A method for constructing a mouse model of lewy body dementia and application thereof

Abstract

The application discloses a method for constructing a Lewy body dementia mouse model and application, and the method comprises the following steps: taking the fontanel point as an origin, determining the stereotactic coordinates of the bilateral NBM of the mouse, and the coordinates are as follows: AP: -0.6 mm, ML: ±2.1 mm, DV: 4.4-4.6 mm; drilling a small hole at the skull position corresponding to the target coordinates, and stereotaxically injecting a suspension of alpha-synuclein PFFs into the bilateral NBM brain regions of the mouse, with the injection volume of each side being 1 microliter and the concentration being 2 micrograms / microliter; and after the injection is completed, the scalp incision is sutured. The application successfully constructs a DLB / PDD animal model which can cover key cognitive brain regions in pathology and stably exhibit cognitive impairment in behavior by precisely injecting PFFs into the bilateral NBM of the mouse.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a method for constructing and applying a Lewy body dementia mouse model. Background Technology

[0002] Lewy body dementia (DLB) and Parkinson's disease dementia (PDD) are two common neurodegenerative diseases, collectively known as the Lewy body dementia spectrum. Their core neuropathological feature is the presence of Lewy bodies and Lewy neurites, primarily composed of abnormally aggregated α-synuclein, within the central nervous system, particularly in the cortical and subcortical regions. The misfolding, aggregation, and intercellular propagation of these pathological proteins are considered key mechanisms in the occurrence and development of the diseases. To delve deeper into the pathophysiological processes of these diseases and develop effective treatment strategies, the ability to construct animal models that accurately mimic the key characteristics of human diseases is crucial.

[0003] In existing animal models, inducing pathology through intracerebral injection of pre-formed fibrous tissue (PFFs) generated from recombinant α-synuclein has become a widely accepted and powerful technique. The core of this method lies in directly introducing pathological α-synuclein with "seed" activity into specific regions of the animal brain. These exogenous "seeds" recruit and promote the pathological aggregation of endogenous normal α-synuclein, thereby mimicking a progressive neurodegenerative process similar to human diseases.

[0004] In this field, the most classic and widely cited existing technique is the model reported by Luk et al. in 2012 (Pathological α-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science. 2012; 338:949–53). This study was the first to systematically demonstrate that injecting α-synuclein PFFs into the dorsal striatum of wild-type C57BL / 6J mice was sufficient to trigger a series of neurodegenerative cascades associated with Parkinson's disease. Specifically, the model successfully reproduced several core pathological and phenotypic features: first, phosphorylated α-synuclein deposition was observed in the injection area and adjacent brain regions, forming Lewy body-like inclusions; second, significant and progressive loss of dopaminergic neurons was observed in the substantia nigra pars compacta; and finally, accompanied by neuronal loss, the mice exhibited persistent and quantifiable motor dysfunction. The successful establishment of this model has provided an extremely valuable tool for studying the initiation and spread mechanisms of synucleinosis, and it has been replicated, verified and improved by a large number of subsequent studies in order to more perfectly reproduce the natural course of the disease.

[0005] However, despite the great success of striatal PFF injection models, exemplified by the work of Luk et al., these animal models for studying DLB / PDD have significant limitations and unresolved shortcomings, mainly in the following aspects:

[0006] First, existing models are severely imbalanced in behavioral assessment, with a severe lack of focus on and validation of cognitive function. The core clinical diagnostic criteria for DLB and PDD are progressively worsening dementia, i.e., significant cognitive impairment, which is often the primary cause affecting patients' quality of life. However, existing intracranial alpha-synuclein inoculation models are designed and applied with a strong bias towards exploring Parkinson's disease-like motor symptoms. Therefore, the behavioral testing protocols in most studies focus primarily on assessing motor function, such as the open field test, swivel bar test, and gait analysis. Systematic assessments of higher cognitive functions such as learning, memory, executive function, and attention are severely lacking. This "emphasis on motor function and neglect of cognition" prevents the use of these models to delve into the neural basis of the most destructive cognitive decline in DLB / PDD, and also greatly limits the preclinical evaluation of treatment strategies to improve cognitive function.

[0007] Second, existing models have blind spots in their pathological research, neglecting a systematic exploration of key cognitively relevant brain regions. Consistent with the bias in behavioral assessments, histological and histochemical examinations of the central nervous system in model animals have largely focused on classical neural circuits related to motor control, namely the dopaminergic pathway from the substantia nigra to the striatum. While researchers typically analyze the pathological changes in the substantia nigra and striatum in detail, this is important, but far from sufficient to explain complex cognitive symptoms. Cognitive impairment in DLB / PDD is considered to be closely related to the pathological load in cortical and subcortical cognitive-related areas, particularly the basal forebrain cholinergic system (such as the nucleus basalis of Meynert, NBM), amygdala, and neocortex. However, in existing striatal PFF injection models, whether these brain regions closely related to cognitive function also exhibit pathological accumulation of α-synuclein, neuroinflammation, and neuronal loss, and the temporal dynamics of their pathological progression, have not yet been systematically and deeply explored. This limitation in research perspective prevents the model from fully reproducing the whole-brain pathological atlas of DLB / PDD, hindering the understanding of how pathological proteins spread from the motor center to the cognitive center, and thus failing to fully reveal the disease mechanism.

[0008] The closest existing animal model of α-synucleinosis, represented by striatal PFF injection, successfully simulates the motor symptoms and pathology of related motor circuits, but it is essentially a model that focuses on "Parkinson's disease" characteristics. It fails to effectively simulate the iconic and impactful cognitive impairments in the Lewy body dementia spectrum, and its pathological studies have not consciously and systematically covered and revealed the lesions in cognitively related brain regions.

[0009] Therefore, there is an urgent need for a new animal model that can more specifically and comprehensively simulate the core characteristics of DLB / PDD—that is, simultaneously exhibiting progressive cognitive impairment and pathology in extensive brain regions (especially cognitive-related brain regions)—to fill this technological gap. This is of vital importance for deciphering the pathogenesis of DLB / PDD and promoting the development of drugs targeting cognitive symptoms.

[0010] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0011] The purpose of this invention is to provide a method for constructing and applying a Lewy body dementia mouse model, which solves the problem that existing animal models are difficult to simulate the core characteristics of DLB / PDD. This invention constructs a DLB / PDD animal model that can pathologically cover key cognitive brain regions and stably exhibit cognitive impairment in behavior.

[0012] To achieve the above objectives, this invention provides a method for constructing a Lewy body dementia mouse model. The method includes: using the anterior fontanelle as the origin, determining the stereotactic coordinates of the bilateral NBMs of the mouse, with the coordinates being: AP: -0.6 mm, ML: ±2.1 mm, DV: 4.4~4.6 mm; drilling a small hole at the skull position corresponding to the target coordinates, and stereotactically injecting a suspension of α-synuclein PFFs into the bilateral NBM brain regions of the mouse, with an injection volume of 1 μL on each side and a concentration of 2 μg / μL; and suturing the scalp incision after the injection.

[0013] In the method of this invention, the concentration of the injected α-synuclein PFF suspension is 2 μg / μL, which is the optimal concentration. At this concentration: (1) the PFF suspension provides sufficient pharmacological effect; (2) the diffusion range of PFF is limited within the NBM boundary; and (3) neurotoxicity or non-specific diffusion caused by high concentration is avoided. In short, this concentration achieves the best balance between effect, specificity, and tissue safety.

[0014] Preferably, the injection involves slowly and vertically lowering the injection needle to the target depth, injecting the PFFs solution into the NBM at a constant and slow flow rate, leaving the needle in place after injection to ensure sufficient diffusion and absorption of the liquid, and then slowly withdrawing the injection needle.

[0015] More preferably, the flow rate is 0.1 μL / min.

[0016] More preferably, the needle retention time is 5 to 15 minutes.

[0017] Preferably, the mice are wild-type C57BL / 6J mice.

[0018] More preferably, the mice are wild-type C57BL / 6J mice aged 6-8 weeks.

[0019] Preferably, the length of the α-synuclein PFFs is less than 100 nm. Controlling the length of the α-synuclein PFFs to less than 100 nm has several advantages. On the one hand, shorter fiber fragments have higher "seed" activity, enabling them to more effectively recruit and promote the pathological aggregation of endogenous normal α-synuclein, thereby more quickly inducing pathological changes similar to human Lewy body dementia. On the other hand, shorter fiber fragments diffuse and distribute more evenly in the brain, which is beneficial for forming more consistent pathological changes in the target brain region and more accurately simulating the disease's development process.

[0020] More preferably, the method for preparing the suspension of α-synuclein PFFs includes: dissolving lyophilized recombinant human full-length α-syn monomers in sterile, endotoxin-free phosphate-buffered saline to prepare a mother liquor; continuously incubating the mother liquor at 37°C and 1000 rpm to promote the aggregation of monomers into PFFs with a β-sheet structure; and sonicating the PFFs solution under ice bath conditions before injection, with the following sonication parameters: power 150~450W, sonication time 1~10 seconds, interval 1~10 seconds, and total duration 1~5 minutes, to break the PFFs into shorter, more "seed" active fiber fragments.

[0021] More preferably, the incubation period is 7 days.

[0022] A second objective of this invention is to provide an application of the method for constructing the Lewy body dementia mouse model, wherein the application is selected from any one or more of the following:

[0023] (1) A mouse model for constructing a screening and evaluation of candidate drugs for the prevention or treatment of Lewy body dementia and / or Parkinson's disease dementia;

[0024] (2) Used to construct a mouse model for studying the initiation and propagation mechanism of α-synuclein pathology in cognitive circuits in Lewy body dementia spectrum diseases;

[0025] (3) To construct a mouse model for studying the synergistic pathogenic mechanism of neuroinflammation and cholinergic system failure associated with cognitive dysfunction;

[0026] (4) To construct mouse models for the discovery of biomarkers associated with the disease progression or treatment of Lewy body dementia and / or Parkinson's disease dementia.

[0027] The method for constructing and applying the Lewy body dementia mouse model of the present invention solves the problem that existing animal models are unable to simulate the core characteristics of DLB / PDD, and has the following advantages:

[0028] This invention successfully constructed a Lewy body dementia mouse model by precisely injecting probiotics (PFFs) into the bilateral forebrain brain (NBM) of mice, exhibiting both progressive cognitive impairment and widespread pathological changes in brain regions (especially cognitive-related brain regions). This model not only demonstrates a significant decline in learning and memory abilities at the behavioral level, but pathological verification also reveals typical DLB / PDD pathological features such as pathological deposition of α-synuclein and a reduction in the number of cholinergic neurons in the NBM and its projected cortical regions. Crucially, this model reveals a specific decrease in ACh levels in cognitive-related brain regions after NBM injection, while motor-related brain regions (such as the striatum) are not significantly affected. This discovery overcomes the limitation of traditional striatal PFF injection models, which "emphasize motor function but neglect cognitive function," and provides an irreplaceable animal model platform for in-depth research into the mechanisms of pathological protein propagation from the basal forebrain to the cortex in DLB / PDD, as well as for developing intervention strategies targeting cognitive symptoms. Compared with existing technologies, this invention achieves comprehensive simulation of the core characteristics of DLB / PDD through innovative targeted selection (NBM rather than striatum) and pathological assessment system, which has significant technical advantages and application value. Attached Figure Description

[0029] Figure 1 Transmission electron microscopy (TEM) image of the PFFs prepared in this invention; scale bar is 100 nm.

[0030] Figure 2 This is an immunofluorescence image of the NBM brain region after injection of PFFs according to the present invention.

[0031] Figure 3 The results of water maze tests for the Lewy body dementia mouse model constructed in this invention are as follows: (A) escape latency; (B) loop crossing; (C) cumulative distance traveled; (D) positioning and navigation test; (E) cumulative distance traveled; (F) space exploration test.

[0032] Figure 4 Results of open field and new object recognition experiments for the Lewy body dementia mouse model constructed in this invention; (A) Total movement distance, percentage of time spent in the central region, and average speed in the open field experiment; (B) Movement trajectory of the mouse in the open field experiment; (C) Discrimination index; (D) Flowchart of the new object recognition experiment; (E) Movement trajectory of the mouse in the new object recognition experiment.

[0033] Figure 5 The following are the results of motor ability testing of the Lewy body dementia mouse model constructed in this invention: (A) Fall latency in the tumbler fatigue test; (B) Turning time in the pole climbing test; (C) Turning time and time to reach the bottom in the pole climbing test; (D) Forelimb muscle strength of the mouse; (E) Forelimb and hindlimb muscle strength of the mouse.

[0034] Figure 6The pathological examination results of the Lewy body dementia mouse model constructed in this invention are as follows: (A) Immunofluorescence map of the NBM and CTX regions; (B) Percentage of NBM cholinergic neurons positive for phosphorylated α-synuclein; (C) Percentage of p-αSYN+ / ChAT+ double positive neurons.

[0035] Figure 7 The results of Ach level detection in various brain regions of the Lewy body dementia mouse model constructed in this invention are as follows: (A) NBM; (B) CTX; (C) mPFC; (D) Hippo; (E) CPU. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] It should be noted that: Unless otherwise specified in the examples, standard conditions or conditions recommended by the manufacturer should be followed. Instruments whose manufacturers are not specified are all commercially available products. Raw materials and reagents whose manufacturers are not specified are all commercially available goods or can be prepared using known methods.

[0038] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0039] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0040] In the description of this invention, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0041] The following is a description of some of the technical terms used in this invention:

[0042] The Nucleus Basalis of Meynert (NBM) is an important component of the cholinergic system in the basal forebrain and plays a crucial regulatory role in cognitive functions, especially higher cognitive processes such as learning, memory, and attention.

[0043] Basal forebrain: A group of structures located on the ventral side of the telencephalon and diencephalon, below the anterior commissure. In a broad sense, it usually refers to nine types of structures, including the preoptic area of ​​the hypothalamus, the septal nuclei, the bed nuclei of the stria terminalis, and the oblique zone nuclei. In a narrow sense, it specifically refers to the gray matter nuclei near the brain surface, such as the globus pallidus of the ventral striatum, the extension of the amygdala, and the basal ganglia of Meynert.

[0044] AP: Anterior-Posterior, meaning the front-to-back direction, is a horizontal direction from the head to the tail, with the anterior fontanelle of the skull as the origin. Forward is positive and backward is negative, and it is used to determine the front-to-back position of a structure.

[0045] ML: Mediolateral, which is a horizontal direction from the inside (closer to the midline) to the outside (away from the midline) with the body midline as the reference, used to locate the left and right hemispheres.

[0046] DV: Dorsoventral, which is perpendicular to the surface of the skull and runs from the back (top of the skull) to the abdomen (bottom of the brain). It is used to determine the depth of structures.

[0047] Escape latency refers to the time it takes for an experimental animal to go from being placed in water to finding and climbing onto a hidden platform.

[0048] Annulus crossing: refers to the number of times an experimental animal crosses the original platform position in the target quadrant.

[0049] Total distance traveled: This refers to the total distance the animal traveled within the testing device on the last day of the training phase.

[0050] Distance in target quadrant: refers to the total distance an animal travels within the target quadrant during a detection experiment.

[0051] Place navigation test: This test teaches animals to use spatial cues to find hidden target platforms in order to assess their spatial learning abilities.

[0052] Spatial probe test: This test assesses an animal's memory retention of the original platform location after the platform is removed, and is used to evaluate spatial memory retention ability.

[0053] Discrimination Index: This objectively reflects an animal's ability to distinguish between new and old objects.

[0054] The fall latency period is a core indicator for assessing an animal's motor coordination and muscle strength, referring to the time from the start of the experiment to the animal's fall.

[0055] The technical problem this invention aims to solve is: how to construct an animal model that can stably reproduce key cognitive impairments in the Lewy body dementia spectrum, and how to induce significant synuclein pathology in key cognitive-related brain regions corresponding to cognitive impairment. While existing striatal PFF injection models can effectively induce motor symptoms, they cannot systematically and reliably simulate progressive cognitive decline, resulting in a lack of an effective platform for evaluating intervention strategies targeting cognitive symptoms in preclinical studies. Furthermore, existing models primarily focus on the substantia nigra-striatal pathway, neglecting brain regions closely related to higher cognitive functions such as learning, memory, and attention, leading to models that cannot fully reflect the whole-brain pathological characteristics of DLB / PDD.

[0056] This invention utilizes stereotactic injection technology to precisely inject PFFs into the bilateral NBMs of mice, thereby specifically inducing α-synucleosis, neuroinflammation, and neuronal dysfunction in the cognitive core hub and its related neural circuits, ultimately constructing an animal model capable of simulating the core cognitive symptoms of DLB / PDD.

[0057] The following embodiments provide a detailed description of the construction method and application of a Lewy body dementia mouse model provided by the present invention.

[0058] Example 1 Preparation of PFFs

[0059] High-purity (>95%) recombinant human full-length α-syn monomer was obtained (purchased from Proteos, Human Alpha-synuclein monomer to generate preformed fibrils, product model RP-003). The lyophilized recombinant human full-length α-syn monomer was dissolved in sterile, endotoxin-free phosphate-buffered saline (PBS) to prepare a stock solution with a concentration of 5 mg / mL.

[0060] The monomeric protein solution prepared above was continuously shaken and incubated at 37°C and 1000 rpm for 7 days to promote the aggregation of monomers into PFFs with β-sheet structures.

[0061] After polymerization, the PFFs solution was aliquoted and stored at -80°C for later use. Shortly before injection, the PFFs solution was briefly sonicated in an ice bath using an ultrasonic homogenizer. The sonication parameters were: power 150W, sonication time 10 seconds, interval 5 seconds, total duration 5 minutes, to break it into shorter, more "seed" fibrous fragments. The fragmented PFFs were morphologically identified using transmission electron microscopy, confirming the formation of a typical fibrous structure. See [link to relevant documentation]. Figure 1 The fragment lengths are mostly within 100nm, with an average of 50nm.

[0062] Example 2: Construction of a Lewy body dementia mouse model

[0063] 1. Laboratory animals and surgical preparation fiber fragments

[0064] Animal selection: Wild-type C57BL / 6J mice aged 6-8 weeks were selected as model animals and were housed under SPF-grade barrier system conditions.

[0065] Anesthesia and restraint: Administer deep anesthesia to the animal (e.g., using 1–3% isoflurane inhalation anesthesia). Restrain the anesthetized animal to a stereotaxic apparatus, ensuring the head remains horizontal.

[0066] Surgical exposure: After routine skin preparation and disinfection, the scalp is incised along the midline of the skull to expose the anterior fontanelle and the surrounding skull surface.

[0067] 2. Localization of NBM target and PFF injection

[0068] Location coordinates: Based on animal brain atlases, the anterior fontanelle was used as the origin to accurately determine the stereoscopic location coordinates of the bilateral NBMs (Meynert basal ganglia). For mice, the coordinate range is approximately: AP (Anteroposterior): -0.6 mm, ML (Mediolateral): ±2.1 mm, DV (Dorsoventral): 4.4~4.6 mm.

[0069] Injection Procedure: Drill a small hole in the skull at the target coordinates using a miniature dental drill. Using a microsyringe (e.g., a 10 μL Hamilton syringe) and its matching pull-type glass capillary needle, aspirate a predetermined volume of PFFs suspension and stereotactically inject it into the bilateral NBM brain regions of the mice, with an injection volume of 1 μL per side and a concentration of 2 μg / μL. Slowly and vertically lower the injection needle to the target depth. Inject the PFFs solution into the NBM at a constant and slow flow rate (range: 0.1 μL / min) using an infusion pump. After injection, leave the needle in place for 5–15 minutes to ensure sufficient diffusion and absorption of the liquid, then slowly withdraw the injection needle.

[0070] Control group setup: Animals in the control group were injected with an equal volume of the solvent (i.e., PBS) of the PFF suspension under the same conditions.

[0071] 3. Postoperative care

[0072] Postoperative care: Suture the scalp incision, place the animal on a 37°C warming pad until it wakes up, then return it to its original cage and house it under SPF-grade barrier system conditions.

[0073] Example 3: Validation of the Lewy body dementia mouse model

[0074] In Example 2 of this invention, a cognitive impairment model (i.e., a Lewy body dementia mouse model) was constructed by injecting PFFs into NBM. Immunofluorescence was used to detect the localization of PFF injection and the uptake of α-synuclein by cholinergic neurons. Figure 2 As shown in the left image (1 day after injection), the green fluorescence represents the dye PKH67, and the red fluorescence represents the cholinergic neuron marker ChAT. This result confirms that the intracerebral injection site of NBM was correct. In the right image (4 weeks after injection), the green fluorescence represents pathologically phosphorylated α-syn, and the red fluorescence represents the cholinergic neuron marker ChAT. Significant co-localization of cholinergic neurons and α-syn in the NBM brain region after injection demonstrates that cholinergic neurons can take up exogenous PFFs in vivo.

[0075] At specific time points after injection (e.g., 2 weeks, 4 weeks, 6 weeks, and 8 weeks after injection), systematic behavioral tests and pathological verification were performed on the model animals, as detailed below:

[0076] 1. Cognitive function assessment

[0077] Working memory and reference memory were assessed using a novel object recognition experiment, a Y-maze experiment, or an eight-armed maze experiment; spatial learning and long-term memory abilities were assessed using a Morris water maze experiment.

[0078] 2. Motor function assessment

[0079] Open field test was used to assess its voluntary activity, and rotarod test, pole climbing test and muscle strength test were used to assess its motor coordination and balance.

[0080] 3. Pathological phenotypic verification

[0081] Animals were euthanized after behavioral testing or at specific time points, and brain tissue was collected for pathological analysis. Lewy-like inclusion bodies in cognitively relevant brain regions such as the NBM and cortex were detected using immunohistochemistry or immunofluorescence staining with specific antibodies against phosphorylated α-synuclein (e.g., pS129). Changes in the number of cholinergic neurons in the NBM region were quantitatively analyzed using Nissl staining or immunostaining with cholinergic neuron-specific markers (ChAT). Glial cell activation in the NBM and related cortical regions was observed using immunostaining with microglia markers (e.g., Iba1). Ach levels in each brain region were detected using an Ach ELISA kit.

[0082] The above cognitive and motor function assessments were conducted using 8 mice in each group. Pathological phenotypic verification was performed using 4 mice in each group. One-way ANOVA was used for comparisons between groups, and data are expressed as mean ± standard deviation. ***P < 0.001, **P < 0.01, and *P < 0.05 were considered statistically significant, while P > 0.05 indicated no statistically significant difference.

[0083] like Figure 3 As shown, the water maze results indicated that mice began to show a decline in learning and memory abilities 4 weeks after NBM PFF injection, and a significant decline in spatial learning and memory abilities occurred after 6 weeks (*P<0.05). Meanwhile, as... Figure 4 As shown, similar results were observed in the new object recognition experiment (**P<0.01, *P<0.05). To avoid the possibility that these results were due to PFFs affecting the mouse's motor ability, the mouse's motor ability was also tested. Figure 5 As shown, there was no significant difference in motor ability among the groups of mice compared with the control group (P > 0.05). Subsequently, the pathological deposition of α-syn in the brains of mice at various time points after PFF injection was examined, and the results are as follows: Figure 6 As shown, consistent with the behavioral results in mice, α-synuclein deposition in NBM tended to worsen after 2, 4, 6, and 8 weeks of NBM injection, while pathological deposition in the corresponding cortical regions projected by NBM also gradually increased. These results demonstrate that the PDD / DLB cognitive impairment model established by bilateral NBM injection of PFFs in mice is successful.

[0084] After validating the behavioral and pathological changes in the cognitive impairment model induced by NBM PFFs injection, the Ach levels in various brain regions of the model were further examined. ELISA results for each brain region are shown below. Figure 7 As shown, 2 weeks after injection, the levels of Ach in the NBM, extensive cortical (CTX) region, and medial prefrontal cortex (mPFC) of mice began to decrease (*P<0.05). At 4 weeks and beyond, the Ach levels in these three brain regions gradually decreased over time, showing a significant difference compared to the control group (**P<0.01). However, no significant changes were detected in the Ach levels of the bilateral hippocampus (Hippo) and bilateral striatum (CPU) of mice (P>0.05).

[0085] Therefore, the above results indicate that in addition to significant changes in behavior and pathology, the PFF injection model of NBM also showed a significant decrease in Ach levels in NBM and its projection brain regions. This result provides new insights into the pathogenesis of cognitive dysfunction in PDD / DLB from the perspective of animal models for the first time.

[0086] In summary, this invention successfully constructed a Lewy body dementia mouse model exhibiting both progressive cognitive impairment and widespread pathological changes in brain regions (especially cognitive-related brain regions) by precisely injecting PFFs into the bilateral NBMs of mice. This invention achieves comprehensive simulation of the core characteristics of DLB / PDD by targeting the NBM rather than the striatum, demonstrating significant technical advantages and application value.

[0087] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for constructing a Lewy body dementia mouse model, characterized in that, This construction method includes: Using the anterior fontanelle as the origin, the stereotactic coordinates of the bilateral NBMs of the mouse were determined as follows: AP: -0.6 mm, ML: ±2.1 mm, DV: 4.4~4.6 mm; A small hole was drilled at the skull location corresponding to the target coordinates. A suspension of α-synuclein PFFs was stereotactically injected into the bilateral NBM brain regions of mice. The injection volume on each side was 1 μL, and the concentration was 2 μg / μL. After the injection, the scalp incision was sutured.

2. The method for constructing the Lewy body dementia mouse model according to claim 1, characterized in that, The injection involves slowly and vertically lowering the injection needle to the target depth, injecting the PFFs solution into the NBM at a constant and slow flow rate, leaving the needle in place after injection to ensure sufficient diffusion and absorption of the liquid, and then slowly withdrawing the injection needle.

3. The method for constructing the Lewy body dementia mouse model according to claim 2, characterized in that, The flow rate is 0.1 μL / min.

4. The method for constructing the Lewy body dementia mouse model according to claim 2, characterized in that, The needle should be left in place for 5 to 15 minutes.

5. The method for constructing the Lewy body dementia mouse model according to claim 1, characterized in that, The mice used were wild-type C57BL / 6J mice.

6. The method for constructing the Lewy body dementia mouse model according to claim 5, characterized in that, The mice used were wild-type C57BL / 6J mice aged 6-8 weeks.

7. The method for constructing the Lewy body dementia mouse model according to claim 1, characterized in that, The length of the α-synuclein PFFs is less than 100 nm.

8. The method for constructing a Lewy body dementia mouse model according to any one of claims 7, characterized in that, The method for preparing the suspension of the α-synuclein PFFs includes: The freeze-dried recombinant human full-length α-syn monomer was dissolved in sterile, endotoxin-free phosphate buffered saline to prepare a mother liquor; The mother liquor was continuously shaken and incubated at 37°C and 1000 rpm to promote the aggregation of monomers into PFFs with β-sheet structures. Just before injection, the PFFs solution was sonicated in an ice bath with the following parameters: power 150~450w, sonication time 1~10 seconds, interval 1~10 seconds, total duration 1~5 minutes, in order to break the PFFs into shorter fiber fragments with more "seed" activity.

9. The method for constructing a Lewy body dementia mouse model according to claim 8, characterized in that, The incubation period is 7 days.

10. The application of the method for constructing the Lewy body dementia mouse model as described in any one of claims 1 to 9, characterized in that, The application is selected from any one or more of the following: (1) A mouse model for constructing a screening and evaluation of candidate drugs for the prevention or treatment of Lewy body dementia and / or Parkinson's disease dementia; (2) Used to construct a mouse model for studying the initiation and propagation mechanism of α-synuclein pathology in cognitive circuits in Lewy body dementia spectrum diseases; (3) To construct a mouse model for studying the synergistic pathogenic mechanism of neuroinflammation and cholinergic system failure associated with cognitive dysfunction; (4) To construct mouse models for discovering biomarkers associated with the disease progression or treatment of Lewy body dementia and / or Parkinson's disease dementia.

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