Preparation of animal model of Parkinson's disease

In order to simulate human PD, the ideal animal model should have the following characteristics: (1) Dopaminergic neurons are normal in number and morphology at birth, gradually decreasing selectively in youth, reducing more than 50%, and easily passing neurochemistry And neurophysiological methods detected; (2) the model should be able to easily detect the damage of motor function, including the main symptoms of PD such as bradykinesia, muscle stiffness and resting tremor; (3) the model should be able to show the Lewy body Formation; (4) If the model is hereditary, the mutation model should have a strong ability to spread based on a single mutation; (5) The model should have a relatively short disease cycle (such as several months) to make drug screening economical. And proceed quickly.

There are two methods for international domestic application: 1 by injecting 6-hydroxydopamine (6-OHDA) 2 into the substantia nigra or medial forebrain of rats by model animals (eg monkeys, mice, pigs, etc.) Method of gavage and stereotactic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTR). The former is a more classical method of model preparation, and it is also carried out earlier in China, but its presence of 1 neuron damage occurs 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 a partially damaged model is very low; compared with the previous method using MPTP gage and stereotactic injection, it has only been in China in recent years. Applying to the preparation of Parkinson's animal model, MPTR has been used in foreign countries to establish an ideal PD model in monkeys and mice. The preparation is usually subcutaneous, intravenous, intraperitoneal and intramuscular, with reliable method and good reproducibility. However, it is highly dangerous and has strict requirements for testers and test conditions.

1. Model rat preparation

In 1968, Ungenstdet first reported the use of 6-dopamine (6-OHDA) in animal models of Parkinson's disease in rats. Some of the characteristics of this animal rotation model are similar to those of human thief Jinsen disease. Recently, people have simulated animal models of Parkinson's disease at different stages according to the injection site and injection dose of 6-OHDA.

Steps for preparing 6-OHDA to prepare Parkinson's disease mice:

1) Experimental selection of 36 healthy male Wistar rats weighing 250-300 g to prepare a rat model of Parkinson's disease

2) Rats were anesthetized with pentobarbital (48 mg/kg) by intraperitoneal injection.

3) Anatomical operation The anesthetized rats were fixed on a Jiangwan Type I C stereotactic instrument, the midline incision was made in the head, the periosteum was removed, and the bleeding was stopped. Referring to the George map, the dental drill is drilled at the top of the skull. The positioning coordinates are: the incisors are parallel to the ear stems, the posterior bony is 3 mm, the lateral opening is 3.0 mm, and the skull surface is 9.0 mm. The self-contained concentric cannula is inserted into the right NS region ( 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-setting teeth.

4) A micro syringe was used to inhale 4 μL of 6-OHDA solution (containing 9.0 μg of 6-OHDA and 8.8 μg of ascorbic acid) at a rate of 1 μg/min, and left for 4 minutes after injection to exit and suture the skin.

5) Record the three major symptoms of Parkinson's disease with reference to the Francois experimental method (including the slow-motion experiment, the grasping test, and the tail-strength test to record changes in the symptoms of tonicity, tremor). EMG changes were recorded to identify whether the rat model was successful. The judgment criteria are as follows: 1 The behavioral delay experiment lasted for 35 min2, the gripping experiment lasted for 55 min, the tail stiffness test lasted for 30 min3, the tremor frequency was above 48 beats/min, and the EMG group discharge frequency was 4-8 beats/s.

2. Preparation of unilateral PD mouse model using 6-OHDA

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

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

3) Referring to the stereotaxic map of the rat brain, take two targets a: 5.0 mm on the anterior caudal side, 1.9 mm on the right side of the midline, 7.4 mm on the ventral side of the dura; b: 5.3 mm on the anterior caudal side, midline 2.5mm on the right side, 6.5mm on the ventral side of the dura mater). 12 μg of 6-OHDA (Sigma) was stereotactically injected into two targets of the right substantia nigra a and b (injection amount of 6 μg per point, concentration of 2 μg/μl, dissolved in physiological saline containing 0.1% ascorbic acid). The injection speed is 0.3μl/min. Note that the needle is left for 15 minutes.

4) After the behavioral test model was established for 3 weeks, the rats were induced to rotate to the left side (uninjured side) with apomorphine (APO) at 0.25 mg/kg, and the number of rotations within half an hour was recorded, and 210 rpm/30 min was selected. Animals above 7 rpm are successful PD models

Preparation of PD animal model using 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine

3, primate PD model

Rhesus monkeys underwent abdominal anesthesia with pentobarbital (40 mg/kg), and the neck skin was incised, and one side of the common carotid artery was exposed by blunt dissection. The animals were injected into the common carotid artery. The 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 in the direction of reverse blood flow. If the animal's PD symptoms are not obvious after the first operation, the second to fourth injections are given every 5 days until the symptoms of PD appear. The primate bilateral and unilateral PD models established by MPTP are very similar to human PD in terms of symptoms, physical evidence, pathology, biochemistry, and response to drug therapy, and are considered to be the most representative PD animal models. .

4. MPTP is used in the mouse model of Parkinson's disease

The sensitivity of mice to MPTP is different between germline and age. Generally, 10 to 12 weeks old, 25~30g adult C57BL brown rats were selected, and MPTP was injected intraperitoneally according to body weight 30~40 mg/kg for 7 days. After the 6th to 7th injections, temporary (2~3h) torso vibration, vertical hair, tail overextension, movement reduction and climbing rod test obstacles appeared.

5. Some attentions when MPTP is used in the production of small country models of the disease

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, and CF-1 and CD-1 are sensitive to MPTP. Worst. The more commonly used mice are C57BL/6 mice.

b) Effect of age on MPTP sensitivity

Older mice are more sensitive to MFTP than younger mice. This difference in sensitivity is not only reflected in the fact that the old mice after cis treatment can show a more significant reduction in the number of dopaminergic neurons in the substantia nigra, but also in the post-treatment. Decreased number of adrenergic neurons in the blue-spot area and changes in typical behavioral behavior of Parkinson's disease observed in aged rats

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

1) Climbing rod test

2) Suspension experiment

3) Swimming experiment

The above quantitative observation methods provide some accurate and objective methods for the study of behavior in the mouse model of Parkinson's disease, and also improve the objectivity of the whole experiment.

7. Other methods for building models

1) The reserpine model can mainly deplete the dopamine (DA) and other catecholamine transmitters stored in the vesicle by inhibiting the reuptake function of noradrenergic neuron terminals. Intra-abdominal injection of a certain dose of reserpine in male Wistar rats (280-300 g) can cause slow bone stiffness, tremor, body flexion, hypokinesia, and other motor symptoms. Neurochemical analysis revealed a significant reduction in the levels of DA and other monoamine transmitters in the rat striatum. therefore. This type of model mimics the clinical manifestations and neurochemical changes of PD to some extent. However, it has obvious shortcomings: First, the dyskinesia caused by drug injection has greater variability between different times and different individuals; secondly, it also causes multiple transmitters to be released and can not cause pathological changes similar to PD. Therefore, the application of this model is limited to the exploration of drugs affecting the state of muscle stiffness, and the study of other motor symptoms has rarely been applied.

2) Fe ³⁺ model In 1991, Ben-Shachar induced significant changes in motor behavior after injection of Fe ^{3+ in the} unilateral substantia nigra of male Sprague-Dawley rats (250-300 g), showing autonomous in a new environment. Reduced exercise, transient stiffness and spontaneous rotation. Amphetamine (AMPH) enhances this same-rotation behavior. Neurochemical studies found that the DA content of the ipsilateral striatum decreased by an average of 95%, and the contents of its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and high vanillic acid (HVA) were also significantly reduced. Histopathology confirmed that tyrosine oxidase (TH) immunostaining positive cells were greatly reduced, and glial cells proliferated actively.

3) Mechanical damage model study found that the medial anterior rib bundle of mechanical injury (MF can cause progressive death of DA neurons in the substantia nigra. The established modeling methods include MFB single-segment and mid-cerebral hemisection, especially in the former There are many applications. The establishment of the MFB cut-off injury model needs to be confirmed on the rat brain stereotaxic instrument, and then cut into the MFB where it is cut with a special Scouten wire cutter. Brechne cuts the male CFHB rats by this method ( After the MFB of 150-180 g), the number of survival of DA-energy neurons was counted at 1, 2, 4, 6, 10, and 16 weeks, respectively, and it was found that about 28% of substantia nigra (SN) neurons died in a week or so, 10 About 70% of the cells died after the week. The average survival of SN neurons was 29%. The retrograde tracing showed that the MFB cut-off success rate was 92%-99%. It can be seen that the method has the following advantages: 1 high success rate Economy. 2 The substantia nigra DA neurons exhibit progressive death, which can simulate the whole process of PD pathological changes. It has certain significance for the study of neuronal regeneration and prevention of PD. The disadvantage is the degree of injury in a short time after cutting. Unstable.