(Stroke. 1996;27:1099-1104.)
© 1996 American Heart Association, Inc.
Articles |
In Vivo Evaluation of Antiplatelet Agents in Gerbil Model of Carotid Artery Thrombosis
From the Fifth Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, and the Division of Cerebral Circulation Research, National Cardiovascular Center, Osaka (H. Naritomi), Japan.
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Abstract |
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Background and Purpose Antiplatelet agents are widely used for the prevention of ischemic stroke. However, their effects on thrombus formation have rarely been evaluated in experimental animals in vivo. We introduce methods for evaluating antithrombotic action in gerbils and the effects of several antiplatelet agents on thrombus formation.
Methods In gerbils 8 to 10 weeks of age, we tightly compressed the unilateral common carotid artery for 2 minutes using the device prepared for this purpose to damage the endothelium. Thrombus formation in the damaged artery was observed directly through the microscope for 30 minutes. In six animals, the damaged artery was examined immediately after the experiments by electron microscopy. We studied the effects of antiplatelets by injecting the drugs intravenously 10 minutes before endothelial damage.
Results In control studies, 70% to 90% of animals developed thrombi after arterial compression. The electron microscopic examination displayed endothelial damage in association with platelet thrombus at the damaged site. Administration of 2 mg/kg aspirin, 3 and 10 mg/kg ticlopidine, and 0.3 and 1.0 mg/kg ibudilast, a novel prostacyclin accelerator, decreased the frequency of thrombus formation significantly, whereas 20 mg/kg aspirin and 20 mg/kg dipyridamole failed to decrease thrombus formation.
Conclusions This model is considered useful for evaluating the antithrombotic effects of drugs because of its feasibility and high reproducibility. The present results support the view that a lower dose of aspirin may prevent cerebral vascular accidents as efficiently as a higher dose of aspirin.
Key Words: aspirin • endothelium • thrombosis • ticlopidine • gerbils
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Introduction |
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Recent studies have established that the interaction of the endothelium and platelets plays an important role in the initiation of arterial thrombosis.1 2 Various other factors, such as leukocytes and circulatory conditions, also appear to be involved in the mechanism of arterial thrombosis. In patients with cerebrovascular diseases, various antiplatelet agents, such as aspirin3 4 and ticlopidine,5 6 are administered to prevent the recurrence of stroke. These drugs act to suppress platelet aggregation in vitro. However, the antithrombotic effects of these drugs have rarely been evaluated in experimental animals in vivo.
Several investigators have induced thrombosis in the pial cerebral arterioles in animals and studied the antithrombotic effects of antiplatelets.7 8 9 However, in clinical cases thrombosis most commonly forms in the extracranial carotid arteries, the circulatory condition of which differs from that of pial arterioles. For these reasons, the effects of antiplatelets on thrombus formation may be studied best in the extracranial artery. Among several experimental models, photochemical methods reported by Dietrich et al10 and Futrell and Riddle11 successfully induced carotid artery thrombosis. Their models, however, appear to be more suitable for studying the occurrence of artery-to-artery embolism in the brain and less appropriate for investigating the development or prevention of carotid artery thrombus. In the present communication we introduce methods for inducing thrombus in the CCA in Mongolian gerbils and results of antiplatelet administration on thrombus formation.
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Materials and Methods |
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Gerbil Preparation
We used 188 male Mongolian gerbils weighing 65 to 85 g for the experiments. All the animals were anesthetized with injections of 45 mg/kg sodium pentobarbital IP. In each animal, either CCA was exposed through a midline incision of the neck and separated from the adjacent tissue. Endothelial damage was induced in the unilateral CCA with the use of a device consisting of paired polyethylene tubes (PE-50; 0.97 mm in external diameter and 4 mm in length) and two surgical threads (4-0 and 5-0), which pass through the inner lumen of the tubes, one (thread A) for arterial compression and the other (thread B) for releasing the tubes. The device was cut incompletely into two joined segments retaining their individual mobility and was placed at either CCA in such a way that the artery was sandwiched between the paired tubes (Fig 1
).
Based on the preliminary data,12 the CCA was compressed
with 400 g tension on thread A for 2 minutes. The compression was
accomplished by sustaining tension on the occluding thread A.
Conversely, the compression was terminated by a brief pull on the
releasing thread B. After the endothelial damage by
arterial compression, thrombus formation at the damage site
was observed for 30 minutes through the microscope. Thrombus was
defined by the manifestation of an adherent mass of platelets
recognized as "white body" (Fig 2).
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After the CCA was exposed, two gerbils showed arterial constriction or slow blood flow. They were excluded from the following study to avoid the influences of hypotension or vascular smooth muscle injury due to the surgery.
Preliminary Tests for Reproducibility
One month before the study, preliminary studies to test the
reproducibility of the methods were performed in two groups of animals
with saline administration 10 minutes before arterial
compression. In both groups, the frequency of thrombus formation after
arterial compression was examined in the same manner as the
study. The preliminary study in one group (n=10) was undertaken 1 month
after that in the other group (n=10).
Examination of Circulatory Changes
Twenty animals were used to study circulatory changes that may
occur after CCA compression. In 10 animals, a PE-10 catheter was
inserted into the femoral artery. Blood pressure was monitored by a
pressure transducer attached to the catheter. Blood flow and velocity
of CCA were measured at the peripheral site of
endothelium damage before and after
arterial compression by a laser flowmeter (ALF21N; Advance
Co Ltd). In the other 10 animals, venous blood was sampled 30 minutes
after arterial compression to measure fibrinogen, PT, and
aPTT.
Pharmacological Examination of Thrombus Formation
The drugs used were aspirin (Sigma), ticlopidine (Sigma),
dipyridamole (Sigma), and ibudilast (KC 404;
3-isobutyryl-2-isopropylpyrazolo-[1,5
]-pyridine; Ketas, Kyorin
Pharmaceutical Co). Aspirin, ticlopidine, and
dipyridamole were stored at -20°C. Aspirin and
dipyridamole were dissolved in redistilled water
containing equimolar sodium hydroxide. Ticlopidine was dissolved in
physiological saline. Ibudilast was dissolved in a
fivefold concentration of polyoxyethylene hydrogenated castor oil
(HCO-60, Nikko Chemicals); 2 and 20 mg/kg aspirin, 3 and 10 mg/kg
ticlopidine, 20 mg/kg dipyridamole, and 0.3 and 1.0
mg/kg ibudilast were dissolved in 100 µL saline or solution on the
day of the experiment. Each drug was injected intravenously
via the femoral vein 10 minutes before arterial compression
in each group (n=10). The control study was performed in each
experiment. In the control groups (n=10), the same volume of
physiological saline was injected.
The experiments were performed in a blinded manner, in which the development of thrombus formation was judged by an author who had no information as to the content of drugs or physiological saline injected. At the conclusion of all experiments, all data were evaluated.
Morphological Examination of Damaged Artery
The damaged carotid artery was obtained from 2 control animals
with thrombus, 2 control animals without thrombus, and 2
sham-operated animals. The damaged artery was also obtained from 1
animal with thrombus despite ticlopidine administration and another
animal without thrombus after ticlopidine administration. After we
confirmed the presence or absence of white body by microscopic
examination, these animals were killed by transcardiac
perfusion with 0.9% sodium chloride, 2%
glutaraldehyde in 0.1 mol/L cacodylate buffer (pH 7.2),
and 0.1 mol/L cacodylate buffer (pH 7.2) for 5 minutes in each
perfusion at a pressure of 50 mm Hg. The brain was removed from the
cranium and immediately immersed in 3% glutaraldehyde
in 0.1 mol/L cacodylate buffer (pH 7.2) for 30 minutes, washed several
times with the same buffer, and postfixed with 2% osmium tetroxide.
After the fixation, they were washed with the same buffer and
dehydrated through a graded ethanol series. They were
critical-point-dried, coated with gold/18.5% palladium, and
examined under a Hitachi S-4000 scanning electron microscope.
Statistical Analysis
Changes in MABP, blood flow, mass, and velocity were compared
with paired Student's t test. Fibrinogen, PT, and aPTT were
compared with unpaired Student's t test. Frequency of
thrombus formation was compared with Fisher's test of exact
probability.
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Results |
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Reproducibility of the Methods
In the preliminary tests, 80% of animals in the first group showed thrombus formation, and 70% in the second group displayed thrombus formation.
Circulatory Changes
Changes in MABP and CCA blood velocity after CCA compression are
shown in Table 1. There were no significant changes in
MABP or CCA blood velocity. Table 2 compares fibrinogen,
PT, and aPTT values obtained before and after CCA compression. None of
these coagulating factors showed significant changes.
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Thrombus Formation in Control and Treated Animals
In the seven control groups, 70% to 90% of animals developed
thrombus (manifested as an adherent mass of platelets described as
white body) after CCA compression. The frequency of thrombus formation
in the control groups is thus almost identical to that obtained in the
two preliminary studies. Thrombus always formed at the site of CCA
compression or a site somewhat peripheral to the compressed
site and was never observed at the proximal areas. Table 3 compares the frequency of thrombus formation in the
control and treated animals. We found that 2 mg/kg aspirin
significantly decreased the frequency of thrombus formation
(P<.01); 20 mg/kg aspirin also decreased the frequency of
thrombus formation, although this decrease was not significant.
Ticlopidine (3 and 10 mg/kg) reduced the frequency of thrombus
formation to the same degree of significance (P<.05 and
P<.01, respectively). On the other hand, 20 mg/kg
dipyridamole failed to decrease the frequency of
thrombus formation. Ibudilast (0.3 and 1.0 mg/kg) reduced the frequency
of thrombus formation significantly (P<.05). The reduced
frequency of thrombus formation by ibudilast was the same as that
obtained by 3 mg/kg ticlopidine.
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Electron Microscopic Findings of Compressed Artery
In normal control animals without CCA compression, no evidence of
cellular damage was discerned. Endothelial cells were
arranged regularly, like paving stones, in the luminal surface of the
artery (Fig 3). No platelet adhered to the vessel
wall. Endothelial cells were fusiform, oriented with
their long axis parallel to the long axis of the vessel. Ovoid
protrusions representing the underlying nuclei were
commonly observed. Numerous microvilli were observed on the
endothelial surface. Vacuolization of the
endothelial cells, rarefaction of the
endothelial cell cytoplasma, and
endothelial luminal membrane rupture were absent.
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In animals with thrombus after CCA compression, multiple-layered
activated platelets with prominent pseudopodia adhered to
the vascular luminal surface of the entire injured area (Fig 4). Partially liberated endothelial
cells were frequently observed. Similar changes were found in two
control animals with thrombus and the animal with thrombus despite
ticlopidine treatment. In contrast, in animals without thrombus after
CCA compression, activated platelets were scattered and
adhered on the surface of the vessel in a monolayer (Fig 5). Endothelial cells were denuded. The
changes were the same in the two control animals without thrombus and
the animal without thrombus due to ticlopidine treatment.
Multiple-layered adhesion of platelets was never observed in
this group.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have developed a new model of carotid artery thrombosis caused by mechanical endothelial damage. Recently, several groups have produced photochemical endothelial damage of the pial artery resulting in development of thrombus.7 8 9 However, since thrombosis most commonly forms in the extracranial carotid arteries in clinical cases, the effects of antiplatelets on thrombus formation may be studied best in the extracranial artery. Moreover, the antithrombotic effects of antiplatelet agents may be better investigated at the site of endothelial damage. Dietrich et al10 and Futrell and Riddle11 developed models of carotid artery thrombosis induced by photochemical irradiation. In their models, platelet thrombus formed in the carotid artery 50 minutes after irradiation. Such models of delayed thrombus development are inappropriate for estimating the antithrombotic action of drugs, particularly short-acting drugs. On the other hand, in the present model white body as the platelet thrombus appeared within 5 minutes after arterial compression in the majority of control animals. Another advantage of the present model is the possibility of observing the manner of platelet thrombus formation through the microscope. The present model is thus considered adequate for screening the efficacy of antiplatelet agents in vivo.
In the preliminary studies to examine reproducibility, two groups of animals showed thrombus formation with frequencies of 80% and 70%. In the pharmacological study, 70% to 90% of control animals showed thrombus formation. Thus, the present methods were shown to induce carotid artery thrombus with high reproducibility. As indicated by recent studies, endothelial injury appears to play an important role in the development of arterial thrombosis.
In clinical cases of cerebral artery thrombosis, endothelial injury may develop gradually under the existence of hypertension or other risk factors for atherothrombosis. In contrast, endothelial injury in the present model was induced mechanically by arterial compression in an abrupt manner. Therefore, the process of endothelial injury essentially differs between the present model and clinical cases, and the manner of thrombus formation after endothelial injury may also differ accordingly. Regardless, the present model is considered useful because it permits the evaluation of antiplatelet effects on endothelial injury–derived thrombus in vivo. The mechanical arterial compression may slightly damage other sites of the arterial wall, such as the media. However, the CCA compression did not significantly alter CCA blood flow and velocity. Slight damage to the media may be considered permissible, since it does not seem to alter arterial flow and may only slightly influence the acute development of thrombus. In the present study we defined the microscopic manifestation of white body in the damaged artery as thrombus. The electron microscopic examinations revealed the presence of multiple-layered platelet adhesion in the animals with white body, supporting the validity of our definition. The electron microscopic examinations showed single-layered or scattered platelet adhesion in animals without white body. Scattered platelet adhesion was also seen in control arteries, which were handled in the same manner as the experimental vessels. This seems to reflect the normal platelet adhesion response observed in electron microscopic studies. Therefore, the absence of white body in animals with antiplatelet administration can be regarded as indicating the prevention of thrombus formation.
In the present pharmacological studies, 3 and 10 mg/kg ticlopidine and 2 mg/kg aspirin significantly prevented thrombus formation. Ticlopidine5 6 and aspirin3 4 are now widely used in clinical cases of ischemic cerebrovascular diseases for the prevention of ischemic stroke. Several clinical trials affirmed their efficacy in the prevention of cerebral ischemic attacks.3 4 5 6 13 14 15 16 17 18 The present results are in accordance with those clinical results. It is of interest that 2 mg/kg aspirin significantly decreased the frequency of thrombus formation, whereas 20 mg/kg aspirin failed to decrease the frequency in the present study. The mode of action of aspirin is thought to inhibit the production of TXA2 derived from platelets. Several clinical trials reported that low-dose aspirin therapy successfully prevented the recurrence of ischemic attacks.15 16 18 On the other hand, the effect of high-dose aspirin is still controversial. One of the explanations for this dissociation is that high-dose aspirin may also inhibit prostacyclin (PGI2) production in the endothelium, thereby diminishing the antiplatelet action. Tohgi et al18 measured serum and urine metabolites of TXA2 and PGI2 in patients with cerebral infarction after 40, 320, and 1280 mg/kg aspirin. They found that 40 mg/kg aspirin inhibited TXA2 production and had no effect on PGI2 production, whereas 320 and 1280 mg/kg aspirin inhibited the production of both TXA2 and PGI2 in a dose-dependent manner. Although there are different pharmacokinetics between intravenous injection and oral administration, the intravenous injection of 2 and 20 mg/kg aspirin used in the present study may correspond to low- and high-dose oral administration of aspirin, respectively. Our results seemingly support the view that high-dose aspirin may exert fewer potential antiplatelet effects than low-dose aspirin in vivo because of inhibition of PGI2 production in the endothelium. However, the different response in only 1 of 10 animals between the 2 and 20 mg/kg groups hardly leads to the conclusion that high-dose aspirin is worse. This view is considered reasonable because the 20% incidence at 20 mg/kg aspirin showed no significant difference from the 70% incidence in the control group, whereas the same 20% incidence at 0.3 mg/kg ibudilast showed a significant difference from the 80% incidence in the control group. While the 20% incidence at 20 mg/kg aspirin was not statistically different from the 70% incidence in the control group, the 20% incidence was also not different from the 10% incidence at 2 mg/kg aspirin. Perhaps a large sample size may show an attenuation at 20 mg/kg aspirin. In addition, the present results cannot be used to distinguish the optimal aspirin dose in clinical use, since the pharmacokinetics of intravenous bolus injection 10 minutes before the insult in the gerbil differ from those in the clinical situation.
Although dipyridamole13 and
ibudilast19 20 are usually not included in a category of
antiplatelet drugs, they are known to have antiplatelet
actions. In the present study dipyridamole
decreased the frequency of thrombus formation to a small extent, and
this inhibitory effect was not significant. This result is
considered reasonable because the antiplatelet action of
dipyridamole appears to be less potent compared with
aspirin or ticlopidine in in vitro studies. In contrast, 0.3 and 1.0
mg/kg ibudilast decreased the frequency of thrombus formation
significantly. The extent of thrombus inhibition by ibudilast was
comparable to that by aspirin and ticlopidine. Ibudilast is a novel
cerebral vasodilator that dilates the isolated canine basilar artery
preconstricted with PGF2 in
vitro.21 The mechanism of its vasodilation is explained by
the acceleration of PGI2 activity. In an experimental study
using rats,20 ibudilast inhibited electroencephalographic
flattening after cerebral ischemia induced by
arachidonic acid injection into the internal carotid
artery. This inhibitory effect may be attributable to
cerebral vasodilatory action or antiplatelet action mediated by
acceleration of PGI2 activity. Ibudilast was shown to have
antiplatelet action in vitro19 that was much less
potent than that of aspirin. This in vitro antiplatelet action
was, however, enhanced enormously by the application of
PGI2. Thus, it appears likely that ibudilast exerts
significant antiplatelet effects only through the existence of
PGI2. Drugs exerting antiplatelet actions through
PGI2 activity, such as ibudilast, may show poor
inhibitory effects on platelet aggregation if tested in
vitro. The present gerbil model is considered potentially useful
for evaluating the effects of such drugs.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by a research grant from the Hyogo College of Medicine and grants-in-aid for scientific research 05770444 and 06770468 from the Japan Ministry of Education.
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Footnotes |
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Reprint requests to Hiroyuki Nishimura, MD, PhD, Fifth Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663, Japan.
Received January 22, 1996; accepted February 12, 1996.
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