偏侧帕金森病猴模型黑质和纹状体一氧化氮合酶表达的变化

目的探讨偏侧帕金森病（PD）猴模型黑质和纹状体一氧化氮合酶（NOS）表达的变化。方法对3只恒河猴经单侧颈内动脉注射1-甲基4-苯基1,2,3,6-四氢吡啶（MPTP）制备成偏侧PD猴模型后,应用还原型尼克酰胺腺嘌呤二核苷酸-黄递酶（NADPH．d）组化染色方法观察偏侧PD猴黑质和纹状体NOS阳性神经元表达的变化,并与正常猴比较。结果偏侧PD猴MPTP毁损侧的黑质和纹状体的NOS阳性神经元数目较毁损对侧和正常猴明显增加（均P〈0．01）,毁损对侧的黑质和纹状体NOS阳性神经元数目与正常猴比较差异无统计学意义。结论偏侧PD猴黑质和纹状体NOS阳性神经元增多,由此引起一氧化氮（NO）合成和释放增多,可能对黑质和纹状体神经元的变性和死亡起重要作用。     

偏侧PD猴模型黑质HMW MAP-2表达的变化

目的探讨偏侧帕金森病（Parkinson＇s disease,PD）猴模型黑质高分子量微管相关蛋白-2（HMW MAP-2）表达的变化。方法对4只恒河猴经单侧颈内动脉注射1-甲基4-苯基1,2,3,6-四氢吡啶（MPTP）制备成偏侧PD猴模型后,应用免疫组化染色方法,观察4只偏侧PD猴模型和2只正常猴黑质HMW MAP-2表达的变化。结果偏侧PD模型猴MPTP毁损侧的黑质的HMW MAP-2免疫活性较毁损侧对侧和正常对照猴明显降低,但MPTP毁损侧对侧的黑质的HMW MAP-2免疫活性未发现明显变化。结论偏侧PD猴模型黑质的HMW MAP-2表达减少可能对黑质神经元的变性和死亡起重要作用。     

偏侧帕金森病猴模型纹状体的质子磁共振波谱研究

目的观察偏侧帕金森病（PD）猴模型纹状体质子磁共振波谱（1H-MRS）的变化,探讨1H-MRS对PD的早期诊断价值。方法对4只恒河猴经右侧颈内动脉注射1-甲基4-苯基1、2、3、6-四氢吡啶（MPTP）制备偏侧PD猴模型前后的双侧纹状体进行1H-MRS检测,对比分析制模术前和术后猴双侧纹状体N-乙酰基天门冬氨酸（NAA）/肌酸复合物（Cr）和胆碱复合物（Cho）/Cr比值的变化。结果4只猴均成功制备成偏侧PD猴模型。偏侧PD猴模型MPTP毁损侧对侧纹状体NAA/Cr和Cho/Cr比值较制模术前无明显改变（P〉0.05）;但偏侧PD猴模型MPTP毁损侧纹状体NAA/Cr比值较制模术前和对侧明显降低（P〈0.05）,Cho/Cr比值较制模术前和对侧明显增高（P〈0.05）。结论H-MRS能够早期发现偏侧PD猴模型纹状体的代谢和生化改变,间接反映偏侧PD猴模型纹状体的神经细胞病理学改变,有助于PD的早期诊断。     

左旋多巴诱发异动症大鼠黑质胶质细胞变化的实验研究

目的研究异动症(LID)大鼠黑质致密部(SNpc)胶质细胞的变化。方法6-羟多巴胺(6-OHDA)立体定位注射制备偏侧帕金森病(PD)大鼠模型,复方左旋多巴(L-dopa)甲酯腹腔内注射治疗4周(10mg.kg-1.d-1,每日2次)诱发异动症大鼠模型。采用免疫组化方法观察大鼠黑质致密部酪氨酸羟化酶(TH)、胶质纤维酸性蛋白(GFAP)、增殖细胞核抗原(PCNA)和S-100蛋白免疫阳性细胞的面密度进行定量分析。结果免疫组化方法证实,PD组、LID组和非LID组大鼠毁损侧SNpc部位的TH免疫阳性面密度较正常组大鼠明显减少(P<0.01),而GFAP、PCNA、S-100免疫阳性面密度则明显增多(P<0.01);LID组和PD组、非LID组相比较,SNpc部位TH的表达进一步减少(P<0.05),GFAP、PCNA、S-100的表达则进一步增加(P<0.05)。结论PD大鼠毁损侧SNpc部位存在明显的胶质细胞增生。LID大鼠毁损侧SNpc部位的多巴胺(DA)能神经元变性及胶质细胞增生较非LID大鼠及PD大鼠更为明显。我们推测SNpc部位的胶质细胞通过其对DA能神经元的神经保护和神经破坏的双重作用,间接影响着LID的发生及其严重程度。     

偏侧帕金森病猴模型的脑血流灌注SPECT活体显象研究

给恒河猴右侧颈内动脉注射神经毒药物甲基-苯基-四氢吡啶(MPTP)后,产生右侧黑质神经元脱失,黑质纹状体多巴胺(DA)浓度明显降低,以及左侧帕金森样症状。投予抗帕金森病(PD)药物治疗显著地改善帕金森样症状。采用单光子发射计算机断层扫描(SPECT),以~(99m)Tc-ECD作为显象剂显示偏侧PD猴模型的脑血流灌注,发现猴模型建立3个月内的损毁侧脑的脑血流灌注明显减低,8个月时的损毁侧脑的脑血流灌注转为正常。结果表明脑血流灌注的变化反映了PD的病理生理变化过程。     

6-羟基多巴胺单侧纹状体注射对大鼠双侧多巴胺能神经元的影响

目的探讨帕金森病大鼠模型中6-羟基多巴胺（6-OHDA）单侧纹状体注射对双侧黑质纹状体多巴胺能神经元的影响。方法大鼠随机分成模型组和对照组，模型组自一侧纹状体注射6-OHDA，对照组注射PBS；用免疫组织化学方法分别检测大鼠双侧黑质和纹状体区酪氨酸羟化酶（TH）阳性细胞和纤维的表达；高效液相色谱检测双侧纹状体多巴胺（DA）及其代谢产物3，4-二羟基苯乙酸（DOPAC）和高香草酸（HVA）含量。结果模型组大鼠双侧（毁损侧与其对侧）黑质致密区TH阳性细胞数量均少于对照组（P〈0．01），模型组双侧纹状体区TH阳性纤维密度均低于对照组；模型组大鼠双侧纹状体区DA含量均低于对照组（P〈0．01）；双侧DOPAC和HVA含量也降低。结论6-羟多巴胺单侧纹状体注射制作的帕金森病大鼠模型的对侧黑质纹状体也有损伤。     

MPTP偏侧恒河猴帕金森病模型研究

目的 对血管介入法、手术夹闭颈外动脉行颈总动脉穿刺、单纯颈总动脉穿刺三种甲基-苯基-四氢吡啶(MPTP)制备偏侧猴帕金森病(Parkinson's disease,PD)模型的方法进行比较,探讨偏侧猴PD模型的制作技术及效果.方法 健康恒河猴6只行单侧颈内动脉MPTP注射,其中2只于DSA下行血管介入法注药;2只采用夹闭颈外动脉颈总动脉注药;2只行单纯颈总动脉注药.术后采用行为学观察和评估,包括运动障碍评分、阿扑吗啡诱发旋转实验等.猴处死后两侧黑质组织切片,NISSL和酪氨酸氢化酶(tyrosine hydroxylase ,TH)免疫组织化学染色.结果 血管介入法和夹闭颈外动脉颈总动脉注药法均一次建模成功;单纯颈总动脉注药法的2只分别于重复注药2次和3次后成功.行为学观察可见猴的自主运动明显减少,运动障碍评分l个月后稳定在6分以上.阿扑吗啡(apomorphine, APO) 试验可诱发出向健侧旋转行为.病理检查可见注药侧黑质内神经细胞缺失减少,残留神经元变性.黑质纹状体区TH阳性多巴胺神经元较正常侧减少65%以上.结论 血管介入法、颈外动脉夹闭颈总动脉注药法和单纯颈总动脉注药法均能够建立满意的偏侧猴PD模型.     

红景天甙促进帕金森病模型小鼠表达内源性胶质细胞源性神经营养因子蛋白保护多巴胺能神经元

目的研究中药单体成分红景天甙(salidroside)对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)诱发的帕金森病(Parkinson disease,PD)小鼠模型的脑保护作用及其可能机制。方法80只C57BL雄性小鼠,分为正常对照组、MPTP模型组、MPTP+红景天甙组(即干预组,分别用3 mg/kg、50 mg／kg和150 mg／kg)及红景天甙对照组(分别用3 mg／kg、50 mg/kg和150 mg／kg),每组10只。应用免疫组化法观察黑质酪氨酸羟化酶(TH)阳性神经元数目变化,蛋白印迹法(Western blot)检测TH、胶质细胞源性神经营养因子(GDNF)、胶质纤维酸性蛋白(GFAP)在纹状体中表达水平的变化。结果在小鼠黑质部位,MPTP组TH阳性神经元数目明显少于正常对照组及干预组(P<0．05)。Western blot结果显示,不同剂量干预组小鼠纹状体中的TH蛋白表达(相对灰度值)(0．8326±0．0930．0．9007±0．0104和1．1114±0．2013)较MPTP组的TH蛋白表达(0．5738±0．01真4)明显增加(P< 0．05);干预组的GDNF蛋白表达(1．3503±0．0573)较MPFP组的蛋白表达(0．1485±0．0118)显著增加(P<0．05);干预组的GFAP蛋白表达(2．4788±0．2093)与MPTP组(1．8431±0．1559)之间差异无统计学意义(P>0．05),但是干预组较正常对照组(1．3695±0.1174)明显增加(P<0．05)。结论红景天甙可以拮抗MPTP诱导的PD模型小鼠黑质多巴胺能神经元的丢失,其神经保护作用可能与促进内源性GDNF分泌增加有关。     

帕金森病功能显像实验研究

目的　应用99mTc TRODAT 1SPECT显像观察偏侧帕金森病 (PD)猴模型两侧纹状体多巴胺转运体 (DAT)密度的变化。方法　 4只恒河猴 ,编号为M1～ 4,M1为正常者 ,M2～M4为右侧颈总动脉注射神经毒素MPTP的PD模型者。静脉注射99mTc TRODAT 1(3 70～ 5 5 0MBq) ,1～ 2小时后进行SPECT断层扫描 ,选取清晰图像进行处理分析。结果　M1两侧纹状体感兴趣区比值为 1 0 0 ,M2～ 4左右侧纹状体感兴趣区比值为 1 10～ 1 2 2 ,而且右侧纹状体与各脑叶感兴趣区的比值均低于左侧。结论　99mTc TRODAT 1SPECT可以在体评估偏侧PD模型猴纹状体DAT的变化 ,有助于定量研究PD的诊断     

抗帕颗粒对帕金森病模型小鼠酪氨酸羟化酶神经元的影响

目的 探讨抗帕颗粒对1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)帕金森病(PD)模型小鼠黑质纹状体酪氨酸羟化酶(TH)阳性神经元的影响。方法 90只健康雄性C57BL/6小鼠,鼠龄8～12周,并随机分为3组,即正常对照组30只、PD模型对照组30只、PD模型干预组30只; MPTP腹腔注射(40 mg·kg^-1·d^-1×7 d)制备小鼠PD模型; 正常对照组及PD模型对照组予生理盐水1 mL·d^-1灌胃,PD模型干预组给予抗帕颗粒40 mg·kg^-1·d^-1灌胃,连续喂养4个月; 黑质纹状体切片、HE染色、免疫组织化学染色TH神经元及Western blotting检测TH蛋白的表达量。结果 ①正常对照组30只(30/30只)最终均存活,PD模型对照组4个月存活27只(27/30只),PD模型干预组4个月存活28只(28/30只); ②PD模型对照组、PD模型干预组小鼠每次注射MPTP后先有短暂兴奋[持续(7.61±2.17)min],表现为四处窜跳,随即出现全身中重度震颤,皮毛及尾巴时有竖立,活动减少,持续(24.23±3.89)min后震颤消失,随后出现活动减少; ③HE染色显示正常对照组大量褐色TH阳性细胞,PD模型对照组TH阳性细胞数明显减少,PD模型干预组TH阳性细胞数有所增加; ④免疫组织化学染色后经Imagepro-Plus 5.1系统分析,正常对照组TH阳性细胞面积为64 145 μm²,高倍镜下可见大量胞质为褐色颗粒的TH阳性细胞; PD模型对照组TH阳性细胞染色面积为40 012 μm²,高倍镜下见TH细胞数明显减少; PD模型干预组TH阳性细胞染色面积为60 952 μm²,高倍镜下见TH阳性细胞数较PD模型对照组增加; ⑤Western blotting检测显示正常对照组TH蛋白表达量与PD模型对照组和PD模型干预组比较均有明显差异(P<0.001),PD模型对照组TH蛋白表达量与PD模型干预组比较也有明显差异(P<0.05)。结论 抗帕颗粒可使PD小鼠黑质纹状体中多巴胺能神经元一定程度地减少丢失,对多巴胺能神经元的数量、形态及功能具有一定的保护作用。     

Chronic Nicotine Treatment Increases nAChRs and Microglial Expression in Monkey Substantia Nigra After Nigrostriatal Damage

Our previous work had shown that long-term nicotine administration improved dopaminergic markers and nicotinic receptors (nAChRs) in the striatum of monkeys with nigrostriatal damage. The present experiments were done to determine whether nicotine treatment also led to changes in the substantia nigra, the region containing dopaminergic cell bodies. Monkeys were chronically treated with nicotine in the drinking water for 6 months after which they were injected with low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydrophridine (MPTP) for a further 6-month period. Nicotine was administered until the monkeys were euthanized 2 months after the last MPTP injection. Nicotine treatment did not affect the dopamine transporter or the number of tyrosine hydroxylase positive cells in the substantia nigra of lesioned monkeys. However, nicotine administration did lead to a greater increase in α3/α6β2* and α4β2* nAChRs in lesioned monkeys compared to controls. Nicotine also significantly elevated microglia and reduced the number of extracellular neuromelanin deposits in the substantia nigra of MPTP-lesioned monkeys. These findings indicate that long-term nicotine treatment modulates expression of several molecular measures in monkey substantia nigra that may result in an improvement in nigral integrity and/or function. These observations may have therapeutic implications for Parkinson’s disease.     

姜黄素对帕金森病小鼠模型黑质多巴胺能神经元损伤的保护作用

目的研究姜黄素对由MPTP诱发的帕金森病小鼠模型的脑保护作用及其可能机制。方法应用免疫组织化学染色法和蛋白质印迹法(Western blotting)分别观察姜黄素干预前后帕金森病小鼠中脑黑质-纹状体系统中酪氨酸羟化酶、胶质纤维酸性蛋白阳性神经元数目的变化,以及酪氨酸羟化酶、诱导型一氧化氮合酶和胶质纤维酸性蛋白表达水平的变化。结果MPTP组小鼠中脑黑质酪氨酸羟化酶和胶质纤维酸性蛋白阳性神经元数目明显减少,与正常对照组及治疗组相比差异有统计学意义(均P<0.05)。经不同剂量(5mg/kg、50mg/kg和150mg/kg)的姜黄素干预治疗后,小鼠中脑纹状体中的酪氨酸羟化酶蛋白表达水平(相对灰度值)明显升高,而黑质中诱导型一氧化氮合酶和胶质纤维酸性蛋白表达水平明显降低,与MPTP组比较差异均有统计学意义(P<0.05);MPTP组与溶剂对照组(MPTP DMSO)之间差异无统计学意义(P>0.05)。结论姜黄素可以有效地拮抗MPTP诱导的帕金森病小鼠模型黑质多巴胺能神经元的丢失,其机制可能与姜黄素降低黑质多巴胺能神经元活性氧含量以及抑制炎症反应等作用有关。     

A nuclear microscopic and histochemical study of iron concentrations and distribution in the midbrain of two age groups of monkeys unilaterally injected with MPTP

The present study was carried out to elucidate the concentration and distribution of iron in the substantia nigra of two age groups of monkeys after experimental hemi-Parkinsonism induced by unilateral internal carotid injections of MPTP. Iron levels and distribution were detected using the nuclear microscope, which is able to provide structural and quantitative elemental analysis of biological tissue down to the parts per million (ppm) level of analytical sensitivity. Five weeks after unilateral lesioning with MPTP, we observed a 30-65% loss of neurons in the injected substantia nigra of each monkey, compared with the contralateral control 'non-lesioned' side. In monkeys less than 7 years of age, the iron was distributed fairly uniformly and showed little evidence of focal deposits. In monkeys greater than 7 years of age, we observed many dense focal deposits of iron in the substantia nigra. A comparison between iron distributions in nuclear microscopic scans and cell distributions in the same sections stained by the Nissl technique showed that areas containing high iron concentrations were present not where large-diameter neurons with abundant Nissl substance (presumed dopaminergic neurons) were located but in a region ventral to these cell bodies, i.e., in the substantia nigra pars reticulata. These distributions were present on the control side as well as the MPTP-injected side. Since a previous study has shown that unilateral MPTP injection results in lesions of the substantia nigra of the same side but negligible injury to the opposite side, this implies that the iron deposits existed in the older monkeys before MPTP injections (i.e. they occurred normally). The accumulation of iron in the substantia nigra with age suggests the possibility of localised damage to neurons through the catalysis of free radicals.     

Neurochemical changes in the substantiae nigrae and caudate nuclei following acute unilateral intranigral infusions of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Acute unilateral intranigral infusions of MPTP at doses (200 micrograms) which produce robust contralateral rotation in the rat induced significant neurochemical changes in the ipsilateral as well as contralateral nigrostriatal systems. There were pronounced increases in the levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the ipsilateral substantia nigra and a significant decrease in the levels of DA in the ipsilateral caudate nucleus while opposite changes occurred in the contralateral substantia nigra and caudate nucleus. The DOPAC:DA and HVA:DA ratios were significantly higher in the ipsilateral caudate nucleus indicating increased activity of the ipsilateral nigrostriatal DA neurones. The levels of noradrenaline and 4-hydroxy-3-methoxyphenylethyline glycol (MHPG) increased and decreased significantly in the ipsilateral and contralateral substantia nigra, respectively, but there were no significant changes in the caudate nuclei. The levels of serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) increased significantly in the ipsilateral substantia nigra and caudate nucleus as well as in the contralateral caudate nucleus but did not increase significantly in the contralateral substantia nigra. The 5-HIAA:5-HT ratio was significantly decreased in the contralateral caudate nucleus indicating a reduced activity of the contralateral nigrostriatal 5-HT neurones. The data thus indicate that MPTP applied to one substantia nigra is capable of producing profound neurochemical changes not only locally but also in the ipsilateral striatum as well as in the contralateral nigrostriatal system. Previous neuropharmacological studies have suggested that the rotation induced by intranigral MPTP may be mediated via dopamine released from dendrites in the pars reticulata in response to MPTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term intracerebral infusion of fibroblast growth factors restores motility and enhances F-DOPA uptake in parkinsonian monkeys

Fibroblast growth factors (FGFs) are important for dopamine neurons in health and disease. Acidic (aFGF) and basic (bFGF) fibroblast growth factors increase the survival and growth of dopamine cells. Nigrostriatal dopamine neurons, the target cells for degeneration in Parkinson's disease, display receptors for basic fibroblast growth factor and these receptors are decreased in the brain of parkinsonian patients. We have investigated the effects of long-term intrastriatal infusion of FGFs in hemiparkinsonian monkeys. All animals were lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 0.4mgkg(-1), into the left internal carotid artery. The monkeys that had persistent asymmetric akinesia and contralateral rotation induced by apomorphine, were selected for chronic, unilateral, intracerebral infusion of neurotrophic factors or vehicle into the striatum ipsilateral to the lesion. Two animals received intrastriatal aFGF or bFGF, 2mugweek(-1), for 6 months. The controls received intrastriatal saline or intraventricular epidermal growth factor (EGF). F-DOPA positron emission tomography scans were performed in each animal before and after the intracerebral infusion of neurotrophic factors. We measured the tyrosine hydroxylase (TH) immunoreactive neurons in the substantia nigra and terminals in the striatum and evaluated the pathological complications related to the treatment or the delivery system. All four animals had, after the lesion with MPTP, a transient but incomplete recovery of akinesia. This period of spontaneous improvement was followed by a progressive deterioration of motor behaviour during the following months. The monkeys treated with FGFs, however, recovered quickly and persistently during the intracerebral infusion. F-DOPA uptake, prior to the intracerebral infusion, was greatly reduced in the lesioned striatum. The post-infusion F-DOPA scans revealed a 60% reduction respect to baseline in the lesioned striatum of the saline and EGF-infused animals. In the animals infused with FGFs, the post-infusion F-DOPA uptake increased more than 400% in the lesioned (and infused) striatum and around 200-300% in the contralateral side, with respect to the pre-infusion scan. The number of TH-positive cells in the substantia nigra correlated well with the uptake of F-DOPA in the post-infusion scan. No severe side-effects were present. Intrastriatal infusion of FGFs restores motor behaviour and increases F-DOPA striatal uptake in hemiparkinsonian monkeys.     

In vivo changes of catecholamines in hemiparkinsonian monkeys measured by microdialysis.

In monkeys, unilateral intracarotid infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces a useful model of hemiparkinsonism. To evaluate MPTP-induced neurochemical changes in vivo, brain microdialysis was employed to measure extracellular levels of dopamine and its metabolites in the neostriatum of normal and hemiparkinsonian rhesus monkeys (Macaca mulatta). The microdialysis probes were implanted bilaterally into the caudate nucleus and putamen at coordinates determined from magnetic resonance imaging. Dopamine and its metabolites were depleted in the MPTP-lesioned side versus the unlesioned side in hemiparkinsonian monkeys. Tyrosine hydroxylase immunocytochemistry revealed a complete unilateral denervation in the caudate nucleus and putamen and a total loss of tyrosine hydroxylase-immunoreactive cells in the substantia nigra pars compacta in those monkeys. Baseline levels of amines in the neostriatum in normal monkeys were not significantly different from those in the normal (non-MPTP-treated) side in hemiparkinsonian monkeys. These data demonstrate that brain microdialysis is a valuable tool for measuring in vivo neurochemical changes in nonhuman primate brains.     

MPTP-Induced Hemiparkinsonism in Nonhuman Primates 6–8 Years after a Single Unilateral Intracarotid Dose

Five female adultMacaca nemestrinamonkeys, given a unilateral intracarotid (ic) infusion of 2.3–3.5 mg of MPTP-HCl, were studied for 6–8 years. Two to 3 days after MPTP, the animals developed hemiparkinsonism characterized by rigidity and flexed posture of the arm contralateral to the side of infusion with episodes of tremor, circling ipsilateral to the lesioned side, a slight balance disturbance, and stooped posture. Rating of parkinsonian features 4 months after ic infusion, and yearly thereafter, did not show any statistically significant changes. The animals maintained their usual appetite and body weight increased normally. Each animal responded to -DOPA methyl ester with decreased parkinsonian signs and symptoms and increased contralateral turning. In contrast, after control vehicle administration, the animals continued to have the same parkinsonian signs and predominant ipsilateral turns. In three of the five monkeys, contralateral turns after vehicle significantly increased after 6–8 years. Unilateral intracarotid MPTP induced asymmetric motor behavior that remained stable after 6–8 years. Animals that showed an increased frequency of contralateral circling after control vehicle showed a decrease in contralateral turns after -DOPA methyl ester, suggesting neuroplastic changes over the years.