α-Synuclein Expression In Substantianigra Chronic Rotenone Parkinson's Disease Mice

Objective1.To study DA neurons in substantia nigra of mice exposed to rotenone for a long-term ,as well as the behavioral changes.2.To study weather or notα-syn is increased in substantia nigra of rotenone PD model mouse , and duration of increasedα-syn.Methods30 male C57BL mice were randomly devided in to 5 groups(n=6).The contrlo group(CP) was given mixture of DMSO and saline (1ml/kg) through subcutaneous injection.The other 4 groups were given rotenone solution per day for 40 days.The dose was 0.5mg/kg for group 1, 1mg/kg for group 2, 1.5mg/kg for group 3,2mg/kg for group 4.3 days after the last injection ,we observed the behavior of mice in each group through pole test,swimming test and open-field test ;the morphology profit of substantia nigra Dopaminergic cell(DAergic cell) was tested with HE staining and Nissl dyeing; We use DAB immunohistochemistry to detect the quanty of tyrosine hydroxylase (TH)-positive neurons in substantia nigra of mice,andα-syn expression. From the above experiment, select the appropriate group exposed proper dose of rotenone as the experimental group for the following experiment and re-modeling. 3 days after the last injection .As before we observed the behavior of mice in each group through swimming test and open-field test ; the morphology profit of substantia nigra DAergic cell was tested with HE staining and Nissl dyeing; We detect the quanty of tyrosine hydroxylase (TH)-positive neurons in substantia nigra of mice through DAB immunohistochemistry. RT-PCR and immunohistochemistry were used to detect the expression ofα-syn in substantia nigra DAergic cell.Results1. Behavioral observations: high mortality occurred in experimental groups injected with rotennone 1.5mg/kg, 2.0mg/kg, respectively 50% and 100%; Mice injected with 1.0mg/kg rotenone had significant tremor,bradycardia and decreased movements ,just according to Parkinson's disease-like symptoms.Swimming test ,pole test and open-field test show that activities of the experimental group(1.0mg/kg) were markedly reduced compared to the control group, and were significantly different (P<0.01). While these did not occurred in the the control group and the group injected with 0.5mg/kg rotenone.2.Morphological examination result:compared to the control group ,irregular morphology and decreased Nissl Body occurred in substantia nigra dopaminergic neurons of the 1.0 mg/kg rotenone injected group. Compared to the control group ,the number of TH positive cells in substantia nigra reduced significently.So we chose the 1.0mg/kg rotenone injected group as the animal model for following experiment.3. DAB immunohistochemistry Results: (1) Compared to the control group ,the number of TH positive cells in substantia nigra reduces by 31% -39% (P<0.05) within the 1.0mg/kg rotenone injected group in the dynamic obsrvation.(2) Compared to the control group the integral optical density (IOD) ofα-syn-positive staining gain increased by 30% (P <0.05) in the experimental group; (3) 2 months after the last injectionα-syn expression in model group in substantia nigra was still higher than in control group.4.RT-PCR results showed that: in the rotenone increasedα-syn mRNA at the 3d after the last injection; and return to normal at 1 month. Conclusion1.Mice exposed to rotenone with the dose 1.0mg/kg for 40 days can produce PD-like clinical manifest and pathology, and the toxin mice model can be used as PD animal model.2.TH-positive DAergic neurons significantly reduced in rotenone handled mouse(1.0mg/kg) substantia nigra compared to the control group;α-syn expression increased and aggregated, and can continue for a longer time (2months).