C57BL小鼠脑增龄性改变和帕金森病发病机制的相关研究
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摘要
第一部分:增龄和MPTP对C57BL小鼠运动功能影响的行为学研究
目的通过构建不同增龄组C57BL小鼠1-甲基,4-苯基,1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)毒性帕金森病模型,观察小鼠运动功能的增龄性变化和增龄对MPTP毒性行为学反应的影响。
方法分别构建3、6、12、16、20月龄组C57BL小鼠,每组20只,雄雌各半。MPTP 30mg/kg.d-1腹腔注射,连续5天,构建小鼠PD模型。分别在造模前、造模后第1、7、14和28天对各增龄组动物的行为学指标进行动态观察。小鼠转棒仪测定小鼠转棒潜伏期;小鼠自发活动箱测定小鼠自发活动量;自制小鼠爬杆测定爬杆时间。
结果6月龄小鼠表现出最长的转棒潜伏期,显著高于其他各组(P<0.05)12月龄后各组转棒潜伏期降低至3月龄水平。6月龄小鼠较其它各组亦表现出较短的爬杆时间(P<0.05)。12月龄组表现出最多的自发活动量。各组动物在MPTP腹腔注射后均立即出现少动、竖尾和激惹等急性期反应。成模后第一天各组均出现转棒潜伏期的明显缩短,爬杆时间的延长和自发活动量的显著减少。对各增龄组动物行为学指标的连续观察显示:3、6月龄小鼠转棒潜伏期和爬杆时间均在成模后第14天已有明显恢复,而12月龄以上各组却恢复缓慢,甚至在第28天仍未完全恢复。各增龄组自发活动在一过性降低后均与一周后恢复。
结论增龄对C57BL小鼠运动功能存在显著影响,6月龄后C57BL小鼠运动功能表现处随龄性衰退;MPTP可明显影响C57BL小鼠运动功能,12月龄以上小鼠对MPTP毒性表现处较高的行为学敏感性。
第二部分:增龄和MPTP对C57BL小鼠黑质-纹状体多巴胺能神经元、多巴胺代谢和氧化应激的影响
目的C57BL小鼠运动功能存在随龄性改变,12月龄以后小鼠运动功能明显衰退,而且在MPTP处理后老龄小鼠亦表现出更加明显的行为学改变,其代偿能力明显低于低龄组。为探讨这一现象相应的解剖病理基础及与黑质-纹状体系统多巴胺(Dopamine,DA)代谢之间存在的联系。
方法雄性C57BL小鼠,分别构建3、6、12、16、20月龄组,每组20只。分为自然增龄组和PD模型组,MPTP 30mg/kg.d-1腹腔注射,连续给药5天,建模。小鼠黑质连续切片,酪氨酸羟化酶(Tyrosine hydroxylase,TH)免疫组织化学染色并计数TH阳性细胞数。高效液相色谱法检测纹状体DA水平。化学比色法检测纹状体组织总活性氧。
结果在自然增龄过程中,小鼠黑质TH阳性细胞计数以6月龄组为最高,各组与6月龄组相比较显示:3月龄组低于6月龄组(P<0.05);16、20月龄组均显著低于6月龄组(P<0.01)。经MPTP处理各组TH阳性细胞计数均显著降低(P<0.01),各月龄组TH阳性细胞计数下降的百分比分别为: 28.3%(3月龄);29.5%(6月龄);35.0%(12月龄);43.7%(16月龄)和60.3%(20月龄)。自然增龄各组纹状体DA水平未见显著差异。MPTP处理各组纹状体DA水平均见显著降低,尤以12、16和20月龄老龄各组为著。自然增龄各组纹状体总活性氧水平在12月龄以上各组明显升高,MPTP处理后各组总活性氧水平均显著升高。
结论增龄过程中C57BL小鼠黑质DA能细胞计数和纹状体DA水平下降;而氧化应激水平升高;MPTP在12月龄以上小鼠引起更加严重的黑质-纹状体DA能系统损害;老龄小鼠对MPTP的高敏感性可能和纹状体氧化应激水平的增龄性升高及抗氧化能力下降有关。
第三部分:C57BL小鼠纹状体DAT、VMAT-2和α-Synuclein表达的增龄模式和对MPTP毒性的反应
目的为进一步观察增龄过程中C57BL小鼠纹状体多巴胺转运体(Dopamine transporter,DAT)、2型囊泡单胺类转运体(Vesicular membrane monoamine transporter type 2,VMAT-2)和α-突触核蛋白(α-Synuclein)表达的变化是否参与老龄小鼠对外源毒素高敏感性的内在机制。
方法雄性C57BL小鼠,分别构建3、6、12、16、20月龄组,每组20只。分为自然增龄组和PD模型组,MPTP 30mg/kg.d-1腹腔注射,连续给药5天,建模。分别以Western-blotting和Real-time PCR法检测自然增龄和MPTP处理各组纹状体DAT、VMAT-2及α-Synuclein蛋白表达及mRNA水平。
结果自然增龄过程DAT总蛋白水平在各增龄组间无显著差异,MPTP处理各增龄组DAT表达水平均较同龄非给药组见明显下降,但以16、20月龄组稍明显,组间差异有显著性,P<0.05;12月龄后小鼠纹状体VMAT-2表达水平明显下降,组间差异有显著性,P<0.05;经MPTP处理各增龄组VMAT-2较同龄非给药组有明显下降P<0.05,但给药后各组组间差异无显著性;DAT和VMAT-2蛋白表达量的比值在6月龄后呈随龄性降低;α-Synuclein的表达呈随龄增高趋势,老龄小鼠纹状体α-Synuclein表达量高于低龄组,组间差异有显著性(P<0.05);MPTP处理后各增龄组α-Synuclein表达较同龄非给药组明显上调,各组与非给药组相比有显著差异(P<0.01)。DAT mRNA水平在自然增龄各组未见显著差异,经MPTP处理后16、20月龄组较同龄非给药组出现异常显著的增高,16月龄组P<0.05,20月龄组P<0.01。VMAT-2 mRNA水平在自然增龄过程中呈现明显下降趋势,各增龄组间比较差异有显著性,P<0.05。MPTP给药后3、16和20月龄组mRNA水平较同龄非给药组出现显著升高,3月龄组P<0.05,16、20月龄组P<0.01。α-Synuclein mRNA水平在自然增龄各组间未见显著差异,但在MPTP处理各组mRNA水平均较同龄的非给药组升高,P<0.05。
结论C57BL小鼠DAT、VMAT-2和α-Synuclein表达存在增龄性变化。高龄小鼠DAT,VMAT-2表达的失衡状态可能介导纹状体多巴胺能神经元的高易损性。老龄小鼠MPTP毒性反应中,DAT、VMAT-2和α-Synuclein在其中发挥重要作用。
第四部分:多巴胺诱导的氧化应激对转人α-Synuclein基因PC12细胞的影响
目的为探讨α-Synuclein在多巴胺诱导的氧化应激中的作用。方法利用转人α-Synuclein的PC12细胞,给予多巴胺150μM处理12h。MTT法检测细胞活力;AnnexinⅤ-PI染色检测细胞凋亡;激光共聚焦显微镜下观察α-Synuclein表达情况;免疫组织化学检测各处理组α-Synuclein表达情况。
结果DA处理(150μM,12h)可显著降低各株细胞的存活率(P<0.01),但转人SNCA-pEGFP-C3 PC12细胞表现出相对较轻的抑制率。DA处理组细胞凋亡明显增多,但镜下观察显示转人SNCA-pEGFP-C3 PC12细胞凋亡数量相对较少。转人SNCA-pEGFP-C3 PC12细胞在给予DA(150μM)后第7分钟开始各感兴趣区荧光强度开始上升,并在给药后30分钟内呈持续上升趋势,较强的荧光强度一直持续至给药后4小时。DA处理后,PC12细胞和转人SNCA-pEGFP-C3 PC12细胞均出现α-Synuclein的高表达,转人SNCA-pEGFP-C3 PC12细胞DA处理后α-Synuclein表达增强,但以外源性α-Synuclein上调为主。两株细胞在未给药状态下α-Synuclein表达于胞浆,免疫阳性着色呈均匀散在分布;在DA处理后,α-Synuclein表达增高,在DA处理的PC12细胞高表达的α-Synuclein呈胞浆分布,免疫阳性着色多分布在核周,
而转人SNCA-pEGFP-C3 PC12细胞α-Synuclein在胞浆中分布呈现局部团块样增强。结论α-Synuclein对DA诱导的氧化应激具有保护性作用。
Part I Behavioral study of influence of aging and MPTP neurotoxin on motor function of C57BL mouse
Objective To investigate the influence of aging and MPTP neurotoxin on motor function of C57BL mice.
Methods MPTP neurotoxin C57BL mice model of Parkinson’s disease was constructed in different aged (3, 6,12,16,20 months old, n=20 in each group, 10 male and 10 female) mice. All mice were treated with five injections of MPTP (30mg/kg, at 24h intervals). Rolling bar tests, spontaneous activity tests and pole climbing tests were all performed before MPTP injection and at the 1st, 7th, 14th, 28th day after MPTP exposure.
Results 6 months old group of mice had significant longer rolling bar test latency than other groups (P<0.05), and also manifested a shortest pole-climbing tim(eP<0.05). 12 months old mice showed a significant more spontaneous activity than other groups. Acute responses to MPTP in mice included hypoactivity, tail erection and irritation. All groups manifested decreased rolling bar latency and spontaneous activity, and increased pole climbing time. Consecutive observation of the behavioral indexes showed: In 3, 6 months old groups’mice, there was an obvious recovery of rolling bar latency and pole climbing time at the 14th day after MPTP exposure. However mice in other older groups did not gain a full recovery even at the 28th day after MPTP exposure. The spontaneous activity recovered at the 7th day after MPTP exposure in all mice.
Conclusion From 6 months old, there is an age-related decline of motor function in C57BL mice. MPTP has a distinct impairment on motor function of C57BL mice. Mice older than 12 months are more sensitive to MPTP neurotoxin than younger ones, and have a deficit in compensation ability of the impairment of motor function.
Part II Age-associated changes of dopaminergic neuron, metabolism of dopamine and oxidative stress of C57BL mice and responses to MPTP neurotoxin
Objective To seek the pathologic basements of ethologic high sensitivity to MPTP in aged C57BL mice.
Methods MPTP neurotoxin C57BL mice model of Parkinson’s disease was constructed in different aged (3, 6,12,16,20 months old, n=20) male mice. All mice were treated with five injections of MPTP (30mg/kg, at 24h intervals). Quantitative morphology and high performance liquid chromatogram (HPLC)was performed to evaluate the number of tyrosine hydroxylase (TH)positive cells of substantia nigra pars compacta (SNc) and striatum dopamine (DA). Level of total reactive oxygen species (ROS) was determined by colorimetric method.
Results In normal aging groups, 6 months old mice had the highest number of SNc TH positive cells. Compare with 6 months old group, the number of 16 and 20 months old mice was significant lower (P<0.01),and so did in 3 months old group(P<0.05). There was a significant decrease of SNc TH positive cells in all MPTP treated mice. The percentage of the decline in each group was 28.3%(3 months), 29.5%(6 months), 35.0%(12 months), 43.7%(16 months) and 60.3%(20 months). There was significant difference of DA level among normal aging groups. All MPTP treated mice manifested a significant decrease of striatum DA level, and more obvious in 12,16 and 20 months old mice. Increased level of striatum total ROS level was observed in mice older than 12 months.and there was an obvious increase of striatum ROS in all MPTP treated groups.
Conclusion There is an age-related decline in SNc dopaminergic neurons and DA level of striatum. MPTP induced more severe dopaminergic neurotoxic impairment in mice older than 12 months. The hypersensitivity to MPTP in aged mice may related to the high level of oxidative stress and the deficit of antioxgen system.
Part III Age-related changes of striatum DAT, VMAT-2 andα-Synuclein of C57BL mice, and responses to MPTP neurotoxin
Objective To investigate whether the age-related changes of striatum dopamine transporter (DAT), vesicular monoamine transporter type 2 (VMAT-2) andα-Synuclein are involved in the mechanism of dopaminergic neuronal hypersensitive to exogenous neurotoxin in aged mice.
Methods MPTP neurotoxin C57BL mice model of Parkinson’s disease was constructed in different aged (3, 6,12,16,20 months old, n=20) male mice. All mice were treated with five injections of MPTP (30mg/kg, at 24h intervals). Western blotting was used to evaluate the protein level of DAT, VMAT-2 andα-Synuclein in normal aging and MPTP treated groups. Real time PCR was performed to evaluate the mRNA level of the three proteins.
Results Despite the steady level of total DAT in normal aging groups, MPTP exposure induced a significant decrease of DAT in each group, and it was more obvious in 16 and 20 months old groups(P<0.05). There was an age-related decline of striatum VMAT-2 level in mice older than 12 months(P<0.05). After MPTP exposure, a significant decline of VMAT-2 level was observed in each group of different aged mice compare with their normal aging accompanier(P<0.05), but there was no difference among all MPTP treated groups. The ratio of DAT and VMAT-2 declined after 6 months old. There was an increased tendency ofα-Synuclein in mice’s striatum during aging(P<0.05). The level ofα-Synuclein was higher in aged mice than in younger ones(P<0.05).α-Synuclein was dramatically increased in each MPTP treated group, there was a significant difference between each MPTP treated group and their normal aging accompanier(P<0.05). DAT mRNA kept steady during normal aging. When treated by MPTP, a dramatic increase of DAT mRNA occurred in 16 month(sP<0.05)and 20 months(P<0.01)old groups compared with their normal aging accompanier. There was an age-related decline of VMAT-2 mRNA in the process of aging, significant difference lay in different aged group(sP<0.05). Compared with normal aging groups, 3,16 and 20 months old groups manifested a significant increase of VMAT-2 mRNA when treated by MPTP(P<0.05 in 3 months old groups, P<0.01 in 16 and 20 months old groups ). Although there was no difference ofα-Synuclein mRNA among normal aging groups, MPTP induced a significant up-regulation ofα-Synuclein mRNA in different aged groups(P<0.05).
Conclusion There are age-related changes in expression of DAT, VMAT-2 andα-Synuclein in striatum of C57BL mice. The high vulnerability of dopaminergic neuron of aged mice may relate to the age-associated imbalance between DAT and VMAT-2 during aging. DAT, VMAT-2 andα-Synuclein play an important role in mechanism of dopaminergic neurotoxin of MPTP.
Part IV Effects of dopamine induced oxidative stress on trans-human SNCA gene PC12 cells
Objective To investigate the role ofα-Synuclein in oxidative stress induced by dopamine.
Methods PC12 cells, transfected by human SNCA(α-Synuclein)gene,were treated by dopamine (150μM,12h). Biology activity of cells was evaluated by MTT, and Annexin V-PI stain to detect the level of apoptosis, confocal microscopy was used to observe the expression ofα-Synuclein, morphology study was performed by immunohistochemistry.
Results Survival rate of each cell strain was significantly decreased by dopamine treatment(P<0.01), but the inhibition ratio was attenuated in trans-hhuman SNCA PC12 cells(hSNCA-PC12 ). Apoptosis degree was increased by DA exposure, but the hSNCA-PC12 cells showed a relative less apoptotic degree after DA exposure. The fluorescence intensity in all ROIs increased at the 7th minute after DA treatment in hSNCA-PC12 cells, and kept increasing in the first 30 minutes. High intensity of fluorescence kept for 4 hours. The expression ofα-Synuclein increased after DA exposure, and the exogenousα-Synuclein was the majority of the increase in hSNCA-PC12 cells. The positive immuno-stain ofα-synuclein distributed in plasm , and was increased by DA exposure. After DA exposure, the distribution of positive stain was peri-nuclear in the PC12 cells, and accumulated into blocks in hSNCA-PC12 cells.
Conclusionα-Synuclein plays a protective role in the dopamine induced oxidative stress.
目的通过构建不同增龄组C57BL小鼠1-甲基,4-苯基,1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)毒性帕金森病模型,观察小鼠运动功能的增龄性变化和增龄对MPTP毒性行为学反应的影响。
方法分别构建3、6、12、16、20月龄组C57BL小鼠,每组20只,雄雌各半。MPTP 30mg/kg.d-1腹腔注射,连续5天,构建小鼠PD模型。分别在造模前、造模后第1、7、14和28天对各增龄组动物的行为学指标进行动态观察。小鼠转棒仪测定小鼠转棒潜伏期;小鼠自发活动箱测定小鼠自发活动量;自制小鼠爬杆测定爬杆时间。
结果6月龄小鼠表现出最长的转棒潜伏期,显著高于其他各组(P<0.05)12月龄后各组转棒潜伏期降低至3月龄水平。6月龄小鼠较其它各组亦表现出较短的爬杆时间(P<0.05)。12月龄组表现出最多的自发活动量。各组动物在MPTP腹腔注射后均立即出现少动、竖尾和激惹等急性期反应。成模后第一天各组均出现转棒潜伏期的明显缩短,爬杆时间的延长和自发活动量的显著减少。对各增龄组动物行为学指标的连续观察显示:3、6月龄小鼠转棒潜伏期和爬杆时间均在成模后第14天已有明显恢复,而12月龄以上各组却恢复缓慢,甚至在第28天仍未完全恢复。各增龄组自发活动在一过性降低后均与一周后恢复。
结论增龄对C57BL小鼠运动功能存在显著影响,6月龄后C57BL小鼠运动功能表现处随龄性衰退;MPTP可明显影响C57BL小鼠运动功能,12月龄以上小鼠对MPTP毒性表现处较高的行为学敏感性。
第二部分:增龄和MPTP对C57BL小鼠黑质-纹状体多巴胺能神经元、多巴胺代谢和氧化应激的影响
目的C57BL小鼠运动功能存在随龄性改变,12月龄以后小鼠运动功能明显衰退,而且在MPTP处理后老龄小鼠亦表现出更加明显的行为学改变,其代偿能力明显低于低龄组。为探讨这一现象相应的解剖病理基础及与黑质-纹状体系统多巴胺(Dopamine,DA)代谢之间存在的联系。
方法雄性C57BL小鼠,分别构建3、6、12、16、20月龄组,每组20只。分为自然增龄组和PD模型组,MPTP 30mg/kg.d-1腹腔注射,连续给药5天,建模。小鼠黑质连续切片,酪氨酸羟化酶(Tyrosine hydroxylase,TH)免疫组织化学染色并计数TH阳性细胞数。高效液相色谱法检测纹状体DA水平。化学比色法检测纹状体组织总活性氧。
结果在自然增龄过程中,小鼠黑质TH阳性细胞计数以6月龄组为最高,各组与6月龄组相比较显示:3月龄组低于6月龄组(P<0.05);16、20月龄组均显著低于6月龄组(P<0.01)。经MPTP处理各组TH阳性细胞计数均显著降低(P<0.01),各月龄组TH阳性细胞计数下降的百分比分别为: 28.3%(3月龄);29.5%(6月龄);35.0%(12月龄);43.7%(16月龄)和60.3%(20月龄)。自然增龄各组纹状体DA水平未见显著差异。MPTP处理各组纹状体DA水平均见显著降低,尤以12、16和20月龄老龄各组为著。自然增龄各组纹状体总活性氧水平在12月龄以上各组明显升高,MPTP处理后各组总活性氧水平均显著升高。
结论增龄过程中C57BL小鼠黑质DA能细胞计数和纹状体DA水平下降;而氧化应激水平升高;MPTP在12月龄以上小鼠引起更加严重的黑质-纹状体DA能系统损害;老龄小鼠对MPTP的高敏感性可能和纹状体氧化应激水平的增龄性升高及抗氧化能力下降有关。
第三部分:C57BL小鼠纹状体DAT、VMAT-2和α-Synuclein表达的增龄模式和对MPTP毒性的反应
目的为进一步观察增龄过程中C57BL小鼠纹状体多巴胺转运体(Dopamine transporter,DAT)、2型囊泡单胺类转运体(Vesicular membrane monoamine transporter type 2,VMAT-2)和α-突触核蛋白(α-Synuclein)表达的变化是否参与老龄小鼠对外源毒素高敏感性的内在机制。
方法雄性C57BL小鼠,分别构建3、6、12、16、20月龄组,每组20只。分为自然增龄组和PD模型组,MPTP 30mg/kg.d-1腹腔注射,连续给药5天,建模。分别以Western-blotting和Real-time PCR法检测自然增龄和MPTP处理各组纹状体DAT、VMAT-2及α-Synuclein蛋白表达及mRNA水平。
结果自然增龄过程DAT总蛋白水平在各增龄组间无显著差异,MPTP处理各增龄组DAT表达水平均较同龄非给药组见明显下降,但以16、20月龄组稍明显,组间差异有显著性,P<0.05;12月龄后小鼠纹状体VMAT-2表达水平明显下降,组间差异有显著性,P<0.05;经MPTP处理各增龄组VMAT-2较同龄非给药组有明显下降P<0.05,但给药后各组组间差异无显著性;DAT和VMAT-2蛋白表达量的比值在6月龄后呈随龄性降低;α-Synuclein的表达呈随龄增高趋势,老龄小鼠纹状体α-Synuclein表达量高于低龄组,组间差异有显著性(P<0.05);MPTP处理后各增龄组α-Synuclein表达较同龄非给药组明显上调,各组与非给药组相比有显著差异(P<0.01)。DAT mRNA水平在自然增龄各组未见显著差异,经MPTP处理后16、20月龄组较同龄非给药组出现异常显著的增高,16月龄组P<0.05,20月龄组P<0.01。VMAT-2 mRNA水平在自然增龄过程中呈现明显下降趋势,各增龄组间比较差异有显著性,P<0.05。MPTP给药后3、16和20月龄组mRNA水平较同龄非给药组出现显著升高,3月龄组P<0.05,16、20月龄组P<0.01。α-Synuclein mRNA水平在自然增龄各组间未见显著差异,但在MPTP处理各组mRNA水平均较同龄的非给药组升高,P<0.05。
结论C57BL小鼠DAT、VMAT-2和α-Synuclein表达存在增龄性变化。高龄小鼠DAT,VMAT-2表达的失衡状态可能介导纹状体多巴胺能神经元的高易损性。老龄小鼠MPTP毒性反应中,DAT、VMAT-2和α-Synuclein在其中发挥重要作用。
第四部分:多巴胺诱导的氧化应激对转人α-Synuclein基因PC12细胞的影响
目的为探讨α-Synuclein在多巴胺诱导的氧化应激中的作用。方法利用转人α-Synuclein的PC12细胞,给予多巴胺150μM处理12h。MTT法检测细胞活力;AnnexinⅤ-PI染色检测细胞凋亡;激光共聚焦显微镜下观察α-Synuclein表达情况;免疫组织化学检测各处理组α-Synuclein表达情况。
结果DA处理(150μM,12h)可显著降低各株细胞的存活率(P<0.01),但转人SNCA-pEGFP-C3 PC12细胞表现出相对较轻的抑制率。DA处理组细胞凋亡明显增多,但镜下观察显示转人SNCA-pEGFP-C3 PC12细胞凋亡数量相对较少。转人SNCA-pEGFP-C3 PC12细胞在给予DA(150μM)后第7分钟开始各感兴趣区荧光强度开始上升,并在给药后30分钟内呈持续上升趋势,较强的荧光强度一直持续至给药后4小时。DA处理后,PC12细胞和转人SNCA-pEGFP-C3 PC12细胞均出现α-Synuclein的高表达,转人SNCA-pEGFP-C3 PC12细胞DA处理后α-Synuclein表达增强,但以外源性α-Synuclein上调为主。两株细胞在未给药状态下α-Synuclein表达于胞浆,免疫阳性着色呈均匀散在分布;在DA处理后,α-Synuclein表达增高,在DA处理的PC12细胞高表达的α-Synuclein呈胞浆分布,免疫阳性着色多分布在核周,
而转人SNCA-pEGFP-C3 PC12细胞α-Synuclein在胞浆中分布呈现局部团块样增强。结论α-Synuclein对DA诱导的氧化应激具有保护性作用。
Part I Behavioral study of influence of aging and MPTP neurotoxin on motor function of C57BL mouse
Objective To investigate the influence of aging and MPTP neurotoxin on motor function of C57BL mice.
Methods MPTP neurotoxin C57BL mice model of Parkinson’s disease was constructed in different aged (3, 6,12,16,20 months old, n=20 in each group, 10 male and 10 female) mice. All mice were treated with five injections of MPTP (30mg/kg, at 24h intervals). Rolling bar tests, spontaneous activity tests and pole climbing tests were all performed before MPTP injection and at the 1st, 7th, 14th, 28th day after MPTP exposure.
Results 6 months old group of mice had significant longer rolling bar test latency than other groups (P<0.05), and also manifested a shortest pole-climbing tim(eP<0.05). 12 months old mice showed a significant more spontaneous activity than other groups. Acute responses to MPTP in mice included hypoactivity, tail erection and irritation. All groups manifested decreased rolling bar latency and spontaneous activity, and increased pole climbing time. Consecutive observation of the behavioral indexes showed: In 3, 6 months old groups’mice, there was an obvious recovery of rolling bar latency and pole climbing time at the 14th day after MPTP exposure. However mice in other older groups did not gain a full recovery even at the 28th day after MPTP exposure. The spontaneous activity recovered at the 7th day after MPTP exposure in all mice.
Conclusion From 6 months old, there is an age-related decline of motor function in C57BL mice. MPTP has a distinct impairment on motor function of C57BL mice. Mice older than 12 months are more sensitive to MPTP neurotoxin than younger ones, and have a deficit in compensation ability of the impairment of motor function.
Part II Age-associated changes of dopaminergic neuron, metabolism of dopamine and oxidative stress of C57BL mice and responses to MPTP neurotoxin
Objective To seek the pathologic basements of ethologic high sensitivity to MPTP in aged C57BL mice.
Methods MPTP neurotoxin C57BL mice model of Parkinson’s disease was constructed in different aged (3, 6,12,16,20 months old, n=20) male mice. All mice were treated with five injections of MPTP (30mg/kg, at 24h intervals). Quantitative morphology and high performance liquid chromatogram (HPLC)was performed to evaluate the number of tyrosine hydroxylase (TH)positive cells of substantia nigra pars compacta (SNc) and striatum dopamine (DA). Level of total reactive oxygen species (ROS) was determined by colorimetric method.
Results In normal aging groups, 6 months old mice had the highest number of SNc TH positive cells. Compare with 6 months old group, the number of 16 and 20 months old mice was significant lower (P<0.01),and so did in 3 months old group(P<0.05). There was a significant decrease of SNc TH positive cells in all MPTP treated mice. The percentage of the decline in each group was 28.3%(3 months), 29.5%(6 months), 35.0%(12 months), 43.7%(16 months) and 60.3%(20 months). There was significant difference of DA level among normal aging groups. All MPTP treated mice manifested a significant decrease of striatum DA level, and more obvious in 12,16 and 20 months old mice. Increased level of striatum total ROS level was observed in mice older than 12 months.and there was an obvious increase of striatum ROS in all MPTP treated groups.
Conclusion There is an age-related decline in SNc dopaminergic neurons and DA level of striatum. MPTP induced more severe dopaminergic neurotoxic impairment in mice older than 12 months. The hypersensitivity to MPTP in aged mice may related to the high level of oxidative stress and the deficit of antioxgen system.
Part III Age-related changes of striatum DAT, VMAT-2 andα-Synuclein of C57BL mice, and responses to MPTP neurotoxin
Objective To investigate whether the age-related changes of striatum dopamine transporter (DAT), vesicular monoamine transporter type 2 (VMAT-2) andα-Synuclein are involved in the mechanism of dopaminergic neuronal hypersensitive to exogenous neurotoxin in aged mice.
Methods MPTP neurotoxin C57BL mice model of Parkinson’s disease was constructed in different aged (3, 6,12,16,20 months old, n=20) male mice. All mice were treated with five injections of MPTP (30mg/kg, at 24h intervals). Western blotting was used to evaluate the protein level of DAT, VMAT-2 andα-Synuclein in normal aging and MPTP treated groups. Real time PCR was performed to evaluate the mRNA level of the three proteins.
Results Despite the steady level of total DAT in normal aging groups, MPTP exposure induced a significant decrease of DAT in each group, and it was more obvious in 16 and 20 months old groups(P<0.05). There was an age-related decline of striatum VMAT-2 level in mice older than 12 months(P<0.05). After MPTP exposure, a significant decline of VMAT-2 level was observed in each group of different aged mice compare with their normal aging accompanier(P<0.05), but there was no difference among all MPTP treated groups. The ratio of DAT and VMAT-2 declined after 6 months old. There was an increased tendency ofα-Synuclein in mice’s striatum during aging(P<0.05). The level ofα-Synuclein was higher in aged mice than in younger ones(P<0.05).α-Synuclein was dramatically increased in each MPTP treated group, there was a significant difference between each MPTP treated group and their normal aging accompanier(P<0.05). DAT mRNA kept steady during normal aging. When treated by MPTP, a dramatic increase of DAT mRNA occurred in 16 month(sP<0.05)and 20 months(P<0.01)old groups compared with their normal aging accompanier. There was an age-related decline of VMAT-2 mRNA in the process of aging, significant difference lay in different aged group(sP<0.05). Compared with normal aging groups, 3,16 and 20 months old groups manifested a significant increase of VMAT-2 mRNA when treated by MPTP(P<0.05 in 3 months old groups, P<0.01 in 16 and 20 months old groups ). Although there was no difference ofα-Synuclein mRNA among normal aging groups, MPTP induced a significant up-regulation ofα-Synuclein mRNA in different aged groups(P<0.05).
Conclusion There are age-related changes in expression of DAT, VMAT-2 andα-Synuclein in striatum of C57BL mice. The high vulnerability of dopaminergic neuron of aged mice may relate to the age-associated imbalance between DAT and VMAT-2 during aging. DAT, VMAT-2 andα-Synuclein play an important role in mechanism of dopaminergic neurotoxin of MPTP.
Part IV Effects of dopamine induced oxidative stress on trans-human SNCA gene PC12 cells
Objective To investigate the role ofα-Synuclein in oxidative stress induced by dopamine.
Methods PC12 cells, transfected by human SNCA(α-Synuclein)gene,were treated by dopamine (150μM,12h). Biology activity of cells was evaluated by MTT, and Annexin V-PI stain to detect the level of apoptosis, confocal microscopy was used to observe the expression ofα-Synuclein, morphology study was performed by immunohistochemistry.
Results Survival rate of each cell strain was significantly decreased by dopamine treatment(P<0.01), but the inhibition ratio was attenuated in trans-hhuman SNCA PC12 cells(hSNCA-PC12 ). Apoptosis degree was increased by DA exposure, but the hSNCA-PC12 cells showed a relative less apoptotic degree after DA exposure. The fluorescence intensity in all ROIs increased at the 7th minute after DA treatment in hSNCA-PC12 cells, and kept increasing in the first 30 minutes. High intensity of fluorescence kept for 4 hours. The expression ofα-Synuclein increased after DA exposure, and the exogenousα-Synuclein was the majority of the increase in hSNCA-PC12 cells. The positive immuno-stain ofα-synuclein distributed in plasm , and was increased by DA exposure. After DA exposure, the distribution of positive stain was peri-nuclear in the PC12 cells, and accumulated into blocks in hSNCA-PC12 cells.
Conclusionα-Synuclein plays a protective role in the dopamine induced oxidative stress.
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[12] Karasawa N, Yamawaki Y, Nagatsu T, etal.Age-associated changes in the dopamine synthesis as determined by GTP cyclohydrolase I inhibitor in the brain of senescence-accelerated mouse-prone inbred strains (SAMP8). Neurosci Res, 1999 , 35: 31-36.
[13] Mahy N, Andres N, Andrade C, etal.Age-related changes of MAO-A and -B distribution in human and mouse brain.Neurobiology (Bp),2000 , 8: 47-54.
[14] Chen EY, Kallwitz E, Leff SE, etal.Age-related decreases in GTP-cyclohydrolase-I immunoreactive neurons in the monkey and human substantia nigra.J Comp Neurol,2000 , 426: 534-548.
[15] Mukherjee J, Christian BT, Dunigan KA, etal.Brain imaging of 18F-fallypride in normal volunteers: blood analysis, distribution, test-retest studies, and preliminary assessment of sensitivity to aging effects on dopamine D-2/D-3 receptors. Synapse, 2002 ,46: 170-188.
[16] Himi T, Cao M and Mori N . Reduced expression of the molecular markers of dopaminergic neuronal atrophy in the aging rat brain . J Gerontol A Biol Sci Med Sci, 1995 , 50: 193-200.
[17] Hebert MA, Larson GA, Zahniser NR, etal.Age-Related Reductions in [3H]WIN 35,428 Binding to the Dopamine Transporter in Nigrostriatal and Mesolimbic BrainRegions of the Fischer 344 Rat.J Pharmacol Exp Ther ,1999,288:1334-1339.
[18] Booij J, Bergmans P, Winogrodzka A, etal.Imaging of dopamine transporters with [123I]FP-CIT SPECT does not suggest a significant effect of age on the symptomatic threshold of disease in Parkinson's disease. Synapse, 2001 ,39: 101-108.
[19] Salvatore M F, Apparsundaram S, Gerhardt GA. Decreased plasma membrane expression of striatal dopamine transporter in aging .Neurobiology of Aging ,2003, 24: 1147–1154.
[20] Tamas A, Lubics A, Szalontay L, etal.Age and gender differences in behavioral and morphological outcome after 6-hydroxydopamine-induced lesion of the substantia nigra in rats.Behav Brain Res, 2005 , 158: 221-229.
[21] Cantuti-Castelvetri I,Shukitt-Hale B, and Joseph JA. Dopamine neurotoxicity: age-dependent behavioral and histological effects.Neurobiol Aging, 2003 ,24:697-706.
[22] Saito Y, Kawashima A, Ruberu NN, etal.Accumulation of phosphorylated alpha- synuclein in aging human brain.J Neuropathol Exp Neurol,2003 ,62: 644-654.
[23] Wakisaka Y, Furuta A,Tanizaki Y, etal.Age-associated prevalence and risk factors of Lewy body pathology in a general population: the Hisayama study.Acta Neuropathol (Berl), 2003 ,106: 374-82.
[24] Li W, Hoffman PN, Stirling W, etal.Axonal transport of human alpha-synuclein slows with aging but is not affected by familial Parkinson's disease-linked mutations.J Neurochem,2004 ,88: 401-410.
[25] Martin WR, Ye FQ, and Allen PS .Increasing striatal iron content associated with normal aging. Mov Disord, 1998 ,13: 281-286.
[26] Graham JM, Paley MN, Grunewald RA, etal.Brain iron deposition in Parkinson's disease imaged using the PRIME magnetic resonance sequence . Brain, 2000 ,123: 2423-2431.
[27] Zecca L, Stroppolo A, Gatti A, etal.The role of iron and copper molecules in the neuronal vulnerability of locus coeruleus and substantia nigra during aging .PNAS, 2004 ,101: 9843 - 9848.
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[1] Zhang ZX, Roman GC, Hong Z,et al.Parkinson's disease in China: prevalence in Beijing, Xian, and Shanghai. Lancet,2005,365(9459): 595-597.
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[9] Greenwood CE, Tatton WG, Seniuk NA, etal. Increased dopamine synthesis in aging substantia nigra neurons.Neurobiol Aging,1991 , 12: 557-565.
[10] Tumer N, Demirel HA, Serova L, etal.Gene expression of catecholamine biosynthetic enzymes following exercise: modulation by age.Neuroscience, 2001 , 103: 703-711.
[11] Halbach O and K Unsicker .Age-related decline in the tyrosine hydroxylase- immunoreactive innervation of the amygdala and dentate gyrus in mice.Cell Tissue Res,2003 , 311: 139-143.
[12] Karasawa N, Yamawaki Y, Nagatsu T, etal.Age-associated changes in the dopamine synthesis as determined by GTP cyclohydrolase I inhibitor in the brain of senescence-accelerated mouse-prone inbred strains (SAMP8). Neurosci Res, 1999 , 35: 31-36.
[13] Mahy N, Andres N, Andrade C, etal.Age-related changes of MAO-A and -B distribution in human and mouse brain.Neurobiology (Bp),2000 , 8: 47-54.
[14] Chen EY, Kallwitz E, Leff SE, etal.Age-related decreases in GTP-cyclohydrolase-I immunoreactive neurons in the monkey and human substantia nigra.J Comp Neurol,2000 , 426: 534-548.
[15] Mukherjee J, Christian BT, Dunigan KA, etal.Brain imaging of 18F-fallypride in normal volunteers: blood analysis, distribution, test-retest studies, and preliminary assessment of sensitivity to aging effects on dopamine D-2/D-3 receptors. Synapse, 2002 ,46: 170-188.
[16] Himi T, Cao M and Mori N . Reduced expression of the molecular markers of dopaminergic neuronal atrophy in the aging rat brain . J Gerontol A Biol Sci Med Sci, 1995 , 50: 193-200.
[17] Hebert MA, Larson GA, Zahniser NR, etal.Age-Related Reductions in [3H]WIN 35,428 Binding to the Dopamine Transporter in Nigrostriatal and Mesolimbic BrainRegions of the Fischer 344 Rat.J Pharmacol Exp Ther ,1999,288:1334-1339.
[18] Booij J, Bergmans P, Winogrodzka A, etal.Imaging of dopamine transporters with [123I]FP-CIT SPECT does not suggest a significant effect of age on the symptomatic threshold of disease in Parkinson's disease. Synapse, 2001 ,39: 101-108.
[19] Salvatore M F, Apparsundaram S, Gerhardt GA. Decreased plasma membrane expression of striatal dopamine transporter in aging .Neurobiology of Aging ,2003, 24: 1147–1154.
[20] Tamas A, Lubics A, Szalontay L, etal.Age and gender differences in behavioral and morphological outcome after 6-hydroxydopamine-induced lesion of the substantia nigra in rats.Behav Brain Res, 2005 , 158: 221-229.
[21] Cantuti-Castelvetri I,Shukitt-Hale B, and Joseph JA. Dopamine neurotoxicity: age-dependent behavioral and histological effects.Neurobiol Aging, 2003 ,24:697-706.
[22] Saito Y, Kawashima A, Ruberu NN, etal.Accumulation of phosphorylated alpha- synuclein in aging human brain.J Neuropathol Exp Neurol,2003 ,62: 644-654.
[23] Wakisaka Y, Furuta A,Tanizaki Y, etal.Age-associated prevalence and risk factors of Lewy body pathology in a general population: the Hisayama study.Acta Neuropathol (Berl), 2003 ,106: 374-82.
[24] Li W, Hoffman PN, Stirling W, etal.Axonal transport of human alpha-synuclein slows with aging but is not affected by familial Parkinson's disease-linked mutations.J Neurochem,2004 ,88: 401-410.
[25] Martin WR, Ye FQ, and Allen PS .Increasing striatal iron content associated with normal aging. Mov Disord, 1998 ,13: 281-286.
[26] Graham JM, Paley MN, Grunewald RA, etal.Brain iron deposition in Parkinson's disease imaged using the PRIME magnetic resonance sequence . Brain, 2000 ,123: 2423-2431.
[27] Zecca L, Stroppolo A, Gatti A, etal.The role of iron and copper molecules in the neuronal vulnerability of locus coeruleus and substantia nigra during aging .PNAS, 2004 ,101: 9843 - 9848.
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