JP2004089148A - Myocardial infarction rabbit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a myocardial infarction rabbit and usable as a model animal for the development of a diagnostic, preventing or treating agent for myocardial infarction or arteriosclerosis or the prevention of the development of the diseases or the stabilization of coronary artery diseases. <P>SOLUTION: The rabbit is produced in a manner to satisfy the requirements that the rabbit has coronary artery lesion rich in macrophage, the serum cholesterol level is ≥800 mg/dl at 12 months old and ≥700 mg/dl at 24 months old, and the luminal constriction ratio of the coronary artery is higher than the sum of the average value and the standard deviation of the ratio of the parental generation. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing a rabbit with myocardial infarction and the produced rabbit with myocardial infarction.
[0002]
[Prior art]
Death due to heart disease is the second leading cause of death in Japan, and it has been pointed out that it is important to respond to ischemic heart disease mainly caused by myocardial infarction, which accounts for about 60% of the death. Regarding the onset of myocardial infarction, it has been pointed out by accumulation of clinical data that occlusion of the coronary artery due to thrombus or the like occurring at the site of atherosclerotic lesion plays an important role. However, since there is no suitable model animal that spontaneously develops myocardial infarction, details of the mechanism of myocardial infarction have not been elucidated.
[0003]
WHHL rabbits (Watanabe heritable hyperlipidemic rabbit) exhibit hypercholesterolemia due to delayed catabolism of LDL in the circulating blood due to abnormalities in the low-density lipoprotein (LDL) receptor gene, and the aorta and coronary arteries immediately after weaning. It is well known as a rabbit that naturally develops atherosclerotic lesions. In WHHL rabbits, severe atherosclerotic lesions develop in the coronary arteries by selective breeding (Reference 1-2). However, the incidence of myocardial infarction was low and myocardial lesions occurred mildly. In order to respond to human hyperlipidemia, research on the development of WHHL rabbits that frequently cause myocardial infarction by selective breeding has been continued, but rabbits with a particularly high incidence have not been obtained.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a method for producing a rabbit with myocardial infarction. The method of the present invention comprises the following conditions 1) to 3):
1) have coronary artery lesions rich in macrophages;
2) The serum cholesterol level is 800 mg / dl or more at 12 months of age and / or 700 mg / dl or more at 24 months of age; and 3) The luminal stenosis rate of the coronary artery is greater than the average of the parental generation + standard error. Breeding using rabbits produced from rabbits that satisfy the high values.
[0005]
Preferably, a rabbit meeting the above conditions 1) -3) further satisfies the condition 4) that a myocardial infarction has developed. In a preferred embodiment, the method of the present invention comprises selectively breeding WHHL rabbits having a serum cholesterol level at 12 months of age of 800 mg / dl or more.
[0006]
Another object of the present invention is to provide a rabbit with myocardial infarction produced by the above method. In a preferred embodiment, the rabbit of the present invention has a cumulative incidence of myocardial infarction up to 35 months of age of 90% or more.
[0007]
The present invention further provides the use of the above-mentioned rabbit with myocardial infarction as a model animal for the development of a diagnostic, prophylactic or therapeutic agent for preventing or developing myocardial infarction or arteriosclerosis, or stabilizing coronary artery disease. The purpose is to provide.
[0008]
[Means for Solving the Problems]
As a cause of the acute coronary syndrome, a hypothesis that an arteriosclerotic lesion of a coronary artery is broken and an obstructive thrombus is generated is considered to be effective (Reference 2-5). A ruptured atherosclerotic lesion is characterized by a thin fibrous cap and a large lipid core directly below it (accumulation of lipids and macrophages). The hypotheses of atherosclerotic lesion rupture are that proteases secreted by macrophages degrade fibrous caps, cytokines secreted by macrophages and lymphocytes suppress smooth muscle cell collagen production, and apoptosis of smooth muscle cells plays an important role Has been pointed out.
[0009]
In view of the above findings, the present inventors have considered that in the development of a WHHL rabbit with onset of myocardial infarction (WHHLMI rabbit), it is important to adopt conditions that facilitate the accumulation of macrophages in atherosclerotic lesions. Then, WHHL rabbits (Watanabe heritable hyperlipidic rabbit) exhibiting hypercholesterolemia were selected and bred based on specific indices, thereby successfully establishing WHHLMI rabbits, and arrived at the present invention.
[0010]
Method for producing rabbit with myocardial infarction The method for producing rabbit with myocardial infarction according to the present invention comprises the following conditions 1) to 3):
1) have coronary artery lesions rich in macrophages;
2) The serum cholesterol level is 800 mg / dl or more at 12 months of age and / or 700 mg / dl or more at 24 months of age; and 3) The luminal stenosis rate of the coronary artery is greater than the average of the parental generation + standard error. Breeding using rabbits produced from rabbits that satisfy the high values.
[0011]
Preferably, a rabbit meeting the above conditions 1) -3) further satisfies the condition 4) that a myocardial infarction has developed.
[0012]
By "myocardial infarction" is meant myocardial necrosis caused by occlusion of a coronary artery. Most myocardial infarction is due to coronary atherosclerosis. It was said to be relatively small in Japan, but it has been increasing rapidly with the rise of serum cholesterol level due to the westernization of lifestyle including dietary habits.
[0013]
In humans, "hypercholesterolemia" refers to hypercholesterolemia, particularly when cholesterol is 220 mg / dl or more among blood lipids. It can be hereditary (familial) or secondary. A typical hereditary type is type IIa hyperlipidemia due to abnormalities in low density lipoprotein (LDL) receptor, which plays an important role in cholesterol metabolism. In addition, hypercholesterolemia is also observed in complex type IIb hyperlipidemia in which triglycerides are increased and type III hyperlipidemia due to apoE abnormality. Secondary cases include those associated with eating habits and obesity, those associated with endocrine diseases such as diabetes and hypothyroidism, kidney diseases, and liver diseases.
[0014]
Although not limited thereto, rabbits that can be used in the method of the present invention are available from Kobe University Faculty of Medicine, Animal Experimental Facility and Kitayama Labes Co., Ltd. WHHL rabbits (Watanabe heritable hyperlipidemic rabbit) are known. The WHHL rabbit was discovered in 1973 as a hypercholesterolemic mutant and was established as a strain in 1979. It causes hypercholesterolemia due to delayed catabolism of LDL in the circulating blood due to abnormalities in the LDL receptor gene, and develops atherosclerotic lesions in the aorta and coronary arteries immediately after weaning. WHHL rabbits are internationally recognized as human model animals for familial hypercholesterolemia (http://www.med.kobe-u.ac.jp/iea/whl.html).
[0015]
Condition 1) "having a macrophage-rich coronary artery lesion" is determined by, for example, but not limited to, the following method.
After the death of the rabbit, the heart is cut out and the coronary arteries are sliced at intervals of 500 μm. The thin sections are immunohistologically stained using a monoclonal antibody specific to rabbit macrophages (for example, RAM-11 available from DAKO A / S, Glastrup, Denmark). Observe under a light microscope to confirm that antibody-positive cells accumulate on the coronary artery lesion surface layer or under the fibrous cap.
[0016]
The condition 2) is “the serum cholesterol level shows 800 mg / dl or more at 12 months of age and / or 700 mg / dl or more at 24 months of age”. In conventional WHHL rabbits, serum control values decrease with aging after 6 months of age. In the method of the present invention, one of the selection criteria is that the decrease in serum cholesterol level after 6 months of age is small. As a preferred embodiment, a rabbit exhibiting hypercholesterolemia whose serum cholesterol level at 12 months of age is 800 mg / dl or more and whose serum cholesterol level is 700 mg / dl or more at 24 months of age may be used as a selection criterion.
[0017]
Condition 3) is that “the luminal stenosis rate of the coronary artery is higher than the average value of the parent generation + standard error”. The luminal stenosis rate of the coronary artery at 12 to 30 months of age is:
[0018]
Embedded image
(Lesion area / (lumen area + lesion area)) × 100%
Calculated according to Methods for measuring the area of a lesion in the coronary artery (portion where a stenosis has occurred) and the lumen area of the coronary artery are known, and can be performed according to the method described in, for example, Atherosclerosis 1992, 96, 43-52. It is. The average of the parent generation is preferably observed for more than 30 parents.
[0019]
Preferably, a rabbit that satisfies the condition of 1) -3) and 4) the onset of myocardial infarction is used. The onset of myocardial infarction includes, for example, histopathological findings, such as necrosis of cardiomyocytes, disappearance of striations, wavy running, edematous changes, infiltration of lymphocytes, fibrosis of myocardium, thinning of ventricular wall, etc. It is judged by being observed. Alternatively, the determination is made by observing an increase or a decrease in ST, appearance of abnormal Q wave, coronary T wave, and the like based on electrocardiogram findings.
[0020]
Rabbits produced from rabbits satisfying the above conditions 1) to 3), more preferably 4) are used for crossing. Such selective mating is not limited, but is repeated for 5 generations or more, more preferably 7 generations or more. The method of breeding and breeding rabbits is not particularly limited, and known methods, for example, methods described in Experimental Animal Techniques, Vol. 14, pp. 4-6, 1979 can be applied.
[0021]
Myocardial infarction-rabbit The present invention provides a myocardial infarction-rabbit in which myocardial infarction occurs spontaneously with a high probability by selective mating based on specific conditions.
[0022]
The rabbit of the present invention has, but is not limited to, an incidence of myocardial infarction up to 35 months of age of 90% or more, more preferably 95% or more. The onset of myocardial infarction can be determined as described above for the condition 4).
[0023]
WHHLMI rabbit Preferred examples of the rabbit having myocardial infarction of the present invention include the WHHLMI (WHHL myocardial infraction) rabbit obtained in the examples of the present invention. WHHLMI rabbits were obtained by continuing selective breeding of rabbits satisfying the above conditions 1) to 3), more preferably 4) among WHHL rabbits for 5 to 7 generations. As described in Example 1, WHHLMI rabbits show a significantly higher incidence of myocardial infarction than WHHL rabbits.
[0024]
The WHHLMI rabbits are maintained by the present inventors at an animal experimental facility attached to Kobe University School of Medicine. . The present inventor also considered depositing a fertilized egg of a WHHLMI rabbit with a depositary institution for filing this patent application, but determined that it was impossible. Specifically, upon depositing a patent application, it is stipulated that the number of fertilized eggs (embryos) frozen (12 or more / ampule) is 25 or more in the same lot, that is, 300 or more fertilized eggs are deposited. In the case of rabbits, it is necessary to sacrifice at least about 20 individuals to secure 300 fertilized eggs. Since the number of female individuals suitable for reproductive age of WHHLMI rabbits maintained at the time of the present application is 40 or less, it is difficult to maintain the strain if at least 20 of them are sacrificed. Rabbits are difficult to maintain inbreeding, and unlike mice and rats, maintenance in small numbers is difficult. We have stored the WHHLMI rabbits and are ready to procure them as needed.
[0025]
Use as a rabbit model animal of myocardial infarction Since the myocardial infarction rabbit of the present invention develops myocardial infarction at a high frequency, it prevents the occurrence of myocardial infarction or arteriosclerosis or the stabilization of coronary artery disease. For the development of diagnostic, prophylactic or therapeutic agents.
[0026]
In particular, the myocardial lesions of the WHHLMI rabbits obtained in the examples described herein resembled myocardial infarction based on human coronary artery multi-branch lesions. Using this WHHLMI rabbit, it is possible to elucidate the pathogenesis of acute coronary syndrome by searching for factors related to rupture of coronary artery lesions and thrombus formation.
[0027]
Myocardial infarction, arteriosclerosis, or the development of a preventive or therapeutic agent for coronary artery multi-branch lesions, by administering a candidate compound, myocardial infarction, arteriosclerosis, or reduction in the incidence of coronary artery lesions, reduction in lesion area, Stability of the lesion is used as an index.
[0028]
【Example】
Hereinafter, the present invention will be described specifically with reference to Examples, but these are not intended to limit the technical scope of the present invention. Modifications and changes can be easily made to the present invention based on the description in the present specification, and they are included in the technical scope of the present invention.
[0029]
Example 1 Preparation of WHHLMI rabbits The present inventors performed selective breeding of WHHL rabbits for 5 to 7 generations using the following conditions 1) to 4) as indices:
1) have coronary artery lesions rich in macrophages;
2) Serum cholesterol levels are greater than 800 mg / dl at 12 months of age and / or greater than 700 mg / dl at 24 months of age;
3) The luminal stenosis rate of the coronary artery is higher than the average of the parental generation + standard error; and 4) Myocardial infarction occurred.
[0030]
The breeding management during the selective mating was the same as the breeding conditions for normal laboratory rabbits, and the feed fed was normal laboratory rabbit feed.
As a result of the selective cross, WHHLMI rabbits that spontaneously develop myocardial infarction with a high probability were obtained.
[0031]
(1) Analysis of Myocardial Lesions Hearts of WHHLMI rabbits and WHHL rabbits (control) were collected within 16 hours after death and immersed in a fixative (formalin or Bouin's fixative). After embedding in paraffin, the sections were cut at 500 μm intervals, and stained for histopathology or immunohistochemistry. Myocardial lesions in WHHLMI rabbits were prevalent in the subventricular subventricular region, and transmural lesions were observed in about 60%. These findings are similar to myocardial infarction associated with multiple coronary artery lesions in humans.
[0032]
FIG. 1 shows a histopathological image of the heart of a 25-month-old male rabbit with acute myocardial infarction associated with old myocardial infarction. Peripheral myocardial lesions in the left ventricle below the endocardium, thinning from the base of the left ventricle to the apex, from the side wall to the posterior wall, and near the posterior wall of the septal wall (FIGS. 1A-D). In the responsible coronary artery, lesions with a stenosis rate of 90% or more were observed in 20 consecutive sections (FIG. 1E). At the site where the myocardial fiber remains adjacent to the fibrotic portion (in the frame of FIG. 1C), disappearance of striations of the myocardial fiber, edema-like change, and wavy running are observed (FIG. 1G-H). In the thinned part, the disappearance of myocardial fibers was also observed. In addition, disappearance or obscuration of myocardial fiber nuclei and necrosis of contractile bodies were also observed. These findings are typical pathological findings in acute myocardial infarction.
[0033]
The electrocardiogram measured immediately before the death of the WHHLMI rabbit shown in FIG. 1 shows typical electrocardiographic findings at the onset of old myocardial infarction, that is, an increase in ST-segment (lead II, lead III, aVF lead) or a decrease in lead (I lead). Lead, aVL lead) and abnormal Q wave (lead III, aVF lead). From the above findings, it was considered that this rabbit died of acute myocardial infarction in combination with old myocardial infarction. Many WHHL rabbits that died after selective breeding showed similar pathological findings.
[0034]
In cases where pathological tissue specimens of WHHLMI rabbits after selective mating do not show old myocardial infarction lesions, loss of myocardial fiber striations, edema-like changes, wavy running, contractile necrosis, loss of myoglobin, and loss of myocardium The findings in the acute phase of myocardial ischemia, such as disappearance, obscuration, and atrophy of cell nuclei, were observed, and more than 90% of luminal stenosis lesions were found in coronary arteries. These findings strongly suggest the development of acute myocardial infarction.
[0035]
(2) Atherosclerotic lesion of responsible coronary artery There were two types of coronary artery lesions in WHHLMI rabbits that spontaneously developed myocardial infarction. One is the occlusion of the lumen due to the accumulation of macrophages on the surface layer of the atherosclerotic lesion, and the other is not showing complete occlusion of the lumen, but a large fibrous cap consisting of a thin fibrous cap and lipids and macrophages immediately below it. A lesion with a lipid core. In the case of the former coronary artery lesion, detachment of the endothelial cells from the macrophage accumulation portion was observed by immunohistological staining. The latter atherosclerotic lesions exhibited the characteristics of typical unstable lesions that may be responsible for acute coronary syndrome.
[0036]
In WHHL rabbits before selective mating, coronary artery lesions were lesions that were low in macrophages and extracellular accumulated lipids and rich in smooth muscle cells and collagen fibers (Reference 6). However, by selective crossing, the coronary artery lesion became a lesion rich in lipid components, and showed a histopathological image more similar to that of a human coronary artery lesion.
[0037]
(3) Summary The WHHLMI rabbit exhibited the following characteristics as compared with the WHHL rabbit before the selection and mating.
[0038]
a) In WHHL rabbits, the cumulative incidence of myocardial infarction before the age of 35 months was 20% or less, while in WHHLMI rabbits, the value was as high as 90%.
b) Coronary artery luminal stenosis rate at 12 to 30 months of age:
[0039]
Embedded image
(Lesion area / (lumen area + lesion area)) × 100%
However, the percentage of WHHL rabbits was 74%, while that of WHHLMI rabbits was 94%.
[0040]
c) In WHHL rabbits, the components of coronary arteriosclerosis lesions were lesions rich in smooth muscle cells and collagen fibers and relatively low in macrophages. In WHHLMI rabbits, macrophages accumulate in large amounts in the superficial portion of arterial lesions and block the coronary artery lumen.
[0041]
d) No abnormal electrocardiogram was observed in WHHL rabbits, but abnormal electrocardiograms showing arrhythmia and typical myocardial infarction and electrocardiographic findings suggesting subintimal infarction were observed in WHHLMI rabbits.
[0042]
e) In WHHL rabbits, the serum cholesterol level decreased with age after 6 months of age, and the average serum cholesterol level at 18 months of age was 529 mg / dl. In WHHLMI rabbits, the decrease in serum cholesterol due to aging was mild, and the average value of serum cholesterol at the age of 18 months was as high as 804 mg / dl. The serum cholesterol of a normal rabbit is about 30 to about 50 mg / dl.
References The following references are incorporated herein by reference.
[0043]
1. Shiomi M .; Outline of WHHL rabbit. http: // www. med. kove-u. ac. jp / iea / whh1-1. html (2002)
2. Shiomi M .; , Ito T .; , Shiraiishi M .; , Watanabe Y .; Inheritability of atherosclerosis and the role of lipoproteins as risk factors in the development of atherosclerosis in WHHL rabbits: Risk factors related to coronary atherosclerosis are different from those related to aortic atherosclerosis. Atherosclerosis 96: 43-52 (1992)
3. Falk E. , Shah P .; K. , Faster V .; Coronary plaque distortion. Circulation 92: 657-671 (1995).
4. Davies M. J. Stability and instability: Two faces of colony atherosclerosis. Circulation 94: 2013-2020 (1996)
5. Libby P.S. Current concepts of the pathogenesis of the acute colony syndromes. Circulation 104: 365-372 (2001).
6. Shiomi M .; , Ito T .; , Tsukada T .; , Yata T .; , Ueda M .; Cell compositions of colony and aortic atherosclerotic lengths diffuser; An immunohistochemical study. Arterioscler Thromb 14: 931-937 (1994)
[Brief description of the drawings]
FIG. 1 is a microscope image of heart and coronary atheroma plaques of a 25 month old male WHHLMI rabbit.
1A-D are views of heart tissue sections (Azan-Mallory staining). FIG. 1E is a coronary lesion of the responsible circumflex (elastic van Gieson staining). FIG. 1F (HE staining) is a high magnification view of the region of the left wall of the left ventricle of FIG. 1C. FIG. 1G is a high magnification view of FIG. 1F. FIG. 1H (immunohistological staining of muscle cell actin) is a high magnification view of the left wall of FIG. 1C.

Claims (7)

A method for producing a rabbit with myocardial infarction, comprising the following conditions 1) -3):
1) have coronary artery lesions rich in macrophages;
2) The serum cholesterol level is 800 mg / dl or more at 12 months of age and / or 700 mg / dl or more at 24 months of age; and 3) The luminal stenosis rate of the coronary artery is greater than the average of the parental generation + standard error. The above-described production method, comprising breeding using a rabbit produced from a rabbit satisfying a high value. The method according to claim 1, wherein the rabbit meeting the conditions of 1) to 3) further satisfies the condition of 4) having developed myocardial infarction. The method according to claim 1 or 2, wherein the mating is repeated for at least 5 generations. The method according to any one of claims 1 to 3, wherein a hereditary hyperlipidemia rabbit (WHHL rabbit) is used. A rabbit with myocardial infarction produced by the method according to any one of claims 1 to 4. The rabbit with myocardial infarction according to claim 5, wherein the cumulative incidence of myocardial infarction up to the age of 35 months is 90% or more. Use of the rabbit with myocardial infarction according to claim 5 or 6 as a model animal for the development of a diagnostic, prophylactic or therapeutic agent for preventing or developing myocardial infarction or arteriosclerosis, or stabilizing coronary artery disease. use.

Images