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In this report, we describe a simple and fast method for creating a murine myocardial infarction model and providing a useful and convenient tool for the research in ischemic heart disease.
We established acute myocardial infarction in the Kunming-strain mouse within 2 minutes by ligating the left anterior descending coronary artery. The model was evaluated by observing the changes in histology and in the serum levels of aspartate aminotransferase and lactate dehydrogenase.
Obvious myocardial necrosis was found in the 24-hr experimental (ligation) group. The average size of the infarction was 44.3% ± 2.9% of the left ventricle. Serum levels of aspartate aminotransferase and lactate dehydrogenase reached their peak in the 24-hr experimental group and were normal in the 72-hr experimental group.
We set forth a simple and quick method for producing acute myocardial infarction experimentally in the mouse. The model can be reproduced in a stable manner, under experimental conditions that are easy to duplicate.
The animal model of myocardial infarction (MI) plays an important role in the prevention, diagnosis, and therapy of human MI. The usual model for acute MI is the mouse, by ligation of the left anterior descending coronary artery (LAD), but the high cost of the animal and the need for specific instruments, such as a respirator, have restricted the use of this model to highly specialized laboratories. Improper use of the respirator and prolonged opening of the chest easily contribute to the death of the animal.1 Therefore, the most difficult problems facing the researcher are how to reduce the cost of the experiment, save operating time, and raise the success rate. In this study, our goal was to establish a convenient, rapid, and reproducible murine model of MI.
Although manipulation of the heart can activate a variety of inflammatory responses that can influence outcomes, the experiment was conducted in an aseptic environment. In addition, we had a control group, so the influence of cardiac manipulation on outcome was very little.
Mice. Male and female Kunming mice, weighing 24 to 30 g, were divided into 4 groups designated as 24, 48, 72, and 96 hours. Each of these groups was paired with a control group.
All procedures were performed according to protocols approved by our Institutional Committee for Use and Care of Laboratory Animals.
Surgical Procedures. The room temperature was controlled at 25 °C. The mice were anesthetized with ether and then fixed in the supine position by tying the legs and the upper jaw. After the left chest was shaved and disinfected with 75% ethanol, the skin was delicately dissected by a lateral 1.5-cm cut along the left side of the sternum. A 4–0 polypropylene suture was passed along the edge of the incision before the skin was dissected. The subcutaneous tissues were detached along the inferior fringe of the left pectoralis major muscles, and the left pectoralis major muscles were then refracted. The 4th intercostal space was exposed and delicately dissected 1 cm with the aid of microforceps. Self-retaining microretractors were then used to separate the 3rd and 4th ribs enough to get adequate exposure of the operating region, but the ribs were kept intact. The heart was squeezed out by pressing the thorax lightly; then the heart was held with the thumb and forefinger of the left hand, and its apex was pointed to the left side of the head. A 5–0 polypropylene suture was passed from the left fringe of the pulmonary infundibulum to the lower right of the left auricle, a distance of about 2 to 3 mm. Rather than ligate the main trunk of the left coronary artery, we ligated the LAD and the great cardiac vein together (Fig. 1). In the control group, we passed the suture but did not ligate. The heart was replaced in the thorax soon after the LAD ligation, and the blood and the air in the thorax were squeezed out by the forefinger. The thorax was closed with the suture that had been prepared before the thoracotomy.
Material Preparation. Blood was collected from each mouse at the conclusion of 24, 48, 72, and 96 hours and was stored for 1 to 2 hours at room temperature. Serum was obtained by centrifugation at 2,000 rpm for 10 min and stored at –70 °C. At the same time, the heart was harvested, washed with physiologic saline, and the ventricles were dissected from the atria, large vessels, and connective tissues.
Staining of the Myocardium. Both ventricles were cut into 3 or 4 pieces and stained with 0.5% nitroblue tetrazolium (NBT) for 5 to 10 min at 37 °C. Then the tissue was washed with physiologic saline in order to rinse away the surplus dye. Images of the sections were taken with a Sony digital camera for later analysis of infarct size. Then the myocardium was fixed in 4% paraformaldehyde at 4 °C for 24 hours and embedded in paraffin. Sections (5 μm thick) were cut from the cross area and stained with hematoxylin and eosin for histologic evaluation of tissue damage.
Determination of Aspartate Aminotransferase and Lactate Dehydrogenase. Excluding the hemolytic serum and any tissue specimens in which the infarct area was either too big (>55%) or too small (<20%), we measured aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) activity as an index of cardiac cellular damage by using a quantitative rapid assay kit (developed by Nanjing Jiancheng Bioengineering Institute; Nanjing, China).
Signs of Myocardial Infarction. Upon initial macroscopic observation, the infarcted region was seen to be off-white. After the myocardium was stained with 0.5% NBT, the infarcted region was still off-white, but the normal region on the periphery of the infarction was blue. Finally, the MI was identified by histologic examination.
Statistical Analysis. Statistical analysis was performed with the SPSS statistical package, version 10.0 (SPSS Inc.; Chicago, Ill), using the t test. Results were expressed as mean ± standard deviation (SD). A P value of <0.05 was considered to be significant.
Our success with the MI model was related to skill, because it followed a learning curve. The death rate was highest within the 1st hours of the surgery because of severe bleeding from intercostal vessels and asphyxiation. In general, our success rate was 60% or more. The entire operative procedure usually took about 2 min. Typically, the whole time that the chest was open did not exceed 30 sec.
Change of AST and LDH Levels in Serum. Serum levels of AST and LDH reached their peak at 24 hours after MI, then declined gradually at 48 hours, and became the same as those of the control group at 72 and 96 hours after MI. In the control group, serum AST and LDH levels also reached their peak at 24 hours after surgery, then fell gradually. Differences between the control and experimental groups, in serum levels of AST and LDH, reached statistical significance at 24 and 48 hours (Table I).
Area of Myocardial Infarction. No MI was found in any control group. In the experimental groups, myocardium of the anterior wall of the left ventricle and of the apex of the heart was off-white. After we excluded 5 specimens (the infarct area exceeded 55% in one and failed to reach 20% in the others), we found that the infarct area in the experimental group ranged between 40.0% and 47.8% (average, 44.3% ± 2.9%). Dilatation of the left ventricle and thinness of the ventricular wall were also found in these specimens (Fig. 2A).
Histologic Change in the Myocardium. In the left ventricular wall, necrosis was found in most of the myocardium and in the papillary muscle. The cardiac muscle fiber became thin and appeared acidophilic. A few remnant cardiac muscle cells, showing vacuolar degeneration, were found only in the endocardium and the epicardium. In the 24-hour experimental group, the nuclei of cardiac muscle cells were still present, but the myocardium appeared acidophilic in the ischemic region. In the 48-hour and 72-hour experimental groups, not only did the nuclei disappear, but inflammatory cells began to infiltrate. The necrotic myocardium was phagocytosed, and a great number of fibroblasts were found in the 72-hour experimental group (Fig. 3).
The LAD is the major vessel that supplies blood to the left ventricle. If the LAD is occluded, it results in MI of the anterior wall of the left ventricle and the anterior portion of the interventricular septum. Occlusion of the LAD in small rodents has proved to be a good model for research in myocardial ischemia.2,3 The mouse is an ideal experimental animal for the study of human illness because of its tame temperament, low cost (of the strain that we use), and physiologic similarity to human beings. The method introduced heretofore had some disadvantages, such as a complicated operative procedure, long operative time, and low success ratio.4 In the present study, we illustrate a simple and fast method to establish the murine MI model and to observe changes in the serum enzymology and in the histology.
Compared with those of control groups, serum levels of AST and LDH clearly rise in experimental groups, reach their peak at 24 hours after MI, and then fall gradually. In the control group, serum levels of AST and LDH also rise 24 hours after surgery, which may be in response to the operative wound. The infarct size of most specimens (about 87.5%) is between 40.0% and 47.8% (average, 44.3% ± 2.9%). Thus, the infarct area of the model is highly stable for the study of MI. The specimens with too large (>55%) or too small (<20%) an infarct area are only about 12.5% of the total specimens, which may relate to a variation in the distribution of the coronary artery.4
The method described in this paper has many advantages: First, it is simple and needs no complicated experimental equipment, such as endotracheal intubation gear and a respirator. Second, the operative procedure takes place mainly outside of the thorax, which causes less hemorrhage and does not require cutting the rib. Third, the duration of the entire operation does not exceed 2 min, and less than 30 sec passes from opening the chest to closing the chest. Fourth, the success ratio is high.
The keys to the operation are these: 1) The duration of anesthesia (30–40 sec) must be controlled strictly; otherwise, the postoperative recovery of respiration will be affected. 2) Thoracotomy must proceed gently along the left side of the sternum, to avoid hemorrhage and damage to the lung. 3) In opening the thorax enough to gain adequate exposure of the operative field, the operator must not allow the microretractors to push the heart out, for fear of cardiac arrest. 4) The left hand, which holds the heart, must be gentle, to avoid ventricular fibrillation. 5) When the suture passes under the LAD, the needle must adhere to the left edge of the pulmonary infundibulum. Because the great cardiac vein might not be visible (or stable in its position), it cannot be used as an indicator of where to ligate the LAD. We use the color difference between the left and right ventricles as an indication of the position of the left edge of the pulmonary infundibulum. The position of the needle insertion is critical to success. If the needle is inserted too far to the right, it will result in hemorrhage and finally in death, because of the very thin ventricular wall at the pulmonary infundibulum. However, if the needle is inserted too far to the left, one can't ligate the LAD satisfactorily. 6) The chest must be de-aired before closing, so that breathing resumes normally.
Address for reprints: Dr. Yin Wu, Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, PRC. E-mail: nc.moc.oohay@35niyuW
Our research was supported by the National High Technology Research and Development Program of China (program 863, no. 2004AA2Z3782); the State Administration of Traditional Chinese Medicine (science and technology program for the optimum return of traditional Chinese medicine); the Social Development Program of Jilin Province (no. 20020638), and the Science and Technology Development Plan project of Jilin Province (no. 20020502).