动物模型,帕金森病 z-bo 2020-08-13 622

　　In order to simulate human PD, an ideal animal model should have the following characteristics: (1) The number and morphology of dopaminergic neurons at birth are normal, and they begin to gradually and selectively decrease in youth, with a decrease of more than 50%, and they are easy to pass neurochemistry. And neurophysiological methods; (2) The model should be able to easily detect the damage of its motor function, including the main symptoms of PD such as slow motion, muscle stiffness and resting tremor; (3) The model should be able to show the Lewy body Formation; (4) If the model is hereditary, it should be based on a single mutation so that the mutation model has a strong transmission capacity; (5) The model should have a relatively short disease cycle (such as several months), so that drug screening can be economical , Proceed quickly.

　　Two methods are widely used internationally and domestically: ①By injecting 6-hydroxydopamine (6-OHDA) into the substantia nigra or medial forebrain tract of rats, ②By treating model animals (such as monkeys, mice, pigs, etc.) 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTR) gavage and stereotactic injection methods. The former is a more classic model preparation method and was also developed earlier in China, but its existence ①The damage of neurons appears earlier, which is related to the progressive death of dopaminergic neurons in the substantia nigra of clinical PD patients Different ② It is difficult to grasp the extent of damage at close range, which makes the success rate of obtaining partially damaged models by this method is very low; compared with the previous methods, MPTP gavage and stereotactic injection methods are used in China. Applied to the preparation of Parkinson's animal models, MPTR has been used abroad to establish ideal PD models in monkeys and mice. The preparation usually uses subcutaneous, intravenous, intraperitoneal injection and intramuscular administration, which are reliable and reproducible. However, it is highly dangerous and has strict requirements for test personnel and test conditions.

　　1. Preparation of model mice

　　In 1968, Ungenstdet first reported the use of 6-dopamine (6-OHDA) to cause Parkinson's disease animal models in rats. Some features of this animal rotation model are similar to human thief Kinsen disease. Recently, people have created animal models that mimic Parkinson's disease at different stages based on the injection site and dosage of 6-OHDA.

　　Inject 6-OHDA to prepare Parkinson's disease mice:

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

　　2) Rats are anesthetized by intraperitoneal injection of pentobarbital (48mg/kg)

　　3) Dissection operation Fix the anesthetized rat on the Jiangwan I-C stereotactic instrument, make a midline incision on the head, peel off the periosteum, and stop bleeding. Refer to the atlas of George and use a dental drill to drill a hole on the top of the skull. The positioning coordinates are: incisors parallel to the ear bars, 3mm behind the front chimney, 3.0mm lateral opening, 9.0mm below the skull surface, and insert the self-made concentric cannula into the right NS area ( The outer diameter of the casing is 0.7mm, the outer diameter of the inner tube is 0.4mm, and the front end of the inner tube is 1.5mm larger than the outer tube. It is fixed with 502 glue and self-setting dental powder.

　　4) Micro-syringe sucks 4μL of 6-OHDA solution (containing 9.0μg 6-OHDA and 8.8μg ascorbic acid) at a speed of 1μg/min. After injection, it is left for 4min to exit and the skin is sutured.

　　5) The three major symptoms of Parkinson's disease were recorded for 21 days with reference to Francois' experimental methods (including slow-moving test, grasping test and tail rigidity test to record changes in rigidity symptoms, and tremor test). And record the changes of EMG to identify whether the rat model is successful. Judging criteria are as follows: ①The slowness of movement experiment lasted for 35min.②The grasping experiment lasted for 55 minutes and the tail rigidity experiment lasted for 30min.③The tremor frequency was above 48 times/min and the EMG group discharge position frequency was 4-8 times/s.

　　2. Using 6-OHDA to prepare a hemilateral PD mouse model

　　1) Male Sprague-Dawley rats, weighing 230-250g

　　2) After the experimental animals were anesthetized with sodium pentobarbital (30mg/kg), they were fixed on a stereotactic instrument (Jiangwan Type I C).

　　3) Refer to the stereotactic map of the rat brain, take two target points a: 5.0mm caudal to bregma, 1.9mm to the right of midline, 7.4mm ventral to the dura; point b: caudal 5.3mm to bregma, midline 2.5mm on the right side and 6.5mm on the ventral side of the dura mater). 12μg of 6-OHDA (Sigma) was stereotactically injected into the two targets of substantia nigra a and b on the right side (the injection volume was 6μg, the concentration was 2μg/μl, and it was dissolved in normal saline containing 0.1% ascorbic acid). The injection speed is 0.3μl/min. Inject the final needle for 15 minutes.

　　4) 3 weeks after the establishment of the behavior detection model, use apomorphine (APO) at 0.25 mg/kg to induce the rat to rotate to the left (uninjured side), record the number of rotations within half an hour, select 210 revolutions / 30 min ( Animals above 7 revolutions/min) are successful PD models

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

　　3. PD model of primate

　　Rhesus monkeys were anesthetized with pentobarbital (40mg/kg) in the abdominal cavity, cut the neck skin, and exposed one common carotid artery after blunt separation. The animals were injected into the common carotid artery, that is, freshly prepared MPTP (Sigma) was dissolved in 2 mL of normal saline at 1.0-1.5 mg/kg body weight, and slowly injected into the blood vessel against the direction of blood flow. If the animals have no obvious PD symptoms after the first operation, they will be given 2-4 injections every 5 days until the PD symptoms appear. The primate bilateral and hemilateral PD models established with MPTP are very similar to human PD in terms of symptoms, physical evidence, pathology, biochemistry, and response to drug treatment, and are considered to be the most representative PD animal model .

　　4. MPTP is used to make Parkinson's disease mouse model

　　The sensitivity of mice to MPTP differs in germline and age. Generally, 10-12 weeks old adult C57BL Rattus norvegicus with a weight of 25-30g are selected, and MPTP is injected intraperitoneally at a weight of 30-40 mg/kg for 7 days. After the 6th to 7th injections, temporary (2~3h) torso shock, vertical hair, tail hyperextension, reduced movement, and obstacles to the climbing test occurred.

　　5. Some precautions when MPTP is used in the making of a small country model of kinsen disease

　　C57BL mice are the most sensitive to MPTP, while CF-W mice, FVB/N mice and Balb/C mice are slightly less sensitive to MPTP than C57BL mice. CF-1 and CD-1 are sensitive to MPTP Worst. The most commonly used mice are C57BL/6 mice

　　B) The effect of rat age on MPTP sensitivity

　　Older mice are more sensitive to MFTP than young mice. This difference in sensitivity is not only reflected in the more obvious reduction in the number of dopaminergic neurons in the substantia nigra after treatment, but also A decrease in the number of adrenergic neurons in the locus coeruleus area and typical behavioral changes in Parkinson’s disease were observed in old mice

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

　　1) Climbing pole test

　　2) Hanging experiment

　　3) Swimming experiment

　　The above several quantitative observation methods provide some more accurate and objective methods for the study of the behavior of Parkinson's disease mouse models, and at the same time improve the objectivity of the whole experiment.

　　7. Other methods of building models

　　1) Reserpine model Reserpine can deplete dopamine (DA) and other catecholamine transmitters stored in vesicles by inhibiting the reuptake function of noradrenergic neuron terminals. Injecting a certain dose of reserpine into the abdominal cavity of male Wistar rats (body weight of 280-300g) can cause slow stiffness, tremor, body flexion, insufficient exercise and other motor symptoms. Neurochemical analysis found that the content of DA and other monoamine transmitters in rat striatum was significantly reduced. therefore. This type of model simulates the clinical manifestations and neurochemical changes of PD to a certain extent. But it has obvious shortcomings: First, the dyskinesia caused by drug injection has great variability at different times and between different individuals; the second is that blood relieving causes the release of multiple transmitters at the same time and cannot cause pathological changes similar to PD. Therefore, the application of this model is limited to experiments that explore the effects of drugs on muscle stiffness, and the research on other motor symptoms is rarely used.

　　2) Fe3+ model In 1991, Ben-Shachar in male SD rats (250~300g) unilaterally injected Fe3+ into the substantia nigra for three weeks, which can cause obvious changes in motor behavior, which is manifested as decreased voluntary movement in the new environment and transient Symptoms of stiffness and spontaneous co-test rotation behavior. Amphetamine (AMPH) can enhance this simultaneous rotation behavior. Neurochemical studies have found that the DA content of ipsilateral striatum is reduced by an average of 95%, and the content of its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) is also significantly reduced. Histopathology confirmed that tyrosine was greatly reduced by TH immunostaining positive cells, and glial cells proliferated actively.

　　3) The study of mechanical injury model found that mechanical injury to the medial anterior costal tract (MF can cause the progressive death of DA neurons in the substantia nigra. The established modeling methods include MFB mitomy and hemisection of the midbrain, especially the former There are many applications. The establishment of the MFB amputation injury model requires accurate positioning on the rat brain stereotaxic instrument, and a special Scouten wire knife is used to penetrate the MFB to cut it off. Brechne uses this method to cut off male CFHB rats ( After 150～180g) MFB, the number of surviving DA neurons was counted at 1, 2, 4, 6, 10, and 16 weeks. It was found that about 28% of substantia nigra (SN) neurons died in about one week. About 70% of the cells died after one week. The average number of SN neurons survived was 29%. Retrograde tracking showed that the success rate of MFB severance was 92% to 99%. It can be seen that this method has the following advantages: ①High success rate, relatively Economical. ②The substantia nigra DA neurons show progressive death, which can simulate the whole process of PD pathological changes, which has certain significance for studying the regeneration of neurons and the prevention of PD. The disadvantage is the degree of damage in a short time after amputation Unstable.