The Mouse Model Of Parkinson's Disease Induced By MPTP And The Expression Of INOS And TNF-α In Some Brain Regions

Part One: The Mouse Model of Parkinson's Disease Induced by MPTPObjective: To observe the changes of morphology and numbers of the TH positive dopaminergic neurons in SNpc and VTA, and the depletion of the TH positive dopaminergic terminals in CPu and FrA in the mouse model of Parkinson's disease induced by MPTP.Methods: (1) Behavioral tests: Using pole test and bar test to observe the degree of bradykinesia and cataleptic symptoms of mice at different time point after administration of MPTP. (2) TH immunofluorescence: 42 C57BL/6 mice, 8 weeks of age, were randomly divided into two groups.①PD model group (36 mice): The mice that received MPTP (20mg/kg i.p) four times at 2-h intervals within a day, total dose are 0.4 ml. After the last injection, these mice were killed at different time points(6h,1d,3d,7d,14d,2m).②Saline group (6 mice): Each mouse received saline (0.1ml i.p) four times at 2-h intervals within a day. The mice were fixed by transaortic perfusion with 4% Polyoxymethylene, and the brains were freezed and cut. Then the coronal serial sections were used for immunofluorescence. (3)Statistical analyses: ONE-WAY ANOVA was used to identify significant differences with the Stata 7.0 software. Values were expressed as Mean±SD. (significance levels:﹡p<0.05,﹡﹡p<0.01,﹡﹡﹡p<0.001).Results: (1) Behavioral tests: Compared with saline group, the mice had a significant prolongation of T-LA and cataleptic effect on day3 and day7 after MPTP treatment (p<0.05). On the 14th day, the mice did not show an obvious change on these two tests. (2)TH immunofluorescence: The TH positive dopaminergic neurons with longer evident immuno-positive processes were intensely stained in the SNpc of saline group. On the model groups, the number of TH positive neurons in the SNpc was reduced 6h after MPTP treatment. On day1 after MPTP injection, the number of TH positive neurons was markedly decreased to 31% (69% lost, p<0.001), and the processes were shorter or disappeared. On the 3rd and 7th day, the numbers of neurons reduced 40% (p<0.01) and 50% (p<0.001) respectively. On day14, only 35% (p<0.05) lost. No different change of the number of TH positive neurons were found between model and saline group (p>0.05) 2 month after MPTP treatment. The morphology of the TH positive dopaminergic neurons in VTA was similar to the neurons in SNpc; the numbers of the neurons in this area also had some changes. Compared with saline group, the number of neurons was markedly decreased (23% lost, p<0.001) on day1 after MPTP treatment. On the 3rd and 7th day, the reduction of the numbers of TH positive neurons were 13% (p<0.05) and 16% (p<0.01) respectively. The numbers of neurons on the other three time points did not find an obvious change between model and saline group (p>0.05). The TH positive terminals in CPu and FrA also had some changes. Compared with saline group, the mean gray values of TH immunofluorescence of the dopaminergic terminals in CPu changed in the model group(23%～62% lost) (p<0.05～p<0.001). The mean gray value on day3 showed a significant reduction (62% lost, p<0.001). In FrA, an obvious reduction (25% lost, p<0.01) of the mean gray value also was found on day3 after MPTP treatment. But no obvious change of the mean gray value were seen on the other four time points between model and saline group (p>0.05).Conclusion: The mouse model of Parkinson's disease induced by MPTP (20mg/kg i.p, four times at 2-h intervals within a day) was successful. The operation is convenient. It can selectively injury the dopaminergic neurons in SNpc and the dopaminergic terminals in CPu. The injury was acute and severe. The behavior of the mouse was also detectable. The changes of neuropathology were similar to human Parkinson's disease. Part Two: The Expression of INOS and TNF-αin Some Brain Regions of the PD MouseObjective: To observe the time-course expression of iNOS and TNF-αmRNA and protein in CPu, SNpc, FrA and VTA of the PD mouse model induced by MPTP.Methods: (1) RT-PCR: 30 mice were randomly divided into 2 groups. One group served as saline control (6 mice) and the others (24 mice) served as MPTP model (including four time points: 6h, 1d, 3d and 14d after the last injection of MPTP). Semi-quantitative RT-PCR method was used to detect the expression of iNOS and TNF-αmRNA in CPu, SNpc, FrA and VTA regions. The relative expression level of iNOS and TNF-αmRNA was indicated by the ratio of the optical density value of iNOS and TNF-αto that of GAPDH. Compared with saline groups, ONE-WAY ANOVA was used to identify significant differences with the Stata 7.0 software. Values were expressed as Mean±SD. (significance levels:﹡p<0.05,﹡﹡p<0.01,﹡﹡﹡p<0.001). (2) Western blot: 30 mice were randomly divided into 2 groups. One group served as saline control (6 mice) and the others (24 mice) served as MPTP administration (including four time points: 12h, 1d, 3d and 14d after the last injection of MPTP). Western blot method was used to detect the expression of iNOS and TNF-αprotein in the four regions. The relative expression level of iNOS and TNF-αprotein was indicated by the ratio of the optical density value of iNOS and TNF-αto that ofβ-actin. The statistic method was similar to that in RT-PCR.Results: (1) RT-PCR: The expression of iNOS and TNF-αmRNA in CPu and SNpc were increased on 6h and day1 after the last injection of MPTP, and a peak was observed on day1, compared with saline group, the change was significant (p<0.001). On the 3rd day after administration, the differences of the expression of iNOS mRNA were still marked, (p<0.001), meanwhile the expression of TNF-αmRNA in CPu and SNpc did not show an obvious change (p>0.05). The expression of iNOS mRNA in VTA and FrA were increased obviously only on day1 and day3, but TNF-αmRNA were not intensively expressed at any time point (p>0.05). (2) Western blot: The expression of iNOS and TNF-αprotein in CPu and SNpc were increased on 12h and day1 after the last injection of MPTP, and reached the peak on day1. On the 3rd day after administration, the differences of the expression of iNOS protein were still obvious, compared with saline group (p<0.01), however there were no obvious expression of TNF-αprotein (p>0.05). The expression of iNOS and TNF-αprotein in VTA and FrA were increased markedly only on day1 after the last injection of MPTP with no obvious expressions at any other time point (p>0.05).Conclusion: The expressions of iNOS and TNF-αmRNA and protein in CPu and SNpc increased significantly at several time points in the mouse model of Parkinson's disease induced by MPTP, and a peak was observed on day1. The expressions of iNOS and TNF-αmRNA and protein in VTA and FrA increased less than those in CPu and SNpc. The intensively expressed of iNOS and TNF-αmay damage the dopaminergic neurons in SNpc and VTA and the dopaminergic terminals in CPu and FrA.