Age-related Changes In Brain Of C57BL Mouse And Mechanism Of Parkinson's Disease

Part I Behavioral study of influence of aging and MPTP neurotoxin on motor function of C57BL mouseObjective 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 neurotoxinObjective 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 neurotoxinObjective 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 cellsObjective 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.