Through the artificial induction of different forms of cardiovascular pathology, the use of experimental animals allows for the study of these without interferences associated with the presence of other diseases or physiological states.
Unmasking the processes underlying the different manifestations of cardiac failure in a detailed way requires the use of simplified models. The interaction between different organs, tissues or cell types is very relevant and contributes to the maintenance of homeostasis naturally. An event of myocardial ischemia will often cause a decrease in ejection fraction, and the consequences at the metabolic level will translate into hormonal and nervous responses that will again affect the development of coronary heart disease. The development of in vitro models has allowed the study of specific pathways in isolated cardiomyocytes (Matter, 1969), cardiac fibroblasts (Leask, 2010) or endothelial cells (Chin, 2011), that are not accessible when these cells are in their natural environment. However, the need to study cellular processes in physiologically relevant settings limits the use of these models. Conversely, the study of ischemic heart disease from human samples is limited by additional factors that interfere with the findings. Thus, age, sex or clinical history are difficult to block in studies in which sample size is limited.
In an animal model, the induced pathology must faithfully reproduce the structural and functional characteristics of human pathology. In ischemic heart disease these involve the chronic narrowing of a coronary artery by deposition of atheroma plaques or occlusion by thrombosis. There are three possible situations arising from the decrease in coronary artery diameter.
Chronic ischemia. The occlusion is complete and the flow is not restored.
Myocardial hibernation. The diameter reduction is partial and, in a prolonged or chronic way, the myocardium has to adapt its metabolism to a deficient supply of oxygen and nutrients.
Ischemia and reperfusion or transient ischemia. The vessel occlusion is complete, but the flow is restored after a period of time.
With this in mind, one of the models initially considered was to induce the formation of atheromatous plaques with diets high in fat and cholesterol. This strategy has been effective in several models (du Toit, 2008; Zhang, 2005). However, the time and position at which the occlusion occurs is random and for this reason, this model is suitable for the study of the evolution of atherosclerosis, but is not practical in experiments aimed to study ischemic heart disease. In addition, the cardioprotective effect of estrogens has been demonstrated in several models of hypercholesterolemia (Kolovou, 2011; Clark, 2011), so that gender is an additional factor to consider that further complicates the development of a model of ischemic heart disease derived from atherosclerosis.
Although it obviates other effects associated with atherosclerosis, surgical induction of occlusion or coronary narrowing greatly facilitates the induction of lesions of predetermined location and size, which lead to more reproducible results. In addition, the surgical induction model has the advantage of being able to fix both the level (total by occlusion or partial by narrowing) and the duration of ischemia (permanent or followed by reperfusion). Both have advantages and disadvantages, and each has to be valued depending on the objectives of the study.
A. TECHNIQUES FOR SURGICAL INDUCTION OF CARDIAC ISCHEMIA
1. Surgical induction of chronic myocardial ischemia or hibernation.
(i) Hydraulic occluder and ameroid constrictor. These systems, based on the total or partial occlusion of a coronary artery branch, are especially used in large animal models. After an incision in the pericardium, the artery is exposed and a hydraulic occluder is placed around it, which is inflated to the desired degree of coronary occlusion. When using an ameroid constrictor, the casein plastic that composes the device is hydrated at body temperature, dilating to obtain constriction of the artery. Since the degree of occlusion can be fixed, both systems are suitable for both chronic ischemia and myocardial hibernation models.
(ii) Coronary ligation. Following a surgical procedure similar to the above, the artery is ligated using a thread or umbilical tissue. This system is used in animal models of very different sizes (Iannini, 1996) but is only suitable for the induction of permanent myocardial ischemia.
(iii) Coronary artery embolism. The strategy of occlusion by coronary embolism is based on the use of microspheres, agarose, polystyrene beads or intracoronary injection of thrombin and autologous blood with fibrinogen to cause coronary obstruction (Sabbah, 1991; Suzuki, 1999). This procedure is performed in large animals, and the effect obtained is that of permanent ischemia. Compared to other systems, it has the advantage of being a percutaneous intervention, which reduces the risk of complications by severe inflammation. As an obvious limitation is the difficulty of accurately controlling the exact location of the occlusion.
2. Surgical induction of ischemia and reperfusion.
(i) Hydraulic occluder and ameroid constrictor. This system has been described above. The duration of occlusion is a crucial aspect in ischemia-reperfusion models. Excessive ischemia time may exceed the limit of myocardial numbness and lead to myocardial infarction.
(ii) Transient coronary ligation. It uses the same procedure as in the case of permanent ligation. The knot is kept closed during the agreed time and removed at the end of the operation. In this model, a small plastic tube is placed between the cord and the vessel, to minimize damage and allow a better restoration of the coronary flow.
(iii) Transient coronary embolism. The need to use highly invasive surgical techniques is a disadvantage since the risk of severe inflammation and other complications makes the survival rate low. In large animals, transient embolism of the coronary artery can be achieved using arterial catheterization in a procedure similar to the reperfusion catheterization performed in patients with acute coronary ischemia events. From a femoral or radial access, a catheter with an apical balloon is advanced to the coronary artery, where it is inflated by a hydraulic system to cause total embolism of the vessel. After the opportune time, the balloon is deflated and the instruments are removed, suturing the arterial route (van Wijngaarden, 1992).
3. Ex vivo model of ischemia.
In 1895, O. Langendorff developed an ex vivo cardiac perfusion system. In this model, the heart of the animal selected model is excised and cannulated, maintaining physiological conditions of temperature and pH. This model has been used with animals ranging from rat (Bachmann, 1993) to pig (Brenner, 2000) and allows the study of the effect of drugs, cytokines and other substances without the interference of endogenous stimuli.
B. ANIMAL MODELS OF ISCHEMIC HEART DISEASE
Several animal models have been used for the study of ischemic heart disease. All of them have advantages that make them valuable or practical, and the selection of the appropriate model must take into account the particular objectives of each study.
(i) Models in non-mammalian animals.
Drosophila melanogaster. Despite the phylogenetic distance that separates it from mammals, the fruit fly presents characteristics that make it a useful model for the study of molecular mechanisms associated with ischemia. The signaling pathways associated with hypoxia are very conserved evolutionarily in D. melanogaster and the catalog of available mutant strains is exhaustive. Obviously, it is an extremely limited model when it comes to going beyond gene regulation, but it has been enormously useful in studies that have revealed, for example, mechanisms of hypoxia tolerance (Vigne, 2009) or regulation of cardiomyogenesis (Cripps, 2002).
Danio renio. Its closed cardiovascular system, rapid development, body transparency and the easiness to establish genetically modified strains make zebrafish a valuable model in the study of heart disease. D. rhenium presents the capacity to regenerate, with little or no scar, the cardiac tissue even when it is extirpated in 20%. After infarction induced by cryogenic damage, cardiac tissue goes through the stages of mammalian inflammation and fibrosis, but then the fibrotic tissue matrix is degraded, and cardiomyocytes proliferate and invade the damaged area to reestablish tissue functionality (Schnabel, 2011; Chablais, 2011). In contrast to D. melanogaster, D. rhenium is not only susceptible to surgery, but also extremely tolerant to it.
(ii) Models in small mammals
Although they have provided very relevant data on the gene regulation of post-ischemia processes and general cardiac development, models in non-mammalian animals are limited by the poor clinical relevance of the results obtained. Much research has focused on small mammal models that have short breeding cycles, require minor care and, above all, allow the extensive use of transgenic individuals.
Rodents. Laboratory rodents like rats and mice have the advantage of being cheap, homogeneous, easy to breed and genetically modifiable, while their use is exposed to little ethical debate. These characterisitic facilitate the use of large sample sizes. The extensive use of rodents has also led to the development of adapted clinical equipment that allows an accurate evaluation of parameters such as cardiac function or infarct sizes. However, there are important physiological differences between these models and humans (Phoon, 2006). The mouse has a body mass 3000-4000 times lower than the average adult, a resting heart rate 5 times higher and its metabolic rate is between 10 and 15 times higher. The action potential of rat and mouse cardiomyocytes is characterized by being very short and lacking the isoelectric plateau phase (Endoh, 2004). The expression of myosin isoforms differs between rodents and humans (Haghighi, 2003; Hasenfuss, 1998) and although the withdrawal of cytosolic Ca2+ occurs due to the activity of Serca2 in rodents and humans, Na+/Ca2+ exchange is less relevant in the former (Bers, 2002).
Rabbits. Models in hyperlipidemic rabbits have been used in several studies of spontaneous myocardial infarction induced by accumulation of intracoronary plaques, showing that the incidence of infarction is greater than 90%. In this model, unfortunately, the average development time is 11 months, and in addition the plaques do not break in a way equivalent to the human (Shiomi, 2003). As in rodents, other differences at the cellular level make the rabbit a rather limited model for the study of ischemic heart disease (Hasenfuss, 1998).
(iii) Models in large mammals
Although models in small mammals are appropriate for the study of processes at the molecular level, several studies show that the conclusions obtained are not always extrapolated to larger animals (ref267). The heart of larger animals is more similar to human at the anatomical and physiological levels and therefore, it is sometimes advisable to scale to models that more accurately reproduce human pathology.
Models in nonhuman primates. Because non-human primates have greater similarities with man than any other mammal, it is tempting to think that they would be the best candidates as a model for cardiovascular disease, but this idea is wrong. Firstly, the use of non-human primates raises enormous criticism regarding animal rights. This is particularly relevant on animals with a great genetic similarity with man. It should also be noted that various primate species are included in the list of endangered species. In addition, there are remarkable logistical difficulties. Their acquisition and maintenance are extremely expensive and also harbor potentially infectious or highly infectious diseases for men, and can be infected by men. Even saving such considerations, non-human primates have important anatomical and physiological differences with man. The heart of many primates is very small and therefore has fewer cardiac cells and a different distribution. These primates also have very rapid heart rates that can reach 200 beats per minute at rest (ref268). Finally, even obviating the aforementioned obstacles, there is no evidence that data obtained in monkeys provided additional data to those already obtained from non-primate mammals.
Canine models. Historically, the canine model has been widely used in the study of cardiac ischemia and myocardial infarction. Reimer et al. carried out in 1979 a study aimed at accurately describing the temporal evolution of the lesion in relation to the ischemia times (ref269). Other studies have used canine ischemia-reperfusion models to evaluate ventricular remodeling and its relationship to the renin-angiotensin system (refs270,271) or the effect of stem cell injection on cardiac function (ref272). However, the coronary vascular system of the dog is characterized by the presence of a significant collateral circulation (refs273,274) which exerts a protective effect on the ischemic damage and alters the development of the post-ischemic processes, making the size of the infarcts induced very unpredictable. This has gradually translated research into alternative models.
Sheep model. In models in sheep and pigs, coronary anatomy and the absence of preformed collateral vessels make it feasible to induce infarctions of predictable size and location that are adequate for the study of post-ischemic ventricular remodelling (refs274,276). The ovine model has been used for studies related to the early expansion of the infarct and the area adjacent to it, showing that border regions extend to adjacent areas of healthy tissue, involving them in the remodeling process (ref277). However, as domestic ruminants, sheep have a gastrointestinal anatomy and a thoracic contour that makes it difficult to obtain ultrasound images and makes an invasive approach advisable (refs276,277). This limits in part the potential of this species as models ischemic heart disease.
Porcine model. Like humans, the pig is an omnivorous animal. Metabolic rate, heart/body mass ratio, and heart rate are comparable, and blood pressure is only slightly higher. The pig's heart has a gross anatomy very similar to humans and has been confirmed in the work of several authors as a model suitable for the study of the pathophysiology of ischemic heart disease (refs279,280), but also studies focused on dilated cardiomyopathy (ref281)or cell theraphy (ref272) support the validity of this model in the cardiovascular field. Because of the similarities found between the hearts of both species, the pig has been used even as a donor in some of the rare experiences of cardiac xenotransplantation (ref282) and cardiac valves are currently used as xenografts in clinical practice (ref283). The main limitations of the use of this animal model have to do with a certain predisposition to the development of arrhythmogenesis in models of ischemic heart disease, which is nevertheless overcome by the administration of supplements with electrolytes and anti-arrhythmogenic agents in non-invasive models (ref284). The limitation represented until recently by the lack of databases and complete catalogs for the pig at the genomic and proteomic level appears increasingly reduced with the sequencing of the porcine genome (ref285,286) and the increase in the number of available antibodies that recognize porcine molecules. For a more detailed discussion on the differences and similarities between the human and the porcine heart, please visit this link.
C. ETHICAL CONSIDERATIONS REGARDING THE USE OF ANIMAL MODELS
Every year, more than 75 million vertebrates are used worldwide for experimental purposes (ref292), being mice and rats the most widely used species. Experiments using animals must be designed under the "Principle of the Three R". 1) Replacement of live animals by in vitro or computerized models. 2) Reduction in the number of animals used for experiments. To this end it is necessary to use of standardized animals that reduce biological variability, and to make previous estimates of statistical power. 3) Refinement, which means guaranteeing maximum animal comfort, by providing sufficient care that covers physiological and ethological animal needs, and avoiding unnecessary suffering through the use of adequate anaesthesia (ref293). In summary, the use of experimental animals should be considered only when there is no other viable alternative. It is necessary to take into account that the discomfort and stress during the experiments, as well as an unreasonable experimental design, are a violation of animal rights, but also inevitably lead to non-specific effects that will ultimately distort the results (ref293).
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