帕金森病动物模型 z-bo 2020-10-14 800

　　In order to simulate human Parkinson's disease, an ideal animal model should have the following characteristics:

　　(1) The number and morphology of dopaminergic neurons at birth are normal, and they are gradually and selectively reduced at a young age, reducing by more than 50%, and they are easy to pass neurochemical reactions. And neurophysiological methods;

　　(2) The model should be able to easily detect impaired motor function, including the main symptoms of PD, such as delay, muscle stiffness and resting tremor;

　　(3) The body of the model Lewy must be able to show the shape;

　　(4) If the model is hereditary, it must be based on a single mutation, so that the mutation model has strong transferability;

　　(5) The model has a relatively short disease cycle (for example, several months), and drug screening should be economical and can be performed quickly.

　　Generally, two methods are used at home and abroad.

　　(1) By treating model animals (monkeys, mice, pigs, etc.), 6-hydroxydopamine (6-OHDA) is injected into the substantive substantia nigra or anteromedial of the rat. ) Perform 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTR) forced feeding and stereotactic injection. The former is a more classic modeling method, developed earlier in China, but its existence neuron damage appears earlier, which is the progressive dopaminergic neurons of PD patients with a large number of substantia nigra in clinical practice.

　　(2) Because it is difficult to grasp the degree of damage at close range, compared with the previous method, the success rate of using this method to obtain a partially damaged model is very low. In contrast, China has adopted MPTP mandatory feeding and stereotactic infusion methods. MPTR is used to create Parkinson's animal models, and has been used to establish ideal PD models in foreign monkeys and mice. This preparation is usually used for reliable and reproducible subcutaneous, intravenous, intraperitoneal and intramuscular administration. However, this is very dangerous and has strict requirements on testers and test conditions.

　　1. Preparation of mouse model

　　In 1968, Ungenstdet first reported the process of using 6-dopamine (6-OHDA) to induce Parkinson's disease in rats. Some features of this animal rotation model resemble thief Kinsen's disease. Recently, people have created animal models that simulate Parkinson's disease at various stages based on the injection site and the dose of 6-OHDA. 6-OHDA was injected to prepare Parkinson's disease rats: 1 In the experiment, 36 healthy male Wistar rats weighing 250-300 g were selected to prepare a Parkinson's disease rat model.

　　2) Intraperitoneal injection of barbital (48 mg/kg) was used for rat anesthesia

　　3) The anesthetized rat was fixed on the Jiangwan Type I C stereotactic fixation device by anatomical surgery, the beginning of the middle was cut, and the periosteum was cut. Peel it off and stop the bleeding. See George Atlas and use a dental drill to make a hole in the upper part of the skull. The placement coordinates are a notch parallel to the ear bar, 3 mm behind the front chimney, 3.0 mm lateral opening, 9.0 mm below the skull surface, and a self-made concentric sleeve inserted into the NS area on the right (outer diameter of the sleeve). The diameter is 0.7mm, the outer diameter of the inner tube is 0.4mm, and the distance between the inner tube and the front end of the outer tube is 1.5mm, fixed with 502 adhesive and self-curing denture powder

　　4) The volume of the microsyringe is 4μL. Aspirate 6-OHDA solution (containing 9.0μg6). -OHDA and 8.8μg ascorbic acid) were injected at a rate of 1μg/min, and then left for 4 minutes to suture the skin.

　　5) For the three main symptoms of Parkinson’s disease, please refer to the Francois test method (including slow exercise test, grip strength and tail stiffness test to record changes in tonic symptoms and tremor test). 21 days were recorded. Then, it records the changes in EMG to identify whether the rat model is successful. The judgment criteria are as follows. (1) The slow motion experiment takes 35 minutes, the grip experiment takes 55 minutes, and the tail stiffness experiment takes 30 minutes. (3) The frequency of vibration is more than 48 times/minute, and the frequency of the discharge position of the EMG group is 4-8 times/second. use

　　2, 6-OHDA

　　1) Create a lateral PD mouse model 1) Weight 230-250 g

　　2) Experimental animals anesthetized with pentopental sodium (30 mg/kg) and a fixed stereotaxic fixture (for harbor type IC).

　　3) Refer to the location map of the rat brain, and take two target points: 5.0 mm from the front B tail, 1.9 mm from the right midline, 7.4 mm from the ventral sclerosing agent, point b: 5.3 from the B tail, 2.5 mm to the right of the midline, 6.5 mm on the ventral side of the dura). Stereotactic injection of 12μg of 6-OHDA (Sigma) at the two target points of the right parenchyma a and b (injection amount 6μg, concentration 2μg/μl, normal physiology, containing 0.1% ascorbic acid). Dissolved in saline). The injection speed is 0.3μl/min. Inject the last needle for 15 minutes.

　　4) Three weeks after the establishment of the behavior detection model, the rat was rotated to the left (full side) with 0.25 mg/kg apomorphine (APO). We recorded the number of revolutions in 30 minutes and chose 210 revolutions. Animals older than 30 minutes (7 revolutions per minute) are successful PD models

　　1- Use 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine

　　3 Preparation of PD animal model. Akagesaru, a primate PD model, was anesthetized in the abdominal cavity of pentobarbital (40 mg/kg). After blunt separation, the neck skin was incised and a common carotid artery was exposed. The animal was injected into the common carotid artery. That is, the newly prepared MPTP (Sigma) is dissolved in 2 mL of physiological saline with a body weight of 1.0 to 1.5 mg/kg, and slowly injected into the blood vessel along the blood flow direction. If the animal has no obvious PD symptoms after the first operation, the injection is given 2-4 times every 5 days until PD symptoms appear. The bilateral and hemilateral PD models of primates established with MPTP are very similar, and are the most representative of human PD in terms of symptoms, physical evidence, pathology, biochemistry, and response to drug treatment. The PD animal model is considered to be.

　　4, MPTP is used to create a mouse model of Parkinson's disease. The sensitivity of mice to MPTP depends on their breed and age. Normally, adult C57BL Rattus norvegicus, 10-12 weeks old, weighing 25-30 g, are injected with MPTP intraperitoneally at a weight of 30-40 mg/kg for 7 days. After the 6th to 7th injections, there was a brief body tremor (2-3 hours), vertical hair, excessive tail extension, reduced movement and impaired climbing test.

　　5. Use MPTP to create a small country model of Kinsen disease

　　"C57BL mice, some precautions are most sensitive to MPTP, while CF-W mice, FVB/N mice and Balb/C mice are sensitive to MPTP. Yes. CF-1 and CD-1 have the lowest sensitivity to MPTP, which is slightly worse than that of C57BL mice. Currently, the most commonly used mice are C57BL/6 mice. b) The effect of mouse age on MPTP sensitivity. Older mice are more sensitive to MFTP than young mice. This difference in sensitivity is not only reflected in the difference in sensitivity after treatment. Older mice may show a substantial decrease in the number of degraded aminergic neurons, a decrease in the number of adrenergic neurons in the site area, and more typical Parkinson's disease behavior. It can also be observed in aged mice after treatment. Learning changes

　　6. Quantitative observation of behavior changes in a mouse model of Parkinson's disease

　　1) Climbing pole test

　　2) Suspension experiment

　　3) Swimming experiment

　　The above-mentioned quantitative observation method is Parkinson's method. The behavioral study of the affected mouse model provides some more accurate and objective methods, and also improves the objectivity of the entire experiment.

　　7. Other methods to build models

　　1) The reserpine model mainly inhibits the reuptake function of noradrenergic neuron terminals, which leads to dopamine (DA) in vesicles and other catecholamine transmitters. Consumption. Injecting a certain amount of Reserpin into the abdominal cavity of male Wistar rats (body weight 280-300 g) slows down the stiffness of the injured muscle and may cause tremor, flexion, lack of exercise and other motor symptoms. .. Neurochemical analysis showed that the content of DA and other monoamine mediators in rat striatum was significantly reduced. therefore. This type of model simulates the clinical manifestations and neurochemical changes of PD to some extent. However, it has obvious disadvantages. First of all, motor dysfunction caused by drug injection varies greatly between individuals at different times. Secondly, blood resuscitation can cause multiple transmitters to be released at the same time, which cannot cause pathological changes similar to PD. Therefore, the application of this model is limited to experiments to study the effects of drugs on muscle stiffness, and the research on other motor symptoms is rarely used.

　　2) Fe3+ model In 1991, Ben-Shachar of a male SD rat (250-300g) unilaterally injected Fe3+ into a large amount of Hegra for 3 weeks. , Short-term stiffness symptoms and voluntary joint testing rotation behavior. Amphetamine (AMPH) can enhance this simultaneous rotation operation. Neurochemical studies have reduced the DA content of the ipsilateral striatum by an average of 95%, as well as the content of its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and high purity acid (HVA). I find. Histopathology confirmed that TH immunostaining positive cells significantly reduced tyrosine and actively proliferated glial cells.

　　3) Research on the mechanical damage model found that mechanical damage to the medial anterior rib bundle (MF can lead to the progressive death of a large number of established Nigra DA neurons. Modeling methods include MFB ridge and hemiectomy. The former is widely used and needs to be Precise positioning on the rat brain stereotactic device to establish the MFB split injury model, followed by the use of a special scout wire knife to pierce the MFB and cut the MFB. Brechne used this method to cut male CFHB. MFB mice (150-180 g) Calculating the number of DA neurons at 1, 2, 4, 6, 10, and 16 weeks of survival revealed that approximately 28% of substantive substantia nigra (SN) neurons died within 1 week. After 10 weeks, approximately 70% of these cells Of people died. The average number of surviving SN neurons was 29%. Retrograde follow-up showed that the success rate of MFB resection was 92% to 99%. (1) More economical than the method with higher success rate (2) Solid Nigra DA neurons It shows progressive death and can simulate the entire process of PD pathological changes. It is of great significance to the study of PD regeneration and prevention. Its disadvantage is that it is damaged short-term after cutting and the degree is unstable.