Meningeal Lymphatic Dysfunction In A Mouse Model Of Parkinson’s Disease Induced By Intrastriatal Injection Of α-synuclein Fibrils And The Underlying Mechanism

BackgroundRecent studies have shown that there are lymphatic-like vessels in the dura mater of mammals and rodents.These vessels are composed of classic lymphatic endothelial cell and have similar functions to peripheral lymphatic vessels.Hence,they are called meningeal lymphatic vessels(mLVs).mLVs can drain both soluble macromolecules and cells from the cerebrospinal fluid to the peripheral deep cervical lymph nodes(dcLNs),which improves the understanding of the fluid balance and waste removal in central nervous system.In addition,more and more animal studies suggest the potential involvement of mLVs in neurological disorders such as neurodegenerative diseases and sequelae of post-traumatic brain injury.For instance,Mesquita et al.found that disruption of mLVs in transgenic mouse models of Alzheimer’s disease(AD)promotes parenchymal amyloid-(3(Aβ)accumulation.Similarly,blocking the meningeal lymphatic drainage in a transgenic mouse model of Parkinson’s disease(PD)increases the phosphorylated α-synuclein(pα-syn)inclusions.However,to date,no evidence has been seen in patients with neurodegenerative diseases to support the findings from animal models.Our group previously used dynamic enhanced magnetic resonance technology and found that patients with idiopathic PD had drainage dysfunction of mLVs and slowed perfusion of dcLNs.Nevertheless,the underlying mechanism needs further exploration.ObjectiveThe current study plans to observe the morphological and functional changes of mLVs in a C57 mouse model of PD induced by bilateral intrastriatal injection ofα-syn preformed fibrils(α-syn PFFs)and explore possible pathological mechanisms.Then,after the bilateral dcLNs are ligated,α-syn PFFs are injected into the bilateral striatum of another C57 mouse model observe the effect of blocking mLVs drainage on the PD-like pathology.Method1.The preparation and validation of mouse α-syn PFFs.2.Eight-week-old C57 mice were randomly divided into control group,α-syn group and ligated α-syn group.Phosphate buffered saline(PBS)or α-syn PFFs were injected into the bilateral striatum of mice in the first two groups,respectively.After bilateral dcLNs were ligated,the α-syn PFFs were injected into the bilateral striatum of mice in ligated α-syn group.3.The mice were sacrificed and perfused at 1,3 and 6 months post injection(mpi).The brain,dura mater and other tissues were preserved.4.The immunohistochemistry(IHC)and Western blot(WB)were used to detect pα-syn inclusions in the nigrostriatal pathway.The immunofluorescence(IF),WB and transmission electron microscopy(TEM)were used to evaluate the morphological and drainage changes of mLVs.5.The motor function of mice was evaluated by rotarod test,wire hang test and open filed test,and the memory function was tested by Y maze test.Result1.The average length of the α-syn PFFs was 44.15 ± 12.83 nm after sonication.2.Compared with the mice in control group,the IHC revealed a gradual increase in the pα-syn inclusions in the nigrostriatal pathway of mice in α-syn group over 1-6 months post-injection.At 6 mpi,there is a loss of dopaminergic neurons in the substantia nigra,and behavioral assessments of these mice indicated impaired motor coordination and balance.3.Compared with the mice in control group,a significant increase in meningeal macrophages with strong pα-syn immunoreactivity was observed in mice in the α-syn group at 6 mpi and this correlated with up-regulated expression of inflammatory cytokines in the meninges.Additionally,a loss of tight junction proteins between meningeal lymphatic endothelial cells was observed.The drainage of mLVs was also impaired.4.Ligation of dcLNs blocked the drainage of mLVs.The mice in ligated α-syn group displayed significantly more pα-syn inclusions in the nigrostriatal pathway than mice in α-syn group at 1,3 and 6 mpi.The mice in ligated α-syn group also exhibited exacerbated motor dysfunction at 3 mpi and memory deficits at 6 mpi.Conclusion1.Bilateral intrastriatal injection of α-syn PFFs could induce the formation and propogation of pα-syn positive inclusions in the nigrostriatal pathway.The phagocytosis of pα-syn by meningeal macrophages causes meningeal inflammation and the latter leads to the impairment of the structure and function of mLVs.2.Bilateral intrastriatal injection of α-syn PFFs after ligation of bilateral dcLNs could aggravate the deposition of pα-syn positive inclusions in the nigrostriatal pathway and exacerbate motor disorders.