动物模型,帕金森 z-bo 2020-07-29 610

　　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, with more than 50% decreasing, young people can easily pass neurochemical reactions and gradually decrease. And neurophysiological methods; (2) The model should be able to easily detect the damage of its motor function, including the key symptoms of PD, such as motor delay, muscle stiffness and resting tremor; (3) The model should be able to display Lewy bodies; (4) ) If the model is hereditary, it should be based on a single mutation, so the mutation model has a strong ability to spread (5) The model should have a relatively short disease cycle (for example, several months) to make drug screening economical, direct.

　　There are two general domestic applications internationally: ①Inject 6-hydroxydopamine (6-OHDA) into the substantia nigra or the inner forebrain ②Model animals (monkeys, mice, pigs, etc.) 1-methyl-4-phenyl- 1,2,3,6-Tetrahydropyridine (MPTR) is injected via oral tube and stereotactic. The former is a more classic model preparation method, developed earlier in 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. (2) It is difficult to understand the degree of damage at a short distance, so the success rate of using this method to obtain a partially damaged model is very low, while China's MPTP is far less successful than the previous method. Use tube irrigation and stereotactic injection methods. MPTR is used to prepare animal models of Parkinson's disease and has been used abroad to establish ideal PD models in monkeys and mice. The preparation is usually used for reliable and reproducible subcutaneous, intravenous, intraperitoneal injection and intramuscular administration. However, this is very dangerous and has strict requirements on testers and test conditions. 1. Preparation of model mice

　　In 1968, Ungenstdet first reported the use of 6-dopamine (6-OHDA) in an animal model of Parkinson's disease in rats. Some features of this animal rotation model resemble human thief marigolds. Recently, people have established animal models that mimic each stage of Parkinson's disease based on the injection site and the dose of 6-OHDA.

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

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

　　2) Rat barbital anesthetized by intraperitoneal injection (48mg/kg)

　　3) The rat anesthetized by the dissection operation is fixed on the Jiangwan I-C stereotaxic device with the head centered. Cut open and peel off the periosteum to stop bleeding. Refer to George's Atlas and use a dental drill to drill a hole in the top of the skull. The positioning coordinates are parallel to the incisor of the ear bar, 3mm behind the front chimney, 3.0mm lateral opening, 9.0mm on the surface of the skull, and a self-made concentric sleeve inserted in the right NS area. (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 adhesive 502 and self-curing denture powder.

　　4) The microsyringe sucked 4μL of 6-OHDA solution (containing 9.0μg6)-OHDA and 8.8μg of ascorbic acid at a rate of 1μg/min, and then left it for 4 minutes to drain the skin and sew the skin.

　　5) The 21 main days of Parkinson’s disease refer to François’ experimental methods, including slow motion test, grip test, tail stiffness test and tremor test to record changes in tension symptoms. Record the EMG changes to identify the success of the rat model. The judgment criteria are as follows. (1) Slow motion lasts for 35 minutes (2) Grip strength test lasts for 55 minutes, and tail stiffness test lasts for 30 minutes (3) Tremor frequency is more than 48 times/min, and the discharge position frequency of EMG group is 4-8 times/sec.. use

　　2,6-PDDA

　　Prepare a lateral PD mouse model

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

　　2) Anesthetize experimental animals with sodium pentobarbital (30 mg/kg), and a fixed stereotaxic device (Jiangwan IC type).

　　3) Referring to the stereotactic map of the rat brain, two target points a: anterior reg caudal 5.0 mm, midline right 1.9 mm, dura ventral 7.4 mm, point b: anterior reg caudal 5.3 mm, midline right 2.5 mm, 6.5 mm on the ventral side of the dura). 12μg of 6-OHDA (Sigma) was stereotactically injected into the two targets of substantia nigra a and b on the right side (injection amount 6μg, concentration 2μg/μl, 0.1% ascorbic acid normally used) 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 behavioral detection model, the rat was rotated to the left (full side) at a speed of 0.25 mg/kg using apomorphine (APO). Record the number of revolutions in 30 minutes and select 210 revolutions. Animals over 30 minutes (7 revolutions/min) have a successful PD model

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

　　Preparation of PD animal model 3. Primate PD model rhesus monkeys were anesthetized with intraperitoneal pentobarbital (40 mg/kg), cut the neck skin, and exposed a common carotid artery by dissecting with a blunt instrument. The animal was injected into the common carotid artery. That is, freshly prepared MPTP (Sigma) was dissolved in 2 mL of physiological saline at 1.0 to 1.5 mg/kg body weight, and slowly injected into the blood vessel along the blood flow direction. If the animal has no obvious PD symptoms after the first operation, 2-4 injections every 5 days until PD symptoms appear. The primate bilateral and hemilateral PD models established by MPTP are very similar to human PD in terms of symptoms, physical evidence, pathology, biochemistry, and response to drug treatment, and are the most representative. It is considered a typical PD animal model.

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

　　The sensitivity of MPTP varies with strain and age. Generally, 10 to 12-week-old adult C57BL Rattus norvegicus weighing 25 to 30 g are selected, and MPTP is injected intraperitoneally at a weight of 30 to 40 mg/kg for 7 days. After the 6th to 7th injections, there was a temporary (2 to 3 hours) trunk shock, vertical hair, excessive tail extension, reduced exercise and impaired climbing tests.

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

　　"C57BL mice are the most sensitive to MPTP, but CF-W, FVB/N and Balb/C mice are MPTP. Compared with C57BL mice, CF-1 and CD-1 are the least sensitive to MPTP. Currently, the most commonly used mice are C57BL/6 mice. b) The effect of mouse age on the sensitivity of MPTP. The sensitivity of old mice to MFTP is higher than that of young mice. This difference in sensitivity can not only be reflected in cis-therapy. In older mice, the number of dopaminergic neurons in the substantia nigra was significantly reduced, while the number of adrenergic neurons in the locus coeruleus area decreased. More typical Parkinson's disease behavior was also observed in old mice after treatment. Learning change

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

　　) 1) Climbing pole test

　　2) Suspension test

　　3) Swimming experiment

　　The above-mentioned quantitative observation method provides several more accurate and objective methods for Parkinson’s behavioral research on Staphylococcus aureus diseased mouse models, and at the same time improves the overall objectivity of the experiment.

　　7. Other methods to build models

　　1) The reserpine model is a dopamine (DA) catecholamine, which is stored in vesicles mainly by inhibiting the reuptake function of noradrenergic neuron terminals. The transmitter is exhausted. Injecting a fixed dose of reserpine into the abdominal cavity of male Wistar rats (body weight 280-300 g) can cause blunt stiffness, which may cause motor symptoms such as tremor, flexion, and lack of exercise. Neurochemical analysis showed 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 some extent. But it has obvious disadvantages. First, the dyskinesia caused by drug injection varies greatly between different periods and individuals. Secondly, the release of blood will release multiple transmitters at the same time, and cannot cause the same pathological changes as PD. Therefore, the application of this model is limited to experiments to study the effects of drugs on muscle stiffness, and other studies of motor symptoms are rarely used.

　　2) Fe3+ model In 1991, a male SD rat (250-300 g) Ben-Shachar injected Fe3+ into the substantia nigra for 3 weeks. , Short-term stiffness symptoms and spontaneous joint testing rotational movement. Amphetamine (AMPH) can enhance this simultaneous rotational movement. Neurochemical studies have shown that the DA content of ipsilateral striatum is reduced by an average of 95%, and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) are also significantly reduced. I already know that histopathology confirmed that TH immunostaining positive cells can significantly reduce tyrosine, while glial cells actively proliferate.

　　3) Mechanical damage model studies have shown that there is mechanical damage to the medial anterior rib (MF may cause the progressive death of DA neurons in the substantia nigra. The established modeling techniques include MFB ridges). In order to establish an MFB amputation injury model, it must be accurately fixed. Mouse stereotaxic equipment, and use a special Scouten to cut the MFC. Using a wire knife, Brechne used this method to cut male CFHB. MFB mice (150-180g) survived 1, 2, 4, 6, 10, and 16 weeks. We found that approximately 28% of substantia nigra (SN) neurons died within one week, and approximately 70% of cells died after 10 weeks. The average S. is 29%, retrograde tracking, and the success rate of MFB extraction is 92%-99%. It can be seen that this method has the following advantages: ① high success rate ② substantia nigra DA neurons show progressive death and can simulate the pathological changes of PD The whole process, which has certain importance in the research of neuron regeneration and PD prevention. It is about to be damaged soon and the degree is unstable.