The Study On The Relationship Of ?-syn Abnormal Expression On Parkinson's Disease In MPTP Induced Mouse Model

Objective1.To establish sub-acute and chronic mouse model of Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP),and determine an appropriate mouse model of PD for this study by comparing behavioral and histopathological changes of two MPTP mouse models of Parkinson's disease.Hopefully,the MPTP mouse model of PD can provide material supporting for a further study of the mechanism of PD.2.To analyze expression difference of ?-synuclein(?-syn)in the brain tissues such as cortex,midbrain,cerebellum,striatum,hippocampus,brain stem in MPTP induced mouse model of PD,and to investigate the relationship between ?-syn which had been demonstrated as a marker of PD in brain tissues and pathologic changes of Parkinson's disease.Methods1.Establishment and validation of MPTP induced mouse model of PD.(1)MPTP induced subacute mouse model of PD,C57/BL mice were intraperitoneally injected(ip)with MPTP 25mg/kg 1 times per day for 7 days;(2)MPTP induced chronic mouse model of PD,C57/BL mice were intraperitoneally injected(ip)with MPTP 25mg/kg 2 times per week for 5 weeks;(3)control model,C57/BL mice were intraperitoneally injected(ip)with physiological saline 2 times per week for 5 weeks.The identification of MPTP induced mouse model of PD.The behavioral change in mice was inspected by climbing pole and suspension test.The striatum in MPTP induced mouse models of PD got isolated and lyzed by ultrasonication followed by detecting the content of DA and 5-HT by HPLC.In the other aspect,the model mice in MPTP induced mouse models of PD were perfused followed by isolating the brain tissues,and then the midbrain tissues were embedded in paraffin for coronal slices and prepared for paraffin sections followed by stain with HE and detection with the tyrosine hydroxylase(TH)by immunohistochemical staining.2.Expression analysis of ?-syn in brain tissues in MPTP induced mouse model of PD: separate brain tissues of mice in MPTP induced mouse models of PD and mice of control model accurately.For all kinds of tissues,the total proteins were extracted by homogenization,and their concentrations were determined by BCA method.The expression of ?-syn in each brain tissue was detected using the monoclonal antibody of ?-syn by Western blotting.In addition,the model mice in MPTP induced mouse models of PD and the control were perfused followed by isolating the brain tissues,and then the brain tissues were embedded in paraffin for coronal slices and prepared for paraffin sections followed by stain with monoclonal antibody of ?-syn by immunohistochemical staining.Results1.The verification and comparison of chronic model and subacute model in MPTP induced mouse model: Mice of chronic model in MPTP induced mouse model showed significantly abnormal behavior in late period of MPTP injection.Comparing with the control group,the behavior of mouse of chronic model group changed significantly and mouse of subacute model group did not.Mice of chronic model group used more time than that of the subacute group in climbing pole experiments.the suspension capability of mouse of chronic model group is weaker than that of subacute model group in suspension experiment.The content of DA decreased significantly in stratium of two groups of model mouse,but compared with mouse of the subacute group model,the content of DA in chronic group was significant lower(P < 0.05)and the change of the content of 5-HT was not significant(P > 0.05).In TH positive cells reduce with the midbrain substantia nigra compacta,chronic group dropped to 17% of control model group and subacute group dropped to 28% of control model group.Nerve cell apoptosis was more significant in chronic group and relatively less in subacute group with HE staining slice of midbrain substantia nigra in mouse.2.Expression of ?-syn in brain tissue of model mice: The expression levels of ?-syn in the brain tissue(cortex,midbrain,cerebellum,striatum,hippocampus,brain stem)of the mice in the chronic model group were significantly higher(increased 47.78%?46.47%?53.95%?37.77%?37.67%?10.87% respectively)than those in the control model group.The expression difference of ?-syn in the cortex,midbrain,cerebellum,striatum and hippocampus was statistically significant(P<0.05).The expression difference of ?-syn in brainstem was not statistically significant(P> 0.05).The relative expression in the cerebellum of the chronic model group mice was the highest,and the expression of ?-syn in the cerebellum was also higher than that in other brain tissues.Compared with the immunohistochemical paraffin sections of the brain tissue of mice in the control model group,the positive expression of ?-syn protein in neurons was significantly higher in cortex,midbrain,cerebellum,striatum and hippocampus in chronic model group and positive staining cells more,cell deformity,aggregation more intensive;the positive staining of ?-syn in brainstem was not significantly different between the two groups.Conclusion1.The chronic model in mouse of Parkinson 's disease induded by MPTP was established and established that was more obvious with similar clinical symptoms expressed pathological change and in human's Parkinson's disease and can be used as a stable model for further study of Parkinson's disease pathophysiology.2.This study explored the distribution of ?-syn in the brain of PD model and analyses their correlation.And we found that it has tissue specificity,which can be used to investigate the relationship between ?-syn and Parkinson's disease,disease progression,prognosis and provide theoretical basis.