A tree shrew parkinsonian animal model modeling method

Abstract

The application discloses a tree shrew Parkinson's animal model modeling method, and belongs to the technical field of animal model construction, and specifically comprises the following steps: (1) under a sterile condition, Oudenone is weighed and dissolved in sterilized ultrapure water, dissolved in a 60 DEG C water bath, and configured into an Oudenone use solution of 20 mg / mL, and the solution is prepared and used immediately; (2) the tree shrew is given intraperitoneal injection of the Oudenone use solution configured in the step (1), the injection dose is 50-100 mg / kg, and the injection time is the first day, the third day and the fifth day; (3) after the administration is completed, normal feeding is carried out for 4 weeks to complete the modeling of Parkinson's disease. It is found for the first time that the Oudenone is injected intraperitoneally, the dose is 50-100 mg / kg, and a stable tree shrew Parkinson's model can be established 4 weeks after the administration is completed.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology for diseases, and particularly relates to a method for creating a tree shrew Parkinson's disease animal model. Background Technology

[0002] Parkinson's disease (PD) is the second most common chronic neurodegenerative disease worldwide after Parkinson's disease, and it is also the fastest-growing neurological disorder globally. The incidence increases with age. PD is a progressive, multifactorial, and heterogeneous disease. PD patients exhibit motor dysfunction, such as resting tremor, rigidity, and bradykinesia, primarily caused by degeneration of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). PD is also accompanied by non-motor symptoms, including olfactory disturbances, sleep disturbances, neuropsychiatric symptoms, cognitive impairment, autonomic dysfunction, and gastrointestinal dysfunction. This pathology occurs alongside Lewy bodies, which are composed of abnormally aggregated α-synuclein. The reasons for DA neuron loss are unclear, but some evidence suggests that these neuropathological processes are related to impaired proteasome protein clearance, mitochondrial dysfunction, and neuroinflammation.

[0003] Currently, the diagnosis of Parkinson's disease (PD) mainly relies on clinical characteristics and imaging examinations. However, when patients present with clinical symptoms, at least 50% of dopaminergic neurons in the substantia nigra have died, and the striatum dopamine content has decreased by more than 80%. Cases diagnosed as early-stage clinically may already be in advanced stages pathologically. Furthermore, there is no effective cure for PD. Current traditional treatments (such as levodopa and dopamine receptor agonists) can only relieve symptoms and are unlikely to stop disease progression; long-term use can easily lead to motor complications. Most novel treatments (such as α-synucleoside targeted drugs and iPSC transplantation) are still in the clinical trial stage and have not yet been widely marketed globally. This is mainly because the pathogenesis of PD is not fully understood, and there are no effective targets available for early diagnosis and treatment.

[0004] Constructing animal models of Parkinson's disease (PD) is an indispensable part of modern neuroscience and medical research. Its core significance lies in providing scientists with a controllable and operable research system to simulate the complex pathological process of human PD, thereby deepening their understanding of disease mechanisms, screening effective drugs, and developing novel therapies. However, animal models also have significant limitations in PD research. Animal models cannot completely simulate human disease; no single model can perfectly replicate all aspects of human PD. Furthermore, experimental animals differ greatly from humans in brain structure, lifespan, and physiology, leading to significant variability when extrapolating experimental results to humans. Therefore, when selecting experimental animals, it is advisable to choose those closely related to humans, which can greatly improve the translation rate of research. Tree shrews, as a newly emerging experimental animal, demonstrate unique value in modern life science research. Evolutionarily closer to primates than traditional rodents (mice and rats), they also possess practical characteristics such as small size, rapid reproduction, and low cost, filling, to some extent, the gap between rodents and non-human primates. Summary of the Invention

[0005] The purpose of this invention is to provide a method for creating a tree shrew Parkinson's disease animal model.

[0006] The objective of this invention is achieved as follows: a method for creating a tree shrew Parkinson's disease animal model, the specific steps of which are as follows:

[0007] (1) Under aseptic conditions, weigh out Oudenone and dissolve it in sterile ultrapure water. Dissolve it in a water bath at 60°C to prepare a 20 mg / mL Oudenone working solution. Prepare and use immediately.

[0008] (2) Administer the Oudenone solution prepared in step (1) to tree shrews via intraperitoneal injection at a dose of 50-100 mg / kg on days 1, 3 and 5.

[0009] (3) After the drug administration was completed, the Parkinson's disease model was established by feeding the animals normally for 4 weeks.

[0010] The beneficial effects of this invention are:

[0011] This invention is the first to discover that intraperitoneal injection of Oudenone at a dose of 50-100 mg / kg can establish a stable tree shrew Parkinson's model 4 weeks after the administration. Attached Figure Description

[0012] Figure 1 The image shows a comparison of TH immunofluorescence staining of the substantia nigra region of the tree shrew brain in the four comparative experiments of this application (×400).

[0013] Figure 2 This is a comparison of the average fluorescence intensity of TH protein in four sets of comparative experiments in this application;

[0014] Figure 3 This is a Western blot comparison of TH protein in the substantia nigra region of tree shrew brain in four sets of comparative experiments in this application;

[0015] Figure 4 This is a comparison chart of the TH gray value ratios in the four sets of comparative experiments in this application;

[0016] Figure 5 This is a Western blotting comparison of p-α-synuclein protein in the substantia nigra region of tree shrew brain in four sets of comparative experiments in this application.

[0017] Figure 6 This is a comparison chart of the gray value ratios of p-α-synuclein in four sets of comparative experiments in this application; Detailed Implementation

[0018] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments are within the protection scope of the present invention.

[0019] 1. Laboratory animals

[0020] Twenty-four ordinary-grade male Burmese tree shrews, weighing 120-150 g, were purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences [SCXK(Yunnan)K2023-0003] and housed in the ordinary-grade animal facility of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK(Yunnan)K2021-0003]. They were housed in stainless steel cages at a room temperature of (24±2)℃, relative humidity of (50±5)%, and a light / dark cycle of 12 h / 12 ​​h.

[0021] 2. Main reagents and consumables

[0022] Reagent Name company Oudenone Mce Tyrosine Hydroxylase (TH) Abcam p-α-synuclein antibody Abcam Anti-rabbit IgG CST Goat Anti-Rabbit IgG H&L (DyLight® 488) Abcam RIPA lysis buffer (strong) Azure Sky Prestained protein marker Thermo Fisher Bovine serum albumin (BSA) Sigma SurperECLPlus Superluminescent Prile Blocking sheep serum Zhongshan Jinqiao ZLI-9021 Fluorescent mounting medium (containing DAPI) Zhongshan Jinqiao ZLI-9557 PVDF membrane Millipore

[0023] 3. Main Instruments

[0024] Instrument Name company Nanophotometer IMPLEN Protein electrophoresis tank, electroporation tank, power supply Bio-Rad ECL luminescence imaging system Tanon Automatic dehydrator Wuhan Junjie Paraffin embedding machine Leica, Germany Rotary slicer Leica, Germany microscope OLYMPUS BX53

[0025] 4. Preparation of reagents

[0026] Oudenone preparation: Under aseptic conditions, weigh out Oudenone and dissolve it in intermediate-sterilized ultrapure water. Dissolve the solution in a 60°C water bath to prepare a 20 mg / mL Oudenone working solution. Prepare and use immediately.

[0027] 5. Experimental Methods

[0028] 5.1 Animal Model Making

[0029] Experimental groups: ① Normal control group, ② 25 mg / kg Oudenone group, ③ 50 mg / kg Oudenone group, ④ 100 mg / kg Oudenone group;

[0030] Groups ②-④ were intraperitoneally injected with the corresponding volume of 20 mg / mL Oudenone solution according to their experimental groupings. The intraperitoneal injections were administered on days 1, 3, and 5. At the same time point, group ① was intraperitoneally injected with 0.75 mL of sterile ultrapure water.

[0031] 5.2 Behavioral Observations of Tree Shrews

[0032] Following intraperitone injection of Oudenone, the behavioral performance of tree shrews in each group was observed daily to determine whether short-term symptoms (2-3 hours) such as resting tremor, bradykinesia, postural instability, limb stiffness, and erection of fur and tail were observed. The presence of overall decreased initiative, including feeding ability and activity levels, and persistent symptoms such as bradykinesia, abnormal gait, clumsy movements, stooped posture, and uncoordinated forepaw movements were also assessed.

[0033] 5.3 Tree shrews collect materials

[0034] After the tree shrews completed drug administration, they were fed normally for 4 weeks before tissue collection. Three tree shrews were randomly selected from each group and injected intramuscularly with an excessive amount of sodium pentobarbital (100 mg / kg). The brains were then fixed via cardiac perfusion with physiological saline and 4% paraformaldehyde solution. The whole brain was removed, the cerebellum was removed, and the brain was fixed in 4% paraformaldehyde for 48 hours before paraffin embedding. Paraffin sections were prepared for subsequent immunofluorescence staining. The remaining three tree shrews from each group were over-anesthetized, and their hearts were perfused with physiological saline. After observing liver whitening, the brain tissue was removed, the substantia nigra was isolated on ice, and the tissue was gently washed with 0.1 mol / L PBS for subsequent Western blotting experiments.

[0035] 5.4 Immunofluorescence detection

[0036] Paraffin sections were dewaxed and hydrated, and antigen retrieval was performed under high pressure with citrate buffer. After blocking with 5% goat serum for 1 hour, primary antibody (Tyrosine Hydroxylase 1:500) was added and incubated overnight at 4°C. After rinsing with PBST, primary antibody (TH 1:500) was added and incubated overnight at 4°C. Secondary antibody labeled with 488 goat anti-rabbit IgG (1:1000) was added and incubated at room temperature in the dark for 2 hours. The sections were then mounted with mounting medium containing DAPI, observed and images were acquired under an upright fluorescence microscope, and the average fluorescence intensity was analyzed using Image-Pro Plus 6.0.

[0037] 5.5 Western Blot Detection

[0038] Melanin tissue from tree shrews in each group was collected, minced, homogenized with RIPA lysis buffer containing protease inhibitors, lysed on ice for 20 min, and centrifuged at 12,000 × g. The supernatant was collected, and protein concentration was determined by the BCA method. Equal volumes of protein samples were separated by SDS-PAGE and transferred to PVDF membranes, blocked with 5% skim milk powder for 1 h. Primary antibodies (rabbit anti-p-α-synuclein antibody 1:1000, rabbit anti-Tyrosine Hydroxylase antibody 1:5000) were added and incubated overnight at 4°C. After washing with TBST, HRP-labeled goat anti-rabbit secondary antibody (1:1000) was added and incubated at room temperature for 2 h. ECL staining was performed, and images were taken using a gel imaging system. ImageJ software was used to analyze the band gray values, and GAPDH was used as an internal reference to calculate the relative expression level of the target protein.

[0039] 5.6 Data Analysis

[0040] Experimental data are expressed as mean ± standard deviation ( The results indicate that GraphPad Prism 9.0.0 was used for analysis and comparison. One-way ANOVA was used for comparisons between groups, and LSD method was used for pairwise comparisons. P < 0.05 was considered statistically significant.

[0041] 6. Experimental Results

[0042] 6.1 Effects of Oudenone on the behavioral performance of tree shrews

[0043] Group 4 (100 mg / kg Oudenone): After the third intraperitoneal injection of Oudenone, tree shrews began to exhibit typical, grossly visible motor symptoms of Parkinson's disease (PD), including resting tremor, bradykinesia, postural instability, limb stiffness, and erection of fur and tail. These grossly visible abnormalities generally lasted for about 20 minutes, but the duration increased with the duration of the model, exceeding 2 hours by day 12. Group 3 (50 mg / kg Oudenone): Typical grossly visible PD motor symptoms appeared on day 8 of the model, lasting about 15 minutes, and exceeding 2 hours by day 17. Group 2 (25 mg / kg Oudenone): Typical grossly visible PD motor symptoms appeared on day 20 of the model, lasting about 5 minutes, and approximately 30 minutes before tissue collection.

[0044] 6.2 Effects of Oudenone on TH expression in the substantia nigra of tree shrew brains

[0045] Tyrosine hydroxylase (TH) is a marker of dopamine neurons, and its expression and distribution reflect changes in dopamine neurons. In the normal control group, TH activity was high and the number of positive neurons was large in the substantia nigra. Compared with the normal control group, TH activity decreased significantly after administration of Oudenone, and the number of positive neurons was significantly reduced, showing a negative correlation with the Oudenone dose.

[0046] Table 1. Effects of Oudenone on TH expression in the substantia nigra of tree shrew brains.

[0047] Experimental Groups Tyrosine Hydroxylase Normal Control 0.090±0.008 25mg / kg Oudenone <![CDATA[0.035±0.006 **** ]]> 50mg / kg Oudenone <![CDATA[0.009±0.002 **** ]]> 100mg / kg Oudenone <![CDATA[0.003±0.001 **** ]]>

[0048] n = 3, Compared with the Normal Control group, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001

[0049] 6.3 Effects of Oudenone on the expression of p-α-synuclein and TH protein in the substantia nigra of the tree shrew brain

[0050] Compared with the Normal Control group, the Oudenone group showed increased p-α-synuclein protein expression, which was positively correlated with dose, and the difference was significant between the 50 mg / kg and 100 mg / kg Oudenone groups (P<0.001). Compared with the Normal Control group, the Oudenone group showed decreased Tyrosine Hydroxylase (TH) protein expression, which was negatively correlated with dose, and the difference was significant between the 50 mg / kg and 100 mg / kg Oudenone groups (P<0.01). Misfolding and abnormal aggregation of α-synuclein to form toxic oligomers and fibers, ultimately forming Lewy bodies, is a hallmark pathological feature of Parkinson's disease. p-α-synuclein is a major component of Lewy bodies.

[0051] Table 2. Effects of Oudenone on the expression of TH and p-α-synuclein proteins in the substantia nigra of tree shrew brain.

[0052] TH p-α-synuclein Normal Control 1.34±0.118 0.45±0.103 25mg / kg Oudenone 1.092±0.167 0.767±0.073 50mg / kg Oudenone <![CDATA[0.737±0.215 ** ]]> <![CDATA[1.218±0.116 *** ]]> 100mg / kg Oudenone <![CDATA[0.423±0.020 **** ]]> <![CDATA[1.482±0.301 **** ]]>

[0053] n = 3, Compared with the Normal Control group, * P<0.05,** P<0.01, *** P<0.001, **** P<0.0001。

Claims

1. A method for establishing a Parkinson's disease animal model in tree shrews, characterized in that: Includes the following steps: (1) Under aseptic conditions, weigh out Oudenone and dissolve it in sterile ultrapure water. Dissolve it in a water bath at 60°C to prepare a 20 mg / mL Oudenone working solution. Prepare and use immediately. (2) Administer the Oudenone solution prepared in step (1) to tree shrews via intraperitoneal injection at a dose of 50-100 mg / kg on days 1, 3 and 5. (3) After the drug administration was completed, the Parkinson's disease model was established by feeding the animals normally for 4 weeks.

2. The method for establishing a tree shrew Parkinson's disease animal model according to claim 1, characterized in that, The mass of the tree shrew is 120-150g.

3. The method for establishing a tree shrew Parkinson's disease animal model according to claim 1, characterized in that, The tree shrews were raised at a room temperature of 24±2℃, a relative humidity of 50±5%, and a light / dark cycle of 12h / 12h.

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