Laboratory
Investigation
Jinfeng Wang, PhD*
Huaben Bo, MS*
Xiangying Meng, PhD
Yin Wu, PhD
Yongli Bao, PhD
Yuxin Li, PhD
Key words: Coronary
vessels/injuries; disease
models, animal; ligation/
methods; mice; myocar-
dial infarction
From: Institute of
Genetics and Cytology,
Northeast Normal
University, Changchun
130024, People’s
Republic of China
*Dr. Wang and Mr. Bo
contributed equally
to this report.
Our research was sup-
ported by the National
High Technology Research
and Development Program
of China (program 863,
no. 2004AA2Z3782); the
State Administration of
Traditional Chinese Medi-
cine (science and tech-
nology program for the
optimum return of tradi-
tional Chinese medicine);
the Social Development
Program of Jilin Province
(no. 20020638), and the
Science and Technology
Development Plan project
of Jilin Province (no.
20020502).
Address for reprints:
Dr. Yin Wu, Institute of
Genetics and Cytology,
Northeast Normal
University, Changchun
130024, PRC
E-mail:
Wuyin53@yahoo.com.cn
© 2006 by the Texas Heart ®
Institute, Houston
290 Experimental Model of Murine MI
A Simple and Fast
Experimental Model
of Myocardial Infarction in the Mouse
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 isch-
emic 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 evaluat-
ed by observing the changes in histology and in the serum levels of aspartate amino-
transferase 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. (Tex Heart Inst J 2006;33:290-3)
T
he 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 suc-
cess 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 re-
sponses that can influence outcomes, the experiment was conducted in an aseptic
environment. In addition, we had a control group, so the influence of cardiac ma-
nipulation on outcome was very little.
Materials and Methods
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 Institu-
tional 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
Volume 33, Number 3, 2006
the left side of the head. A 5-0 polypropylene suture was
passed from the left fringe of the pulmonary infundibu-
lum 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 tetra-
zolium (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
Fig. 1 The pattern of left coronary arteries in the mouse, and
the position of the ligation.
Texas Heart Institute Journal
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 Lac-
tate 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 aspar-
tate 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 macro-
scopic 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 exami-
nation.
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.
Results
Our success with the MI model was related to skill, be-
cause 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 asphyxia-
tion. 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, myo-
cardium 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. 2).
Experimental Model of Murine MI 291
TABLE I. Alterations in Serum AST and LDH in 4 Experimental and 4 Control Groups (Mean ± SD)

No. No.
Hrs. Group Mice AST LDH

24 Control 8 34.74 ± 8.59 6,873.52 ± 1,756.27

Experimental 8 125.45 ± 39.12* 11,964.22 ± 1,671.80*

48 Control 8 22.11 ± 3.87 6,253.89 ± 1,050.79

Experimental 8 36.03 ± 11.32** 8,722.16 ± 1,586.04**

72 Control 7 22.75 ± 4.04 5,888.23 ± 1,208.07

Experimental 7 23.16 ± 5.75 6,102.37 ± 1,357.06

96 Control 8 21.39 ± 5.16 5,372.93 ± 957.50

Experimental 7 21.67 ± 2.84 6,044.60 ± 775.70

Comparison with the control group: *P <0.01; **P <0.05

AST = aspartate aminotransferase; LDH = lactate dehydrogynase

Histologic Change in the Myocardium. In the left ven-
tricular wall, necrosis was found in most of the myo-
cardium and in the papillary muscle. The cardiac muscle
fiber became thin and appeared acidophilic. A few rem-
nant 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 ap-
peared acidophilic in the ischemic region. In the 48-hour
and 72-hour experimental groups, not only did the nu-
clei disappear, but inflammatory cells began to infiltrate.
The necrotic myocardium was phagocytosed, and a great
number of fibroblasts were found in the 72-hour experi-
mental group (Fig. 3).
Discussion
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 ill-

A B

Fig. 2 A) In the ligated heart, the anterior wall of the left ventricle and the apex of the heart were off-white and bulged outward.
The short arrow indicates the ligated positions and the ligated thread, while the long arrow indicates the left ventricle. B) Cross-
sections of heart stained by 0.5% nitroblue tetrazolium. The top row comprises cross-sections from control groups. On the bot-
tom row, the arrows indicate infarcted myocardium, left ventricular dilatation, and thinness of the ventricular wall, all from experi-
mental groups.

292 Experimental Model of Murine MI Volume 33, Number 3, 2006


Fig. 3 Histologic features of myocardial damage in the 72-hr
experimental group. Myocardial infarction is observed in the
left ventricular wall: central myofibers appear to be acidophilic
and the nucleus has disappeared; inflammatory cells have
begun to infiltrate the periphery; and the necrotic myocardium
has begun to be phagocytosed (H & E stain, orig. ×200).
ness 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 proce-
dure, 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 speci-
mens, which may relate to a variation in the distribu-
tion of the coronary artery.4
The method described in this paper has many advan-
tages: First, it is simple and needs no complicated ex-
perimental 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
Texas Heart Institute Journal
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 gen-
tly along the left side of the sternum, to avoid hemor-
rhage 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 ven-
tricular 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 posi-
tion 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 nor-
mally.
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