Animal Models In Myocardial Ischemia

Despite tremendous advances in treatment options, atherosclerotic coronary vascular disease remains the leading cause of death worldwide. As a result, this disease continues to be an active area of cardiovascular research. Originally defined by the Greeks as a lack of blood flow, the modern definition of ischemia emphasizes both the imbalance between oxygen supply and demand and the inadequate removal of waste products. Impaired oxygen delivery results in a reduction in oxidative phosphorylation, resulting in myocardial dependence on anaerobic glycolysis for the production of high-energy phosphates. This shift in metabolism produces excess lactate, which then accumulates in the myocardium. As impaired adenosine triphosphate production and acidosis prevail, there is a resultant decline in cardiac contractility. Ultimately, if not reversed, myocardial infarction occurs, with permanent cellular loss and impaired cardiac function.

Multiple experimental techniques have been developed for the study of ischemia. Currently, scientists consistently use isolated myocytes to examine single-cell responses; isolated perfused hearts and whole animal models allow a better understanding of the whole organ response. Regardless of the model type, experimental animals remain a crucial tool in the area of research (see also Chapter 12).

6.1. Experimental Methods for Creating Ischemia

The ideal model for ischemic investigations would theoretically be in the intact chronically instrumented awake animal because acute surgical trauma and anesthetic agents both depress cardiac function (2). The awake animal model also has the major advantage that it can be used in studies requiring physiological stress (e.g., stress produced by exercise). However, the high cost of the implanted transducers and probes as well as difficulties with measurement techniques often preclude the use of such methodologies.

To date, the majority of studies use anesthetized animal models for the study of ischemia in either closed or open chest models. Closed chest models have the advantage that tissue trauma is minimized, but in such models, direct access to the heart for metabolite measurement is a major limitation. In contrast, the open chest preparation has the advantage that regional function and metabolism can be studied in detail. The open chest models suffer from drawbacks that include a greater susceptibility to temperature variations, and potential for surgical trauma may considerably alter cardiac function (Fig. 7).

Multiple techniques have been used to create models of myocardial ischemia for research purposes, including permanent occlusions, temporary occlusions, or progressive occlusions. Methods to produce complete permanent occlusions include surgical coronary ligation or radiological embolization. Furthermore, permanent or temporary partial coronary occlusions are commonly induced by ligation, balloon occlusion, or clamping. Typically, models of progressive coronary artery occlusions use either balloon/catheter occlusion or ameroid constrictors (Fig. 7).

Regardless of the method chosen, the researcher must be aware that the concentric experimental lesions created differ from those of naturally occurring atherosclerotic coronary vascular disease, which are typically eccentric. Normally, such eccentric stenoses remain vasoactive and are capable of altering coronary blood flow by changing their lumen diameter. It should be noted that no such vasoactivity remains in experimentally created concentric lesions, which prohibits humeral agents from altering regional coronary flow (Fig. 8).

Fig. 9. In a left anterior descending ligation model in the dog, both infarct size as measured by cardiac magnetic resonance imaging (MRI) and peak troponin I are plotted. (A) and (B) demonstrate good correlation with peak troponin I and both infarct size and mass. In contrast, although a trend between ejection fraction and troponin I and infarct size is evident, correlation is not strong, suggesting that infarct size alone does not determine the effect on cardiac function.

Fig. 9. In a left anterior descending ligation model in the dog, both infarct size as measured by cardiac magnetic resonance imaging (MRI) and peak troponin I are plotted. (A) and (B) demonstrate good correlation with peak troponin I and both infarct size and mass. In contrast, although a trend between ejection fraction and troponin I and infarct size is evident, correlation is not strong, suggesting that infarct size alone does not determine the effect on cardiac function.

Historical experience has shown that an induced occlusion of the left anterior descending coronary artery is favored over that in the left circumflex coronary artery for the production of regional myocardial ischemia. It is generally accepted that occlusion of the left anterior descending coronary artery results in a larger area of myocardial ischemia and therefore greater impairment of global left ventricular function. However, estimates of infarction size alone have not correlated well with ventricular function (Fig. 9) (28).

In fact, it has been demonstrated that, for the same amount of ischemic myocardium, the compensatory increase by the nonischemic myocardium is different for the left anterior descending coronary artery and the left circumflex coronary arteries (29). Therefore, in an ideal model, both infarct size and its location must be similar to achieve the same degree of impairment in left ventricular global function.

6.2. Localizing and Quantifying Myocardial Ischemia

Blood samples collected from the coronary sinus or from a regional coronary vein are commonly obtained and used for metabolic studies. Yet, such results must be interpreted with the knowledge that these samples include contaminated blood from adjacent noninjured myocardium. However, it should be noted that the use of coronary venous samples for studying metabolism is decreasing because of recent developments in micro-dialysis, magnetic resonance imaging, nuclear magnetic resonance spectroscopy, and positron emission tomography (30-32).

The size and location of myocardial infarction can be determined by triphenyltetrazolium chloride (TTC) staining, which has been the gold standard for quantifying the extent of myocardial infarction in pathological specimens (Fig. 10) (33). In addition, the assessment of localized tissue blood flow using microspheres (radioactive or colored) remains the gold standard. However, newer noninvasive methods of determining blood flow in the live animal that allow for repeated follow-up determinations of flow are continually under development and improvement, including spectroscopy and MRI.

6.3. Specific Animal Models for Ischemia Investigations

Both large and small animal models have been developed for the study of myocardial ischemia. The advantage of large animal models relates primarily to their similarity in physiology to

Fig. 10. Triphenyltetrazolium chloride (TTC) staining in canine infarct model showing paleness of myocardium in left anterior coronary artery distribution. Photo courtesy of Robert P. Gallegos.

Table 4 Definition of Graft Types humans and ease of instrumentation. However, disadvantages include significantly greater care and cost issues, which may make small animal models more attractive, particularly when large numbers of animals are required to achieve significant statistical power.

Traditionally, the dog has been the most frequently utilized animal species for in vivo experimental ischemia studies. The dog is considered a strong model for the condition of chronic ischemia because these animals have a well-developed coronary collateral circulation, similar to humans with chronic ischemia (progressive heart failure). Furthermore, the ease in handling of this species and the lack of significant growth in the adult dog are strengths of this model by allowing long-term follow-up. However, it should be noted that the significant variability in this collateralization may also hamper efforts to create consistent sizes of ischemic regions between animals or may result in a minimized ischemic zone.

The pig heart is more similar to the relatively healthy human heart in that there is limited collateral blood flow; this makes the swine heart ideal for acute ischemia studies. However, long-term follow-up using the swine model generally is considered problematic; ifjuvenile animals are utilized, significant changes in animal weight will result in both increased difficulties with handling as well as alterations in basic cardiac physiology. More specifically, in consideration of ratios of heart to body weight, in a healthy person the ratio is about 5 g/kg; for pigs weighing between 25 and 30 kg, the ratio is similar to that in humans, but for animals exceeding 100 kg, it is only half that value (29). Importantly, such ratio changes must be considered when interpreting experimental results.

Small animals have also been used as models for investigations of regional myocardial ischemia. Note that it is well established that the collateral circulation of the rat is sparse, and that the rabbit may show intraspecies differences (34). In addition, the guinea pig has such an extensive collateral network that normal perfusion is maintained after a coronary artery occlu-

Autograft Transplant from one site to another in the same individual

Isograft Transplant from a donor to a genetically identical individual (monozygotic twin) Syngraft Transplant from a donor to a recipient with no detectable genetic difference (inbred strain) Allograft Transplant from a donor to a genetically different

(homograft) individual of the same species Xenograft Transplant from a donor to a recipient of another (heterograft) species sion, and infarction does not often develop. Another problem with using these animals is that the small vessel diameters may delay or prevent instantaneous reperfusion following transient vessel occlusions. This is further hampered by the inability to make quantitative assessments of coronary blood flows in these small vessels to verify reperfusion. Nevertheless, the use of small animal models remains quite important for such studies, including recent investigations involving stem cell research and those that require large numbers of animals.

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