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帕金森,动物模型
z-bo
2020-07-17
703
To simulate human PD, the ideal animal model should have the following characteristics: (1) The number and morphology of dopaminergic neurons at birth are normal, and gradually decrease gradually during youth, the reduction is more than 50%, and it is easy to pass neurochemistry And neurophysiological methods; (2) the model should be able to easily detect the impairment of its motor function, including the main symptoms of PD such as bradykinesia, muscle rigidity, and resting tremor; (3) the model should be able to show Lewy bodies Formation; (4) If the model is hereditary, the mutation model should have a strong transmission ability based on a single mutation; (5) The model should have a relatively short disease period (such as several months) to make drug screening economical And proceed quickly.
There are two methods that are widely used internationally and domestically: ① by injecting 6-hydroxydopamine (6-OHDA) in the substantia nigra or medial forebrain tract of rats ② through model animals (such as: monkeys, mice, pigs, etc.) Tube feeding and stereotactic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The former is a more classic model preparation method, and it was also carried out earlier in China, but its existence ① the damage of neurons appeared earlier, which is related to the progressive death of midbrain dopaminergic neurons in clinical PD patients. Different ② close-range damage is difficult to grasp the degree of damage, which makes the success rate of using this method to obtain partially damaged models very low; compared with the former, the MPTP tube feeding method and the stereotactic injection method have only been used in China in recent years. Using the preparation of Parkinson's animal model, MPTP has been used to establish a more ideal PD model in monkeys and mice abroad. The preparation usually uses subcutaneous, intravenous, intraperitoneal injection and intramuscular administration. It has reliable methods and good repeatability. However, the danger is high, and there are strict requirements for test personnel and test conditions [1].
Model preparation
Application of 6-dopamine to prepare PD model
In 1968, Ungenstdet first reported the use of 6-pass dopamine (6-OHDA) to cause Parkinson's disease animal models in rats. Some features of this animal rotation model are similar to those of human thieves. Recently, people have created animal models that simulate Parkinson's disease at different stages based on the injection site and injection dose of 6-OHDA.
Injection of 6-OHDA to prepare Parkinson's disease rats [2]:
1) The experiment selected 36 healthy male Wistar rats weighing 250 ~ 300g to prepare a rat model of Parkinson's disease
2) Rats are anesthetized by intraperitoneal injection of pentobarbital (48mg/kg)
3) Dissecting operation Anesthetized rats were fixed on Jiangwan Type I C stereotaxic instrument with a mid-head incision, peeling off the periosteum, and hemostasis. According to the George spectrum, use a dental drill to drill the top of the skull, the positioning coordinates are: incisor parallel to the ear rod, 3mm behind the anterior chamber, 3.0mm laterally, 9.0mm below the skull surface, insert the self-made concentric sleeve into the right NS area ( The outer diameter of the sleeve is 0.7mm, the outer diameter of the inner tube is 0.4mm, and the inner tube is 1.5mm longer than the front end of the outer tube). It is fixed with 502 glue and self-curing dental tray powder.
4) A micro-syringe draws 4µL of 6-OHDA solution (containing 9.0µg 6-OHDA and 8.8µg ascorbic acid) at a rate of 1µg/min. After leaving the injection, it is left for 4 minutes to exit and suture the skin.
5) Record the three major symptoms of Parkinson's disease with reference to Francois's experimental methods (including bradykinesia test, gripping test and tail rigidity test to record changes in rigidity symptoms, tremor test) on 21 days. And record the changes in EMG to identify whether the rat model was established successfully. The judging criteria are as follows: ① the action delay experiment lasts for 35 minutes ② the grip experiment lasts for 55 minutes and the tail rigidity experiment lasts for 30 minutes ③ the tremor frequency is above 48 times/minute and the frequency of EMG group discharge position is 4-8 times/second.
Using 6-OHDA to prepare the lateral PD mouse model [3]:
1) Male Sprague-Dawley rats weighing 230～250g
2) After the animals were anesthetized with pentobarbital sodium (30 mg/kg), they were fixed on a stereotaxic instrument (Jiangwan Type I C).
3) According to the stereotaxic map of the rat brain, take two target points a: 5.0mm lateral to the bregma, 1.9mm to the right of the midline, and 7.4mm ventral to the dura; point b: 5.3mm to the bregma, midline 2.5mm on the right, 6.5mm on the ventral side of the dura). 12μg 6-OHDA (Sigma) was stereotactically injected into two targets on the right substantia nigra a and b (injection volume 6μg per point, concentration 2μg/μl, dissolved in normal saline containing 0.1% ascorbic acid). Injection speed 0.3µl/min. Note the retention needle 15min.
4) Three weeks after the establishment of the behavior detection model, apomorphine (APO) was used to induce the rat to rotate to the left (undamaged side) at 0.25 mg/kg, record the number of rotations within half an hour, and select 210 rpm/30 min ( 7 rounds/min) The above animals are successful PD models
Apply 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine to prepare PD animal model
Primate PD model [4]
Rhesus monkey under pentobarbital (40mg/kg) abdominal anesthesia, cut the neck skin, and exposed the common carotid artery by blunt separation. 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 blood vessels in the direction of blood flow. If the animal's PD symptoms are not obvious after the first operation, they are given 2 to 4 injections every 5 days until the symptoms of PD appear. The primate bilateral and lateral PD models established with MPTP are very similar to the characteristics of 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 models. .
MPTP is used to make Parkinson's disease mouse model［5］
The sensitivity of mice to MPTP differs in germline and age. Adult C57BL rats aged 10-12 weeks old and weighing 25-30g are generally selected, and MPTP is injected intraperitoneally at a weight of 30-40 mg/kg for 7 consecutive days. After the 6th to 7th injections, temporary (2~3h) trunk shocks, vertical hairs, tail overstretching, decreased movements and obstacles in the climbing test appeared.
Some precautions when MPTP is used in the production of a small country model of Kimson’s disease [6]
a) Effects of mouse germline on MPTP sensitivity C57BL mice are 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 the least sensitive to MPTP. The most commonly used mice are C57BL/6 mice
b) The effect of rat age on MPTP sensitivity Old rats are more sensitive to MPTP than young rats. This difference in sensitivity is not only manifested in the aged rats after cis-treatment can present more obvious numbers of substantia nigra dopaminergic neurons. Decreased, and the number of adrenal neurons in the blue spot area and the more typical behavioral changes of Parkinson's disease can also be observed in aging mice after cis-treatment
Other methods for building models
Reserpine model Reserpine can mainly deplete the dopamine (DA) and other catecholamine transmitters stored in the vesicles by inhibiting the reuptake function of norepinephrine neuron terminals. Male Wistar rats (weight 280-300g) injected intraperitoneally with a certain dose of reserpine can cause bone injury muscle slowing, tremor, body flexion, too little exercise and other sports symptoms. Neurochemical analysis found that the content of DA and other monoamine transmitters in the striatum of the rat was significantly reduced. therefore. Such models mimic the clinical manifestations and neurochemical changes of PD to a certain extent. But it has obvious shortcomings: First, the movement disorder caused by drug injection varies greatly at different times and between different individuals; second, the blood circulation causes multiple transmitters to be released at the same time and cannot cause PD-like pathological changes. Therefore, the application of this model is limited to the experiment of exploring the effect of drugs on muscle stiffness, and the study of other motor symptoms has been rarely applied.
Fe3+ model In 1991, Ben-Shachar injected Fe3+ into the unilateral substantia nigra of male SD rats (250~300g) for three weeks, which showed obvious changes in motor behavior, which showed reduced voluntary movement in a new environment and short stiffness. Symptoms and spontaneous rotation of the same test. Amphetamine (AMPH) can enhance this co-rotation behavior. Neurochemical studies have found that the DA content of the ipsilateral striatum is reduced by an average of 95%, and the contents of its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and high vanillic acid (HVA) are also significantly reduced. Histopathology confirmed that the number of tyrosine-mediated enzyme (TH) immunostaining positive cells decreased greatly, and the proliferation of glial cells was active.
A mechanical injury model study found that mechanical damage to the medial anterior rib bundle (MF can cause DA neurons in the substantia nigra to progressively die. The established modeling methods include MFB singulation and midbrain hemisection, especially the former. More. The establishment of MFB excision injury model needs to be accurately fixed on the rat brain stereotaxic instrument, and use a special Scouten wire knife to go to the location where the MFB is located. Brechne uses this method to cut off male CFHB rats (150 ~ After 180g) of MFB, the number of surviving DA neurons was counted at 1, 2, 4, 6, 10, and 16 weeks, respectively, and it was found that about 28% of the substantia nigra (SN) neurons died in about one week. After 10 weeks About 70% of the cells die. The average number of SN neuron survival is 29%. Retrograde tracking shows that the success rate of MFB cutting is 92% to 99%. It can be seen that the method has the following advantages: ①High success rate, more 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 that the degree of damage is unstable in a short time after cutting off .
Quantitative observation of behavioral changes in mice model of Parkinson's disease
1) Climbing pole test
2) Suspension experiment
3) Swimming experiment
The above quantitative observation methods provide some more accurate and objective methods for studying the behavior of Parkinson's disease mouse model, and also improve the objectivity of the entire experiment.