A method for establishing a cynomolgus monkey animal model of Parkinson's disease

Abstract

A method for establishing a cynomolgus monkey animal model of Parkinson's disease belongs to animal experimental technology. Using cynomolgus monkeys as model animals, AAV-SNCA is first injected into the substantia nigra by stereotaxic method, with a total injection volume of 42-48 μl; after an interval of 85-95 days, PAR is injected into the same position again, with a total injection volume of 23-27 μl. The method for establishing an animal model of the present invention is safe and reliable, simulates the pathological, imaging and behavioral results of PD, and has good application prospects.

Description

Technical Field

[0001] The invention belongs to animal experiment technology, and specifically relates to a method for establishing a Parkinson's disease cynomolgus monkey animal model. Background Art

[0002] Parkinson's disease (PD) is the second largest neurodegenerative disease in the world. Its main clinical manifestations are motor symptoms such as resting tremor, bradykinesia, muscle rigidity and gait instability, as well as non-motor symptoms such as sleep disorders, depression and cognitive changes. The prevalence of PD in people over 65 years old is about 2%. With the aging of the population, the incidence of the disease continues to increase, and it has become a heavy medical and economic burden on the country and families. The typical pathological characteristics of PD (including sporadic and hereditary PD) are the progressive death of dopaminergic neurons in the substantia nigra of the midbrain and the formation of Lewy bodies in the central nervous system. Among them, Lewy bodies are mainly composed of α-syn inclusions and their aggregates, so PD is considered to be an α-synuclein disease. Although there are many hypotheses and conjectures about the pathogenic mechanism of PD, the accuracy and reliability of these theories still need to be verified. Due to the unclear pathogenic mechanism, the research on disease treatment methods has been largely limited. In order to further study the pathogenic mechanism of PD and develop effective treatment measures that can change the progression of the disease, it is necessary to establish a suitable animal model. At present, the most commonly used rodent model in PD research is the one established by the drug 6-OHDA. However, due to the large differences between rodents and humans in genetic similarity and brain structure, the experimental data obtained from using rodents as research subjects have limited clinical guidance significance. The brain anatomical structure, physiological function, immune and metabolic systems of cynomolgus monkeys are closer to those of humans, and the genetic homology between cynomolgus monkeys and humans is highly similar. They have advanced brain functions, including complex cognitive processes and emotional expressions. The use of cynomolgus monkeys as a model for PD disease mechanism research and preclinical drug research has higher value. For many years, the PD non-human primate model established by MPTP has been recognized as the gold standard model. However, the PD model established by MPTP cannot replicate the multi-system pathological manifestations of PD, directly leading to the death of midbrain dopaminergic neurons, and cannot replicate the formation of intracellular α-syn aggregates. It cannot simulate the disease process of clinical patients. Therefore, from the perspective of etiology, the establishment of a cynomolgus monkey PD model can simulate the pathogenesis of PD, which is an urgent need for treatment research and preclinical drug evaluation. Using the cynomolgus macaque PD model in the study of stem cell transplantation strategies can better simulate the spread of pa-syn after cell transplantation into the pathological brain environment, as well as the survival of cells in an inflammatory environment after transplantation, providing a better disease model for clinical transformation. It can also provide a window for screening neuroprotective drugs in the early stages of the disease. Summary of the invention

[0003] The purpose of the present invention is to establish a method for establishing a Parkinson's disease cynomolgus monkey animal model with a high modeling success rate, pathological, behavioral and imaging characteristics, and better for treatment research and drug evaluation.

[0004] Overexpression of α-syn by adenovirus (AAV) can cause neuronal loss and aggregation of α-syn after injection into the brain, which can simulate the pathological characteristics of PD to a certain extent (death of dopaminergic neurons in the substantia nigra), but it is difficult to simulate some PD-like motor defects (such as resting tremor, etc.). PARP-1 was found in the autopsy brain of PD patients. Its metabolite PAR can interact with the amino terminus of α-syn to accelerate the fibrillation of α-syn, transforming α-syn into a structure that is more resistant to proteinase K digestion, more toxic, easier to spread and more phosphorylated. Injection of a-syn fibers combined with PAR can accelerate the obvious PD progression in mice. Therefore, we established a PD monkey model by injecting AAV virus overexpressing α-syn and injecting PAR at the same time, aiming to quickly and efficiently obtain a PD monkey model with clinical pathology, behavior and imaging characteristics. At present, this combined modeling method has not been reported in crab-eating macaques, so its use in modeling of non-human primates requires rigorous experimental research.

[0005] The method of the present invention is as follows: using cynomolgus monkeys as model animals, using brain stereotaxic positioning, first injecting AAV-SNCA in the substantia nigra, with a total injection volume of 42-48 μl; after an interval of 85-95 days, injecting PAR in the same position, with a total injection volume of 23-27 μl.

[0006] Preferably, the method of the present invention comprises the following steps: deeply anesthetizing the cynomolgus monkey, fixing the head, removing the hair, disinfecting, exposing the skull with a scalpel, determining the position of the AP0 point by a locator, determining the injection position on the skull surface according to the position of the striatum measured by MRI and marking it, first injecting AAV into the substantia nigra with a total amount of 45 μl, and then injecting tolfenamic acid, Vetoquinol S.A, 4 mg / kg, im; after an interval of 3 months, injecting PAR into the same position with a total amount of 25 μl, and injecting tolfenamic acid (4 mg / kg, im) in the same manner after the operation. After the virus and PAR injection, each animal is fed with a special feed and has free access to water.

[0007] The advantages and positive effects of the present invention are:

[0008] 1. The method for establishing the animal model of the present invention is safe and reliable, simulates the pathological, imaging and behavioral results of PD, and has good application prospects.

[0009] 2. The establishment of an AAV+PAR-induced PD cynomolgus monkey model can be used to study the pathogenesis and disease progression of PD, explore the impact of the disease environment after cell transplantation on treatment, screen neuroprotective drugs, and conduct preclinical drug evaluation, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The Kurlan scores of the cynomolgus monkeys after injection in the example.

[0011] Figure 2 The imaging examination comparison of a cynomolgus monkey before and after modeling in the embodiment. A in the figure shows that there is no significant difference in the statistics of dopamine transporter uptake on the left and right sides of the striatum before virus and PAR injection, and B shows that the virus and PAR were injected into the right substantia nigra. 18 months after the injection, the dopamine transporter uptake in the right striatum was significantly reduced compared with the ipsilateral side before modeling, and significantly reduced compared with the contralateral side without injection, indicating the death of dopaminergic neurons.

[0012] Figure 3 This is an immunohistochemical staining image of dopaminergic neurons in the substantia nigra of the brain tissue of a cynomolgus monkey after modeling in the embodiment.

[0013] Figure 4 The figure is an immunohistochemical staining image of Lewy bodies in the substantia nigra and striatum of the brain tissue of a cynomolgus monkey after modeling in the example.

[0014] Figure 5 The figure is an immunohistochemical staining image of glial cells in the substantia nigra and striatum of the brain tissue of a cynomolgus monkey after modeling in the example.

[0015] The present invention is further described below in conjunction with the accompanying drawings, and the present invention includes but is not limited to the contents listed in the embodiments. DETAILED DESCRIPTION

[0016] See the Examples below.

[0017] 1. Model establishment

[0018] Four elderly male cynomolgus monkeys weighing 6-8 kg were selected as model animals. AAV-SNCA and PAR were injected into the model animals by stereotaxic brain positioning. Before the operation, the experimental monkeys were deeply anesthetized with ketamine hydrochloride injection (0.2 mL / Kg, im), atropine hydrochloride injection (0.02 mg / Kg, im), and Su Mian Xin II injection (2 mg / Kg, im), and penicillin (Harbin Pharmaceutical Group, 5×105 units, im) was injected to prevent infection. After the experimental monkeys were thoroughly anesthetized, they were transferred to the operating table and the head was fixed with a brain positioning device; then the hair was removed with a scalpel and disinfected. The skull was exposed with a scalpel, and the AP0 point was located by a locator. The injection site on the skull surface was determined and marked based on the striatum location measured by MRI. AAV was injected into the substantia nigra. The injection sites were: SN1: 18.4 mm anterior-posterior; 45.86 mm lateral; 13.9 ± 0.5 mm depth (5 μl of virus was injected every 0.5 mm) SN2: 17.2 mm anterior-posterior; 47 mm lateral; 13.4 ± 0.5 mm depth (5 μl of virus was injected every 0.5 mm)

[0019] SN3: 15.5mm anterior to posterior; 48.01mm lateral; 14.6±0.5mm depth, AAV-SNCA (BrainVTA, rAAV-hSyn-SNCA-P2A-EGFP-3flag-WPRE-hGH Pa, 9-3562-K210308, 1.00E+14vg / ml), injection of tolfenamic acid (Vetoquinol SA, 4mg / kg, im) after surgery to reduce animal infection and relieve pain. PAR was injected again 3 months after surgery, and the injection site was exactly the same as the site of virus injection, with a total volume of 25μl, and tolfenamic acid (4mg / kg, im) was injected after the surgery. After virus and PAR injection, each animal was fed with special feed and had free access to water.

[0020] (II) Diagnostic evaluation and results of PD in cynomolgus monkeys

[0021] Including behavioral evaluation, imaging, and pathological histological diagnosis.

[0022] 1. Behavioral evaluation is mainly based on PD Coulomb score

[0023] See Figure 1 The behavior was recorded every 3 months after modeling, and the recorded videos were scored by Kurlan. The scoring results showed that the model monkeys had higher scores after modeling than before modeling. Figure 1 The horizontal axis is time (month) and the vertical axis is score.

[0024] 2. Imaging examination

[0025] See Figure 2 Under ketamine muscle anesthesia, the head was scanned using SPECT, and the images were saved for analysis. The results showed that compared with before modeling, the number of dopamine transporters in the model monkey's lateral brain was significantly reduced.

[0026] 3. Histopathological analysis

[0027] See Figure 3-Figure 5 After euthanasia, a monkey was selected for experiment, and the brain tissue was peeled off, fixed and dehydrated. Immunohistochemistry was used to detect the reduction of dopaminergic neurons, the formation of Lewy bodies and the proliferation of glial cells. Pathological section analysis results:

[0028] The experimental monkeys showed a significant loss of dopaminergic neurons in the substantia nigra on the virus-injected side, accompanied by the appearance of Lewy bodies, which also caused a persistent inflammatory response, and microglia were activated and infiltrated.

Claims

1. A method for establishing a Parkinson's disease cynomolgus monkey animal model, characterized in that: Using cynomolgus monkeys as model animals, AAV-SNCA was first injected into the substantia nigra using stereotaxic methods, with a total injection volume of 42-48 μl; after an interval of 85-95 days, PAR was injected into the same location again, with a total injection volume of 23-27 μl.

2. The method for establishing a cynomolgus monkey animal model of Parkinson's disease according to claim 1, characterized in that: The cynomolgus monkeys were deeply anesthetized, their heads were fixed, hair was removed, and disinfected. The skull was exposed with a scalpel, and the AP0 point was located using a locator. The injection site on the skull surface was determined and marked based on the striatum location measured by MRI. AAV-SNCA was first injected into the substantia nigra with a total volume of 45 μl, followed by an injection of tolfenamic acid, Vetoquinol SA, 4 mg / kg, im. After an interval of 3 months, PAR was injected into the same location with a total volume of 25 μl, and after the operation, 4 mg / kg, im of tolfenamic acid was injected. After the virus and PAR injections, each animal was fed with feed and had free access to water.

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