【作者】 张晓梅；

【导师】 刘立；

【作者基本信息】 第四军医大学 ， 航空航天与航海医学， 2007， 硕士

【摘要】 帕金森病(Parkinson’s disease,PD)是中老年人最常见的中枢神经系统退行性疾病之一,发病率仅次于阿尔茨海默病(Alzheimer’s disease,AD)。PD的病变特征是黑质——纹状体多巴胺(Dopamine,DA)能系统功能受损,但发病机制至今尚不明确。目前认为PD是由遗传、环境及老龄等因素共同作用的结果。百草枯(Paraquat,PQ)是一种除草剂,代森锰(Maneb,MB)是一种杀真菌剂,他们属于农业化学药品范围,在很多地理区域常联合使用。PQ、MB都具有一定的中脑DA能神经元损伤效应,但目前明确的观点是两者合用时能够产生更稳定的协同效应,且与人类散发性PD的多重环境危险因素暴露条件相近。因此,PQ和MB联合(PQMB)诱导DA能系统损伤是研究散发性PD的有效手段之一。饮食限制(Dietary restriction,DR)能够显著延长实验动物的生存期,诱导大鼠和小鼠脑内神经元产生良性应激反应,具有神经保护作用并能改善多种老年退行性疾病的症状[1]。是否DR对PQMB引起的DA能系统损伤也具有保护作用?另外,既往已有实验表明星形胶质细胞(Astrocyte,Ast)过度激活在PD进展中起到一定的负面影响。在PQMB诱导的PD环境模型中,中脑Ast反应如何?DR与胶质细胞活化的关系如何?目前报道很少。本实验拟采用DR对PQMB诱导的PD小鼠模型施加干预,旨在探讨DR在其中的保护作用,并观察Ast的标志物——胶质细胞原纤维酸性蛋白(Glial fibrillary acidic protein,GFAP)的表达与DR之间的关系,揭示DR可能的保护机制。实验内容如下:实验一目的:探讨PQMB对小鼠黑质DA能神经元及Ast活化程度的影响。方法:将C57BL/6J小鼠随机分为药物注射组(PQMB组)和生理盐水组(Saline组)。PQMB组按PQ 8 mg/kg,MB 24 mg/kg剂量行腹腔注射,共6周。Saline组腹腔注射0.9%无菌生理盐水,注射时间、剂量和方法同PQMB组。在PQMB首次注射前(Blank group)和末次注射后处死动物,行黑质抗酪氨酸羟化酶(Tyrosine hydroxylase,TH)、GFAP的免疫组化染色,观察PQMB暴露对中脑DA能神经元和Ast的影响。结果: PQMB组小鼠黑质致密部(Substantial nigra pars compact,SNpc)TH阳性神经元数目为116.50±7.44,显著少于Saline组的178.83±5.68(P < 0.001),以Blank组的神经元数目作为基数进行标准化(后同),PQMB组神经元百分比为70.2±4.4%,减少了近30%。细胞形态呈现突起细小,分支减少等不健康表现。PQMB组黑质Ast分布稀疏,胞体小、突起细而短,染色浅淡,Ast的GFAP免疫阳性产物表达少于Saline组。PQMB组相对灰度值(△Gray Value,△GV)为45.98±1.46,低于Saline组(P < 0.001)。结论: PQMB暴露对小鼠SNpc DA能神经元有轻度的损伤作用,模拟了人类PD的特征性病理学改变,同时对黑质区Ast也有一定的损伤作用。实验二目的: PQMB对小鼠黑质的持续损伤效应及DR对其的保护作用。方法:按照实验一的方法将注射过PQMB的动物随机分为PQMB并随意(adlibitum,AL)进食组(PQMB/AL),PQMB并饮食限制组(PQMB/DR)。以DR干预后第0周,3周、5周和8周为观察时间点,行抗TH、GFAP免疫组化染色,选取第5周的部分切片行TH/GFAP和Fos/TH的双重标记染色。结果: PQMB/AL组小鼠SNpc TH阳性神经元数目呈持续减少趋势,第8周时为82.64±5.10(相对数为49.8±3.0 %),明显少于0周的116.50±7.44(相对数为70.2±4.4 %)(P < 0.05)。而PQMB/DR组,DA能神经元数目缺失明显减缓,自DR干预5周起,PQMB/DR与PQMB/AL比较有显著差异(P < 0.05)。PQMB/AL组小鼠Ast的活化程度经过短暂降低后开始上升,第3周时恢复到对照组水平,之后呈升高趋势,第5、8周△GV分别为69.02±1.25和63.3±1.67,均高于同期对照组的51.66±0.92和53.04±1.12(P < 0.01),而在PQMB/DR组GFAP的表达明显受到抑制,第5、8周△GV分别为48.92±1.28和47.5±2.68,均低于同期PQMB/AL组数值(P < 0.01)。结论: PQMB PD模型小鼠能够模拟人类PD DA能神经元渐进性缺失的特点,而DR可减缓这种持续损伤进程,DR对Ast活性的抑制可能是实现这一保护作用的机制之一。实验三目的: DR对PQMB联合暴露引起的小鼠运动功能损伤的影响。方法:动物处理及分组同实验第一、二部分,在初次注射PQMB前(-6周)和末次注射后各时间点(0、3、5、8周)行自发活动实验和爬杆实验等行为学检测。结果:动物经PQMB注射后出现急性行为学改变,如运动减少、后肢张开、僵直、步态不稳、弓背、竖尾、竖毛等。后期出现动作迟缓、肢体僵硬、步态不稳等症状。0周时PQMB/AL组小鼠的水平运动距离(935.01±146.27 cm)小于Saline/AL组(1903.38±203.14 cm),两组比较差异显著(P < 0.01)。同期爬杆实验中的Tturn和TLA分别为2.59±0.21 s和13.85±0.69 s,较Saline/AL组的0.90±0.12 s和6.34±0.37 s显著延长,两组间差异显著(P < 0.01)。随着时间延长,PQMB/AL组小鼠行为学测试数据没有改善的趋势,而PQMB/DR组的水平运动距离和爬杆测试中的Tturn值在DR 5周后明显改善,TLA在DR 8周后改善明显,但行为学指标均未能达到对照组的水平。结论: PQMB可引起小鼠运动功能减退,而较长时间DR干预后小鼠的运动功能有好转的趋势。更多还原

【Abstract】 Parkinson’s disease (PD), a profound movement disorder resulting from nigrostriatal dopaminergic (DA) system degeneration, has been linked to living in a rural environment, farming, and occupational exposure to agricultural chemicals, suggesting an environmental exposure basis for the disease. Exposure to paraquat (PQ) as a risk factor for sporadic PD has been associated with Parkinsonism. Other agricultural chemicals, including dithiocarbamate fungicides such as maneb (MB), are widely used in the same geographical regions and also impact dopamine systems, suggesting that mixtures may be more relevant to sporadic PD.The chemical constitution of PQ is extremely similar to MPP+, the active metabolite of MPTP which is known as a neurotoxin and their pathogenesis mechanism is also similar. Additionally, it was shown that MB is able to alter the biodisposition of DA and PQ, resulting in a prolonged exposure to reactive oxygen species (ROS) and reactive nitrogen species generating compounds. MB is capable of converting a non-toxic dose of PQ and other xenobiotics into a toxic dose through alterations in toxicokinetics.Epidemiological studies demonstrate long-term co-exposure to PQ and MB obviously increases the risk of PD onset. Experiments indicate that PQ selectively kills dopaminergic neurons in the substantia nigra and produces some of the syndrom of PD in laboratory animals, while, it is clear that MB enhances the PQ toxicity.Dietary restriction has been shown to have several health benefits including increased insulin sensitivity, stress resistance, reduced morbidity in some disease, and increased life span, while the mechanism remains unclear. Studies reveal it plays significantly therapeutical effects on Huntington disease (HD). Whether DR has the protective effects on the damage of dopaminergic neurons induced by systemic exposure to combination of PQ and MB? There is few related report yet. This study plans to use the combination of PQ and MB to set up the PD model mimicing the environmental toxin related PD, to demonstrate whether DR has the protective effects on nerve injury induced by PQ and MB, and to reveal the possible mechanism of its protective effects. Experimental contents as follows:Experiment 1Objective: To investigate the insult of PQ and MB on DA neurons in SNpc and the expression of glial fibrillary acidic protein (GFAP) in substantia nigra in C57BL/6J mice. Methods: Animals were divided into saline and PQMB group randomly. All were injected with either Vehicle (0.9%) or a combination of PQ (8 mg/kg) and MB (24 mg/kg) twice a week (Tuesday and Thursday, 9:00 am) for 6 weeks intraperitoneally. Before the first injection and after the last injection, the mice were perfused and the brains were removed and cut with the common practice. Expression of tyrosine hydroxylase (TH) and GFAP in substantial nigra were investigated utilizing the immunohistochemical methods. Results: The survival number of TH positive cells in SNpc in PQMB group was 116.50±7.44, which was less than that of the saline group (178.83±5.68) (P < 0.001). And the DA neurons did not appear as healthy as those in saline-treated animals, with fewer dendrites and less intense TH immunoreactivity within cells. The reactive level of Asts decreased in PQMB group and the△GV of GFAP was 45.98±1.46, which is lower than that of saline group (P < 0.001). Conclusion: The results suggested that combination of PQ and MB can induce loss of DA neurons in SNpc in mice to some extent, which is the characteristic change of pathology in PD patient. PQ and MB can also insult Ast in area of substantia nigra.Experiment 2Objective: To explore the persistent neurotoxic effects of combinded use of PQ and MB on mice, and to observe the protective function of DR to this damage. Methods: The PQ and MB combination injected mice were divided into two groups randomly; one group had access to food ad libitum (PQMB/AL), while another fed on alternate days only (PQMB/DR). Animals were tested on three different time-points, the follow0, 3, 5, 8 weeks, the mice were perfused and the brains were removed and cut with the common practice. Expression of TH in SNpc was investigated utilizing the immunohistochemical methods. Expression of GFAP was localized on frozen sections by immunohistochemical methods and BX51 microscopy, and GFAP content (△Gray Value,△GV) was evaluated by Leica Q570c True colour image analysis system. Results: immunohistochemical results showed that the number of TH positive neurons in SNpc reduced continuously and irreversably at the end of 8 weeks, the survival TH positive cells in SNpc in PQMB/AL group decreased sharply to 82.64±5.10 and was lower than that of 0 week in the samel group (P < 0.05). While, the dietary restriction seemed to be able to slow down this process, the statistical significance for the number of the survival neurons could be found between the PQMB/DR and PQMB/AL group from the 5th week (P < 0.05). The glial reactivity was different among the PQMB groups and Saline groups. The expression level of GFAP in PQMB/Al group reduced first, upregulated to the same level to the control group in 3 weeks, then△GV reached 69.02±1.25 and 63.3±1.67 in 5 and 8 weeks, which was much higher than that of control group (51.66±0.92 and 53.04±1.12), respectively (P < 0.01). While, DR reduced the expression level significantly and the△GV was 48.92±1.28 and 47.5±2.68 in 5 and 8 weeks respectively, which was lower than it of the PQMB/AL group simultaneously (P < 0.01). Conclusion: the nigrostriatal dopaminergic neurotoxicity of the PQ and MB combination was progressive and irreversible. DR can limit the TH neurons necrosis or apoptosis and relieve the reaction of Asts induced by PQ and MB in mice brain, which may be one of the conceivable mechanisms for the neuroprotection of DR.Experiment 3Objective: To explore the effects of DR on behavioral alterations in mice induced by combination of PQ and MB. Methods: Mice were divided into saline and PQMB group randomly. All were injected with either saline (0.9%) or a combination of PQ (8 mg/kg) and MB (24 mg/kg) twice a week (Tuesday and Thursday, 9:00 am) for 6 weeks intraperitoneally. On the last injection, the PQMB treated mice were divided into two groups randomly; one group had access to food ad libitum (PQMB/AL), while another fed on alternate days only (PQMB/DR). The saline treated mice were divided into two groups randomly too, Saline/AL and Saline/DR. The pole test and the open-field test were used to analyze the behavioral abnormalities. Animals were tested on five different time-points, time just before the first injection (-6 weeks), time after the last-injection (0 week) and the following 3, 5, 8 weeks. Results: Injection of PQMB caused behavioral syndromes including restlessness, straub tail, hindlimb abduction, tremor and instability of gait in C57BL/6J mice. Symptoms of Bradykinesia, akinesia and instability of gait appeared after several weeks of PQMB administration. In locomotor activity test, the horizontal movement distance in PQMB/AL group was 935.01±146.27 cm in 0 week, which was lower than that of saline group (1903.38±203.14 cm) (P < 0.01). In the pole test, Tturn (defined as the time taken until mice turned completely downward) and TLA (defined as the time until mice had climbed down to the floor) were 2.59±0.21 s and 13.85±0.69 s respectively in 0 week after the last injection of PQMB, which showed significantly prolongation compared with the saline group (0.90±0.12 s; 6.34±0.37 s) (P < 0.01). And motor deficits were persistent in PQMB group over time, while the horizontal movement distance in locomotor activity test increased (P < 0.05) and TLA in the pole test decreased after 8 weeks’dietary restriction (P < 0.01) and Tturn decreased significantly in PQMB/DR group from the 5th week after the last injection (P < 0.01). Conclusion: PQMB can induce behavioral alteration in C57BL/6J mice and motor deficits were persistent over time. Mid or long term of Dietary restriction may have protection effects on those motor deficits.更多还原