A novel model for evaluating thrombolytic therapy in dogs with ST-elevation myocardial infarction
Zhang et al. BMC Cardiovascular Disorders
A novel model for evaluating thrombolytic therapy in dogs with ST-elevation myocardial infarction
Hong Zhang 0 2
Yong-chun Cui 0 2
Yi Tian 2
Wei-min Yuan 2
Jian-zhong Yang 2
Peng Peng 2
Kai Li 2
Xiao-peng Liu 2
Dong Zhang 2
Ai-li Wu 2
Zhou Zhou 1
Yue Tang 2
0 Equal contributors
1 Center of Clinical Laboratory, State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing 100037 , China
2 Animal Experiment Center & Beijing Key Laboratory of Pre-clinical Research and Evaluation for Cardiovascular Implant Materials , Beijing 100037 , People's Republic of China
Background: There is still no standard large animal model for evaluating the effectiveness of potential thrombolytic therapies. Here, we aimed to develop a new beagle model with ST-elevation myocardial infarction (STEMI) by injecting autologous emboli with similar components of coronary thrombus. Methods: 18 male beagles were included and divided into three groups: red embolus group (n = 6), white embolus group (n = 6) or white embolus + rt-PA group (n = 6). Autologous emboli were infused into the mid-distal region of the left anterior descending coronary artery. The composition of embolus was examined by scanning electron microscope (SEM). Coronary angiography was performed to verify the status of embolism. Myocardial infarct size was measured by 2, 3, 5- triphenyltetrazolium chloride (TTC) staining. Results: Red thrombus was characteristic of loose reticular structure of erythrocytes under SEM, while the white embolus had compacted structure that mainly consisted of a dense mass of fibrin. Coronary angiography showed the recanalization rate was 2/6 in the red embolus group versus 0/6 in the white embolus group in three hours after occlusion. Arrhythmia, resolution of ST-segment elevation and lower T wave on the electrocardiogram appeared in the red embolus group but not in the white embolus group. Another six dogs with white thrombi were treated with rt-PA. Five out of six dogs exhibited coronary recanalization after two hours of therapy, compared to zero dogs without rt-PA treatment. The size of myocardial infarction in rt-PA group reduced significantly compared with white embolus group using TTC staining method. Conclusions: The white embolism model was more convenient experimentally and had a higher uniformity, stability and success rate. The major innovation of our study is that we applied fibrin-rich white thrombi to establish beagle model possessing features of clinically observed coronary thrombi in time window of intravenous thrombolysis of STEMI. This model can be used to evaluate new thrombolytic drugs for the treatment of STEMI.
Thrombolytic therapy; White thrombus; Coronary artery occlusion
Thrombolytic therapies are critical in salvaging ST-segment
elevation (STEMI), which accounted for 25 % to 40 % of
cases in myocardial infarction [
]. When percutaneous
coronary intervention (PCI) cannot be administered in a timely
manner [anticipated first medical contact (FMC) to device
time > 120 min], thrombolytic therapies were
recommended for STEMI according to the most recent ACCF/
AHA and ESC guidelines [
]. Pre-hospital fibrinolysis is
an important intervention to salvage ischemic
myocardium, improve prognosis and offer additional time for
clinical treatment . Most patients can benefit from
thrombolysis, however, the specificity, effectiveness and
safety of thrombolytic drugs are still required to be
]. To develop new drugs with faster effects
and fewer side effects, it is essential to establish an animal
model of the coronary artery embolism mimicking clinical
status, especially the thrombus composition, good
uniformity and repeatability. However, the coronary thrombi
in previous animal models mostly were red or mixed
emboli, which were different from that of clinical settings.
The composition of the coronary thrombi in time window
of thrombolysis was not clarified until the coronary
thrombus suction technique was used. Recently, we
understood the coronary thrombi in STEMI patients are
mainly composed of fibrin with a small portion of platelets
that decrease over time, a few erythrocytes, cholesterol
crystals and leukocytes . This kind of fibrin-rich
thrombi is similar to those in cerebrovascular thrombosis.
Kirchhof et al. used white embolus to make rabbit model
of cerebral embolism to evaluate thrombolytic drugs [
which has not been applied to the heart. The aim of
present work was to set up an ideal arterial thrombus
model that reflected the clinical syndrome in patients with
STEMI. Therefore, we compared red and white embolism
models via catheter injection into coronary arteries in
animal models and investigated the effectiveness of
Twenty-one adult male beagles (12 to 17 kg) were used in
this study, which was approved by the animal welfare and
the ethical review committee of Fuwai Hospital, Chinese
Academy of Medical Sciences (permission number
2013-230-BJK02). The animal procedures of this experiment were
performed according to the guidelines from Directive 2010/
63/EU of the European Parliament on the protection of
animals used for scientific purposes.
Preparation of emboli
Based on previous methods [
], we do some modification.
Four hours before the operation, 3 ml autologous venous
blood was collected from each experimental animal to
prepare the individual matched emboli. For white
embolus: the venous blood without anticoagulant was
centrifuged at 1500 rpm, for 5 min at 4 °C. After extracting the
supernatant and injecting it into a silicone tube (2.5 mm
in diameter), the blood clots were put into a 37 °C water
bath for 0.5 h. The blood clots were extruded into a sterile
plate by needle tubing for automatic retraction for 3 h
(about 1.2 mm in diameter) and were cut into 5 mm long
cylinders. For the red embolus: the blood in the syringe
was directly injected into a silicone tube, followed by
incubation at 37 °C for 0.5 h. The subsequent processes were
identical to those used to make the white embolus. We
also measured the concentrations of fibrin, platelets and
erythrocytes in whole blood or supernatant. Fibrin
parameters were determined by an automatic coagulation
analyzer (STA-Revolution, Stago), platelets and
erythrocytes were measured by an automated hematology
analyzer (XE-2100, Sysmex).
Coronary artery thrombosis embolism model
Animals were anesthesied with ketamine (35 mg/kg) and
diazepam (15 mg/kg) and maintained with the same drugs
(dose = 1/2 of induction) administered once every hour.
Fentanyl (0.03 mg/kg) was used for analgesia during the
operation and post-operation. After anesthesia, animals
were affixed to the operation table in the supine position
and given endotracheal intubation for assisted respiration
in synchronized intermittent mandatory ventilation
(SIMV) mode (Savina, Draeger Medical AG&Co.KG,
Germany). The parameters include tidal volume (10 ml/
kg), breathing rate (20 times/min), expiration/inspiration
(E/I) ratio (1:1.5–2) and oxygen saturation (55 %). The
arrhythmias were also monitored (M8005A, Philips
Medizin Systeme Boeblingen GmbH, Germany). The
branchiocephalic vein of the left forelimb was used for heparin and
rt-PA injection. An artery sheath catheter was inserted in
the axillary artery branch of right forelimb. Under
fluroscopic guidance of C arm X-ray (9800–12, Beijing
Tongyong Medical Equipment Co., Ltd, China), a 5 F
catheter was inserted into the left coronary artery to
obtain a coronary angiogram. Keeping the 5 F catheter
positioned at the left anterior descending artery near the first
diagonal branch, one embolus were injected to occlude
blood flow through the mid-distal region of the LAD.
Because the catheter cannot be inserted too deeply, the
embolus might reach the diagonal branch. So, the
operator must carefully handle and avoid the embolus flowing
into the vessels with lower pressure.
After LAD occlusion for 60 min, 1000 U heparin sodium
injection was henceforth given once every two hours. The
rt-PA infusion (0.4 mg/kg) was given as a loading dose,
and the thrombolytic agent was continuously infused over
30 min (1.2 mg/kg) afterwards. The remainder of the
rtPA was continuously infused over 60 min (0.8 mg/kg).
This protocol was according with drug specification and
the dose used in dogs was based on the equivalence of
clinical safe dosage [
Measurements of coronary perfusion
The animals received electrocardiogram (ECG)
examination before embolus injection, at the moment of injection
and every 15 min for three hours after injection to record
the changes of ST-segment, T wave and other variations to
estimate the statuses of embolism. For coronary angiogram,
animals received right anterior oblique, anteroposterior and
left anterior oblique coronary angiography before injection
of the embolus, at the moment of injection and every
30 min for three hours after injection to evaluate the degree
of occlusion and/or autolysis. Coronary angiogram was also
carried out at 10, 20, 30, 60, 90 and 120 min after using
rtPA or at the time of occurrence of arrhythmia or
electrocardiographic changes to evaluate the thrombolytic effects.
Reperfusion time was defined as the time when
recanalization was verified by coronary angiogram [
Autologous emboli were analyzed by scanning electron
microscope (SEM). After three hours automatic
retraction, specimens were washed three times with phosphate
buffer, fixed for 120 min in 2 % glutaraldehyde and
rinsed three times with phosphate buffer. Samples were
then fixed for 120 min with osmic acid, rinsed, and
dehydrated in a graded series of ethanol concentrations
(50 %, 70 %, 90 % and 100 %) over a period of 40 min
and further dehydrated in a graded series of
concentrations (50 %, 70 %, 90 %, 100 %) of isoamyl acetate-ethanol
solvent. The clots were dried with hexamethyldisilazane
for 10 min and fractured naturally through pulling to
obtain a fracture surface for analysis. Finally, clots were
coated with gold-palladium prior to examination in a
scanning electron microscope (TM-1000, HITACHI).
The infarct size was determined by
2,3,5-triphenyltetrazolium chloride (TTC) staining. The animals were
anesthetized by intravenously injecting Ketamine (35 mg/kg)
combined with diazepam (1.5 mg/kg) and euthanized
through injecting 10 % potassium chloride (15-20 ml)
after that. The hearts were excised and cut
crosssectionally into plates with 10 mm thick and stained with
2,3,5-triphenyltetrazolium chloride (TTC) [
infarct size was identified as the non-TTC-stained area and
the infarct ratio (%) is the ratio of area of infarction size to
area of left ventricular. We also dissected LAD to observe
the situation of thrombolysis. The skin, mucosa, hearts,
brains, lungs, livers, spleens and kidneys were subjected
for microscopic examination to estimate the bleeding risk
according to the previous study [
Data were analyzed by SPSS 10.0 software (Chicago, IL:
SPSS Inc.) and presented as mean ± SEM. The frequency
of recanalization was statistically analysed with Fisher’s
exact, 2-tailed test. Infarct size ratio was evaluated by
unpaired Student’s t-Test. P < 0.05 indicated a significant
A total of twenty-one dogs were used in the experiment.
One animal died of ventricular fibrillation due to the
extended duration of the catheter in the LAD. Two
animals had diagonal branch embolisms. These three
animals were excluded from the study. Finally, eighteen
animals were divided into three groups, including red
embolus group (n = 6), white embolus group (n = 6) and
white embolus + rt-PA group (n = 6).
The ECG of all animals was normal before the operation.
Transient premature ventricular fibrillation occurred in
two cases in the red embolus group and four cases in the
rt-PA group and was reversed to sinus rhythm after
defibrillation. Furthermore, these six animals were present
coronary recanalization (reperfusion) according to coronary
angiogram as described below. ST-segment and T wave of
lead V1-V4 elevated after injection of autologous emboli.
ST-segment resolution and lower T wave appeared on the
ECG in the red embolus group at 120 min, indicating that
the embolism autolyzed. Neither of these was observed in
the white embolus group. ST-segment resolution and a
lower T wave were observed after the administration of
rtPA to dissolve the white embolus (Fig. 1).
The preoperative coronary angiograms showed that all
coronary arteries in the experimental animals were
normal. Three hours after LAD occlusion, two animals
in red embolus group appeared to have recanalization
and the embolism position in white embolus group was
still in the mid-distal LAD. These results indicated the
white embolus might be better for subsequent
experiments. In addition, five out of six animals (5/6) received
rt-PA had recanalization flow compared with none in
control group (0/6; p = 0.015) after two hours (Fig. 2).
The average time to reperfusion was 43.2 ± 7.4 min in
the rt-PA group (Table 1).
SEM examination was carried out to identify the
characteristics of different autologous emboli (Fig. 3). Blood
parameters of whole blood and supernatant after emboli
preparation were shown in Table 2. White emboli were
more rigid than whole blood emboli because the white
emboli contained more fibrin and less erythrocytes
under same volume.
After autopsy, no obvious signs of bleeding were seen in
the skin, mucosa, heart, brain, lungs, liver, spleen, kidneys
or other important organs (data not shown).
For infarct size measurement, individual slices were
photographed in color using Image J, and the extent
of myocardial necrosis was determined by quantifying
the unstained sections of the heart. Infarct size ratios
were 11.61 ± 0.64 % and 4.48 ± 0.52 % in the white
embolus group and rt-PA group respectively (Fig. 4).
The situations of thrombolysis detected through
white embolus group P value
coronary artery dissection were in accordance with
the results of coronary angiogram.
About 70 % of cases of acute coronary thrombosis are
associated with a disrupted atherosclerotic plaque and
about 30 % of them are only with superficial intimal
]. In case of plaque rupture or endothelial
damage, the exposure of collagen and tissue factor
triggers the activation of platelets and coagulation factors,
which result in thrombus formation [
different components of thrombus in coronary artery can
affect the pathological process of STEMI and the
thrombolytic effect directly. In recent years, the
pathological analysis of aspirated intracoronary thrombi
demonstrated that about 65 % of patients had platelet-rich
(white) thrombi, particularly in the early hours of AMI,
the remaining 35 % of cases had erythrocyte-rich (red)
thrombi with low thrombolysis in myocardial infarction
(TIMI) flow . Silvain et al. found that intracoronary
thrombi were mainly composed of fibrin with the
median ischemic time of 175 min. The fibrin content
increased with the ischemic time, whereas the platelet
content decreased and the erythrocyte content had no
]. The results showed that there were
fibrinrich thrombi in the early time (<3 h) after onset of
STEMI. The white thrombi in our experiments are
similar to the fibrin-rich thrombi in patients with STEMI.
Erythrocytes may contribute more to thrombus
composition at later stages but not in the time window
of acute reperfusion of STEMI. Red thrombus are
composed of fewer massed platelets and more erythrocytes
]. As previously reported, the ratio of erythrocytes
determines the size of the pores between cellulose meshes.
The softer thrombi with larger pores [
] are easier to be
penetrated by fibrinogenase to dissolve. In this study,
after injecting of red thrombi into coronary artery of
dogs, the fibrinolytic system was activated, and the
thrombi started dissolved through coronary angiogram,
ECG and occurrence of arrhythmia. Whereas white
thrombus with compacted structure has strong ability to
resist fibrinolysis and no thrombolytic phenomenon
occurred obviously. Recent experiments have reported the
high stability of white emboli used in animal models in
agreement with our observations [
]. Overall, the
white thrombus is more stable in animal model and has
similar components of STEMI patients in early time of
onset of this disease. These results made the animal model
suitable for subsequent application in evaluating
The new model has better uniformity not only in
embolus preparation but also in animal model establishment.
Firstly, the white thrombi have identical components. To
mimic this kind of fibrin-rich white thrombus,
centrifugation conditions of 1500 rpm at 4 °C for 5 min were
chosen. The blood parameters, mainly including platelet
and fibrinogen, in the supernatant are at the similar levels.
To avoid affecting the antifibrinolytic ability of thrombi,
we did not use any anticoagulants in emboli preparation
]. By operating quickly, we could guarantee that the
supernatant did not coagulate before the silicone tube
shaping step. From the SEM analysis, it was clear that
white emboli with compacted structure mainly consist of
a dense mass of fibrin. Secondly, the size, length and
number of thrombi can be controlled and the locations of
thrombi in coronary artery were similar. Through the
guidance of catheter positioning, emboli were sent to
anterior descending coronary artery and could be stuck in
LAD with similar diameters. This model mimics the
clinical situation that the fresh clot was broken off and carried
through the flow into distal coronary artery at early stage
of myocardial infarction. Thrombolytic therapy in
myocardial infarction is a dynamic process due to the progressive
embolus dissolution. In our experiment, we found the
white embolus moved forward for a limited distance after
using rt-PA during early stage, then it formed eccentrically
clot in the distal section of coronary artery. The white
embolus could be completely dissolved and LAD became
recanalized in a certain time period.
The anti-fibrinolytic ability of the embolus is very
important. Looser or more rigid thrombi do not behave
like the endogenous thrombi in STEMI and therefore
may not provide accurate information concerning the
efficacy and safety of different thrombolytic drugs. There
are no universal criteria to evaluate autologous emboli,
and the different methods resulted in different
experimental outcomes [
]. In current experiments, we used
the autologous white embolism model to examine the
thrombolytic actions of rt-PA. This drug is commonly
used in clinical practice because of its rapid clearance
and ability to be co-administered with heparin. The
average time to reperfusion was 43.2 ± 7.4 min and the
patency rate in this model was 5/6(83 %) in 90 min,
which was similar to those in clinical study (73–84 %)
8, 19, 20
]. The size ratio of myocardial infarct in rt-PA
group reduced significantly compared to control group,
which confirmed that use of thrombolytic drugs timely
could improve the prognosis of patients with STEMI.
As determined by microscopic examination, there was
no obvious bleeding-induced damage to skin, mucosa,
heart, brain, lungs, liver, spleen, kidneys or other
important organs. It may be related to the safer dose adopted
in healthy animals. There is no perfect system to predict
bleeding risk associated with thrombolysis in clinical
treatment, so the animal experiments to evaluate bleeding risk
is needed for further studies. Because the time window of
thrombolytic treatment is relatively short [
19, 21, 22
thrombolysis is very important for developing new
thrombolytic agents. In conclusion, our model can be used
to compare the rates and time of recanalization among
different thrombolytic drugs in their safe dose ranges.
Catheter-based delivery of the autologous emboli was
shown to be effective in our study. We selected the
axillary artery branch instead of carotid artery or femoral
artery as the puncture path for the first time, which
could shorten the length of interventional devices in the
body, reduce the risk of postoperative infection and
decrease hemorrhage at the puncture point. In terms of
detection, coronary angiography was effective at
instantaneously ascertaining the degree of coronary artery
stenosis, allowing the progress of thrombolysis to be
carefully monitored. Because of its gentle temperament
and homogenous genetic background, the beagle is
widely used in preclinical drug evaluation. However, the
diameter of coronary arteries in beagle is small and no
specialized coronary artery catheters are currently
available for these animals. Our protocol resolved this
At present, the methods for evaluating thrombolytic
therapy in large animal models mainly include electrical
], open chest thrombosis injection [
occlusion and thrombin injection [
] and copper
coilinduced coronary thrombosis [
]. The first two methods
require open chest operation, with complex operations
and a larger trauma. The thrombus in the last two models
may have different compositions from those seen in
STEMI patients. Although the thrombus induced by
electrical injury have similar composition to human coronary
artery thrombi, this method takes a long time (3.2 ± 0.4 h)
to complete coronary occlusion and the size of thrombus
cannot be controlled [
]. The present model was a
simple, efficient and inexpensive method with a smaller
trauma. Furthermore, the artificially produced fibrin-rich
white thrombus has uniform size and clinical features of
coronary thrombi in time window of intravenous
thrombolysis of STEMI.
We established, for the first time to our knowledge, the
coronary artery embolism model with white thrombus
which had better stability, uniformity and a higher
success rate. Importantly, the model produced thrombi with
characteristics similar to those in STEMI patients in
time window of thrombolytic therapy and was amenable
to evaluation of thrombolytic therapies. This model can
be used to evaluate new thrombolytic drugs for the
treatment of STEMI.
ECG: Electrocardiogram; E/I: Expiration/inspiration; FMC: First medical contact;
LAD: Left anterior descending artery; PCI: Percutaneous coronary
intervention; rt-PA: Recombinant tissue plasminogen activator; SEM: Scanning
electron microscope; SIMV: Synchronized intermittent mandatory ventilation;
STEMI: ST-segment elevation; TIMI: thrombolysis in myocardial infarction;
TTC: 2,3,5-triphenyltetrazolium chloride.
The authors declare that they have no competing interests.
HZ and YCC participated in the design of the study and drafted the
manuscript. YT, WMY, JZY and PP participated in the operation. KL
performed the data collection. XPL and DZ performed the statistical analysis.
ALW participated in its coordination. ZZ and YT helped to draft the
manuscript. All authors read and approved the final manuscript.
This work was supported by Beijing Municipal Science & Technology
Commission (Project No: Z101107052210004).
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