Method for establishing a unilateral single-hole double-point Parkinson's rat model and unilateral single-hole double-point Parkinson's rat model

Abstract

The present invention belongs to the technical field of Parkinson's treatment, and specifically relates to a method for establishing a unilateral single-hole double-point Parkinson's rat model and a unilateral single-hole double-point Parkinson's rat model. The method for establishing a unilateral single-hole double-point Parkinson's rat model includes a brain localization step and a fixed-point injection step. In the fixed-point injection step, based on the brain stereotaxic atlas of rats, the site coordinates of the first injection point and the second injection point are determined unilaterally in the brain of the rat. A single hole is opened on this side based on the site coordinates of the first injection point and the second injection point, and a micro-injection needle is used for fixed-point injection. The Parkinson's rat model and the method for establishing the model provided by this application adopt the method of unilateral hole opening and double-point injection to construct the Parkinson's animal model. The success rate reaches 80% in the second week after modeling and 100% in the third week. It not only significantly improves the success rate of modeling, but also can reduce the mechanical damage of the model, improve the safety of modeling, shorten the modeling duration, and effectively improve the modeling efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of Parkinson's treatment, and specifically relates to a method for establishing a unilateral single-hole double-point Parkinson's rat model and a unilateral single-hole double-point Parkinson's rat model. Background Art

[0002] Parkinson's disease (PD) is a common chronic central neurodegenerative disease mainly characterized by the degeneration of the nigrostriatal pathway. Its main pathological features are: ① the degeneration and death of dopamine (DA)-ergic neurons in the substantia nigra of the midbrain, accompanied by a significant decrease in the DA content in the striatum; ② Lewy bodies produced by the misfolding of alpha-synuclein (α-synuclein). It can affect different neurotransmitter systems and different nervous system regions. The pathogenesis of PD has not been fully elucidated, and there is currently no effective drug to cure or reverse the disease. To better understand the pathological mechanism of Parkinson's disease and seek more effective therapeutic drugs, it is crucial to establish an animal model of Parkinson's disease.

[0003] According to different treatment methods, PD animal models can be divided into three categories: neurotoxin models, transgenic models, and combined models of the two. Neurotoxin models are the earliest animal models used in PD research and are also one of the most commonly used means to evaluate the therapeutic effect of PD. This type of model is simple to operate, has a low cost, good result repeatability, and obvious advantages over other models, and is widely used in actual research. The most commonly used neurotoxins currently are: 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and pesticide neurotoxins such as rotenone and paraquat. Transgenic models use genetic methods to modify specific species with PD-related genes to create transgenic animal models related to PD. Transgenic models and combined models of the two have a high cost and a small application range, and are often used to study the pathogenesis and pathological changes of PD at the gene and molecular level.

[0004] 6-OHDA is a substance structurally similar to catecholamine. It was first used in the study of PD animal models in 1960. The characteristics of this model are rapid disease course, simple operation, high repeatability, stability and safety, and it is widely used. It can be used for experimental studies at the molecular level, cellular level, and evaluation of drug prevention and treatment effects of PD. The model is mainly established by stereotactic injection of 6-OHDA. Commonly used brain regions include the striatum, substantia nigra pars compacta, ventral tegmental area of the midbrain, and medial forebrain bundle. After modeling, the success rate of the model is verified by apomorphine-induced rotation on the healthy side (usually 7 rotations per minute is used as the standard for rats). According to research, the number of rotations induced by apomorphine in animals is proportional to the degree of damage to dopaminergic neurons in the substantia nigra. That is, when the damage to dopaminergic neurons is mild, the compensatory effect of the nigrostriatal pathway causes no rotation phenomenon after the application of apomorphine. Only when the dopaminergic neurons are damaged to a certain extent will the apomorphine-induced rotation behavior appear. When injected unilaterally, the motor symptoms are more obvious and generally no bilateral motor disorders occur, which is conducive to the observation of experimenters. The injection methods include unilateral single-point injection method and unilateral multi-point injection method. The degree of damage of the unilateral single-point method model varies, and the success rate generally does not exceed 50%. The unilateral multi-point method usually combines two brain regions or uses different coordinates in a single brain region, and it is one of the most commonly used models. According to previous domestic and foreign literature reports, the success rate of the unilateral two-point method ranges from 32% to 92%, and the success rate of most studies is less than 80%, and it takes 4 weeks after modeling to succeed. Summary of the Invention

[0005] In view of this, the present invention provides a method for establishing a unilateral single-hole double-point Parkinson's rat model and a unilateral single-hole double-point Parkinson's rat model to solve the technical problems existing in the prior art.

[0006] The method for establishing a unilateral single-hole double-point Parkinson's rat model provided by the present invention to solve its technical problems is as follows:

[0007] A method for establishing a unilateral single-hole double-point Parkinson's rat model, characterized by comprising:

[0008] Brain localization step: Keep the top of the rat's skull horizontal. Taking the tip of the microinjection needle as the reference, measure the ordinate of the center points of the bregma and the lambda, and adjust the bregma and the lambda to the same horizontal level;

[0009] Fixed-point injection procedure: According to the brain stereotaxic atlas of rats, determine the site coordinates of the first injection point and the second injection point on one side of the rat's brain. Make a single hole on this side according to the site coordinates of the first injection point and the second injection point, and use a microinjection needle for fixed-point injection of 6-OHDA. Among them: The site coordinates of the first injection point are: 4.2 mm posterior to bregma, 1.2 mm to the right of the midline, 8.0 mm below the skull surface; The site coordinates of the second injection point are: 4.9 mm posterior to bregma, 0.9 mm to the right of the midline, 8.2 mm below the skull surface; Or, the site coordinates of the first injection point are: 4.2 mm posterior to bregma, 1.2 mm to the left of the midline, 8.0 mm below the skull surface; The site coordinates of the second injection point are: 4.9 mm posterior to bregma, 0.9 mm to the left of the midline, 8.2 mm below the skull surface; The concentration of 6-OHDA is 2.5 μg / μL, and the injection doses of the first injection point and the second injection point are both 5.5 - 6.5 μL.

[0010] Preferably, in the fixed-point injection procedure, the injection speed is 1 μL / min, and the needle retention time after injection is 5 min.

[0011] Preferably, the injection doses of the first injection point and the second injection point are both 6.0 μL.

[0012] Preferably, the first injection point and the second injection point are respectively located in the medial forebrain bundle and the ventral tegmental area of the midbrain.

[0013] Preferably, the method for establishing a unilateral single-hole double-point Parkinson's animal model further includes a model detection step. Two weeks after the completion of the fixed-point injection step, inject 5 mg / mL of apomorphine subcutaneously at the top of the skull according to the rat's body weight, with an injection volume of 1 mL / kg. Observe the behavioral changes, continuously time for 30 minutes. If the rotation is towards the healthy side and the number of rotations is greater than 7 r / min, it is considered that the model establishment is successful; if the rotation direction is incorrect or the number of rotations is insufficient, it is considered that the model establishment fails.

[0014] The unilateral single-hole double-point Parkinson's rat model provided by the present invention to solve its technical problems is:

[0015] A unilateral single-hole double-point Parkinson's rat model, characterized in that: this unilateral single-hole double-point Parkinson's rat model is made by using the method for establishing a unilateral single-hole double-point Parkinson's rat model described in any of the foregoing technical solutions, and has the following characteristics: There are model-making hole-opening marks on the right side of the skull of the rat model between 4.2 mm and 4.9 mm posterior to bregma and between 0.9 mm and 1.2 mm to the right of the midline, and there are only such model-making hole-opening marks on the skull of the rat model; Or, there are model-making hole-opening marks on the left side of the skull of the rat model between 4.2 mm and 4.9 mm posterior to bregma and between 0.9 mm and 1.2 mm to the left of the midline, and there are only such model-making hole-opening marks on the skull of the rat model.

[0016] Preferably, the aperture of the hole-making trace is less than or equal to 1.0 mm.

[0017] Beneficial technical effects:

[0018] The Parkinson's rat model and its modeling method provided by this application use the method of unilateral hole opening and double-point injection to construct the Parkinson's animal model. The success rate reaches 80% in the second week after modeling and 100% in the third week. It not only significantly improves the modeling success rate, but also can reduce the mechanical damage of the model, improve the modeling safety, shorten the modeling time, and effectively improve the modeling efficiency.

[0019] The following combines the specification drawings and specific implementation manners to introduce the technical solutions and technical effects of this application in detail. Description of the Drawings

[0020] Figure 1 : Coordinate diagram of the first injection point

[0021] Figure 2 : Coordinate diagram of the second injection point

[0022] Figure 3 : Comparison of TH staining in the left and right brain regions of the rat model. Specific Embodiment

[0023] The method for establishing a unilateral single-hole double-point Parkinson's rat model provided by the present invention includes a brain positioning step and a fixed-point injection step. In the brain positioning step, keeping the top of the rat's skull horizontal, taking the tip of the microinjection needle as the reference, measuring the vertical coordinates of the midpoints of the anterior fontanelle and the posterior fontanelle, and adjusting the anterior fontanelle and the posterior fontanelle to the same horizontal level. In the fixed-point injection step, according to the brain stereotaxic atlas of the rat, the site coordinates of the first injection point and the second injection point are determined on one side of the rat's brain. A single hole is opened on this side according to the site coordinates of the first injection point and the second injection point, and 6-OHDA is injected at a fixed point with a microinjection needle; wherein: the site coordinates of the first injection point are: 4.2 mm behind the anterior fontanelle, 1.2 mm to the right of the midline, 8.0 mm below the skull surface; the site coordinates of the second injection point are: 4.9 mm behind the anterior fontanelle, 0.9 mm to the right of the midline, 8.2 mm below the skull surface; or, the site coordinates of the first injection point are: 4.2 mm behind the anterior fontanelle, 1.2 mm to the left of the midline, 8.0 mm below the skull surface; the site coordinates of the second injection point are: 4.9 mm behind the anterior fontanelle, 0.9 mm to the left of the midline, 8.2 mm below the skull surface; the concentration of 6-OHDA is 2.5 μg / μL, and the injection doses of the first injection point and the second injection point are both 5.5 - 6.5 μL.

[0024] Please refer to Figures 1 - 3 , and the following takes the right brain side hole opening and modeling as an example to describe the technical solution of this application in detail.

[0025] 1. Anesthetize the SD rats and remove the hair on the top of the skull with a razor.

[0026] 2. Locate with a stereotaxic apparatus for the brain: Fix the head of the SD rats with ear rods, and fix the mouth and nose of the SD rats with the front fixator to keep the top of the skull horizontal.

[0027] 3. Disinfect the skin preparation area on the top of the skull with alcohol, incise the skin, and expose the anterior fontanelle, posterior fontanelle and sagittal suture of the skull. Taking the tip of the microinjection needle as the standard, measure the ordinate of the central points of the anterior and posterior fontanelles to keep the anterior and posterior fontanelles on the same horizontal plane for preparing the brain region positioning.

[0028] 4. After adjusting the anterior and posterior fontanelles to the same level, adjust the tip of the microinjection needle to the central point of the anterior fontanelle, zero it, and then adjust the site coordinates of the first injection point and the second injection point. Obtain the sites of the ventral tegmental area (the second injection point) and the medial forebrain bundle (the first injection point) of the SD rats according to the stereotaxic atlas of the experimental rats. The site coordinates of the first injection point are: 4.2 mm posterior to the anterior fontanelle, 1.2 mm to the right of the midline, 8.0 mm below the skull surface, as Figure 1 shown; the site coordinates of the second injection point are: 4.9 mm posterior to the anterior fontanelle, 0.9 mm to the right of the midline, 8.2 mm below the skull surface, as Figure 2 shown. And make a mark at this coordinate point with a marker pen, and drill a hole with a dental drill with a diameter of about 1 mm.

[0029] 5. Aspirate 6 μL of 6-OHDA with a concentration of 2.5 μg / μL with the microinjection needle, and then inject it at a rate of 1 μL / min. After the injection is completed, keep the needle in place for 5 minutes. The injection dose and method for the second brain region are the same. After injecting the drugs into the two brain regions, suture the skin.

[0030] 6. Two or three weeks later, inject apomorphine subcutaneously at the top of the skull according to the body weight of the SD rats at a dose of 5 mg / mL and an injection volume of 1 mL / kg, and observe the behavioral changes. Continuously time for 30 minutes after injecting apomorphine. If the rotation is towards the healthy side and the number of turns is greater than 7 r / min, it is considered that the modeling is successful. If the rotation direction is incorrect or the number of turns is insufficient, it is considered that the modeling fails.

[0031] Experimental results:

[0032]

[0033] As can be seen from the above table, the method for constructing a Parkinson's rat model with unilateral single-hole double-points provided by this application uses the method of unilateral hole opening and double-point injection to construct a Parkinson's animal model. The success rate reaches 80% in the second week after modeling and 100% in the third week. It not only significantly improves the modeling success rate, but also can effectively shorten the modeling duration and improve the modeling efficiency. At the same time, it can also reduce the mechanical damage to the rat model and increase the modeling safety.

[0034] Please refer toFigure 3 , there are obvious differences in the substantia nigra striatum regions of the left and right brains of the rat model. There is no positive staining in the substantia nigra striatum region on the right side (the damaged side), and no obvious dopamine neurons are seen. It shows that the rat model constructed by the method for establishing a unilateral single-hole double-site Parkinson's rat model provided by this application also has technical effects such as high quality reliability and high coincidence with Parkinson's symptoms. Therefore, the unilateral single-hole double-site Parkinson's rat model provided by this application, compared with other Parkinson's rat models, also has the following characteristics: There are modeling opening traces between 4.2 mm and 4.9 mm behind the anterior fontanelle and between 0.9 mm and 1.2 mm to the right of the midline on the right side of the cranial bone of the rat model, and there are only such modeling opening traces on the cranial bone of the rat model; or, there are modeling opening traces between 4.2 mm and 4.9 mm behind the anterior fontanelle and between 0.9 mm and 1.2 mm to the left of the midline on the left side of the cranial bone of the rat model, and there are only such modeling opening traces on the cranial bone of the rat model. The aperture of the modeling opening trace is less than or equal to 1.0 mm.

[0035] The technical solutions and technical effects of the present invention have been elaborated in detail above in combination with the accompanying drawings of the specification and specific embodiments. It should be noted that the specific embodiments disclosed in the specification are only the preferred embodiments of the present invention, and those skilled in the art can also develop other embodiments on this basis; any simple deformation and equivalent replacement that do not deviate from the innovative concept of the present invention are covered by the present invention and belong to the protection scope of this patent.

Claims

1. A method for establishing a Parkinson's disease rat model with unilateral single-hole and double-point positions, characterized in that, Comprising: Brain localization step: Keep the top of the rat's skull horizontal. Taking the tip of the microinjection needle as a reference, measure the vertical coordinates of the central points of the bregma and the lambda, and adjust the bregma and the lambda to the same level. Fixed-point injection step: Determine the site coordinates of the first injection point and the second injection point unilaterally in the rat's brain according to the brain stereotaxic atlas of the rat. Open a single hole on this side according to the site coordinates of the first injection point and the second injection point, and perform 6-OHDA fixed-point injection with a microinjection needle; wherein: the site coordinates of the first injection point are: 4.2 mm posterior to the bregma, 1.2 mm lateral to the right of the midline, 8.0 mm below the skull surface; the site coordinates of the second injection point are: 4.9 mm posterior to the bregma, 0.9 mm lateral to the right of the midline, 8.2 mm below the skull surface; or, the site coordinates of the first injection point are: 4.2 mm posterior to the bregma, 1.2 mm lateral to the left of the midline, 8.0 mm below the skull surface; the site coordinates of the second injection point are: 4.9 mm posterior to the bregma, 0.9 mm lateral to the left of the midline, 8.2 mm below the skull surface; the concentration of 6-OHDA is 2.5 μg / μL, and the injection doses of the first injection point and the second injection point are both 5.5 - 6.5 μL.

2. The method for establishing a unilateral single-hole double-point Parkinson's rat model according to claim 1, wherein: In the fixed-point injection step, the injection speed is 1 μL / min, and the needle retention time after injection is 5 min.

3. The method for establishing a unilateral single - hole double - point Parkinson's rat model according to claim 1, wherein: The injection doses of the first injection point and the second injection point are both 6.0 μL.

4. The method for establishing a unilateral single-hole double-point Parkinson's rat model according to claim 1, wherein: The first injection point and the second injection point are respectively located in the medial forebrain bundle and the ventral tegmental area of the midbrain.

5. The method for establishing a unilateral single-hole double-point Parkinson's rat model according to claim 1, characterized in that: The method for establishing a unilateral single-hole double-point Parkinson's animal model further comprises: Model detection step: 2 weeks after the completion of the fixed-point injection step, inject apomorphine with a concentration of 5 mg / mL subcutaneously at the top of the skull according to the rat body weight, with an injection volume of 1 mL / kg, observe the behavioral changes, continuously time for 30 minutes, and those with a rotation towards the healthy side and a rotation speed greater than 7 r / min are regarded as successful model establishment, and those with incorrect rotation direction or insufficient rotation speed are regarded as failed model establishment.

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