛋白印迹法观察各组小鼠黑质酪氨酸羟化酶(TH)和磷酸化p38蛋白激酶(p-p38 protein kinase,p-p38MAPK)之间表达变化的关系。结果:模型组小鼠黒质区p38MAPK显著活化,同时伴有TH阳性神经元明显丢失;SB239063 15 mg/kg与25 mg/kg组均可明显减少TH神经元丢失,而5 mg/kg组无显著影响;免疫荧光双标记结果显示p38MAPK与TH阳性神经元存在共表达。结论:p38MAPK对PD模型小鼠中脑黑质多巴胺能神经元丢失可能有重要调控作用,SB239063对多巴胺神经元具有一定的神经保护作用。     

慢性帕金森病模型小鼠的行为学表现和多巴胺能神经细胞及转运体的数量变化

目的:研究慢性帕金森病模型小鼠行为学、黑质致密部多巴胺神经细胞和纹状体多巴胺转运体(DAT)的变化。方法:利用MPTP和二丙苯磺胺联合注射小鼠5周(每周2次,共10次)制作帕金森病小鼠慢性模型,通过转棒法检测小鼠行为学表现,免疫组织化学方法测定黑质内多巴胺能神经细胞的变化,放射自显影技术测定纹状体内DAT的密度。结果:帕金森病小鼠在转棒试验中行为发生明显改变,模型组行为学得分明显低于对照组(895±238 vs 1202±252,P0.001),酪氨酸羟化酶(TH)阳性细胞数也明显低于对照组(10.4±6.5 vs22.4±8.0,P0.001)。模型组动物纹状体DAT放射自显影灰度值明显低于对照组(0.2123±0.0316 vs 0.3034±0.0588,P0.001)。线性回归表明,模型组小鼠黑质致密部TH阳性细胞数及纹状体内DAT密度与对应行为学得分呈明显正相关(r=0.6354,P0.01)。结论:MPTP和二丙苯磺胺所致的帕金森病小鼠,在行为学、纹状体DAT密度和黑质致密部多巴胺神经细胞量上均有明显改变,提示这种小鼠模型可用于研究帕金森病的发病过程和药物治疗。     

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。     

MPTP诱导的帕金森病模型小鼠味觉偏好变化

目的:探讨1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)对帕金森病(PD)模型小鼠味觉的影响。方法:将雄性C57BL/6J小鼠分成对照组(control)和MPTP组,通过转棒、爬杆、嗅觉和悬尾实验检测小鼠行为,组织免疫荧光染色检测小鼠黑质致密部(SNpc)多巴胺(DA)能神经元。采用双瓶偏好实验检测小鼠对蔗糖、糖精、氯化钠及奎宁溶液的偏好率。利用real time RT-PCR检测小鼠舌上皮TAS1R1、TAS1R2和TAS1R3的mRNA表达的变化。结果:与control组相比,MPTP组小鼠转棒时间显著性降低,爬杆时间、找到隐藏食物的时间以及悬尾实验中静止时间均显著增加,免疫荧光结果显示MPTP处理可显著减少SNpc的DA能神经元数量。通过双瓶偏好实验发现MPTP可诱导小鼠对蔗糖、糖精、氯化钠的偏好率增加;但对苦味剂奎宁的偏好率没有影响。qRT-PCR分析发现MPTP抑制了小鼠舌上皮味觉受体TAS1R1和TAS1R2 mRNA的表达,而对TAS1R3 mRNA的表达没有影响。结论:MPTP诱导的PD模型小鼠对蔗糖、糖精、氯化钠的饮用偏好率增加,而对奎宁的偏好率没有影响,...     

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.     

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     

线粒体转录终止因子蛋白家族的表达以及在MPTP/MPP~+诱导下的表达变化

目的探讨线粒体转录终止因子(MTERFs)蛋白家族在组织和细胞中的表达以及在神经毒素MPTP/MPP+诱导下的表达变化。方法取正常C57BL小黑鼠的脑、肝、肾组织,以及人神经母细胞瘤细胞SH-SY5Y、人心肌细胞HCM、人肝细胞L-02,检测MTERFs的蛋白表达情况;通过MPTP/MPP+构建帕金森病(PD)动物/细胞模型,检测MTERFs的蛋白表达变化情况。结果 MTERFs在小鼠脑中和SH-SY5Y细胞中蛋白表达水平较高;MPTP/MPP+诱导的PD模型中,MTERF3蛋白表达水平明显降低。结论在小鼠脑和SH-SY5Y细胞中,MTERFs表达较高;MPTP/MPP+诱导下MTERF3蛋白表达明显降低,影响了线粒体转录水平,这可能参与了帕金森病发生的过程。     

Nrf3基因在帕金森病猴模型黑质中表达的研究

本研究目的在于采用 m RNA差异显示技术及改进的硝酸银核酸染色技术 ,寻找 1-甲基 -4 -苯基 -1,2 ,3,6 -四氢吡啶诱导侧与对照侧中脑黑质细胞之间的差异表达基因。用 1-甲基 -4 -苯基 -1,2 ,3,6 -四氢吡啶诱发单侧帕金森病猴模型后 ,将两侧黑质m RNA用 3’端三个锚定引物与 30种随机引物进行逆转录 -聚合酶链反应 ,经聚丙烯酰胺凝胶电泳及硝酸银染色后显示扩增产物条带。结果 :对获得的差异表达基因片段的序列分析、同源性比较及功能分析证明 ,差异基因中的一个 (14 9bp)为 Nrf3基因片段。此结果提示 :Nrf3可能参与小胶质细胞的生长 ,促进对变性神经元的吞噬作用 ,可为从分子水平了解人类帕金森病的发生机理提供依据     

Relationship between the severity of hypokinesia induced by neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and neurochemical changes in brain structures of C57Bl/6 mice

The dynamics of hypokinesia in male C57Bl/6 mice induced by single administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine was studied on the model of parkinsonian syndrome. The neurochemical effect of this neurotoxin was evaluated at the peak of locomotor disorders. Severe hypokinesia was accompanied by an increase in serotonin content and decrease in the rate of serotonin biodegradation in the striatum, hippocampus, and frontal cortex. The content of dopamine metabolite 3,4-dihydroxyphenylacetic acid and dopamine turnover decreased in the striatum, but increased in the hippocampus and frontal cortex. Norepinephrine content decreased in the hypothalamus and cortex. Aspartate content decreased in the hypothalamus and hippocampus. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 7, pp. 58–61, July, 2008     

The importance of the ‘4–5’ double bond for neurotoxicity in primates of the pyridine derivative MPTP

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a newly recognized neurotoxin which selectively damages the cells of the substantia nigra in primates. It has been effective in producing an animal model for Parkinson's disease. We now report that saturating the ‘4–5’ double bond in the tetrahydropyridine ring of MPTP prevents its metabolic oxidation to 1-methyl-4-phenylpyridinium ion (MPP⁺) and removes its behavioral and neurotoxic effects. We believe that this represents an important step towards elucidating the mechanism of action of this compound.     

Potentiation by reserpine and tetrabenazine of brain catecholamine depletions by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in the mouse; evidence for subcellular sequestration as basis for cellular resistance to the toxicant

Administration to mice of the neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) decreased striatal dopamine and, to a lesser extent, hippocampal noradrenaline levels when measured 2 weeks after the last dose of MPTP. Reserpine and tetrabenazine, inhibitors of the catecholamine vesicular transporter, potentiated the catecholamine depletions produced by MPTP in the hippocampus and striatum, respectively. These results are compatible with our hypothesis that sequestration of the toxic MPTP metabolite MPP⁺ (1-methyl-4-phenylpyridinium) in the catecholamine storage vesicle retards the catecholaminergic toxicity of MPTP.     

Dopaminergic toxicity after the stereotaxic administration of the 1-methyl-4-phenylpyridinium ion (MPP+) to rats

The administration of the 1-methyl-4-phenylpyridinium ion (MPP+) stereotaxically into the left neostriatum or left median forebrain bundle of female rats resulted in a very large and highly significant loss of dopamine and of its metabolites in the left neostriatum. The effect of MPP+ on neostriatal dopamine content was in general considerably greater than its effect on serotonin or on several amino acids. These results are consistent with the premise that MPP+, formed from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by the enzyme monoamine oxidase B, may be responsible for the toxicity observed after MPTP administration.     

Effect of complex phytoadaptogen on MPTP-induced parkinson's syndrome in mice

Oral administration of 10% solution of Phytomix-40 (multicomponent plant phytoadaptogen) to C57Bl/6 mice with MPTP-induced Parkinson's syndrome alleviated symptoms (oligokinesia and muscle rigidity), compensated for the deficiency of dopamine and its metabolites (DOPAC and homovanillic acid), and reduced the level of lipid peroxides in the striatum. In vitro Phytomix-40 in a concentration of 3.3×10⁻² g/liter exhibited a pronounced antioxidant effect (5-fold decreased MDA level in mouse brain homogenate in Fe²⁺-ascorbate-dependent LPO). __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 141, No. 5, pp. 495–498, May, 2006     

Septohippocampal connections to field CA1 of the rat identified with field potential analysis and retrograde labeling by horseradish peroxidase

Electrical stimulation of the medial septal nucleus produced field potentials in the hippocampal CA1 region of the rat. The laminar field-potential analysis suggested that the electromotive force of the septum-induced responses might be attributable mainly to excitatory postsynaptic potential currents generated in the stratum oriens (layer of distribution of basal dendrites of hippocampal pyramidal cells). Neural cell bodies in the medial septum-diagonal band complex were retrogradely labeled with horseradish peroxidase (HRP) injected into the stratum oriens of CA1, but not with HRP injected into other CA1 strata. Thus the medial septal nucleus was indicated to send excitatory inputs to basal dendrites of CA1 pyramidal cells.     

Dopaminergic toxicity of rotenone and the 1-methyl-4-phenylpyridinium ion after their stereotaxic administration to rats: implication for the mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity

The 1-methyl-4-phenyl-pyridinium ion (MPP+) is the four electron oxidation product of the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6 -tetrahydropyridine (MPTP). MPP+ can be formed by the oxidation of MPTP by monoamine oxidase B to the intermediate dihydropyridinium species, MPDP+, which is spontaneously transformed to MPP+. In the present study, MPP+, like the mitochondrial toxin rotenone, inhibited pyruvate-malate respiration in isolated mitochondrial preparations. Moreover, the stereotaxic administration of both MPP+ and rotenone caused damage to the dopaminergic nigrostriatal pathway. These data clearly demonstrate that a mitochondrial toxin, administered stereotaxically, is extremely neurotoxic. The data lend support to the concept that MPTP-induced neurotoxicity may be due to the detrimental actions of enzymatically formed MPP+ on mitochondrial function.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism in the common marmoset

The administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (1–4 mg/kg i.p.) for 4 days induced dose-dependent parkinsonism in the common marmoset within 48 h. MPTP produced profound akinesia, rigidity of the trunk and limbs, postural abnormalities, loss of vocalization and, in some cases, postural tremor. In a single animal the administration of -DOPA in conjunction with a peripheral decarboxylase inhibitor, reversed the parkinsonian symptoms. Subsequent biochemical analysis showed a profound loss of dopamine and [³H]dopamine uptake in the caudate-putamen, but no change in specific [³H]spiperone binding.     

Deficits in behavioral initiation and execution processes in monkeys with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to two monkeys led to hypokinesia, tremor, rigidity, adipsia and aphagia. Quantitative assessment of hypokinesia revealed increased reaction time, delayed onset of muscle activity and prolonged movement time in a forelimb reaching task after selective degeneration of the nigrostriatal dopamine (DA) system sparing mesocortical dopamine neurons. The losses of pars compacta cells of substantia nigra, of striatal [3H]mazindol binding and of striatal DA content (more than 90%) quantitatively paralleled the severity of behavioral deficits. Additional monoamine systems were affected with stronger MPTP effects.