| BackgroundPure autonomic failure is an extremely rare,idiopathic,slowly progressive autonomic nervous system disease that selectively involves the myelinated and unmyelinated autonomic nerve fibers.The main features of pure autonomic failure are orthostatic hypotension,sweating disorders,gastrointestinal,urogenital dysfunction,erectile dysfunction and other autonomic dysfunction,involving cardiovascular disease,endocrine disease,neurodegenerative disease,infectious disease,oncology and many other fields.The clinical phenotype is complex and difficult to diagnosis.α-Synucleinopathies include pure autonomic failure,multiple system atrophy and Parkinson’s disease,which are caused by abnormal deposition ofα-syn-positive inclusions on neurons,nerve fibers or glial cells.The autopsy results of patients with pure autonomic failure show that a large number of eosinophilic Lewy bodies accumulation in sympathetic ganglia and loss of neuron are detected in early pure autonomic failure patients,suggesting the formation of α-syn positive inclusions in the sympathetic ganglia may participate in the initiation of pure autonomic failure.Previous studies have confirmed that α-syn-positive inclusions formed and spread in the nervous system as a mode of "prion protein".Based on the above research,we intend to inoculate α-syn preformed fibrils into bilateral celiac ganglia of TgM83+/-mice to induce the formation of α-syn-positive inclusions and spread through the autonomic pathway to both the central nervous system and the autonomic innervation of peripheral organs.It is the first time to establish a pure autonomic failure animal model in the world,which is helpful to verify the diffusion theory of α-syn-positive inclusions and probes the diffusion pathway ofα-syn-positive inclusions in pure autonomic failure.The pathological mechanism of pure autonomic failure is revealed,and the research gap of pure autonomic failure animal models is filled.ObjectiveInoculation of α-syn preformed fibrils in celiac ganglia induces the formation ofα-syn-positive inclusions in TgM83+/-mice and spread through the autonomic pathway to both the central nervous system and the autonomic innervation of peripheral organs.The mice exhibit various autonomic dysfunctions.And it can be used as a mouse model of pure autonomic failure.Method1.The α-syn monomer resuspended at a concentration of 1 mg/ml was agitated by a magnetic stirrer for 7 days at 37 ℃ and 350 rpm,and an α-syn preformed fibrils was obtained by ultrasonic treatment.The nature of the α-syn preformed fibrils forms was examined using a transmission electron microscope.Under the operating microscope,α-syn preformed fibrils or PBS buffer(4.0 μl on each side)were injected into bilateral celiac ganglia in 2-month-old male TgM83+/-mice,and the gastrointestinal function of the mice were evaluated monthly by activated carbon gavage.The sweating function of the mice were evaluated by the starch iodine assay,and the motor function indexes of the mice were respectively evaluated by the rotarod test,the footprint test,the hanging wire test and the beam walking test.Immunohistochemistry,Western blot and immunofluorescence were performed on the brain,spinal cord,bilateral celiac ganglia,gastrointestinal tissues to detect the content of α-syn-positive inclusions.2.All of the statistical data were analyzed using statistical software such as SPSS 21.0 and GraphPad Prism 7.The mice were divided into experimental group and control group.Paired or unpaired Student’s t test was performed to compare the results of immunohistochemistry,sweat function,and behavioral evaluation.The survival curve was calculated by the log-rank test and Gehan-Breslow-Wilcoxon test.Immunohistochemical results are analyzed by ImageJ software.P≤0.05 indicates that the difference between the two groups is statistically significant.Measurement data are expressed as mean ±error of the mean((?)±s).test standard α=0.05.Result1.The α-syn preformed fibrils(47.8±23.7 nm)prepared in vitro were inoculated into bilateral celiac ganglia of the experimental group of mice.8 weeks later,in the various segments of the central autonomic pathway(3rd thoracic spinal cord,the 11th thoracic spinal cord,medulla,pons(lower),pons(upper)and midbrain)exhibited the distribution of phosphorylated α-syn.The formation of α-syn-positive inclusions was detected in neurons related to autonomic nerve.Intermediolateral nucleus and Lamina VII of the thoracic spinal cord showed abundant phosphorylated α-syn.α-Syn-positive inclusions aggregated in neurons in autonomic related regions,which is significantly different from the control group.The unilateral celiac ganglia injected with α-syn preformed fibrils mice showed more abundant phosphorylated α-syn in the ipsilateral side of the brain and spinal cord after 8 weeks.The reticular structure was firstly invaded in the brain and spread to the bilateral brain and spinal cord 12 weeks later.2.Compared with the control group,the mice in the experimental group were found to exhibit phosphorylated α-syn neuronal bodies and axonal profiles in the stellate ganglia and celiac ganglia for 4 weeks after the inoculating α-syn preformed fibrils.Two months later,in autonomic effector organs(gastrointestinal tract and skin)showed a few scattered phosphorylated α-syn inclusions.Over time,phosphorylatedα-syn inclusions in the celiac ganglia and autonomic effector organs became more condensed and intense.In unilateral inoculating of α-syn preformed fibrils mice,phosphorylated α-syn inclusions were detected in autonomic effector organs at about 12 weeks.Compared with the control group,the survival time of mice in the experimental group was significantly reduced.The median survival times of the experimental group mice and the control group mice unilaterally are 24 weeks and 100 weeks of age respectively.3.Compared with the control group,the mean gastrointestinal transit of the mice in the experimental group was significantly reduced and the defecation interval time was significantly prolonged after 14 weeks of induction of α-syn preformed fibrils.The unilaterally α-syn preformed fibrils injected mice developed constipation at about 16 weeks,which were much delayed compared with the bilaterally injected mice.The increase in sweat gland number was significantly attenuated in the experimental group of mice after α-syn preformed fibrils induction for 14 weeks,and the mice with unilateral injection of α-syn preformed fibrils developed hypohidrosis at around 18 weeks.motor dysfunction was not displayed in the experimental and control groups.4.There was no phosphorylated α-syn in the dorsal motor nucleus of the vagus within 28 weeks after cutting off the vagus nerve distributed near the gastrointestinal tract.In the sham operation group,the vagus nerve dorsal motor nucleus region and other regions and nuclei existed phosphorylated α-syn as early as 8 weeks.ConclusionInoculation of α-synuclein preformed fibrils via ganglia ganglia induces the formation of pathological α-syn in TgM83+/-mice and spreads through the autonomic pathway to both the central nervous system and the autonomic innervation of peripheral organs.Mice exhibit autonomic dysfunction without motor dysfunction,which is consistent with the characteristics of pure autonomic failure.This mouse model may help to define the mechanistic link between transmission of pathologicalα-syn and assess autonomic dysfunction in pure autonomic failure. |
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