疾病动物模型,帕金森病 z-bio 2021-03-08 331

　　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 gradually decrease selectively at the beginning of adolescence, reducing by more than 50. (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 model must be able to show the formation of Lewy bodies; (4) If the model is hereditary, it must be based on a single mutation, so that the mutation model has a strong transmission ability. 5) The model runs fast and the disease cycle is short (for example, several months), so drug screening is relatively economical.

　　There are two general domestic and international use methods. ① By treating model animals (monkeys, mice, pigs, etc.), 6-hydroxydopamine (6-OHDA) is injected into the substantia nigra or the medial forebrain tract of rats. ) Intravenous oral and stereotactic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTR). The former is a more classic modeling method developed in early China, but its existence (1) Neuronal damage appears earlier and is related to the progressive death of dopaminergic neurons in the substantia nigra of clinical PD patients. Because it is difficult to understand the degree of near-distance injury, the success rate of using this method to obtain partial injury models is very low. In China, compared with previous methods, MPTP uses forced oral and stereotactic infusion methods. MPTR is a preparation of Parkinson's disease animal models. It has been used abroad to establish ideal PD models in monkeys and mice. It is usually a reliable and reproducible subcutaneous, intravenous and intraperitoneal injection, and intramuscular injection. 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 use of 6-dopamine (6-OHDA) to create an animal model of Parkinson's disease in rats. Some features of this animal rotation model are similar to those of human thief Kinsen's disease. Recently, people have created animal models that mimic Parkinson's disease at different stages based on the different injection sites and injection doses of 6-OHDA.

　　Prepare Parkinson's disease rats with 6-OHDA infusion:

　　1) In the experiment, 36 healthy male Wistar rats weighing 250-300 g were selected to establish a rat model of Parkinson's disease.

　　2) Rats were anesthetized by intraperitoneal injection of barbital (48) (mg/kg))

　　3) The anesthetized rat is fixed on the Jiangwan IC stereotactic fixation device by anatomical surgery, the head is cut in the middle, and the periosteum is removed, and the skin is peeled off to stop the bleeding. Please refer to the George Atlas and use a dental drill to drill a hole in the upper part of the skull. The placement coordinates are the incision parallel to the ear bar, 3 mm behind the front chimney, 3.0 mm side opening, and 9.0 mm below the surface of the skull. , Insert your own concentric sleeve into the NS area on the right. The outer diameter of the sleeve is 0.7 mm, the outer diameter of the inner tube is 0.4 mm, and the distance between the inner tube and the front end of the outer tube is 1.5 mm. .. Fix with 502 glue and self-curing denture powder. 4) The microsyringe draws 4μL of 6-OHDA solution (containing 9.0μg6)-OHDA and 8.8μg of ascorbic acid at a rate of 1μg/min. After injection, let it stand for 4 minutes and suture the skin.

　　5) The three main symptoms of Parkinson's disease are François's experimental methods (including slow exercise test, grip test and tail stiffness test to record changes in tonic symptoms and tremor test), reference records for 21 days. Then, it records the changes in EMG to recognize the success of the rat model. The judgment criteria are as follows. (1) Motor retardation test for 35 minutes (2) Grip strength test for 55 minutes, and tail stiffness test for 30 minutes (3) Tremor frequency is 48 times/min or more, and EMG group discharge position frequency is 4 to 8 times per second. use

　　2, 6-OHDA

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

　　2) Anesthetize experimental animals with sodium pentobarbital (30 mg/kg), stereotactic fixation and fixation device (Jiangwan IC).

　　3) Refer to the rat brain location map, two target points a: reg forearm 5.0 mm, midline right 1.9 mm, ventral dura mater 7.4 mm, point b: take one target point for two tail vertebrae. The anterior reg is 5.3 mm, the right side of the midline is 2.5 mm, and the ventral dura is 6.5 mm. Stereotactic injection of 12 μg of 6-OHDA (Sigma) into the two targets of the substantia nigra a and b on the right (the injection volume is 6 μg/point, the concentration is 2 μg/μl, saline containing 0.1% ascorbic acid). )). The injection speed is 0.3μl/min. Inject the last needle for 15 minutes.

　　4) Three weeks after the establishment of the behavioral detection model, use 0.25mg/kg of apomorphine (APO) to rotate the rat to the left (full side). Record the number of revolutions in 30 minutes. Animals over 30 minutes (7 revolutions per minute) are successful PD models

　　Use 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine 1 to prepare PD animal model

　　3. Primate PD model

　　Rhesus monkeys were anesthetized with pentobarbital (40 mg/kg) in the abdominal cavity, an incision was made in the skin of the neck, and a common carotid artery was separated and exposed with a blunt instrument. The animal was injected into the common carotid artery. That is, the newly prepared MPTP (Sigma) was dissolved in 2 mL of saline at 1.0-1.5 mg/kg body weight, and slowly injected into the blood vessel along the blood direction. flow. 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 unilateral 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.

　　4, MPTP is used to create a mouse model of Parkinson's disease

　　The sensitivity of

　　mice to MPTP depends on the germline and age. In general, adult C57BL adult rats aged 10-12 weeks with a body weight of 25-30 g are selected, and MPTP is injected intraperitoneally at 30-40 mg/kg body weight for 7 days. After the 6th to 7th injections, there was a temporary (2-3 hours) trunk shock, vertical hair, excessive tail extension, reduced movement and impaired climbing tests.

　　5. Precautions when using MPTP to create a small country model of Kinsen's disease

　　C57BL mice are the most sensitive to MPTP, but CF-W mice, FVB/N mice and Balb/C mice have slightly worse MPTP. CF-Compared with C57BL mice, Figure 1 and CD-1 sensitivity to MPTP lowest. 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 Sith. Treatment of aging mice may show a significant reduction in the number of degraded aminergic neurons, a decrease in the number of adrenergic neurons in the cerebellar trajectory area, and more typical Parkinson’s disease behavior. It can also be seen in older mice after treatment. Observed in. Parkinson's disease mouse model

　　1) Climbing pole test

　　2) Suspension experiment

　　3) Swimming experiment

　　Behavioral Study of Diseased Mouse Model

　　6. Learning quantitative observation is a more accurate objective method, and at the same time it improves the objectivity of the whole experiment.

　　7. Other ways to build a model

　　1) The reserpine model mainly inhibits the reuptake function of the noradrenergic neuron terminal, which leads to the accumulation of dopamine (DA) in vesicles and other catecholamine transmitters and gradually consumes it. Injecting a fixed dose of reserpine into the peritoneum of male Wistar rats (body weight 280-300 g) can cause stiffness, tremor, flexion, lack of exercise and other motor symptoms. Neurochemical analysis showed that the content of DA and other monoamine transmitters in rat striatum was significantly reduced. and so. This type of model simulates the clinical manifestations and neurochemical changes of PD to a certain extent. However, there are obvious shortcomings. First, the dyskinesia caused by lethal injections varies greatly from person to person at different times. Secondly, refusal of blood will result in the simultaneous release of multiple transmitters, and will not cause pathological changes similar to PD. .. Therefore, the application of this model is limited to experiments that study the effects of drugs on muscle stiffness, and it is rarely used to study other motor symptoms.

　　2) Fe3+ model In 1991, male SD rats (250-300 g) Ben-Shachar unilaterally injected Fe3+ into the substantia nigra for 3 weeks. This may cause major changes in sports behavior. New environment, short-term stiffness symptoms, voluntary collaborative testing of alternate behaviors. Amphetamine (AMPH) can enhance this simultaneous rotation operation. Neurochemical studies have shown that the DA content of the ipsilateral striatum is reduced by an average of 95%, and its biotransformants 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) are also significantly reduced. Histopathology confirmed that TH immunostaining positive cells significantly reduced tyrosine and actively proliferated glial cells.

　　3) Mechanical damage model research found that mechanical damage to the medial anterior rib cage (MF can lead to the progressive death of DA neurons in the substantia nigra. The established modeling methods include MFB ridge and semi-resection, especially the former widely used. MFB cutting injury model It needs to be accurately fixed on the rat brain stereotactic fixation device, and it is a special scout wire knife used to pierce the MFB and cut the MFB. Brechne used this method to cut the male CFHB.? 180 g) Cut with MFB, 1 , 2, 4, 6, 10 and. Counting the DA neurons that survived within 16 weeks, we found that about 28% of the substantia nigra (SN) neurons died within 1 week after 10 weeks. , About 70% of the cells die. The average number of surviving SN neurons was 29%. Retrograde tracking shows that the success rate of MFB cutting is between 92% and 99%. You can see that this method has the following advantages: ①It is more economical than a higher success rate. (2) The DA neurons of the substantia nigra show progressive death, which can simulate the whole process of the pathological changes of PD, and has certain significance in the research of neuron regeneration and PD prevention.