动物模型,帕金森病 z-bo 2020-08-11 547

　　1. Make a PD model by depleting monoamine neurotransmitters

　　(1) Reserpine model

　　Reserpine is an alkaloid. By irreversibly blocking the monoamine transport in the capsule consumes central and peripheral monoamine, thereby affecting the monoamine recycling of intracellular vesicles, it was used to make a rat PD model in the 1950s. When rodents are injected with reserpine, the DA, 5-HT and norepinephrine (NA) taken in the capsule are blocked and degraded in the cytoplasm, which rapidly reduces the monoamine level, resulting in muscle Stiffness and other symptoms of PD.

　　Preparation method: Using male Wistar rats, intraperitoneal injection of a certain dose of reserpine can cause skeletal muscle stiffness, tremor, and abnormal posture. The model making method is simple. Its greatest contribution to humans is to reveal that levodopa (L-DOPA) can be used as a potential drug for the treatment of PD. The disadvantage is that the symptoms of PD can only be simulated briefly, and the pathological changes of spontaneous PD cannot be fully replicated.

　　(2) methamphetamine model

　　Amphetamines are a class of potentially addictive neurostimulants that can promote DA release. Large-dose application is neurotoxic to rodents and non-human primates. Like reserpine, it can cause The depletion of DA in DA neurons has less effect on substantia nigra neurons. Boirean and other studies have shown that the drug can be taken into cells through DA transporters, and the excitatory amino acid receptor antagonist MK.801 can block its toxic effects. In vitro experiments suggest that energy metabolism disorders, oxidative stress and excitatory amino acids are all Related to its toxic effects. Production method: Use adult rats to subcutaneously inject methamphetamine at a weight of 10-25 mg/kg once a day for 4 consecutive days. Excessive activity can occur temporarily after medication, followed by reduced activity. After 4 to 5 days, there may be "hallucination-like" behaviors, manifested as shaking, licking, and biting fur. One week later, it was found that the activity of tyrosine hydroxylase (TH) in the substantia nigra and striatum was reduced, and the DA content was reduced, but the loss of DA neurons in the substantia nigra was not obvious. The model is simple to make and is an acute injury model. The duration and stability of symptoms are not yet ideal, and there is no characteristic histopathological changes of PD. It is mainly used to study the physiological and biochemical changes of the striatum after PD dopamine depletion, and can also be used for neuroprotection research.

　　2. Neurotoxin making PD model

　　Research on the brains of PD patients shows that there is inhibition of mitochondrial complex I function during oxidative stress, so this may be an important part of the substantia nigra DA neurodegeneration. The research on MPTP, 6-hydroxydopamine (6-OHDA) and Rotenone has provided hope for exploring the molecular mechanism of dopamine neuron death.

　　MPTP was discovered in a group of patients with subacute severe Parkinson's disease caused by drug addiction in 1982. This symptom is caused by these people taking some drugs that contain a structure similar to synthetic morphine. The drug is contaminated by some of the by-products MPTP during processing, which is highly lipophilic and can pass through the blood-brain barrier. Monoamine oxidase in glial cells can convert MPTP into 1-methyl-4-phenylpyridine (MPP+), which can be taken up by DA transporters and accumulated in DA neurons. Mice lacking this transporter can avoid the toxic damage of MPTP. The absorbed MPP+ is enriched in mitochondria, and they can inhibit the function of complex I in the mitochondrial electron transport chain, thereby reducing the production of ATP and generating reactive oxygen free radicals to induce DA neuron apoptosis. Interestingly, MPTP can even kill some of the lowest level invertebrates with a central nervous system such as flatworms and trituria neurons. This suggests that MPTP is neurotoxic to invertebrates and primates. Therefore, in primates, such as humans, monkeys, and baboons, MPTP can cause irreversible and severe Parkinson's disease symptoms, which are indistinguishable from sporadic PD. This includes the degeneration of DA neurons in the substantia nigra, and the appearance of micro-inclusion bodies [such as eosinophilic inclusion bodies and α-synuclein polymer (not typical Lewy bodies)]. These micro-inclusion bodies are observed under an electron microscope. It consists of a high-density granular core and a radiating filament around it. Primates treated with MPTP showed a better response to levodopa (L-3,4 dihydroxydopa) and other DA receptor agonists. However, the main disadvantage of using MPTP toxins to make PD models is that PD is a chronically developing disease, while MPTP causes an acute or subacute pathogenesis. Chronic administration of MPTP at different dose levels in primates has been shown to produce slowly progressing symptoms of Parkinson’s disease, the loss of dopamine fibers in the striatum is uneven, and it shows the selection of DA neurons in the substantia nigra性loss.

　　6-OHDA is another drug used to make animal models of Parkinson's disease. The most commonly used method is to inject 6-OHDA unilaterally into the substantia nigra or the central forebrain tract, which can cause DA neuron cells to die quickly in a short period of time. This is similar to the acute MPTP model. Another 6-OHDA model directly injects this toxin into the striatum to cause degeneration of substantia nigra neurons. This method can slowly damage some neurons within 4 weeks, so it has been used to simulate the chronic course of PD.

　　After injection of 6-OHDA into the substantia nigra, these drugs selectively accumulate in DA neurons and produce toxic effects on them. This process may be accomplished by generating free radicals. 6-OHDA is a highly effective toxicant to rats, mice, cats and primates, and is widely used to make unilateral injury models. Extensive DA depletion in rats can be assessed by the rotational movement induced by the administration of amphetamine and anhydromorphine. The main advantage of this model is that it can measure the reduction in exercise (rotation), so this model has been proved to be very useful in pharmacological research to observe whether drugs are effective on dopamine and its receptors.

　　Rotenone is a natural insecticide, which originates from the roots of some special plants, and is a high-affinity specific inhibitor of mitochondrial complex I. Betarbet et al. confirmed that long-term and systematic administration of rottenone through jugular plexus cannulation in rats can produce many characteristics of PD, including the degeneration of dopaminergic neurons in the substantia nigra striatum and the formation of Lewy bodies in DA neurons. These inclusion bodies can be stained by ubiquitin and α-synuclein antibody. Under electron microscope, the high-density core is surrounded by fibrous material, which is very similar to the structure of Lewy body. The experimental rats showed signs of bradykinesia, unstable posture, unstable gait and some tremors. These symptoms were improved after administration of the DA receptor agonist anhydromorphine, suggesting that this slow administration of rotenone is a better method. Good and improved simple administration method.

　　In the substantia nigra of rats, injection of MPP+, 6-OHDA and Rotenone alone caused different motor function inhibition. MPP+, 6-OHDA and Rotenone are dopaminergic neurotoxins, but the mechanism of the toxic effects of these toxins on rat brain tissue is still not very clear, and further research is needed.

　　Three, gene model

　　Most PD is sporadic, and genetic factors do not play a major role. In the PD population, which accounts for a small number of familial PD cases, genetic factors play a key role. The genes that have been found to cause familial PD include Ot-synuclein, Parkin, and ubiq-uitin c-erminal hydrolase L1. Among them, Ot-synuclein protein is the main component of the Lewy body, and its gene mutation can lead to familial PD dopaminergic neurons Of degeneration.

　　(1) Transgenic model

　　Masliah et al. found that transgenic mice that highly express human α-synuclein have some of the characteristics of PD, such as the loss of DA nerve endings in the striatum, the formation of Ot-synuclein and ubiquitin positive inclusion bodies in the cytoplasm, and motor dysfunction. These transgenic mouse inclusion bodies are different from human Lewy bodies, which are mainly characterized by lack of fiber-like structure. Sometimes inclusion bodies can also be seen in the nucleus, which is obviously different from human PD. Some transgenic mice have only inclusion body formation and movement disorders. Without DA neuron degeneration, the brainstem neuron disease of these mice changed significantly. The wild type and mutant α-synuclein transgenic mice have similar pathological changes. The Ot-synuclein transgenic flies produced by Feany et al. possess many important characteristics of PD. Including the loss of DA neurons, the formation of fiber-like inclusions in nerve cells, and motor dysfunction. Due to the thorough study of the genetic law of Drosophila and its short life span, this model is of great value for understanding the role of certain new proteins in the pathogenesis of PD. For example, this model can be used to discover inhibitory genes that prevent DA neuron degeneration and susceptibility genes that promote Ot-synuclein gene expression.

　　(2) Gene knockout model

　　Compared with wild-type mice, Ot-synuclein knockout mice showed no obvious pathological characteristics. Histological examination revealed that TH-positive DA neurons in the substantia nigra were not significantly different from normal wild-type DA neurons. Morphological differences. The difference between the two is that DA in the striatum of knockout mice is reduced by 18%. These evidences indicate that Ot-synuclein is not necessary for the growth and development of mouse brain tissue and for maintaining the integrity of DA neurons. It plays a regulatory role in the release of DA from presynaptic vesicles. Recently, Goldberg et al. established a Parkin gene knockout mouse model. They also have no obvious clinical and pathological manifestations. These relative normalities may come from adaptive changes during growth. Perhaps knocking out the Parkin gene after embryogenesis can lead to more changes. Serious performance. The gene model provides a good method for studying the pathogenesis of PD. But not all genetic models can show typical characteristics of PD such as substantia nigra DA neuron degeneration, in humans. PD is a mutated gene, but it may be expressed normally in mice. Its disadvantages are complex preparation process, high technical requirements and high cost. Despite the above limitations, transgenic Ot-synuclein mice provide a perfect model for studying the formation of Ot-synuclein-positive protein aggregates. Moreover, transgenic mice provide a good model for verifying the interaction between genetic mutations and environmental factors in PD. In order to make animal models that are closer to human PD in terms of pathology, biochemistry, and symptoms, researchers have made unremitting efforts. The ideal PD animal model should have the following characteristics:

　　①Remission: There should be a certain time interval between the establishment of the PD animal model and the death of the animal. Through treatment, the symptoms of model animals can be alleviated, and the animals can survive for a long time, so as to observe the efficacy and facilitate evaluation. ②Repeatability: Repeatability is the hallmark of all successful animal models. And under certain conditions, the application has the unity of both isolated single cell and in vivo animal models. ③ Consistency with human PD: the symptoms of animal models (motor symptoms and non-motor symptoms including psychiatric symptoms and autonomic symptoms) and the hallmark pathological changes of PD (substantia nigra DA neuron apoptosis and Lewy small Body formation) should be similar to PD patients. ④ If the model is hereditary, it should be based on a single mutation to make the mutation model have the ability to pass down. ⑤The model should have a relatively short disease cycle (such as several months) to make drug screening more economical and faster. ⑥Simple operation, easy to master, low risk. At present, there is no model that fully conforms to the ideal model. However, with the continuous deepening of PD research, the best PD model will be found in the near future, and the foundation will be laid for the ultimate victory of PD.