Modeling method for animal with cytopathology specificity, fast eye movement and sleep behavior disorder

Abstract

The invention relates to the field of animal models, in particular to a preparation method of a fast eye movement sleep behavior disorder animal model with cytopathology specificity, which comprises the following steps: injecting adeno-associated virus for inducing neuronal overexpression alpha-synuclein into a capped karyobrain area on the back and the outer side of a non-human tested animal, and injecting the neuron-derived extracellular vesicles or the oligodendroglia-derived extracellular vesicles into the capped karyocerebral region on the back outer side of the non-human tested animal again. The model prepared by the method disclosed by the invention has the disease characteristics of fast eye movement sleep and has the characteristic that Parkinson-like phenotype is transformed. The model disclosed by the invention can be used for researching the etiology and pathological mechanism of transformation of rapid eye movement sleep behavior disorder to Parkinson's disease or multi-system atrophy and screening prevention and treatment drugs, and has remarkable significance for clinically providing effective drug treatment targets and prevention and treatment means.

Description

Technical Field

[0001] This invention relates to the field of animal models, and more specifically, to a method for establishing a model of cell pathology-specific rapid eye movement sleep behavior disorder in animals. Background Technology

[0002] Rapid eye movement (REM) sleep behavior disorder (RBD) is a parasomnia characterized by REM sleep without atonia (RSWA) during REM sleep. RBD patients often experience RSWA and dream enactment behavior (DEB), which frequently leads to accidental injuries and decreased sleep quality, impacting daily life. Furthermore, RBD is a specific early marker of Parkinson's disease (PD) and multiple system atrophy (MSA). Epidemiological studies show that 70-90% of RBD patients will progress to PD or MSA within 10-15 years of diagnosis. Therefore, research into the pathogenesis of RBD is of significant importance, especially simulating the transformation from RBD to PD or MSA in animals, which is crucial for elucidating the disease's development and progression.

[0003] Misfolding and aggregation of α-synuclein (α-syn) are common pathological features of both neuronal dysplasia (PD) and microsporous acne (MSA). However, the cells in which α-syn misfolding and aggregation occur differ significantly between PD and MSA: in PD, α-syn pathologically aggregates within neurons, while in MSA, it aggregates within oligodendrocytes. These different pathological characteristics dictate that animal models characterizing the transformation of recurrent neuronal dysplasia (RBD) into PD or MSA should possess cell pathological specificity: for animal models transforming from RBD to PD, the pathological feature should be the aggregation of α-syn within neurons; for animal models transforming from RBD to MSA, the pathological feature should be the aggregation of α-syn within oligodendrocytes. However, current RBD animal models lack cell pathological specificity. One model is the transgenic mouse overexpressing α-syn (SNCA). A53T, through transgenic technology, caused systemic overexpression of pathological α-syn protein in model mice (TAGUCHI T, IKUNO M, HONDO M, et al. α-Synuclein BACtransgenic mice exhibit RBD-like behaviour and hyposmia: a prodromal Parkinson's disease model[J]. Brain, 2020,143(1):249-265.). Although the model mice exhibited the key sleep phenotype RBD and pathological α-syn protein in the sublaterodorsal tegmental nucleus (SLD), a nucleus that governs REM sleep atony, it did not possess the characteristics of cellular pathology specificity. In addition, another classic model involves injecting pre-prepared α-synucleinopathy fibrils into mice with substantia nigra (SLD) to induce neuronal loss and pathological accumulation of α-synucleinopathy, as well as the appearance of sleep disorder phenotypes associated with Recurrent Parkinson's Disease (RBD) (Shen Y, Yu WB, Shen B, et al. Propagated α-synucleinopathy recapitulates REM sleep behaviour disorder followed by parkinsonian phenotypes in mice. Brain. 2020. 143(11):3374-3392.). This model mice even exhibit characteristics of Parkinson's disease-like transformation, including behavioral tests revealing decreased motor coordination, olfactory disturbances, and constipation—non-motor symptoms of Parkinson's disease. Histopathological examination revealed the loss of dopaminergic neurons in the substantia nigra, a reduction in dopaminergic nerve fibers in the striatum, and pathological accumulation of α-synucleinopathy in the substantia nigra, striatum, olfactory bulb, and intestines. However, this model is mainly characterized by the appearance of pathological α-synuclein within neurons, and therefore lacks cellular pathology specificity, making it insufficient to characterize the transformation of RBD into PD or MSA. Therefore, a cellular pathology-specific animal model of RBD is still urgently needed.

[0004] Chinese patent document CN113016719A discloses a method for preparing a synuclein-pathological REM sleep behavior disorder model. This involves stereotactic injection of pre-fabricated fibrils (PFFs) capable of inducing synuclein pathology into the SLD nucleus. Utilizing the pathological induction properties of these fibrils through "dissemination-nucleation," a mouse RBD model that can revert to a Parkinson's phenotype is constructed. Chinese patent document CN113005150A discloses a novel method for preparing an animal REM sleep behavior disorder model. This involves stereotactic injection of a retrotransporter into the ventral giant cell reticular nucleus (GiV). The retrotransporter can be retrotransported from the GiV to the SLD nucleus and express a chromogenic marker and Cre recombinase. The chromogenic marker can label neurons in the SLD nucleus that have fiber connections to downstream neurons.

[0005] However, no modeling method for a cell pathology-specific rapid eye movement sleep behavior disorder has been reported to date. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing a cell pathology-specific animal model of rapid eye movement (REM) sleep behavior disorder, as well as the related applications of the aforementioned animal model of REM sleep behavior disorder.

[0007] The inventors discovered in their research that by using an adeno-associated virus (AAV) vector as a tool, they were able to induce overexpression of α-synuclein (α-syn) in the sublaterodorsal tegmental nucleus (SLD) of mice in both neurons and oligodendrocytes. This allowed them to construct neuronal pathology with α-syn overexpression and oligodendrocyte pathology with α-syn overexpression in mouse SLDs. One month after AAV injection, neuropathic mice with SLD neuronal overexpression of α-syn were injected again with neuronal extracellular vesicles (NEVs) derived from the plasma of Parkinson's disease (PD) patients, and oligodendrocyte pathological mice with SLD oligodendrocyte overexpression of α-syn were injected with oligodendrocyte extracellular vesicles (OEVs) derived from the plasma of multiple system atrophy (MSA) patients. After injecting PD-NEV into neuropathic mice and MSA-OEVs into oligodendrocyte pathological mice, both types of mice showed corresponding cell-specific pathology and neuronal loss in the SLD brain region, as well as the RBD sleep disorder phenotype, motor and nonmotor symptoms common to both PD and MSA, and neurodegenerative changes in the substantia nigra-striatal dopaminergic system.

[0008] In a first aspect, the present invention provides a method for preparing a cell pathology-specific animal model of rapid eye movement sleep behavior disorder, wherein the cell pathology is neuronal pathology or oligodendrocyte pathology.

[0009] The preparation method involves injecting adeno-associated virus (AAV) that induces neuronal overexpression of α-synuclein (α-syn) into the dorsolateral tegmental nucleus (SLD) brain region of a non-human test animal. One month after the AAV injection, neuronal-derived extracellular vesicles or oligodendrocyte-derived extracellular vesicles are injected again into the SLD brain region of the non-human test animal.

[0010] Furthermore, the non-human test animal is a mouse.

[0011] Furthermore, the neuronal extracellular vesicles are derived from PD-NEV, a neuronal extracellular vesicle from a Parkinson's disease (PD) patient; and the oligodendrocyte extracellular vesicles are derived from MSA-OEV, an oligodendrocyte extracellular vesicle from a multiple system atrophy (MSA) patient.

[0012] Furthermore, the neuronal extracellular vesicle PD-NEV is derived from the plasma of Parkinson's disease (PD) patients; the oligodendrocyte extracellular vesicle MSA-OEV is derived from the plasma of MSA patients.

[0013] Furthermore, the AAV, PD-NEV, and MSA-OEV were injected into the bilateral SLD brain regions of mice using stereotactic injection.

[0014] Furthermore, the AAV is used in conjunction with PD-NEV or MSA-OEV to prepare animal models of rapid eye movement sleep behavior disorder.

[0015] In one specific embodiment of the present invention, the method for preparing the cell pathology-specific rapid eye movement sleep behavior disorder animal model includes the following steps:

[0016] In male C57BL / 6J mice, retrograde tracer virus (AAV-Retro-Cre-hSyn-EGFP) was injected into the mouse giant cell reticular nucleus (ventral part, GiV) to retrogradely label neurons in the SLD brain region. Simultaneously, AAV (AAV-DIO-mCherry-hSNCA), which induces neuronal overexpression of α-syn, was injected into the mouse SLD brain region to induce neuronal overexpression of α-syn protein (neuronal cell pathology specific). Four weeks later, NEV from PD patient plasma was injected into the mouse SLD brain region again. After another four weeks, a neuronal cell pathology specific REM sleep behavior disorder animal model was obtained.

[0017] In one specific embodiment of the present invention, the method for preparing the cell pathology-specific rapid eye movement sleep behavior disorder animal model includes the following steps:

[0018] C57BL / 6J male mice were injected with AAV (AAV-DIO-mCherry-hSNCA and AAV / mMBP-eGFP-Cre) that induce oligodendrocyte overexpression of α-syn to the SLD brain region of mice, inducing oligodendrocyte overexpression of α-syn protein (oligodendrocyte pathology specific). Four weeks later, OEV from MSA patient plasma was injected into the SLD brain region of mice again. After another four weeks, an oligodendrocyte pathology specific REM sleep behavior disorder animal model was obtained.

[0019] Furthermore, the aforementioned cell pathology-specific animal model of rapid eye movement sleep behavior disorder exhibits neuronal or oligodendrocyte cell pathology-specific α-syn protein expression.

[0020] Furthermore, neuronal loss was observed in the SLD brain region of the aforementioned cell pathology-specific rapid eye movement sleep behavior disorder animal model.

[0021] Furthermore, the aforementioned cell pathology-specific animal model of REM sleep behavior disorder exhibits increased muscle tone during REM sleep.

[0022] Furthermore, the motor coordination ability of the aforementioned cell pathology-specific rapid eye movement sleep behavior disorder animal model is reduced.

[0023] Furthermore, the striatum and substantia nigra of the aforementioned cell pathology-specific rapid eye movement sleep behavior disorder animal model showed neurodegenerative lesions of the dopaminergic system.

[0024] In a second aspect, the present invention provides the application of adeno-associated virus (AAV) overexpressing α-synuclein (α-syn) in combination with PD-NEV or MSA-OEV in the preparation of animal models of rapid eye movement sleep behavior disorder.

[0025] A third aspect of the present invention provides the use of a cell pathology-specific rapid eye movement sleep behavior disorder animal model prepared using the preparation method described above in screening and / or preparing drugs for the prevention and treatment of cell pathology-specific rapid eye movement sleep behavior disorder.

[0026] A fourth aspect of the present invention provides a method for screening drugs to prevent and treat cytopathologically specific rapid eye movement sleep behavior disorder, comprising the following steps:

[0027] The drug is administered to an animal model prepared using the above method and compared with an animal model that has not been given the drug. The drug that leads to improvement or cure of symptoms of REM sleep behavior disorder after administration is the therapeutic drug.

[0028] The advantages of this invention are:

[0029] The model prepared by the method of this invention is characterized by cytopathological overexpression of α-syn protein in key brain regions, specifically neuronal or oligodendrocyte pathologies of the subarachnoid process (SLD). Furthermore, the model exhibits disease characteristics such as increased muscle tone and hypotonia during REM sleep, neuronal loss in the key brain region SLD, and the transformation to a Parkinsonian phenotype (decreased motor coordination and dopaminergic neurodegeneration of the substantia nigra-striatum). This model can be used to study the etiology and pathological mechanisms of RBD transforming into PD or MSA, and to screen drugs for preventing and treating RBD-PD or MSA transformation. It is of significant importance for providing effective drug targets and preventative measures for clinical practice. Attached Figure Description

[0030] Figure 1 A. Electron micrographs of extracellular vesicles in neurons and oligodendrocytes. Top image scale bar: 200 nm; Bottom image scale bar: 50 nm. B. Schematic diagram of nanoparticle tracking analysis of extracellular vesicles in neurons and oligodendrocytes. C. Western blot image verifying marker proteins of extracellular vesicles in neurons and oligodendrocytes.

[0031] Figure 2 A. Neuropathological and oligodendrocyte pathological findings of SLD mice in the neuronal pathological group, oligodendrocyte pathological group, and control group at 1, 3, and 5 months after modeling. Thumbnail scale bar: 40 micrometers; magnified scale bar: 20 micrometers. B. Statistical analysis of the number of neurons in SLD mice in the neuronal pathological group, oligodendrocyte pathological group, and control group at 1, 3, and 5 months after modeling. One-way ANOVA was used for comparisons among the three groups. P < 0.01, P < 0.001, P <0.0001. C. Statistical analysis of pS129 α-syn pathological findings in the neuronal pathological group, oligodendrocyte pathological group, and control group of mice at 1, 3, and 5 months after modeling. One-way ANOVA was used for comparisons among the three groups; P < 0.05, P < 0.01, P < 0.001, P < 0.0001.

[0032] Figure 3Sleep phases and EEG / EMG waveforms during RSWA episodes in mice of the neuronal pathology group, oligodendrocyte pathology group, and control group at 1, 3, and 5 months after modeling (arrows indicate RSWA). REM episode: REM sleep phase; RSWA episode: RSWA phase; RBD episode: RBD phase.

[0033] Figure 4 Statistical analysis of electromyography (EMG) intensity during REM sleep in mice of the neuronal pathology group, oligodendrocyte pathology group, and control group at 1, 3, and 5 months after modeling. EMG: electromyography; REM: rapid eye movement (REM) sleep; NREM: non-rapid eye movement (NREM) sleep. One-way ANOVA was used for comparisons among the three groups. P < 0.05, P < 0.01, P < 0.0001.

[0034] Figure 5 Statistical analysis of the rotarod test results of the neuronal pathological group, oligodendrocyte pathological group, and control mice at 1, 3, and 5 months after modeling.

[0035] Figure 6 A. Immunohistochemical staining of TH in striatal dopaminergic nerve fibers of mice in the neuronal pathological group, oligodendrocyte pathological group, and control group at 1, 3, and 5 months after modeling. Thumbnail scale bar: 500 micrometers. Enlarged view scale bar: 50 micrometers. B. Statistical analysis of the relative intensity of TH signal in striatal dopaminergic nerve fibers of mice in the neuronal pathological group, oligodendrocyte pathological group, and control group at 1, 3, and 5 months after modeling. One-way ANOVA was used for comparison among the three groups. P <0.05, P < 0.01, P < 0.001, P < 0.0001.

[0036] Figure 7 A. Immunohistochemical staining of TH staining on dopaminergic neurons in the substantia nigra of mice in the neuronal pathological group, oligodendrocyte pathological group, and control group at 1, 3, and 5 months after modeling. Thumbnail scale bar: 250 micrometers. Enlarged view scale bar: 50 micrometers. B. Statistical analysis of the number of dopaminergic neurons in the substantia nigra of mice in the neuronal pathological group, oligodendrocyte pathological group, and control group at 1, 3, and 5 months after modeling. One-way ANOVA was used for comparisons among the three groups. P < 0.05, P < 0.01, P < 0.001, P < 0.0001. Detailed Implementation

[0037] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0038] Example 1: Acquisition and identification of extracellular vesicles of neurons from Parkinson's disease patients and extracellular vesicles of oligodendrocytes from patients with multiple system atrophy

[0039] 1.1 Experimental Materials

[0040] Peripheral blood samples from patients with Parkinson's disease and multiple system atrophy; L1CAM antibody (abcam, ab80832); CNPase antibody (abcam, ab237961); normal mouse IgG (Santa-Cruz, sc-2025), magnetic bead conjugation kit (Thermo, 14302D); PBS; fetal bovine serum albumin (BSA); Flotilin-1 antibody (abcam, ab41927); CD63 antibody (abcam, ab134045); Synaptophysin antibody (abcam, ab32127); Calnexin antibody (abcam, ab213243); Cytochrom c antibody (Proteintech, 66264-1-lg); protease inhibitors, RIPA lysis buffer, TBST.

[0041] 1.2 Experimental Methods

[0042] 1.2.1 Capture extracellular vesicles (NEVs) of neurons in patients with Parkinson's disease (PD) and extracellular vesicles (OEVs) of oligodendrocytes in patients with multiple system atrophy (MSA).

[0043] 1.2.1.1 Antibody-Magnetic Bead Conjugation

[0044] (1) Weighing of magnetic beads: Weigh 5 mg of magnetic beads using a precision balance. For every 5 mg of magnetic beads, 50 mg of antibody is used for incubation and conjugation. The purchased antibody concentration is 1 mg / mL.

[0045] (2) Washing magnetic beads: Add 1 mL of C1 solution to wash 5 mg of magnetic beads, centrifuge briefly, place on a magnetic rack, and remove the supernatant.

[0046] (3) Antibody conjugation: Add 200 μL of C1 solution, 50 μL of antibody, and 250 μL of C2 solution. After mixing, seal with sealing film and incubate at 37°C for 24 h on a vertical mixing shaker.

[0047] 1.2.1.2 Washing of magnetic bead-antibody complex

[0048] (1) Remove the coupled magnetic bead-antibody complex from the shaker, briefly separate it, place it on the magnetic rack for 1 minute, and carefully remove the supernatant.

[0049] (2) HB rinse: Add 800 μL of HB buffer, mix gently, place the coupling reaction tube on a magnetic rack for 1 minute, and carefully remove the supernatant.

[0050] (3) LB cleaning: Add 800 μL of LB buffer, mix gently, place on a magnetic rack for 1 minute, and carefully remove the supernatant.

[0051] (4) SB cleaning: Add 800 μL of SB buffer, mix gently, place on a magnetic rack for 1 minute, and carefully remove the supernatant.

[0052] (5) Repeat the SB cleaning step, and clean the coupled magnetic beads 3 times in total.

[0053] (6) Add 500 μL of SB, mix gently, and divide the magnetic beads into 100 μL tubes, for a total of 5 tubes.

[0054] (7) Seal the tube with sealing film and store it in an environment of 4°C for later use.

[0055] 1.2.1.3 Immunoprecipitation to capture extracellular vesicles

[0056] (1) Thaw the plasma rapidly in a 37°C water bath. The plasma aliquoting condition is 510 μL per tube.

[0057] (2) Add the working solution concentration of protease inhibitor to the plasma and mix well.

[0058] (3) Centrifuge the plasma at 4°C, 2,000×g for 15 minutes.

[0059] (4) After centrifugation, take 480 μL of plasma supernatant and transfer it to a new EP tube.

[0060] (5) Centrifuge further at 4°C, 12,000×g for 30 minutes.

[0061] (6) During the 30 minutes of centrifugation, simultaneously process the antibody-magnetic bead complex:

[0062] (i) After removing it from the 4°C environment, briefly separate it and place it on a magnetic rack for 1 minute to remove the supernatant.

[0063] (ii) Add 1 mL of pre-cooled PBS, mix gently, place on a magnetic rack for 1 minute, remove the supernatant, and set aside.

[0064] (7) Take 450 μL of the supernatant from the centrifuged plasma, add the antibody-magnetic bead complex, and then add 2 times the volume of PBS (900 μL), and mix gently.

[0065] (8) Wrap the container with sealing film and then place it in a cold room at 4°C and mix it with a vertically rotating shaker for 24 hours.

[0066] 1.2.1.4 Elution of extracellular vesicles

[0067] (1) Briefly separate the tube and place it on the magnetic rack for 1 minute to remove the supernatant.

[0068] (2) Wash the magnetic beads four times with 0.1% BSA / PBS (w / v):

[0069] (i) Add 1 mL of 0.1% BSA / PBS and mix gently.

[0070] (ii) Briefly leave.

[0071] (iii) Place the magnetic beads on the magnetic rack for 1 minute to remove the supernatant.

[0072] (3) Transfer the magnetic beads using 400 μL of 0.1% BSA / PBS:

[0073] (i) Resuspend the magnetic beads in 200 μL of 0.1% BSA / PBS and mix well.

[0074] (ii) Briefly transfer the magnetic bead mixture to a new EP tube.

[0075] (iii) Add 200 μL of 0.1% BSA / PBS, mix again, and transfer to a new EP tube.

[0076] (4) Place the new EP tube on the magnetic rack for 1 minute and remove the supernatant. At this time, the extracellular vesicles are bound to the antibody-magnetic bead complex.

[0077] Choose different operations based on the objectives of the following experiment:

[0078] To obtain extracellular vesicle lysates, add 110 μL of RIPA strong lysis buffer to each EP tube and incubate at room temperature on a Vortex shaker at speed 5-6 for 1 hour.

[0079] To obtain intact extracellular vesicles, add 70 μL of 0.1 M glycine solution (pH=3) to each EP tube, mix well, and incubate at room temperature on a vortex shaker at speed 5-6 for 15 minutes. Afterward, briefly detach and place on a magnetic rack for 1 minute. The supernatant is the eluent for eluting extracellular vesicles. Collect the supernatant and add 5 μL of 1 M Tris solution (pH=7) to neutralize the pH. Repeat this step twice, collecting the supernatant from both washes.

[0080] 1.2.2 Identification of extracellular vesicles

[0081] 1.2.2.1 Transmission Electron Microscopy (TEM)

[0082] (1) Hydrophilization of copper mesh: Place the copper mesh in the glow discharge apparatus and wait for it to automatically vacuum-discharge to complete before taking out the copper mesh.

[0083] (2) Sample addition: Use reverse forceps to pick up the copper grid, add 8 μL of extracellular vesicle sample onto the copper grid, and let it stand at room temperature for 30 minutes.

[0084] (3) Staining: Use a lint-free paper to absorb the residual sample liquid, add 2% uranium acetate staining solution, incubate for 45 seconds, and then wipe it off with a lint-free paper.

[0085] (4) Drying: Dry the copper mesh under the illumination of a warm lamp. After it is dry, it can be used for electron microscopy.

[0086] (5) Sample loading for electron microscopy: Place the copper mesh into the sample loading rod, wait for the vacuum to stabilize, and gradually increase the voltage. Each time the voltage is increased, wait for the emission to stabilize (80kV-100kV-120kV). Turn on the filament, and after the relevant electron microscopy parameters (Gun / Col) values ​​stabilize, close the valve and observe the sample.

[0087] 1.2.2.2 Nanoparticle Tracking Analysis (NTA)

[0088] Extracellular vesicle samples were diluted at ratios of 1:10 to 1:40 (using filtered PBS solution) to determine the optimal loading concentration for recording. Nanoparticle tracking analysis was performed using ZetaView 8.05.05 SP2 (ParticleMetrix, Germany). Each sample was measured three times, with each recording lasting 20 seconds. The final particle concentration and size were the average of the three measurements.

[0089] 1.2.2.3 Western Blot (WB)

[0090] Extracellular vesicle samples obtained from 5 ml of plasma were used for Western blotting of proteins. Protein concentration was measured using a BCA kit (CST, 7780S) for quantification. 10 μg of protein was loaded into each well. Protein samples were diluted with 2 × Laemmli loading buffer (Merck, S3401) and boiled in a metal bath for 10 minutes.

[0091] (1) Sample loading: Load 10 μg of protein into each well, along with the protein marker, and replenish the different volumes with 1× loading buffer. Use a 4-20% precast gel.

[0092] (2) Electrophoresis: constant voltage 80V electrophoresis, bromophenol blue until it reaches near the bottom of the gel.

[0093] (3) Transfer: The eBlot rapid transfer instrument was used. First, the PVDF membrane was activated with methanol for 1 minute. Then, the gel and PVDF membrane were equilibrated with buffer solution. After clamping with a foam board and removing air bubbles, a "sandwich" structure was formed. The membrane was then inserted into the transfer instrument and transferred for 15 minutes.

[0094] (4) Sealing: Immerse the membrane in 5% skim milk / TBST, shake it on a shaker at room temperature, and seal it for 1 hour.

[0095] (5) Primary antibody: Dilute the primary antibody according to the recommended ratio in the instructions and dissolve it in 5% skim milk / TBST. Incubate overnight for 12 hours on a shaker in a 4°C cold storage.

[0096] The primary antibody dilution ratios are as follows: L1CAM, 1:500; CNPase, 1:200; Flotilin-1, 1:900; CD63, 1:1000; Calnexin, 1:500; cytochrom c, 1:5000; Synaptophysin, 1:20000.

[0097] (6) Washing: Wash the membrane 3 times with TBST on a shaker at room temperature, 5 minutes each time.

[0098] (7) Secondary antibody: Dilute the secondary antibody with TBST at a ratio of 1:5000 and incubate on a shaker at room temperature for 1 hour.

[0099] (8) Washing: Wash the membrane 3 times with TBST on a shaker at room temperature, 5 minutes each time.

[0100] (9) Development: Add ECL developer to the film and expose it automatically using an Invitrogen iBright 1500 exposure machine.

[0101] 1.3 Experimental Results

[0102] See results Figure 1 Negative staining-transmission electron microscopy results showed that the extracted PD-NEV and MSA-OEV were saucer-like vesicle structures. Figure 1 A). NTA results showed that the most prominent particles of PD-NEV and MSA-OEV were around 150 nm in diameter, and their distribution was consistent with the characteristics of extracellular vesicles. Figure 1 B). Western blot results showed that PD-NEV captured with L1CAM antibody and MSA-OEV captured with CNPase antibody were both rich in extracellular vesicle marker proteins such as Flotilin-1 and CD63; NEV was rich in two neuronal marker proteins, L1CAM and synaptophysin; and OEV was rich in CNPase, an oligodendrocyte marker protein. Neither NEV nor OEV contained negative proteins that extracellular vesicles should contain, such as the endoplasmic reticulum marker Calnexin and the mitochondrial marker cytochrome c. Figure 1 C).

[0103] 1.4 Discussion of Results

[0104] Extracellular vesicles from neurons and oligodendrocytes were captured in their plasma using immunoprecipitation with antibodies against neurons and oligodendrocyte-specific marker proteins. The capture of the target extracellular vesicles was confirmed using established methods—negative staining-transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Notably, the captured extracellular vesicles, based on negative staining-transmission electron microscopy and nanoparticle tracking analysis, exhibited diameters primarily consistent with exosomes, ranging from 30 to 150 nm. Western blotting analysis revealed that the captured extracellular vesicles were enriched with CD63 and Flotilin-1, also marker proteins of exosomes. In addition to being rich in the L1CAM protein used for capture, the neuronal-derived extracellular vesicles were also rich in another neuronal marker protein—synaptophysin. This protein is also primarily found on the neuronal membrane and in the cytoplasm, thus confirming the presence of this protein and further validating that the L1CAM-positive extracellular vesicles originated from neurons. Therefore, we can use patient-derived cell-specific extracellular vesicles as pathological seeds to induce pathological development in mouse model mice.

[0105] Example 2: SLD cell-specific pathological mice constructed by extracellular vesicle injection of adeno-associated virus vector and immunofluorescence pathological analysis

[0106] 2.1 Experimental Materials

[0107] Animal modeling: C57BL / 6J mice (18-20g body weight, 8 weeks old); adeno-associated virus (AAV) vectors: AAV2 / Retro-hSyn-Cre-EGFP (Shanghai Taiertu Biotechnology Co., Ltd., S0237-2R); AAV2 / 9-DIO-hSNCA (Shanghai Taiertu Biotechnology Co., Ltd., S0877-9), AAV2 / mMBP-eGFP-iCre (Shanghai Taiertu Biotechnology Co., Ltd., S0830-O); isoflurane; erythromycin eye ointment; medical surgical sutures; cotton swabs.

[0108] Immunofluorescence: OCT; blade; 4% paraformaldehyde (4% PFA); sucrose; Triton-X-100; BSA; PBS; pS129α-syn antibody; NeuN antibody; MBP antibody; anti-fluorescence quenching mounting medium (containing DAPI); glass slide; coverslip.

[0109] 2.2 Experimental Methods

[0110] 2.2.1 Constructing a cell pathology-specific animal model

[0111] 2.2.1.1 Stereoscopic injection of AAV to establish the basis of neuronal and oligodendrocyte pathology in mice

[0112] (1) Anesthesia: Mice were anesthetized with isoflurane.

[0113] (2) Skin preparation: Use depilatory cream to remove the hair on the mouse's head to expose a clean surgical field.

[0114] (3) Fixation: Place the anesthetized mouse into the fixation frame of the stereotaxic apparatus, cover it with a gas anesthesia mask, and adjust the anesthetic gas rate to 1.5 mL / min. Heat the surgical heating blanket to 37°C to keep the mouse warm. Adjust the position of the head clamp appropriately so that the mouse skull is roughly level and symmetrical to the naked eye. Apply erythromycin eye ointment to the mouse's eyes to protect them.

[0115] (4) Cutting: Cut open the scalp of the mouse with scissors, pull back the scalp to expose the skull, wipe away the blood with a wet cotton swab, wipe away the surface fascia with a dry cotton swab, and expose the clean anterior fontanelle (Bregma point) and posterior fontanelle (Lambda point) for correction.

[0116] (5) Calibration: Align the tip of the microinjection needle with the Bregma point on the mouse skull and zero out x, y, and z. Measure the z value at x = ±2.00 mm and y = 0.00 mm so that the z value is the same when the needle tip is at these two points (based on experience, the z value is often -0.18 to -0.22 mm at this point, with slight variation among different mice, most commonly -0.20 mm). Then adjust the z values ​​of the lambda and Bregma points to be aligned, so that the z value of the Bregma point is also 0.00 mm.

[0117] (6) Drilling: Use a cranial drill to drill open the target points: x=±0.25mm, y=-6.72mm; x=±0.75mm, y=-5.20mm; expose brain tissue.

[0118] (7) Aspirate the injection material: AAV is aspirated into the microsyringe; all AAV is diluted with glycerol / PBS to a titer of 10. 12 vg / ml.

[0119] (8) Injection: For mice with neuronal pathology, retrograde tracer AAV (AAV2 / Retro-hSyn-Cre-EGFP) was injected into both sides of the GiV at coordinates x=±0.25mm, y=-6.72mm, z=-5.60mm. 200 nmol was injected into each side. Injection rate: 200 nmol / min. Additionally, AAV2 / 9-DIO-hSNCA was injected into both sides of the SLD at coordinates x=±0.75mm, y=-5.20mm, z=-4.10mm. 300 nmol was injected into each side. Injection rate: 200 nmol / min. For mice with oligodendrocyte pathology, AAV2 / mMBP-eGFP-iCre was first injected into both sides of the SLD at coordinates x=±0.75mm, y=-5.20mm, z=-4.10mm. 300 nmol was injected into each side. Injection rate setting: 200 nl / min. Leave the injection needle in place for 10 minutes, then slowly withdraw it. Inject AAV2 / 9-DIO-hSNCA again into both SLDs, 300 nl per side. Injection rate setting: 200 nl / min.

[0120] (9) Stop the injection: After the injection is completed, keep the microsyringe in place for 10 minutes so that the injected material can be stably absorbed in place for a period of time.

[0121] (10) Needle withdrawal: After stopping the needle for 10 minutes, slowly raise the micro-injector away from the brain tissue.

[0122] (11) Suturing: Clean the surgical field with cotton swabs, suture the mouse scalp with interrupted continuous suture, and apply antibiotics.

[0123] (12) Recovery: The anesthetized and comatose mice were placed in a separate cage on a heat-insulating blanket to keep them warm. After the mice had been awake for a period of time, they were returned to their original cages.

[0124] 2.2.1.2 Stereoscopic injection of PD-NEV into neuropathic mice and injection of MSA-OEV into oligodendrocyte mice

[0125] One month after AAV injection, mice underwent another stereotactic injection procedure. Whether the mice were neuronal pathology mice or oligodendrocyte pathology mice, the injection was performed in both SLDs.

[0126] (1) Anesthesia: Mice were anesthetized with isoflurane.

[0127] (2) Skin preparation: Use depilatory cream to remove the hair on the mouse's head to expose a clean surgical field.

[0128] (3) Fixation: Place the anesthetized mouse into the fixation frame of the stereotaxic apparatus, cover it with a gas anesthesia mask, and adjust the anesthetic gas rate to 1.5 mL / min. Heat the surgical heating blanket to 37°C to keep the mouse warm. Adjust the position of the head clamp appropriately so that the mouse skull is roughly level and symmetrical to the naked eye. Apply erythromycin eye ointment to the mouse's eyes to protect them.

[0129] (4) Cutting: Cut open the scalp of the mouse with scissors, pull back the scalp to expose the skull, wipe away the blood with a wet cotton swab, wipe away the surface fascia with a dry cotton swab, and expose the clean anterior fontanelle (Bregma point) and posterior fontanelle (Lambda point) for correction.

[0130] (5) Calibration: Align the tip of the microinjection needle with the Bregma point on the mouse skull and zero out x, y, and z. Measure the z value at x = ±2.00 mm and y = 0.00 mm so that the z value is the same when the needle tip is at these two points (based on experience, the z value is often -0.18 to -0.22 mm at this point, with slight variation among different mice, most commonly -0.20 mm). Then adjust the z values ​​of the lambda and Bregma points to be aligned, so that the z value of the Bregma point is also 0.00 mm.

[0131] (6) Drilling: Use a cranial drill to drill open the target point: x=±0.75mm, y=-5.20mm, to expose the brain tissue.

[0132] (7) Aspirate the injection: Aspirate PD-NEV (for mice in the neuronal pathology group) or MSA-OEV (for mice in the oligodendrocyte pathology group) or PBS (for mice in the control group) into the microsyringe. The injection volume is 2.0 μl (total protein concentration of PD-NEV and MSA-OEV is 0.5 μg / μl).

[0133] (20) Injection: The injection points are bilateral SLDs, with injection coordinates of x=±0.75mm, y=-5.20mm, and z=-4.10mm. A total of 2.0μl was injected. Injection speed setting: 200nL / min.

[0134] (21) Stop the injection: After the injection is completed, keep the microsyringe in place for 10 minutes so that the injected material can be stably absorbed in place for a period of time.

[0135] (22) Needle withdrawal: After stopping the needle for 10 minutes, slowly raise the micro-injector away from the brain tissue.

[0136] (23) Suturing: Clean the surgical field with cotton swabs, suture the mouse scalp with interrupted continuous suture, and apply antibiotics.

[0137] (24) Recovery: The anesthetized and comatose mice were placed in a separate cage on a heat-insulating blanket to keep them warm. After the mice had been awake for a period of time, they were returned to their original cages.

[0138] 2.2.2 Immunofluorescence analysis of pathology

[0139] 2.2.2.1 Brain harvesting from mice via perfusion

[0140] (1) Anesthesia: Gently grasp the mouse and place it in the anesthesia box. Use a cotton swab to apply isoflurane and place it in the anesthesia box containing the mouse.

[0141] (2) Fixing the mouse's exposed field of vision: After the mouse is anesthetized, the limbs are fixed with thumbtacks, and the skin and muscles of the chest are cut open with small scissors, as well as the chest cavity and the rib cage to expose the heart, where the left ventricle and right atrium can be seen.

[0142] (3) Perfusion hemostasis: Insert the soft intravenous injection needle connected to the syringe containing 20ml PBS into the left ventricle of the mouse. Blood can be seen flowing back from the ventricle to the needle tip. Inject a portion of PBS, then cut open the right atrium and inject PBS again, so that all the blood of the mouse flows out.

[0143] (4) Perfusion of paraformaldehyde: After PBS injection, keep the needle inserted into the ventricle and replace the syringe with a 20ml syringe filled with 4% paraformaldehyde. Slowly push the needle to allow 4% paraformaldehyde to enter the mouse circulatory system. It can be seen that the mouse's leg muscles trembled and its tail was raised due to the stimulation of paraformaldehyde, proving that paraformaldehyde successfully entered the circulatory system and exerted a fixation effect on the mouse's organs and tissues.

[0144] (5) Brain removal and fixation: The head of the mouse was cut off with large scissors, and the skull was gradually peeled open with hemostatic forceps to expose the brain of the mouse. The connection with the optic nerve and other nerves was severed to obtain complete mouse brain tissue. The brain tissue was placed in a centrifuge tube containing 4% paraformaldehyde and stored at 4°C for 48 hours.

[0145] 2.2.2.2 Gradient dehydration of brain tissue

[0146] Mouse brain tissue was sequentially placed in 10%-20%-30% sucrose / PBS solutions (w / v) for glycemic gradient dehydration.

[0147] 2.2.3.3 Frozen sections

[0148] The dehydrated mouse brain tissue was embedded in OCT and then sectioned. The section thickness was selected to be 25 μm. The sections were stored in 24-well plates containing PBS solution and stored at 4°C.

[0149] 2.2.3.4 Immunofluorescence

[0150] (1) Blocking: Take out the brain slices and put them into blocking solution: 5% BSA / PBST, and block at room temperature for 1 hour.

[0151] (2) Primary antibody incubation: The primary antibody dilution solution was 5% BSA / 0.3% Triton-X-100 / PBST. The primary antibodies used included mouse NeuN antibody (abcam, ab104224, 1:1000), rat MBP antibody (abcam, ab7349, 1:200), and rabbit pS129 α-syn antibody (abcam, ab51253, 1:1000). The antibodies were incubated overnight for 12 hours on a shaker in a cold storage at 4°C.

[0152] (3) Washing: Wash 3 times with PBST, 5 minutes each time.

[0153] (4) Secondary antibody incubation: 488 and 568 fluorescent secondary antibodies were diluted 1:1000 in PBST and incubated for 1 hour in a shaker at room temperature in the dark.

[0154] (5) Washing: Wash 3 times with PBST for 5 minutes each time.

[0155] (6) Mounting: Mount the floating brain slices onto a glass slide.

[0156] (7) Nucleus staining and mounting: Add anti-fluorescence quenching mounting medium, cover with a coverslip. Seal the edges with nail polish. Protect from light and air dry naturally.

[0157] (8) Scanning: Use a PANNORAMIC 250 Flash III DX scanner to automatically scan the fluorescence information of the slide or take pictures with an SP8 microscope.

[0158] (9) Processing: After obtaining the scanned image, use ImageJ to process the image.

[0159] To count the number of neurons in the SLD region, a brain slice of AP -5.20 mm was taken, the SLD brain region was delineated, and the number of NeuN-positive neurons was counted.

[0160] To quantify the pathology of pS129 α-syn, a threshold was set, and the area was calculated using the particle analysis module of ImageJ.

[0161] (1) Set the image file to 8-bit format.

[0162] (2) Adjust the threshold to a value between 100 and 255, and observe whether it best reflects the visible effect. After application, the image is a grayscale image with a black background.

[0163] (3) Select Region of Interest (ROI): Freely select the target brain region and add it to the list for analysis.

[0164] (4) Analyze particles: Input the set particle size, such as 1 square micrometer, and analyze the particle data within the ROI, i.e. pS129 α-syn pathological aggregates.

[0165] 2.3 Experimental Results

[0166] Immunofluorescence experiments revealed that one month after modeling, co-localization signals of pS129 α-syn and NeuN were observed in the SLD of mice with neuronal pathology injected with PD-NEV, indicating the presence of neuronal cell-specific pathology. Conversely, co-localization signals of MBP and pS129 α-syn were observed in mice with oligodendrocyte pathology injected with MSA-OEV, indicating the presence of oligodendrocyte cell-specific pathology. Figure 2 A). One month after modeling, compared with the control group, mice in both the neuronal pathology group and the oligodendrocyte pathology group showed significant loss of neurons in the SLD region (neuronal pathology group vs. control group: P < 0.001; oligodendrocyte pathology group vs. control group: P < 0.001). P < 0.01). Three and five months after modeling, the loss of neurons was more severe in both the neuronal pathology group and the oligodendrocyte pathology group (at 3 and 5 months, both experimental groups vs. the control group showed significant neuronal loss). P < 0.0001) Figure 2 B). Furthermore, at 3 and 5 months post-modeling, although the degree of neuronal loss was similar in both groups, the pathological burden of pS129 α-syn in the oligodendrocyte pathology group was significantly greater than that in the neuronal pathology group (3 months post-injection). P < 0.0001; 5 months after injection, P < 0.01)( Figure 2 C).

[0167] 2.4 Discussion of Results

[0168] Since PD and MSA represent neuronal and oligodendrocyte pathologies, respectively, and PD patients' neurons contain extracellular vesicles with pathological α-syn, these extracellular vesicles from PD patients and MSA patients' oligodendrocytes were used as pathological induction seeds. The experimental results show that, through basic modeling, using AAV to induce an overexpression of α-syn in neurons or oligodendrocytes of mice with SLD, followed by the application of pathological seeds from patients (PD-NEV, MSA-OEV), can significantly induce neuronal and oligodendrocyte pathologies. Furthermore, both cell-specific pathological modeling mice exhibited significant SLD neuronal loss and α-syn pathology, which are currently the core pathological features of RBD animal models. These results indicate that the two cell-specific pathological mouse models of this invention possess different RBD cell-specific pathologies and the core pathological features of RBD.

[0169] Example 3: Detection of sleep characteristics in cell-specific pathological mice

[0170] 3.1 Experimental Materials

[0171] PTFE-coated silver wires, stainless steel electrode wires, stainless steel screws, and acrylic resin dental bases.

[0172] 3.2 Experimental Methods

[0173] 3.2.1 Electroencephalogram (EEG) and electromyogram (EMG) electrode implantation

[0174] The EEG electrode consists of four PTFE-coated silver wires, while the EMG electrode consists of two stainless steel wires. After anesthesia, scalp preparation, and fixation of the mice, a 1mm diameter hole was drilled in each of the frontal, parietal, and occipital bones. The EEG electrode was connected to a screw through the implantation hole, and the EMG electrode was inserted into the bilateral neck muscles of the mouse. The EEG and EMG electrodes were secured in a miniature connector within the electrode assembly, which was then fixed to the skull surface using a dental acrylic base. One week after implantation, video polysomnography was performed on the mice.

[0175] 3.2.2 Video-polysomnography (vPSG) and data analysis

[0176] Before formal recording, mice were fitted with electrode components and connected to the device for 72 hours to acclimatize. During formal vPSG recording, each mouse was continuously recorded for 24 hours (7:00 AM to 7:00 AM the following day). Cortical EEG and neck muscle EMG signals were digitized at a sampling rate of 1000Hz using Medusa software (Eage BioSignal Technology, China), and then converted into visual analysis using the Accusleep component in Matlab, while simultaneously recording mouse behavior videos. Fast Fourier Transform (FFT) analysis was performed using MATLAB software to extract EEG spectral data, and the raw frequency data was converted into EEG spectrograms according to standard EEG rhythms (δ wave: 0.65–4Hz, θ wave: 6–10Hz, α wave: 12–14Hz, β wave: 15–25Hz). Mouse states were manually labeled using Accusleep software (divided into 5-second intervals), including wakefulness, REM sleep, and NREM sleep, and the frequency, average duration, percentage, and transition frequency of each state were calculated. EMG signal quantification was performed using MATLAB software to calculate neck muscle tension intensity, thereby determining the average electromyographic (EMG) values ​​during REM and NREM sleep periods. (REM) / EMG (NREM) ).

[0177] 3.3 Experimental Results

[0178] One month after modeling, mice in both the neuronal pathology group and the oligodendrocyte pathology group exhibited significant REM sleep without atonia (RSWA), a core manifestation of RBD. The frequency of RSWA was even more pronounced at 3 and 5 months after modeling. Figure 3 Quantitative analysis of electromyography (EMG) intensity during REM sleep revealed that, compared to the control group, mice in the neuronal pathology group and the oligodendrocyte pathology group showed significantly increased relative EMG intensity during REM sleep (1, 3, and 5 months post-modeling, P < 0.05 compared to the control group). Figure 4 ).

[0179] 3.4 Discussion of Results

[0180] Elevated electromyography (EMG) during RSWA and REM phases is a core manifestation of RBD. Through the above sleep analysis, we found significant increases in RSWA and REM phase EMG in both neuropathological and oligodendrocyte pathological mice, confirming that the two cell pathology-specific animal models of this invention possess the core characteristics of RBD.

[0181] Example 4: Motor behavior assessment of Parkinson's phenotype in cell-specific pathological RBD mice

[0182] 4.1 Experimental Materials

[0183] The rotator was purchased from Jiangsu Sansbio Company.

[0184] 4.2 Experimental Methods

[0185] 4.2.1 Rotating bar experiment

[0186] Mice underwent training on a rotundus fatigue tester for three consecutive days. During the training period, mice completed three adaptive training sessions daily on a constantly accelerating rotundus (accelerating from 4 rpm to 40 rpm within 5 minutes), with 15-minute intervals between each session. The same procedure was performed on the test day, and the latency of the mice falling off the rotundus was recorded. The average of the three latency periods was taken as the final data for each mouse.

[0187] 4.3 Experimental Results

[0188] The results of the rotator experiment showed that ( Figure 5 One month after modeling, compared with the control group, the neuronal pathology group mice showed a significant shortening of the drop latency time. P < 0.05 indicates a decline in motor coordination. Three months after modeling, compared with the control group, the drop latency time of mice in the neuropathological group was significantly shortened ( P < 0.001), and the shedding latency time of mice in the oligodendrocyte pathology group was also significantly shortened ( P < 0.05). Five months after modeling, compared with the control group, the drop latency time of mice in the neuropathological group was significantly shortened ( P < 0.0001), the shedding latency time of mice in the oligodendrocyte pathology group was also significantly shortened ( P < 0.01), while the drop latency of mice in the neuronal pathology group was significantly shorter than that in mice in the oligodendrocyte pathology group ( P < 0.01). This suggests that the motor coordination ability of mice in both cell pathology groups was significantly reduced, while the reduction in motor ability was more significant in the neuronal pathology group.

[0189] 4.4 Discussion of Results

[0190] The above behavioral results indicate that mice in the neuronal pathology group showed a significant decline in motor coordination one month after modeling. As time progressed, mice in the oligodendrocyte pathology group also showed a decline in motor coordination three months after modeling. Five months after modeling, both cell-specific pathology groups exhibited a Parkinson's disease phenotype of motor coordination decline, with the decline in motor coordination being more severe in the neuronal pathology group than in the oligodendrocyte pathology group. This replicates the characteristic of decreased motor coordination observed in the clinical transformation of RBD patients to PD and MSA.

[0191] Example 5: Histopathological verification of the Parkinson's phenotype in cell-specific pathological RBD mice

[0192] 5.1 Experimental Materials

[0193] BSA; PBS; PBST; mouse TH primary antibody (Merck, MAB318, 1:1000); Triton-X-100; anti-mouse secondary antibody (Vector laboratories, BA-1000, BA-9200, 1:1000); avidin-biotin kit (Vectorlaboratories, PK-4001, 1:500); DAB (Vector Laboratories, SK-4105); hematoxylin; ethanol; xylene; neutral resin; glass slide; coverslip.

[0194] 5.2 Experimental Methods

[0195] Immunohistochemistry was used to stain the striatum and substantia nigra of mice to detect the density of dopaminergic nerve fibers in the striatum and the number of dopaminergic neurons in the substantia nigra at various time points.

[0196] (1) Blocking: Take out the brain slices and put them into blocking solution: 5% BSA / PBST, and block at room temperature for 1 hour.

[0197] (2) Primary antibody incubation: TH primary antibody was diluted 1:1000 in 5% BSA / 0.3% Triton-X-100 / PBST. Incubated overnight for 15 hours on a shaker in a cold storage at 4°C.

[0198] (3) Washing: Wash 3 times with PBST, 5 minutes each time.

[0199] (4) Secondary antibody incubation: The anti-mouse secondary antibody was diluted 1:1000 in PBST and incubated on a shaker at room temperature for 1 hour.

[0200] (5) Washing: Wash 3 times with PBST for 5 minutes each time.

[0201] (6) Avidin-Biotin incubation: Dilute 1:500 in PBS and incubate on a shaker at room temperature for 30 minutes.

[0202] (7) Washing: Wash twice with PBS for 5 minutes each time.

[0203] (8) DAB staining: DAB was prepared into a working solution and the brain slices were incubated at room temperature for 2 minutes.

[0204] (9) Termination of reaction: Wash with PBS to terminate the reaction.

[0205] (10) Mounting: Mount the brain slices onto a glass slide.

[0206] (11) Hematoxylin staining: After rinsing the air-dried brain slices with water for 5 minutes, stain them with hematoxylin for 3 minutes.

[0207] (12) Differentiation and blueing: After rinsing the brain slices with water, soak them in 0.1% hydrochloric acid / pure water for 1 minute, and then rinse them with running tap water for 2 hours.

[0208] (13) Dehydration and clearing: 75% ethanol-80% ethanol-95% ethanol-95% ethanol-100% ethanol-100% ethanol, 1 minute each time; xylene 3 times, 5 minutes each time.

[0209] (14) Mounting: Neutral resin for mounting.

[0210] (15) Analysis: ImageJ was used to analyze the optical density of the striatum and count the number of TH-positive neurons in the substantia nigra region.

[0211] 5.3 Experimental Results

[0212] The results of the striatal TH staining experiment showed that ( Figure 6 A), one month after modeling, compared with the control group, the neuropathological group of mice showed a significant decrease in striatal dopaminergic nerve fiber density ( P < 0.05). Three months after modeling, compared with the control group, the striatal dopaminergic nerve fiber density in the neuropathological group mice was significantly reduced ( P < 0.001), and the density of dopaminergic nerve fibers in the striatum of mice in the oligodendrocyte pathology group was also significantly reduced ( P < 0.01). Five months after modeling, compared with the control group, the density of striatal dopaminergic nerve fibers in both the neuronal pathology group and the oligodendrocyte pathology group was significantly reduced ( P < 0.001 indicates that the striatal system of both pathological mice has significant neurodegenerative lesions, and the density of striatal dopaminergic nerve fibers in the neuronal pathological group is significantly lower than that in the oligodendrocyte pathological group. P < 0.01)( Figure 6 B).

[0213] The results of the TH staining experiment on the substantia nigra showed that ( Figure 7 A), one month after modeling, compared with the control group, the neuropathological group of mice showed a significant reduction in the number of dopaminergic neurons in the substantia nigra (A). P < 0.05. At 3 and 5 months after modeling, compared with the control group, the number of dopaminergic neurons in the substantia nigra of mice in both the neuronal pathology group and the oligodendrocyte pathology group was significantly reduced ( P< 0.01 indicates significant neurodegenerative changes in the substantia nigra of both pathological mice. Furthermore, at 3 and 5 months post-modeling, the number of dopaminergic neurons in the substantia nigra of the neuropathological group was significantly lower than that of the oligodendrocyte pathological group (…). P < 0.01, together with the results of striatal staining, suggests that the degree of neurodegenerative changes in the substantia nigra-striatal dopaminergic system was more severe in the neuronal pathology group than in the oligodendrocyte pathology group. Figure 7 B).

[0214] 5.4 Discussion of Results

[0215] The immunohistochemical results above indicate that mice in the neuronal pathology group showed significant degenerative changes in the nigrostriatal system one month after modeling. As time progressed, mice in the oligodendrocyte pathology group also showed significant degenerative changes in the nigrostriatal system three months after modeling. Five months after modeling, both cell-specific pathology groups showed significant neurodegenerative changes in the nigrostriatal system. Furthermore, the degree of degeneration in the nigrostriatal system was more severe in the neuronal pathology group than in the oligodendrocyte pathology group, replicating the characteristics of neurodegenerative changes in the nigrostriatal system observed in RBD patients progressing to PD and MSA in clinical practice.

[0216] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A method for preparing a cell pathology-specific animal model of rapid eye movement sleep behavior disorder, characterized in that, The cellular pathology is neuronal pathology or oligodendrocyte pathology; the preparation method is to inject adeno-associated virus that induces neuronal overexpression of α-synuclein into the dorsolateral tegmental nucleus brain region of a non-human test animal, and one month after the adeno-associated virus injection, to inject neuronal-derived extracellular vesicles or oligodendrocyte-derived extracellular vesicles into the dorsolateral tegmental nucleus brain region of the non-human test animal again.

2. The method for preparing a cell pathology-specific rapid eye movement sleep behavior disorder animal model according to claim 1, characterized in that, The non-human test animal mentioned above is a mouse.

3. The method for preparing a cell pathology-specific rapid eye movement sleep behavior disorder animal model according to claim 1, characterized in that, The neuronal extracellular vesicles mentioned are PD-NEV, derived from neuronal extracellular vesicles from Parkinson's disease patients; the oligodendrocyte extracellular vesicles mentioned are MSA-OEV, derived from oligodendrocyte extracellular vesicles from patients with multiple system atrophy.

4. The method for preparing a cell pathology-specific rapid eye movement sleep behavior disorder animal model according to claim 3, characterized in that, The adeno-associated virus (AAV), neuronal extracellular vesicle PD-NEV, and oligodendrocyte extracellular vesicle MSA-OEV that induce neuronal overexpression of α-synuclein are injected into the bilateral dorsolateral tegmental nucleus brain region via stereotactic injection.

5. The method for preparing a cell pathology-specific rapid eye movement sleep behavior disorder animal model according to claim 3, characterized in that, The adeno-associated virus that induces neuronal overexpression of α-synuclein is used in combination with neuronal extracellular vesicle PD-NEV or oligodendrocyte extracellular vesicle MSA-OEV to prepare an animal model of rapid eye movement sleep behavior disorder.

6. Application of adeno-associated virus (AAV) that induces neuronal overexpression of α-synuclein in combination with neuronal extracellular vesicles PD-NEV or oligodendrocyte extracellular vesicles MSA-OEV in the preparation of animal models of rapid eye movement sleep behavior disorder.

7. The use of the cell pathology-specific rapid eye movement sleep behavior disorder animal model prepared by the methods described in claims 1-5 in screening and / or preparing drugs for the prevention and treatment of cell pathology-specific rapid eye movement sleep behavior disorder.

8. A method for screening drugs to prevent and treat cytopathologically specific rapid eye movement sleep behavior disorder, characterized in that, Includes the following steps: A therapeutic drug is a drug that is administered to an animal model prepared using the methods described in claims 1-5 and compared with an animal model that has not been administered the drug, and that results in improvement or cure of symptoms of REM sleep behavior disorder after administration.

Images