| Backgroundα-synuclein(α-syn)positive inclusion bodies are the main pathological features of Parkinson’s disease(PD).Injecting α-syn fibrils(α-syn PFF)into the bilateral striatum of animals to simulate the pathological progression of PD has become the most important method to study the progression of PD.It is well known that the formation of α-syn inclusion bodies in the central nervous system and projection and dissemination along neuronal axons are the main process of the pathological formation of PD,and the clearance of α-syn inclusion bodies in brain tissues has become the main target for the treatment of PD.The cerebrospinal fluid(CSF)enters the brain parenchyma through the perivascular space of the arteries,mixes with the Interstitialfluid(ISF),and drains into the perivascular space of the veins.This system is called the glymphatic system.The glymphatic system is a new way to clear metabolic waste from the brain,which plays an important role in many neurological diseases.Aquaporin4(AQP4)in the foot process of astrocytes mediates the rapid flow exchange system of CSF-ISF,which plays a key role in the transport of the lymphoid system.However,few studies have explored whether the expression of AQP4 is correlated with the glymphatic clearance function and α-syn.ObjectiveIn our study,a model of Parkinson’s disease in AQP4-deficient and C57BL/J mice with injection of α-syn fibrils in bilateral striatum,build different genetic background of PD mice model,to observe the effects of exogenous α-syn fibers on AQP4-deficient transgenic mice,and explore the relationship between the accumulation of α-syn and glymphatic dysfunction,and to explore the potential pathological mechanism of PDMethods1.α-syn fibers were prepared and length was measured by ImageJ software.2.AQP4+/-and WT mice aged 6-8 weeks were randomly divided into 4 groups.The experimental group(AQP4-PFF group and WT-PFF group)was injected withα-syn PFF in bilateral striatum.Control group(AQP4-PBS group and WT-PBS group)were injected with Phosphatebufferedsaline(PBS).3.Brain tissue was collected after 1 month,3 months and 6 months of perfusion,respectively.4.Immunohistochemistry(IHC)was used to detect the distribution of pathological α-syn positive inclusion body deposition,the number of TH positive cells in the brain.Western blotting(WB)was used to detect pathological pα-syn positive inclusion body deposition5.The motor function of mice was tested by rotatord test to detect the coordination ability of mice.The muscle tension of mice was detected by wire hang test.The spontaneous activity of mice was detected in the open field.6.Immunofluorescence(IF)was used to detect the expression of AQP4 in the brain of four groups of mice.7.AQP4+/-and C57BL/J mice aged 6-8 weeks were randomly divided into 4 groups with 6 mice in each group:the experimental group(AQP4-PFF group and WT-PFF group)and the control group(AQP4-PBS group and WT-PBS group),AlexaFluor594 labeled α-syn was inj ected into the striatum of the four groups of mice,respectively.Post after 120 min,the samples were collected and frozen sections were taken to detect the difference in the clearance of glymphatic system between the four groups.Fluorescent Evans blue(EB)was injected into the cerebral cisterns of four groups of mice,and left for 30 minutes.The samples were collected and frozen sections were made to detect the difference in the function of glymphatic drainage between four groups of mice.Results1.The average length of the prepared α-syn PFF was measured to be 46.92±26.2nm2.1 month after α-syn PFF injection,a small amount of α-syn inclusion bodies appeared in the striatum,the nigra,the amygdala,and the cortex of mice.In the experimental group,there was no significant difference in α-syn accumulation between AQP4+/-mice and WT mice.3 months after injection,a large number ofα-syn inclusion bodies appeared in the striatum,substantia nigra,amygdala and cortex of mice in the experimental group,but the accumulation of α-syn in AQP4+/mice was significantly higher than that in the WT group.6 months after injection,the pathologic α-syn accumulation in α-syn PFF group was significantly increased,but the inclusion bodies of AQP4+/-mice was still significantly higher than that in WT group.However,at the same time,we found that the pathological accumulation in the substantia nigra was significantly reduced 6 months after injection compared with that in 3 months,but the pathological accumulation of AQP4+/-mice was still higher than that in WT group.3.6 months after the injection of α-syn PFF,the four groups of mice were analyzed by western blot,and α-syn deposition was found in the cortex and striatum of the experimental group.Compared with the WT-PFF group,the accumulation ofα-syn in the cortex and striatum of the four groups of mice was significantly increased in the AQP4+/-PFF group(P<0.0001).4.The immunoreactive neurons in the substantia nigra of mice in the experimental group decreased in a time dependent manner at 1,3 and 6 months after the injection of α-syn PFF.The results of stereological quantitative analysis showed that there was no significant difference in the TH positive neurons in the substantia nigra of the four groups after 1 month of injection,but the number of TH positive neurons in the brain of the experimental group was significantly decreased from 3 to 6 months after injection(P<0.05).Compared with WT-PFF group,the decrease of TH positive neurons was more obvious in AQP4+/-PFF group(P<0.001).5.Motor function of mice was detected.There was no significant difference in motor function among each group 1 month after injection.3 months after injection,the behavioral impairment of AQP4+/-PFF mice was significantly earlier than that of WT-PFFS mice(P<0.05).After 6 months of injection,the behavioral disorders of mice in the AQP4+/-PFF group were significantly worse than those in the WT-PFF mice(P<0.01).6.There were significant differences in the expression of AQP4 in the four groups of mice.Although the AQP4 expression of AQP4+/-mice was decreased compared with WT-PBS,the AQP4 expression of AQP4+/-mice was further decreased after the injection of α-syn PFF.7.Low AQP4 expression can impair the transport function of glymphatic system.On the one hand,the low expression of AQP4 impaired the function of glymphatic drainage.Compared with WT group,the function of glymphatic drainage was slightly impaired in AQP4+/-mice(P<0.05),and the AQP4+/-PFF group was the most severely impaired(P<0.01).On the other hand,the low expression of AQP4 impaired glymphatic clearance.Compared with WT group,the glymphatic clearance was slightly impaired in AQP4+/-mice(P<0.05),and the damage was the most serious in AQP4+/-PFF group(P<0.01).Conclusions1.Injecting exogenous α-syn fibers into the striatum can induce the distribution and dissemination of α-syn positive inclusion bodies in the brain.2.The low expression of AQP4 can accelerate the pathological accumulation ofα-syn and lead to the aggravation of the loss of dopamine neurons,leading to the early appearance of motor function symptoms.3.The low expression of AQP4 can cause the dysfunction of glymphatic transport,which may be the main factor for the clearance of α-syn. |
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