Parkinson-like Phenotype And Pathological Characteristics In Mice Induced By Intrastriatal Inoculation Of ?-syn PFFs

BackgroundParkinson's disease(PD)is a neurodegenerative disorder,which clinical manifestations are mainly including resting tremor,bradykinesia,limb rigidity,and impaired posture balance.It's pathological features are the selective degeneration of dopamine neurons in the substantia nigra pars compacta(SNpc)of the midbrain[1,2]and Lewy-bodies/Lewy-neurites(LBs/LNs)mainly containing misfolded ?-synuclein(?-Syn)in neurons[3-5].It was studied that most sporadic and familial PD patients have?-Syn pathological manifestations in the brain[6],but the pathological process is still unclear[7,8].Misfolded ?-Syn is selectively distributed in specific areas and cell populations of the central nervous system,and its distribution area is closely related to clinical symptoms[9].To date,the exact relationship between misfolded ?-Syn deposition and loss of degeneration of dopamine neurons in the mesencephalic substantia is still controversial.Many animal models of PD have been widely used to study the pathogenesis and pathophysiology of this neurodegenerative disease.The animal model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)is a classic experimental animal model of PD.MPTP is a fat-soluble toxin that can smoothly pass through the blood-brain barrier and selectively kill dopamine energy neurons in the nigrostriatal through oxidative stress and inhibition of the mitochondrial respiratory chain.This model can well simulate the dyskinesia symptoms of PD patients,but the deficiency is the lack of pathological features of?-Syn deposition in the brain.Transgenic animal models that overexpress human?-Syn have some pathological features of PD,such as ?-Syn deposition in dopamine neurons,and progressive loss of dopamine neurons.However,the model itself has a fixed genetic background,and most of PD are sporadic diseases,and its research on?-Syn pathological dissemination pathways is still defective.Therefore,it is particularly important to construct a wild-type PD animal model that can study the?-Syn pathological dissemination process.It has proven that misfolded ?-Syn aggregates can act as "seeds" in brain tissue and induce normal ?-Syn to fold abnormally and spread from one cell to another[11,12].Exogenous ?-Syn pre-formed fibrils(PFFs)can deposit and induce endogenous ?-Syn to misfold fibrosis in vitro cultured wild-type primary nerves[13,14].In this study,We will oberserve Parkinson-like phenotype and pathological characteristics in wild-type C57BL/6J mice induced by intrastriatal inoculation of ?-Syn PFFs.ObjectiveIn this study,wild-type C57BL/6J mice were used as research objects.By using stereotactic injection technology,mouse ?-Syn PFFs were injected into the right striatum of mice to construct a PD wild-type mouse model.We will study the pathology dissemination of ?-Syn positive inclusion bodies and behavioral phenotypes in mice.Methods1.Prepare the ?-Syn PFFs in advance and observe its morphology under a transmission electron microscope.2.By using stereotactic technology,we inject ?-Syn PFFs 5?L into the right striatum of mice.The mice were perfused at three time points:30 days post injection,90 days post injection,and 180 days post injection.We observed the pathology of phosphorylated ?-Syn in the brain tissue sections.3.The histopathological manifestations of ?-Syn were observed by tissue immunochemical staining,and the number of dopamine neurons in the substantia nigra and astrocytes in the pontine were observed by tissue immunofluorescence staining.4.Observe and statistically analyze the behavioral changes of the mice through the Rotary Rod Test,Wire Hanging Test,and Tail Suspension Test.Results1.Using a transmission electron microscope,it was observed that the measured ?-Syn PFFs diameter after ultrasonic treatment was 67.56±41.86 nm,showing less cross-entanglement.2.At 30 days post injection of mouse ?-Syn PFFs,the pathology of phosphorylated?-Syn in brain tissue sections of experimental mice was mainly distributed in the right striatum,right substantia nigra,and right amygdala.The right cortex showed a light brown LBs/LNs-like pathology.At 90 days post injection of mouse ?-Syn PFFs,phosphorylated ?-Syn pathology was observed on both sides of the amygdala of mice in the experimental group.At 180 days post injection of mouse ?-Syn PFFs,Pathology of granular,plaque-like LBs were observed in the right striatum,the right substantia nigra,the right amygdala,and the right cortex of experimental group.In contrast,no phosphorylated ?-Syn pathology was observed in brain tissue sections of mice injected with the PBS control group,.3.After injection 30 days,90 days,and 180 days of mouse ?-Syn PFFs,the TH immunoreactive areas in the right substantia nigra of the experimental group were respectively(19.01±3.09)%Area,(13.93±1.60)%Area and(9.78±1.81)%Area.One-way ANOVA method was used for analysis of variance(P<0.0001),the difference was statistically significant,and then Sidak method was used for post hoc multiple comparisons(PFF 30d vs PFF 90d,P=0.0044;PFF 30d vs PFF 180d,P<0.0001;PFF 90d vs PFF 180d,P=0.0187).And after PBS injection 180 days,the TH immunoreactive area in the substantia nigra of the control group was(31.04±2.61)%Area.Comparing with the experimental group injected with mouse?-Syn PFFs,we used two independent sample T tests(PFF 180d vs PBS 180d,P<0.0001).The dopaminergic neurons in the right substantia nigra of the experimental group were reduced by approximately 21.26%,and the difference was statistically significant.4.At 30 days,90 days,180 days post injection of mouse ?-Syn PFFs,tissue immunohistochemical showed the number of astrocytes in pontine sections of the experimental group was(7.83±1.47),(29.67±3.98),(49.17±4.22).One-way ANOVA method was used for analysis of variance(P<0.0001),the difference was statistically significant.And then Sidak method was used for post hoc multiple comparison(PFF 30d vs PFF 90d,P<0.0001;PFF 30d vs PFF 180d,P<0.0001;PFF 90d vs PFF 180d,P<0.0001),the differences are statistically significant.5.Compared with mice in the PBS control group,behavioral statistical analysis of?-Syn PFFs experimental group showed the limb motor coordination function and limb muscle strength progressive deterioration,but no significant depression occurred.Conclusion1.Injecting mouse ?-Syn PFFs into the striatum of mice can induce the formation of?-Syn positive inclusion bodies and spread along the striatum projection pathway.2.Compared with mice in the PBS control group,the dopaminergic neurons in the right substantia nigra of the ?-Syn PFFs experimental group mice progressively decreased.3.Over time,the number of astrocytes in the pontine of the ?-Syn PFFs experimental group gradually increased.4.Injection of mouse ?-Syn PFFs in mice striatum can induce PD-like behavioral phenotype in mice.