动物模型,心肌梗死 z-bo 2020-08-12 487

The rupture of atherosclerotic plaque in the coronary arteries and the accompanying arterial thrombosis is the final end point of the long and complicated pathophysiological process of atherosclerosis, which is difficult to perform in humans in any other animal. Fully copied out. However, based on the recognition of the central role of unstable plaque in acute coronary syndromes in recent years, people have still conducted a lot of research on the ideal animal model, although so far there is no model that can meet all our requirements. However, the existing models have made a lot of contributions to the research progress in this field. Here is a brief introduction to the relevant models currently reported.

  1. Choice of experimental animals

(1) Mice: Because the mouse model is easy to operate, there are more in-depth studies on the physiological and genetic information of mice. There have been a large number of gene knockout and transgenic mouse models, so most of them are in the field of atherosclerosis. Researchers like to use mouse models, and mouse models also account for the vast majority of animal models related to plaque rupture reported so far.

　　 Despite this, the limitations of the mouse model should still be recognized. Under natural conditions, mice will not develop atherosclerosis. The lipid composition of mice is completely different from that of humans. Most of the cholesterol is transported by particles similar to high-density lipoproteins. In addition, the weight of mice is about 25g, which is only about 1/3000 of the average weight of humans, and the size of mouse cells is similar to that of human cells, which means that the number of cells in a certain section of the mouse coronary artery Only 1/3000 of the number of cells in a similar section of a human coronary artery. Reflected on the histological level, the arterial endothelial cells of mice are directly attached to the inner elastic membrane, and there are only a few layers of smooth muscle cells in the middle layer of the arterial wall. Compared with humans, atherosclerotic lesions in mice often stretch along the inner elastic membrane, and media remodeling and aneurysms are also more common. In addition, in mice, it is difficult to distinguish plaque erosion (peeling of endothelial cells) and fibrous cap rupture. Although typical eccentric atherosclerotic plaques can appear in genetically engineered mouse models, lesions with multiple necrotic cores, with or without independent fibrous caps, are more often formed. As Calara et al. pointed out, the rupture of these lesions may not simulate human plaque rupture, which limits the application of mouse models in studying the mechanism of plaque rupture.

　　 In addition to the biological differences between mice and humans, one must also be very cautious when interpreting the research results obtained from genetically engineered mouse models. First, when two different genetic engineering models are used to study the effects of the third genetic modification, problems may arise. For example, to study the effect of type A scavenger receptor genes in different genetic engineering models of atherosclerosis: Suzuki et al. reported that in mice with ApoE gene knockout, type A scavenger receptor gene knockout can make atherosclerosis The sclerosing lesions were reduced by 60%; and de Winther et al. found that in the ApoE3 Leiden mouse model, the inactivation of this gene increased the atherosclerotic lesions. This difference in research results may be related to the role of ApoE in the blood vessel wall.

　　 On the other hand, different mouse strains used to establish transgenes or gene knockouts also have genetic heterogeneity, which may cause mice treated with the same genetic engineering to have different phenotypes due to different genetic backgrounds. For example, in C57BL/6 and FVB mice that were also knocked out of the ApoE gene, the former formed 7 times larger atherosclerotic plaques than the latter. For this reason, a better method is to use inbred mice for research, so that the study group and the control group only differ in target genes. When publishing relevant studies, the genetic background of the model mice must be described in detail, and this point should also be taken into consideration when evaluating the results of the study.

　　(2) Other animals: In atherosclerosis-related research, rabbits have been one of the commonly used model animals for a long time. Feeding rabbits with a large amount of cholesterol and fat will form lipid-rich arterial lesions, which have certain characteristics of atherosclerotic lesions. In fact, before 1990, Anitschkow and others first discovered the accumulation of cholesterol in the aorta in rabbits fed a high-cholesterol diet. The cholesterol levels of rabbits on this diet may be several times higher than that of humans. This lesion in rabbits looks similar to human atherosclerotic plaques, but contains more fat and macrophages.

　　 Under the condition of a high cholesterol diet, many primates will develop atherosclerotic lesions similar to humans. However, many primates are protected species and the price is very expensive. Therefore, they are currently only used for some complex problems such as mental and psychological research. They are not routinely used in the field of coronary heart disease research. They are only used in clinical trials of new therapies. In the previous demonstration, it was used in accordance with the requirements of the drug regulatory department.

　　Pig is currently the most commonly used atherosclerosis model animal. Atherosclerosis can occur in pigs under normal diet. When fed on a high-cholesterol diet, their plasma cholesterol levels and atherosclerotic lesions are very similar to humans. Belgian white pigs may even experience sudden cardiac death due to coronary artery disease under stress. However, the purchase and long-term breeding of pigs are expensive, and the needs of research should be fully evaluated before use.

　　 Among the commonly used model animals, dogs and rats lack susceptibility to atherosclerosis and are not suitable for related research. However, in recent years, some transgenic rat models have appeared, which can cause lesions similar to human atherosclerosis, and may be used as model animals in the future.

　　2. Modeling method of plaque instability and plaque rupture

　　(1) Mouse model: So far, there is no unified way to build a model. The existing models have some defects. Here is a brief introduction based on the existing literature.

　　 Existing models all use genetically engineered mice, mainly ApoE knockout mice.

　　 In 1998, Reddick et al. reported the formation of thrombus in the artery after the aorta was clamped with forceps in ApoE knockout mice, causing vascular injury. For the first time, evidence of plaque rupture was found indirectly in an animal model.

Subsequently, Rosenfeld et al. found that collagen-rich fibrolipid lesions and xanthomas were formed in the head and arm stems in elderly ApoE knockout mice (60 weeks old). These tumor-like lesions had necrotic inner cores and showed signs of intramural hemorrhage. This bleeding may be caused by a ruptured plaque. Moreover, after 42 weeks, the atherosclerotic lesions of the mice were layered, suggesting that the lesions may have repeatedly ruptured. In subsequent studies, Rosenfeld et al. also reported that in 30-week-old ApoE knockout mice, high-dose simvastatin [50mg/(kg·d)] can reduce the bleeding and calcification in the plaque of the brachiocephalic artery. They believe that this indicates that simvastatin has a stabilizing effect on advanced plaques.

Calara et al. observed 82 ApoE and LDL receptor gene knockout mice on a high-cholesterol diet for a period of 12 months. Three of them had aortic plaque rupture and/or thrombosis; another 33 ApoE genes Knockout mice were fed with a regular diet. After 20 months of observation, 18 developed coronary atherosclerosis, and 3 of them showed blood-filled tubes in coronary lesions, suggesting that plaque rupture may have occurred And thrombus. In this model, the incidence of plaque rupture is only 5%, far less than the actual situation in humans.

　　Williams et al. used C57BL6/129SvJ mice to knock out the ApoE gene and fed a diet containing 21% lard and 0.15% cholesterol for 14 months. Most mice showed rupture of atherosclerotic plaque and thrombosis in the lumen at the branch of the brachiocephalic trunk to the right common carotid artery. Among 98 mice, 51 had acute rupture of brachiocephalic artery plaque, and 64 had sudden death. In all mice that died suddenly, thrombosis was found in the blood vessels. 75% of the animals with acute plaque rupture had thrombosis in the blood vessels, while no intravascular thrombosis was found in the animals without acute plaque rupture. The analysis of sudden death found that there was no difference in the proportion of plaque ruptures in the mice that experienced sudden death and the mice that survived to the end of the study. This suggests that the plaque rupture of the head and arm stem is not the cause of the sudden death of these mice. The author's analysis may It is due to unstable plaques that also exist in the coronary arteries that caused sudden death, and some mice in the study did have myocardial infarction, but there is no clear evidence to support this.

In another study, in ApoE gene knockout mice, a silica gel ring was placed outside the carotid artery to cause damage. After the formation of plaque, the p53 gene was locally transferred to promote cell apoptosis and reduce the cell and extracellular matrix in the fibrous cap. Content: After using phenylephrine to cause hypertension in mice, 40% of the mice transferred into the p53 gene have plaque rupture. Due to a variety of mechanical and biological intervention factors, this model may only be suitable for studying the mechanical process of plaque rupture, and it is difficult to provide information about the pathophysiological mechanism.

　　 Some researchers in Northern Europe used ApoE and LDLR double knockout mice to establish a model of myocardial infarction. ApoE-/-/LDLR-/- mice were fed a "Western diet", including corn starch, glucose, sucrose, cocoa butter, cellulose, minerals, vitamins, 0.15% cholesterol and 21% fat. After 6 months, the serum cholesterol of mice can reach (27.8±1.3) mmol/L. Subsequent exertion of mental stress or systemic hypoxia on the mice induces reversible, endothelial-dependent ischemia, which can eventually lead to myocardial infarction in 50% of the mice (determined by troponin T, electrocardiogram and histology). Later studies showed that administration of thrombin inhibitors before systemic hypoxia can reduce the area of myocardial infarction in mice, suggesting that irreversible myocardial necrosis occurred after the initial ischemic phase triggered the process of thrombosis. This model simulates a variety of factors and specific phenotypic changes that promote coronary occlusion before clinical myocardial infarction, including coronary atherosclerosis, vasospasm, thrombosis, typical ECG changes, and protein leakage in myocardial cells . This model can help understand the mechanism by which resting atherosclerotic plaques develop to acute myocardial infarction. In this model, the extent of myocardial infarction in mice is still difficult to determine, because the occurrence of infarctions is scattered and small. Only after the animals are sacrificed can the location of myocardial infarction be determined by tissue sections, which is similar to the typical clinical myocardial infarction There is still a certain gap.

　　Takeshi et al. proposed another plaque rupture model in an article published in 2006. They ligated the common carotid artery on one side of the ApoE knockout mice. After 4 weeks, they ligated the proximal segment and placed a polyethylene cuff, and then executed. Pathological examination found that the ligation caused obvious intimal hyperplasia of the carotid artery, forming a lesion rich in lipids and fibers, including a small number of macrophages, T cells and fibroblasts. After treatment with a polyethylene cuff, it caused hemorrhage in the plaque, rupture of the plaque, and thrombosis in the blood vessel lumen. A few days later, the decrease of intimal fiber components and the appearance of apoptotic cells can be seen.

　　 Other methods that have been used include thrombin injection to cause thrombus, application of adenovirus or FeCl3 treatment, etc., which are not ideal methods to simulate the pathophysiological process of plaque rupture.

　　 (2) Non-mouse model: Rekhter et al. reported a plaque rupture model in rabbits in 1998. The purpose of establishing this model is not to study the pathophysiological mechanism of unstable plaque, but to evaluate the mechanical properties of plaque. In rabbits on a high-cholesterol/high-fat diet, two balloon catheters are used to create lesions in the thoracic aorta, and the other built-in balloon is repeatedly filled and deflated to cause the lesion to rupture. This model may be suitable for studying the mechanical process of plaque rupture, but it is difficult to provide useful information on the pathophysiological mechanism of plaque rupture.

　　 In 2003, Masashi et al. in Japan reported a spontaneous myocardial infarction model in rabbits. The research group has used Watanabe hereditary hyperlipidemia rabbits (WHHL) with coronary atherosclerosis tendency for many years, and after 6 years of screening, 5-7 generations of screening, bred WHHL rabbits with myocardial infarction tendency . The screening conditions for WHHL rabbits include: ① Myocardial infarction has occurred; ② Coronary plaque is rich in macrophages; ③ The degree of coronary stenosis exceeds the average value of the parents’ generation plus standard errors: ④ Plasma cholesterol levels are At 12 months of age, >21mmol/L, at 24 months of age, >18mmol/L. Compared with WHHL rabbits, the plasma cholesterol level of WHHLMI rabbits is 1.4 times higher, and the degree of coronary artery stenosis is significantly increased. More than 90% of WHHLMI rabbits have >90% coronary luminal stenosis. From the age of 11 months, WHHLMI rabbits can die suddenly, and the electrocardiogram recorded before death can reveal the typical pattern of myocardial infarction. Until the age of 35 months, 97% of the dead WHHLMI rabbits can find myocardial infarction histologically. Compared with WHHL rabbits, the incidence of myocardial infarction increased 4.1 times. Myocardial infarction is widely distributed in the left ventricle, right ventricle, and interventricular septum. Subendocardial, transmural, and subepicardial myocardial infarction can be seen histologically; in most WHHLMI rabbits, old myocardial infarction lesions and fresh lesions can be found At the same time, the old lesions often have calcification and mainly subendocardial infarction, similar to the performance of old human myocardial infarction. Fresh myocardial infarctions in WHHLMI rabbits are usually small in scope, and a small number of them die suddenly after the first myocardial infarction. These animals may die of malignant arrhythmia caused by myocardial infarction or severe myocardial ischemia. Atherosclerotic lesions can be found in the corresponding blood vessels of all myocardial infarction lesions. The most serious lesion is LCX, followed by LSP, LAD and RCA. In LCX, 73% of the sections can see atherosclerotic plaques with a lumen area narrowing more than 90%, while the sections with a lumen area narrowing more than 70% reach 97.2%. Typical plaques are mostly located in large branches of the coronary artery, and some lesions are vulnerable plaques: they have thin fibrous caps, rich in macrophages, lipids, and calcifications, but only a few smooth muscle cells. Plaques can be found Internal bleeding. But despite this, no real plaque rupture and thrombosis were found in WHHLMI rabbits. There may be several reasons: on the one hand, the simple existence of vulnerable plaque is only the basis for plaque rupture, and many other factors such as MMPs, Tissue factor, Lp(a), etc. may be involved in the process of plaque rupture and thrombosis; on the other hand, the feeding environment of WHHLMI rabbits is very simple and lacks environmental stimuli that may induce plaque rupture; in addition, the thrombosis of WHHLMI rabbits There may be key links in the process that are different from humans. The author points out that WHHLMI rabbits may have several uses in future research: ①The fact that WHHLMI rabbits have vulnerable plaques but not plaque rupture shows that the occurrence of plaque rupture and thrombosis does not only depend on vulnerable plaques. The existence of related inducing factors cannot be ignored. Therefore, based on WHHLMI rabbits, adding other influencing factors in the establishment of animal models may more accurately simulate the pathophysiological process of human myocardial infarction. The production of transgenic rabbits makes this animal The establishment of the model becomes possible. ②WHHLMI rabbits have both old and fresh myocardial infarction lesions, which can simulate the situation of surviving patients after myocardial infarction, and may be helpful for studying the treatment of these patients. ③WHHLMI rabbits can be used to study treatment methods to inhibit the generation of vulnerable plaques.

(3) Application of plaque instability and plaque rupture models: plaque instability and plaque rupture models undoubtedly occupy a very important position in the related research of acute coronary syndrome, mainly myocardial infarction, but so far Not a very satisfactory model appeared. ① The incidence of plaque rupture in the existing models is very different and has a certain degree of randomness. In some models, the incidence of plaque rupture is very low, such as only 5% in Calara's model, which is far from the actual situation of humans. Cannot meet the needs of research. ②The location of plaque rupture in some models is selective. For example, in the model of Rosenfeld and Williams, plaque rupture is limited to the brachiocephalic artery. Williams believes that this suggests that there is a large vascular wall in this part of the mouse artery. Tension, which may be different from the basis of human coronary plaque rupture. ③There is a common problem in the study of multiple animal models, that is, there is no clear evidence of platelet-rich and fibrin-rich thrombosis at the site where plaque rupture is presumed; this is very important because of human myocardial infarction Neither plaque rupture nor cerebral infarction is caused by the rupture itself, but by platelet-rich and fibrin-rich thrombi that block blood vessels. This may be the reason why the sudden death of mice in the Williams model is not related to plaque rupture. In the absence of thrombosis, intraplaque hemorrhage is considered to be evidence that plaque rupture has occurred, but this is only a speculation and is not certain. There is a clear gap with the AHA definition of plaque rupture. Plaque rupture refers to "the rupture of the fibrous cap, and the thrombus covered on it is continuous with the lipid core", in which thrombosis is an indispensable component closely related to plaque rupture, which is carried out in animal models lacking this component. The conclusions of the research, especially the efficacy research, need to be carefully evaluated. ④In the plaque rupture model, myocardial infarction in animals is often a recurrent small-area myocardial infarction. Unlike most clinical acute myocardial infarction cases, it is not suitable for research on acute myocardial infarction. ⑤ Existing models often need to feed animals for a long time under the condition of feeding high cholesterol/high fat diet, which is expensive and difficult to feed, which greatly increases the difficulty and cost of research. Therefore, it is still too early to apply models of myocardial infarction caused by plaque rupture in preclinical studies of the evaluation of myocardial infarction treatment methods, but the research using these models may itself provide us with useful information for our understanding of myocardial infarction. With the deepening of understanding, it will also help to form a more ideal animal model.