The inhibitory effect of dilazep on in vivo accumulation of platelets onto the damaged aorta in rabbit

THROMBOSIS RESEARCH29; 37-42, 1983 0049-3848/83/010037-06$03.00/O Printed Copyright (c) 1983 Pergamon Press Ltd.

in the USA. All rights reserved.

THE INHIBITORY EFFECT OF DILAZEP ON IN VIVO ACCUMULATION

OF PLATELETS ONTO THE DAMAGED-IN

RABBIT

II. Morphological Analysis

A. Sumiyoshi and T. Hayashi Department

of Pathology, (Received

ABSTRACT

Miyazaki Medical College, Miyazaki 889-16, Japan

6.5.1982; in revised form 29.9.1982. Accepted by Editor N. Aoki)

The inhibitory effect of dilazep on platelet adhesion and aggregation onto the damaged aorta was morphologically investigated using an animal model of thrombosis in which polyethylene tubing was inserted into the rabbit aorta in order to injure the intima. Rabbits were divided into 2 groups, control and dilazep-treated groups. The aortas 60 minutes after insertion of tubing were examined by light, scanning and transmission electron microscopy. An amount of adhered and aggregated platelets onto the exposed subendothelial surface of the aorta in rabbits pretreated with dilazep 100 ug/kg was significantly less than in control rabbits. Fibrin formation was also sparse in the mural thrombi in dilazep-treated group compared to that in control group. The results show that dilazep has the definite inhibitory effect in early mural thrombogenesis following the endothelial denudation in rabbit.

INTRODUCTION The problem regarding prevention of thrombosis has come to occupy an important position in the therapy and/or prevention of vascular occlusive diseases including atherosclerosis. Platelets are well-known to play a key part in the initiation and growth of thrombi. Therefore, drugs which inhibit interaction of platelet and vascular wall, and platelet aggregation, are considered to be beneficial in medical practice. However, basic research in vivo on anti-

Key words : thrombosis,

platelet,

damaged

37

aorta,

antiplatelet

drug,

dilazep

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platelet drugs is seldom found, although their practical applications have been rather extensively studied. Moreover, it has been shown that the preventive effects of antiplatelet drugs on in vivo thrombus formation are not always correlated with inhibitory effect of these on in vitro platelet aggregation. Therefore, the true effect of antiplatelet drugs should be evaluated in vivo in an experimental animal model. Hence, the authors presented the quantitative analysis of the inhibitory effect of dilazep and aspirin on in vivo accumulation of platelets to the damaged aorta using 51Cr-labeled patelets(l).The summary of the result was as follows: Radioactivity of the damaged aortas in rabbits pretreated intravenously with 50 or 100 ug/kg of dilazep as well as 10 mg/kg of aspirin was significantly lower than in control rabbits, and dilazep and aspirin seemed to possess the definite inhibitory effect on platelet accumulation onto the denuded subendothelium in vivo. The present report showed the results of morphological analysis of an antithrombotic effect of dilazep in the experiment similar to the study referred to in the previous report.

METHODS Twelve male New Zealand white rabbits keighing between 2.0 and 2.3 kg were divided into 2 groups, control and dilazep-treated groups. They were anesthetized with intravenous administration of sodium pentobarbital in a dose of 25 mg/kg. Five minutes before induction of intimal injuries, dilazep, 1,4-Bis [ 3- ( 3,4,5-trimethoxybenzoyloxy) propyll -perhydro- 1,4-diazepine dihydrochloride monohydrate, 100 u g/O. 2 ml of saline/kg (Dilazep was kindly supplied from Kowa Co. , Ltd. , Tokyo, Japan. ) were intravenously injected in dilazeptreated group and saline was similarly injected in control group. The intimal injuries were induced by insertion of polyethylene tubing (Intramedic, PE 50) in the aorta up to the mid portion of descending thoracic aorta via the femoral artery as previously described (2). Evans blue dye solution (4% in saline) 0.5 ml/kg were given intravenously 45 minutes after insertion of tubing to visualize the injured areas. Sixty minutes after insertion of tubing, rabbits were heparinized with a dose of 500 U/kg and tubing was immediately removed. Subsequently rabbits were perfused with 0.1 M phosphate buffered saline with heparin 1,000 U/l, pH 7.4, at a pressure of 75 mmHg. The aorta was then excised intact, opened along the lesser curvature of the arch and the ventral

s.m.a.

r.a.

A3

A2

Al

T3

T2

Tl

FIG. 1 Schema of tissue sampling from the damaged aorta. r .a. : renal artery, s.m.8.: superior mesenteric artery. Numbers, Tl , T2, T3, Al, A2, and A3, indicate the aortic segments examined.

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side of the thoracic and abdominal aorta, pinned out, and fixed by immersion in a combination fixative of 4%neutralized formaldehyde and 1%glutaraldehyde in a 200-mOsm phosphate buffer, pH 7.4, at 4OC. Six cross-sectional tissue blocks, 3 from the thoracic and 3 from the abdominal aorta, were obtained equally from each animal as shown in FIG. 1, postfixed with 2.%060~ in 0.1 M phosphate buffer, pH 7.4, and embedded in Epon 812 as usual. For light microscopy, specimens, 1 urn thick, were cut on a Reichert Ultracut microtome and stained with hematoxylin and eosin after removing Epon by 1%KOH in absolute ethanol. Selected Epon blocks were then cut for transmission electron microscopy. The ultrathin sections were stained with uranium and lead as usual and examined with a JEM-100s microscope at 80 kV. For scanning electron microscopy, selected samples from the blue stained areas of the aorta fixed with a combination fixative were dehydrated, critical-point-dried using liquid CO, , sputter-coated with gold, and examined with a JSM-25s microscope.

RESULTS AND DISCUSSION The rabbit aorta revealed sharply demarcated irregular streaks stained with Evans blue dye which indicated the endotheIia1 denudation and increased permeability. The internal elastic lamina appeared intact by light and electron microscopic examination. The pattern and extent of aortic intimal injuries estimated by discoloration of Evans blue dye were roughly similar in all rabbits. Platelets adhered to the subendothelial surface where the endothelium was completely removed. Platelets were never found to adhere onto the intact endothelium (3). The distribution and density of platelets adhered to the exposed subendothelial surface of the damaged aorta varied widely from segment to segment examined in the same animal and from microscopic field to field even in one cross-section (FIG. 2A, 2C) or in a low-power scanning micrograph (FIG. 2B). There were different types of platelet accumulation on the exposed subendothelium: no platelet, scattered platelets, monolayer to several layers of platelets, and medium-sized or large lumps of platelets. The last were associated with leukocytes and occasional fibrin formation in them, and considered to be mural platelet thrombi(FIG. 2A, 2B). These raised mural platelet thrombi were frequently noted in sections from control rabbits than from dilazep-treated rabbits. Based on the density and amount of adhered andlor aggregated platelets onto injured sites, the semiquantitative scores were light-microscopically evaluated at a field of the maximumlesion of each cross-sectional segment and arbitrarily expressed on a five point scale as l+, 2+, 3+, 4+, and 5+. The results were summarized in TABLE 1. The amount of platelets attached to the damaged aorta in dilazep-treated rabbits was significantly less than in control rabbits in general. Thus, the results of the present morphological study confirmed the results of the previous quantitative radioisotopic study on the inhibitory effect of dilazep on in vivo accumulation of platelets onto the damaged aorta (1)) and showed thaxep did not completely inhibit the platelet adherence to the de-endothelialized subendothelium but significantly inhibited the platelet aggregation which gave rise to the rised mural thrombi. And further, this study provides evidence that this experimental method is an appropriate one for the semiquantitative analysis of antithrombotic effect of drugs in vivo without using isotopes. In the present study, fibrin formation was noted at the base of large mural thrombi in control rabbits, where platelets showed prominent degranulation and degradation (FIG. 3). On the other hand, platelets in dilazep-treated rabbits had a tendency to keep their shape and granules fairly normal (FIG. 4).

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FIG. 2 Light and scanning electron micrographs of representative injured areas one hour after injury. A and B are from the aorta of a control rabbit, and C and D from the aorta of a dilazep-treated rabbit. The de-endothelialized surface in control rabbit is covered with thickly adhered platelets forming a large raised platelet thrombus in the center. Most of the denuded subendothelial surface in dilazep-treated rabbit are covered with one to two layers of platelets and occasional small aggregates of platelets.

TABLE 1 Effect of dilazep on platelet adhesion and aggregation onto damaged aorta. Comparison of corresponding segments between two groups. Scores of lesion* Segment

Control group

Dilazep-treated

group

P**

Tl (n=6)

3.167 f: 0.833

2.500 + 0.500

T2 (n=6)

2.500 t 0.671

1.667 + 0.333

< 0.05

A3 (n=6)

3.000 rt:0.775

1.333 f 0.211

< 0.01

2.833 + 0.520

2.083 + 0.313

< 0.01

Tl + T2 (n=12)

*Values are average f S.E.

**Student’s t-test.

N.S.

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FIG. 3 (left) and FIG. 4 (right) Transmission electron micrographs of a large platelet thrombus in a control rabbit (FIG. 3) and of a representative lesion from a dilazep-treated rabbit (FIG. 4). FIG. 3 shows prominent degranulation and degradation of platelets associated with many fibrin threads (F) on and near the internal elastic lamina (E) . In FIG. 4, one to two layers of platelets cover the de-endothelialized surface, and some platelets adhered retain their granules and show a minimal shape change.

These findings suggest that dilazep may inhibit platelet aggregation by preventing a release reaction of platelet constituents and shape change by its membrane stabilizing action (5) and its action as a Ca++ antagonist (6). Platelet aggregates on the damaged aorta are reversible. The formation of fibrin plays a major role in stabilizing platelet aggregates on the wall (3,’ 4). Adhered platelets release their granule constituents and activate the arachidonate pathway which causes further platelet aggregation (7). Intimal damage together with platelet aggregation and release reaction provides a site for the activation of clotting factors (8) resulting in the generation of thrombin. The thrombin converts fibrinogen into fibrin and also causes further platelet aggregation and release reaction (9). This relation between platelet aggregation and blood coagulation is mutually intimate. Therefore, the sparsity of fibrin threads in dilazep-treated rabbits may result from an antiplatelet effect of dilazep. Moreover, some thrombi appear to undergo resolution following their formation. The fibrinolytic pathway may operate in this resolution of thrombi. This effect may also play a role in diminishing mural thrombi and fibrin threads in dilazeptreated rabbits, since it has been reported that dilazep has an enhancing effect of fibrinolysis by increasing the release of plasminogen activator from vascular bed and decreasing the plasmin inhibitory activities (10, 11). There is a growing interest on the potential use of drugs to prevent thrombosis, atherosclerosis and its complications (4, 12). The use of antiplatelet drugs to be effective in vivo may modify the development of thrombosis, and also the initiation and progression of atherosclerosis. Since dilazep has both the inhibitory effect on thrombogenesis as described in this report, and the enhancing effect of fibrinolysis, it is anticipated that this drug may be useful to prevent and reduce the thrombotic and atherosclerotic diseases.

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ACKNOWLEDGEMENT This work was supported by a Research Grant for Cardiovascular (55A-1) from the Ministry of Health and Welfare, Japan.

Diseases

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