A method for constructing a tree shrew Parkinson's model

Abstract

The present invention discloses a method for constructing a Parkinson's disease model in tree shrews, comprising the following steps: (1) weighing ceramide and dissolving it in sterilized ultrapure water to prepare a solution with a concentration of 0.6 mg / mL; (2) administering 4 μg / g of ceramide to the tree shrews through the tail vein once a day for 7 consecutive days, and then raising the tree shrews normally for 2 weeks after the administration to complete the modeling. In this application, a stable Parkinson's disease model can be established in tree shrews by injecting ceramide into the tail vein at a dose of 4 μg / g for 1 week and then raising the tree shrews normally for 2 weeks after the administration.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease animal model construction, and in particular relates to a method for constructing a tree shrew Parkinson's disease model. Background Art

[0002] Parkinson's disease (PD), also known as parkinsonism, is a clinical syndrome caused by multiple factors. It is the second most common neurodegenerative disease worldwide and one of the leading causes of neurological dysfunction. The classic pathological manifestations of PD are the loss or even death of dopamine (DA) neurons in the substantia nigra compacta, leading to a significant decrease in striatal DA content and the abnormal aggregation of α-synuclein (α-syn) to form Lewy bodies, which contribute to the disease. Clinical manifestations include motor symptoms (bradykinesia, rigidity, resting tremor, and postural balance disorders) and non-motor symptoms (hyposmia, constipation, sleep disturbances, and depression).

[0003] Studies have shown that the pathogenesis of PD is complex, and its cause and pathogenesis are still unclear. However, a large amount of evidence shows that oxidative stress, neuroinflammation, apoptosis, mitochondrial dysfunction and proteasome dysfunction play an important role in the pathogenesis of PD.

[0004] Parkinson's disease animal models are powerful tools for elucidating the pathogenesis of Parkinson's disease and exploring new pharmacological interventions. Current animal models for Parkinson's disease are categorized into two main types: neurotoxin-induced and genetically engineered. Neurotoxin-based models of Parkinson's disease can be established and widely used by introducing neurotoxins such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), paraquat, and rotenone. However, existing animal models for Parkinson's disease require a long development time and primarily utilize rodents (rat and mice). Therefore, developing animal models that are more closely related to humans and require a shorter development time is crucial. Summary of the Invention

[0005] The present invention aims to provide a method for constructing a Parkinson's disease model in tree shrews. Tree shrews are highly similar to primates and even humans in terms of physiology, anatomy, neural development, and psychological stress patterns, and are widely used in biomedical research.

[0006] The object of the present invention is achieved by: (1) weighing ceramide and dissolving it in sterilized ultrapure water to prepare a solution with a concentration of 0.6 mg / mL;

[0007] (2) The tree shrews were given the solution described in step (1) by tail vein injection at a dose of 4 μg / g, once a day for 7 consecutive days. After the end of the administration, the tree shrews were raised normally for 2 weeks to complete the modeling.

[0008] The beneficial effects of the present invention are:

[0009] The present application establishes a stable Parkinson's disease model in tree shrews by injecting ceramide into the tail vein at a dose of 4 μg / g for 1 week and feeding the tree shrews normally for 2 weeks after the end of the administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The changes in TH protein expression in the four groups of tree shrews during the experiment of this application;

[0011] Figure 2 The changes in p-α-synuclein protein expression in four groups of tree shrews during the experiment of this application;

[0012] Figure 3 These are the TH immunofluorescence results of four groups of tree shrews during the experiment of this application;

[0013] Figure 4 This is the change in the average fluorescence intensity of TH immunofluorescence in the four groups of tree shrews during the experiment of this application. DETAILED DESCRIPTION

[0014] The following will clearly and completely describe the technical solution of the present invention in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, but not all of the embodiments. All other embodiments fall within the scope of protection of the present invention.

[0015] 1. Experimental Animals

[0016] Fifteen 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 house of Yunnan Luoyu Biotechnology Co., Ltd. [SYXK (Yunnan) K2021-0003]. They were housed in stainless steel cages at a room temperature of (24 ± 2) °C, a relative humidity of (50 ± 5)%, and a 12 h / 12 h light / dark cycle.

[0017] 2. Main reagents and consumables

[0018] Reagent name company Ceramide Sigma 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) Blue Sky Prestained protein marker Thermo Fisher Bovine serum albumin (BSA) Sigma SurperECLPlus Ultrasensitive Luminescence Prile Fluorescent mounting medium (containing DAPI) Zhongshan Jinqiao ZLI-9557 blocking sheep serum Zhongshan Jinqiao ZLI-9021 PVDF membrane Millipore

[0019] 3. Main instruments

[0020] Instrument name company Nanophotometer ultra-micro-spectrophotometer IMPLEN Protein electrophoresis tank, electrotransfer tank, power supply Bio-Rad ECL Luminescence Imager Tanon Fluorescence microscopy OLYMPUS BX53

[0021] 4. Preparation of reagents

[0022] Ceramide preparation: Weigh the required ceramide and dissolve it in sterile ultrapure water to prepare a solution with a concentration of 0.6 mg / mL.

[0023] 5. Experimental methods

[0024] 5.1 Animal model preparation

[0025] Experimental groups: ① normal group, ② ceramide 2 μg / g group, ③ ceramide 4 μg / g group, ④ ceramide 8 μg / g group; groups ②-④ were given tail vein injections of corresponding doses of ceramide solution according to the body weight of tree shrews according to the experimental groups, once a day for 7 consecutive days; group ① was injected with an equal volume of sterile ultrapure water through the tail vein.

[0026] 5.2 Behavioral Observations of Tree Shrews

[0027] After each injection, the tree shrews in each group were observed for short-term symptoms (2-3 hours) such as involuntary tremors, slow movement, unstable posture, limb stiffness, and erect hair and tail. The tree shrews were also observed for persistent symptoms such as decreased overall activity (including feeding ability and activity) and slow movement, unsteady gait, clumsy movements, hunched back, and uncoordinated forepaw movements.

[0028] 5.3 Detection

[0029] After the end of drug administration (7 days), the tree shrews were housed normally for 2 weeks before sampling. After anesthesia, the thorax was opened and transcardially perfused with normal saline. After observing the liver for blanching, the tree shrew brain tissue was removed and the substantia nigra was isolated on ice. The tissue was gently rinsed with 0.1 mol / L PBS. For every 100 mg of fresh tissue, 500 μL of RIPA lysis buffer (containing 50 μL of protease inhibitors) was added. The tissue was thoroughly minced with sterile ophthalmic scissors and homogenized with an ultrasonic cell disruptor for 20 seconds in an ice bath. The cells were then lysed for 20 minutes on ice. The cells were centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was collected and approximately 360 μL of supernatant was aspirated into each tube. 90 μL of 5× loading buffer (1 / 4 the volume of the supernatant) was added to each tube. The cells were thoroughly mixed, centrifuged briefly for 10 seconds, and then boiled in a 100°C metal bath for 10 minutes to fully denature the proteins. After the samples cooled, protein concentration was measured. Then, SDS-PAGE protein electrophoresis was performed using a constant voltage of 80V until the lower edge of the sample touched the separation gel (about 30 minutes), then a constant voltage of 130V was used to continue electrophoresis for nearly 2 hours, until the bromophenol blue just overflowed the lower edge of the separation gel; the membrane was transferred using a wet transfer at 280mA / 1h, and 5% skim milk was added for blocking at room temperature for 2 hours. Primary antibodies (rabbit anti-p-α-synuclein antibody 1:1000, rabbit anti-Tyrosine Hydroxylase antibody 1:5000) were added and incubated overnight in a silent mixer at 4°C. The membrane was washed three times with TBST buffer and incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:1000) at room temperature for 2 hours. The membrane was washed three times with TBST buffer and developed using a chemiluminescence method with ECL reagent on a gel imaging system.

[0030] 5.4 Immunofluorescence detection

[0031] After the end of drug administration (7 days), the tree shrews were housed normally for 2 weeks before sampling. After anesthesia, the tree shrews were transcardially perfused with saline and then 4% paraformaldehyde solution. The brains were removed and placed in 4% paraformaldehyde for paraffin section preparation and subsequent immunofluorescence staining.

[0032] Immunofluorescence staining: Dewax the paraffin sections and hydrate them. Then, immerse the sections in an antigen retrieval box containing pH 6.0 citrate buffer in a microwave oven for antigen retrieval. Block with 5% goat serum for 1 h, then add the primary antibody (TH 1:500) and incubate at 4°C overnight. Then, add 488-labeled goat anti-rabbit IgG (1:1000) and incubate at room temperature for 2 h in the dark. Mount the sections with a mounting medium containing DAPI and observe and photograph under an upright fluorescence microscope.

[0033] 6. Experimental Results

[0034] 6.1 Ceramide-induced behavioral manifestations in tree shrews

[0035] After three injections of 4μg / g and 8μg / g ceramide, tree shrews began to exhibit typical macroscopic motor symptoms of Parkinson's disease (PD), including involuntary tremors, bradykinesia, postural instability, limb stiffness, and erect hair and tail. These abnormalities typically appeared 1-3 minutes after ceramide injection and generally returned to normal within 2 hours. PD motor symptoms became more pronounced with increasing injection frequency. After six injections of 2μg / g ceramide, typical PD motor symptoms began to appear. The control group showed no abnormalities.

[0036] 6.2 Detection

[0037] Compared with the normal group, the protein expression of p-α-synuclein in the ceramide group was increased and positively correlated with the dose, and the differences were significant (p<0.05). Compared with the normal group, the protein expression of Tyrosine Hydroxylase (TH) in the ceramide group was decreased and negatively correlated with the dose, but there was no significant difference between the 4μg / g and 8μg / g groups.

[0038] Table 1 Changes in p-α-synuclein and TH protein expression in the four groups of tree shrews during the experiment

[0039] TH (tyrosine hydroxylase) p-α-synuclein NC 1.442±0.058 0.399±0.098 2μg / g Ceramide <![CDATA[1.202±0.056 b ]]> <![CDATA[0.619±0.015 b ]]> 4μg / g Ceramide <![CDATA[0.529±0.089 b,d ]]> <![CDATA[1.116±0.077 b,d ]]> 8μg / g Ceramide <![CDATA[0.458±0.109 b,d ]]> <![CDATA[1.274±0.051 b,d,e ]]>

[0040] n = 3, , a P <0.05, b P <0.01, compared with the NC group; c P <0.05, d P <0.01, compared with the 2 μg / g Ceramide group; e P <0.05, compared with the 4μg / g Ceramide group.

[0041] 6.3 TH immunofluorescence results

[0042] Compared with the normal group, the mean fluorescence intensity of Tyrosine Hydroxylase (TH) immunofluorescence in the ceramide group was weakened, proving that ceramide induced dopamine neuron damage in tree shrews, and there was little difference between the 4μg / g and 8μg / g groups.

Claims

1. A method for constructing a tree shrew Parkinson's disease model, characterized by: The steps include: (1) Weigh ceramide and dissolve it in sterile ultrapure water to prepare a solution with a concentration of 0.6 mg / mL; (2) The tree shrews were given the solution described in step (1) by tail vein injection at a dose of 4 μg / g, once a day for 7 consecutive days. After the end of the administration, the tree shrews were raised normally for 2 weeks to complete the modeling.

2. The method for constructing a tree shrew Parkinson's disease model according to claim 1, wherein: The tree shrew is a common male Burmese tree shrew.

3. The method for constructing a tree shrew Parkinson's disease model according to claim 1, wherein: The tree shrews have a body mass of 120-150 g and are kept at a room temperature of 24±2° C., a relative humidity of 50±5%, and a light / dark cycle of 12 h / 12 h.

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