Source: Practical Experimental Techniques in Neurobiology, Fourth Military Medical University Press
Operation method
basic program
Principle
Parkinson disease (PD) is a common progressive degenerative disease of the basal ganglia neurons in middle-aged and old-aged people, which mainly involves the nigrostriatal and striatal dopamine transporter systems. 6-tibial dopamine (6-0HDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are two major dopaminergic neurotoxicants, which can be good mimic the clinical manifestations and pathological changes in PD patients. Since 6-OHDA cannot pass the blood-brain barrier, it needs to be injected into the brain in a stereotactic manner. Currently, most of them are injected into the dense part of the substantia nigra (SNc), the striatum (cs), the ventral tegmental area (VTA) of the midbrain, and the medial forebrain bundle (MFB), etc.; MPTP has a high degree of lipophilicity, can easily pass the blood-brain barrier, and concentrates in the substantia nigra, and it is usually injected via the peritoneal cavity or subcutaneous injection.
Materials and Instruments
Male SD rats weighing 200-250g Male C57 BL mice weighing 20-25g Move 1.6-OHDA-induced PD modeling in rats: (1) SD rats were weighed and kept at 5-6 per cage. At the time point of experimental design, rats were anesthetized with 1% sodium pentobarbital, and the operation could be started when the animals were unconscious. (2) The rats were fixed with a stereotaxic apparatus to both ears and incisors. The skin was scratched from the sagittal suture, the cotton ball was used to stop bleeding, positioning, drilling, and picking through the meninges (taking the unilateral destruction of the dense part of the substantia nigra as an example, the injection coordinates were determined with reference to the Paxinos atlas, anterior posterior to the cranium of 5.2 mm, left or right parietal opening of 2.2 mm, and the depth of the needle was 7.8 mm). (3) Micro syringe injection was performed, injecting at a concentration of 2µg/µL and an injection rate of 1µL/min and leaving the needle in place for 10min,withdrawing the needle at a rate of 1mm/min. Hemostasis was achieved and the wound was sutured. During the awakening of the experimental animals, keep them warm and observe all vital signs.156 Chapter 4, Production of Common Animal Models of Neurological Diseases In the weeks following the surgery, observe the animals for feeding, body weight, and behavioral changes. The method of unilateral destruction is technically mature, easier to operate, has a low mortality rate, is a relatively stable model, is easy to differentiate between minor abnormalities of bilateral limb movements, and can compare the pre and post-surgical performance of the affected nigrostriatum and its performance with that of the normal side. Bilateral destruction is a larger range of neurochemical and behavioral damage, more similar to human PD. however, it is more damaging to special areas such as the hypothalamus of the basal ganglia, has a high mortality rate, and is more cumbersome to operate, and is currently less commonly used in experimental studies of PD. : 2.Fabrication of MPTP-induced PD model in mice: C57/BL mice were weighed, about 5-6 mice per cage. The head, neck and tail of the squad were fixed by wearing gloves, and injected via intraperitoneal or subcutaneous injection, and the dose of single injection was 20-30 mg/kg.According to the experimental requirements and purposes, it can be divided into fast model and slow model. The fast model can be divided into single injection and multiple injections. The single injection dose is 30mg/kg, the model has light damage and is easy to recover; the multiple injection dose is 20mg/kg, every 2h, 3-4 times in total, the model has fast and serious damage, the damage to DA neurons can be completed within 3d. The slow model requires multiple injections, one injection per day, a single injection dose of 20mg/kg, continuous injection for 5-7d. The survival rate of this model is higher, and the experimental period is longer. 3 Result determination: the PD model can be evaluated by body weight test, rotating rod experiment, open field experiment and hanging test. The degree of damage to nigrostriatal dopamine neurons can be better assessed by apomorphine (or amphetamine)-triggered rotational movements toward the healthy side (or injured side). The successful model group will be characterized by significantly slower weight gain and reduced voluntary activity compared to the control group. In addition, the obvious reduction of TH expression in the substantia nigra and striatum observed by immunohistochemistry or molecular biology is also a gold standard for determining the success of the PD model. Caveat 1.MPTP is highly toxic, when weighing and dissolving and injecting, be sure to do a good job of personal protection; MPTP, 6-OHDA and other reagents are used now. 2. The experimental grouping and animals are more, must be well labeled, so as to avoid confusion, resulting in experimental failure. 3. After modeling, SD rats must pay attention to heat preservation and sputum suction. According to the nocturnal habits of rats, the survival rate of modeling in the afternoon or evening is higher. 4. Mice have a strong stress reaction, so they should wear thick gloves when grasping to avoid being bitten. 5. Mice will show a series of behavioral changes such as trembling, oral vesiculation and poor limb balance within 30 min after MPTP injection, which may be due to the toxic effects of MPTP on the kidneys, liver and other organs, resulting in some physiological indexes such as blood pressure, body temperature and liver function changes. Therefore, behavioral tests should be performed after the 3rd or 4th day after the last injection of MPTP to more objectively detect the behavioral changes in the mouse PD model. 6. 6-OHDA PD model should be injected slowly, too fast can lead to the spread of the injection range; microinjector selection to avoid the injection needle is too thick to cause the increase of the damaged area. 7. When conducting behavioral experiments, avoid the interference of external environment (noisy, bright light, etc.) as much as possible. Common Problems The success of modeling determines the quality of the results of each relevant index. Preparation of PD model selection needs to consider the purpose and use of modeling, the location of the target (SN, CS, vTA, MFB), the amount of drug dose and the size of the infiltration range, the degree of damage (partial, complete), the safety (mortality) and effectiveness of the animal (success rate), the difficulty of the operation, accuracy and stability and so on. Therefore, the specific application of the two models should be selected: ① Selection of modeling method according to drug properties 6-OHDA needs to be stereotactically injected into the target site, MPTP via intraperitoneal or subcutaneous injection; ② according to drug sensitivity selection of animals rats are sensitive to 6-OHDA, the preparation of the model according to the aforementioned factors are considered comprehensively, primates are the most sensitive to MPTP but restricted, and commonly used mice; ③ Selection of models according to the application purpose and use of 6-OHDA injection target site selection flexibility, adjustable injection dose, stable and reliable changes in the behavior of the animal, detection of good quantitative, mostly used in the preclinical drug research and pharmacological efficacy determination of PD, neuroprotection, cell transplantation and gene therapy and other aspects of the study, the MPTP dosing regimen is varied, the induced production of symptoms and pathological and biochemical changes in terms of the more with human PD MPTP is mainly used for research on the etiology and pathogenesis of PD and drug toxicology test. 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6-OHDA and MPTP (Sigma)
Stereotaxic (NarishigeJAPAN) Cranial drill Attractor 1µL or 5µL micro-pull syringe