Effect of thiouracil upon canine arterial elastic tissue

Atherosclerosis, 22 (1975) 369-377

0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

EFFECT ELASTIC

OF THIOURACIL TISSUE * 9* *

ALBERT0

TRILLO* * *t

UPON

CANINE

369

ARTERIAL

Department of Pathology, The University of Western Ontario, London, Ontario (Canada) (Received November 19th, 1974) (Accepted June 9th, 1975)

SUMMARY

Unlike

some other

mammalian

species,

the dog is relatively

resistant

to the

development of elevated levels of serum cholesterol after prolonged cholesterol feeding. This may be overcome by suppressing thyroid activity with thiouracil. Information regarding possible activity of thiouracil itself upon the arterial tissues is almost nonexistent. The present investigation was undertaken to test whether this drug has any such action, especially upon the arterial Destructive changes were observed in arterial

elastic tissues. elastic tissues

in dogs

given

thiouracil for three and six months. The changes consisted of accentuation of the elastic fibrillar components, formation and subsequent coalescence of clefts, and fragmentation and ultimate “dissolution” of the elastic elements. The results suggest that thiouracil

may exert a damaging

effect upon

the arterial

elastic fibers;

thus, it

is possible that one of the mechanisms by which thiouracil and cholesterol administration induces experimental atherosclerosis in the dog is by elastic tissue destruction, possibly promoting the subsequent lipid accumulation in the arterial wall.

Key words:

Antithyroid drug - Atherosclerosis - Elastic lamella - Internal elastic Iamella - Lipid degeneration

* Presented in part at the 29th Annual Meeting of the Electron Microscopy Society of America, Boston, Mass., August, 1971. by Grants-in-aid T-11 from the Ontario Heart Foundation, Toronto, Ontario, and MT-1037 from the Medical Research Council of Canada. *** This work was carried out while the author was a recipient of an Ontario Heart Foundation Fellowship. t Present address : Department of Pathology, The Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, N.C. 27103, U.S.A.

** Supported

370

A. TRILL0

INTRODUCTION

Since

Anitschkows

demonstrated

in his pioneering

work

on experimental

atherosclerosis that arterial lesions similar to those in man could be produced rabbits fed a high cholesterol diet, numerous experiments have been designed produce

atherosclerotic

lesions in several animal

species, particularly

animals having a diet similar to that of man. Early attempts at inducing atherosclerotic

lesions

in to

in omnivorous

in dogs by feeding

them a

high cholesterol diet failed due to a natural resistance of these animals to sustained elevated cholesterol serum levels for a long period of time. Steiner and Kendallz5 suggested that in omnivorous animals, particularly the dog, the mechanisms regulating cholesterol metabolism may continue to function effectively in spite of a high cholesterol intake.

These authors

showed that the important

role of the thyroid

in the regu-

lation of serum cholesterol levels may be altered by the use of an antithyroid drug such as thiouracil. When a high cholesterol diet was given concomitantly with thiouracil, the dogs maintained serum cholesterol levels as high as 2000 mg/dl. Light-

and electron-microscopic

studies

sidered similar to those of human atherosclerosis were largely produced

experimentally

of canine

arterial

lesions that are con-

are numerous4~9,10,12~l~,l*~19~25,26 and

by employing

thiouracil

along with diets having

added fats or cholesterol. However, to date information regarding the effect of thiouracil itself upon the arterial wall is scanty and inconclusivesp2s. The present investigation was undertaken to assess by electron microscopy the possible effects of thiouracil administration on the mural arterial elements, specifically upon the elastic tissues. MATERIAL AND METHODS

Four

mongrel

male dogs, approximately

24 months

of age (estimated

on the

basis of wear and tear of their nails and teeth), were used in the present work. The animals weighed from 12 to 14 kg and were divided into two groups of two animals each. The dogs were fed Purina dog chow supplemented by thiouracil* at a dosage of 100 mg/kg/day for three and six months respectively. At the end of each feeding period the animals were anaesthetized with pentobarbital and the arteries were fixed in situ by perfusing the entire circulatory system with a 3 % phosphate-btiered glutaraldehyde solution infused under pressure which was maintained at 140-155 mm Hg. Following perfusion, the common carotid and femoral arteries were removed and cut into ring-shaped cross sections measuring approximately two mm in width; these were post-fixed in 1% phosphate-buffered according to established osmium tetroxide and processed for electron microscopy techniques.

* Propyl Thyrocil obtained from Frost

Laboratories.

Fig. 1. Carotid artery from a normal dog. The internal elastic lamina (IEL) appears homogeneous, although in most areas it shows a fibrillar structure (arrow heads). x 25,000. Fig. 2. Carotid artery of a normal dog. The elastic lamella (EL) consists of an amorphous matrix embedded in which are delicate fibrils (arrow heads). x 22,500. Fig. 3. Carotid artery from a dog given thiouracil for three months. The elastic lamella (EL) shows an accentuation of its fibrillar structure and appears split by collagen fibrils (COL) and fine fibritlar material (arrows). X 19,000.

Fig. 4. Femoral artery from a dog given thiouracil for three months. The internal elastic lamina (IEL) shows numerous fissures partially filled with fine fibrillar material (arrows). x 19,500. Fig. 5. Carotid artery from a dog given thiouracil for three months. The internal elastic lamina (IEL) shows wide fissures (arrows) now containing fibrillar material and collagen fibrils (COL). x 42,000. Fig. 6. Carotid artery from a dog given thiouracil for three months. A wide cleft in the medial elastic lamella (EL) contains collagen fibrils (COL) and electron-dense osmiophilic bodies (OB). x 41,300. Fig. 7. Carotid artery from a dog given thiouracil for six months. The internal elastic lamina (IEL) shows a “spongy” appearance, representing a process of rarefication. The numerous clefts contain fine fibrillar material (arrow heads). x 38,000.

EFFECT

OF THIOURACIL

Controls

UPON

CANINE

were the carotid

ARTERIAL

ELASTIC

and femoral

arteries

373

TISSUE

of four

untreated

dogs also

utilized in another studyss, and obtained and processed as above. One micron thick sections were cut with glass knives on either a Porter MT-l,

or Richert

microscopy. selected original

ultramicrotome

and stained

From each ring-shaped

for electron microscopy. large blocks, re-embedded

tissues were doubly

stained

a Philips EM-300 briefly elsewhere27.

electron

cross arterial

with alkaline

toluidine

Blum

blue for light

section, three to four segments

were

The selected segments were removed from the and thin-sectioned with a diamond knife. The

with uranyl microscope.

acetate

and lead citrate

Preliminary

and examined

observations

were

with

reported

RESULTS

The observations made concerned largely the changes of elastic tissue elements in the inner arterial wall and thus this report will be limited to these arterial elements. Moreover, since the observations in both the common carotid and femoral arteries were similar, they will be reported without reference to either the one or the other vessel. Arteries from untreated animals The internal elastic lamina appeared as a ribbon-like structure measuring 1500-2000 nm in width. The texture of the lamina varied considerably from area to area; it appeared homogeneous or almost amorphous; more often, however, it was traversed by electron-dense filaments largely oriented longitudinally (Figs. 1 and 2). The medial elastic lamellae, although having fenestrations, appeared usually as almost continuous

sheets of elastic substance.

was much the same as that of the internal lamellae was formed (Fig. 2).

by SOA thick

The appearance

elastic lamina.

filaments

embedded

of the elastic lamellae

The internal

structure

in an amorphous

of the matrix

Arteries from experimental animals In dogs treated with thiouracil for three months, the earliest changes observed in the elastic tissues consisted of marked accentuation of the filamentous components, and the formation of clefts or fissures; the latter appeared occupied either by a condensation of fibrillar material or by collagen fibrils (Figs. 3-5). More advanced changes were represented by widening of the clefts and by the presence within them of clumps of electron-dense material, collagen fibrils, and large clusters of osmiophilic electron-dense material (Fig. 6). In animals given thiouracil for six months, the changes in the elastic elements were more conspicuous. The elastic fibers acquired a spongy appearance owing to marked rarefication of the elastic matrix, and enlargement and coalescence of the clefts. The margins of the rarefied areas appeared more electron-dense than the remaining elastic matrix as a result of condensation of finely fibrillar material which

Fig. 8. Tissues as in Fig. 6. Higher magnification of a portion of the internal elastic lamina (IEL) showing a detail of the fibrillar material filling the clefts (arrow heads). The margins of the rarefied areas are more electron-dense than the remaining matrix. Collagen fibrils (COL) are present within the elastic substance. x 55,000. Fig. 9. Carotid artery from a dog given thiouracil for six months. Both the internal elastic lamina (IEL) and elastic lamella (EL) appear fragmented and have frayed edges. The fragments of elastic substance are surrounded by fine fibrils (arrow heads) and small electron-dense clusters (brackets). x 33,000.

EFFECT

OF THIOURACIL

UPON

CANINE

ARTERIAL

in some areas was seen in apparent

ELASTIC

continuity

(Figs. 7 and 8). In these areas, collagen

375

TISSUE

with the fibrils occupying

fibrils were often closely apposed

edges of the elastic fibers (Fig. 8). The fibrillar meshwork occupying to be composed of fibrils morphologically similar to the connective The individual

fibril units had an average

diameter

of 40 A, and in most instances of the elastic

this resulted

fragments

partially

surrounded

of irregularly-outlined

by a loose meshwork

to the frayed

the clefts appeared tissue microfibrils.

they possessed an electron-dense thicker end (Fig. 8). At a still further stage, complete fragmentation in the appearance

the clefts

fibers ensued;

of elastic

material

of small fibrils (Fig. 9).

DISCUSSION

The ultrastructure of normal elastic and muscular arteries lian species has been the subject of numerous studiessJsJO-24,st on the elastic structures

are similar

to those reported

of several mammaand observations

in the present

study.

Among the changes seen in experimental cholesterol atherosclerosis that have been attributed to the influence of normal or abnormal function of endocrine organs, those which interfere the most definitive.

with the thyroid

function

Experimentally-induced

are the best known

hypothyroidism

and apparently

tends to accelerate

the

development of experimental cholesterol atherosclerosislo. Production of cholesterol-induced atherosclerosis in the dog has been, with some exceptions, unsuccessfulspz4 unless an antithyroid drug such as thiouracil is usedsJ0,25. Surprising, however, is the scarcity of information regarding the possible effects of thiouracil administration upon the arterial structures and yet, the effect of this substance on arterial tissues, especially on elastic fibers, is of paramount importance in assessing The concept

the experimental that degeneration

lesions. of elastic

fibers may be one of the causative

factors in the development of atherosclerotic lesions has been proposed by several investigatorsl,s,7JlJ4. On the basis of electron-microscopical studies of early lesions in human aortae, Haust et ~1.13 and Haust14 suggested that there are at least two additional sources of lipid in the early lesions: one derived from altered elastic tissue of the intima, the other probably derived from the insudate from blood. Kramsch et al.” concluded that the prerequisite for lipid accumulation in the elastic fibers appeared to be an altered chemical composition of the elastin protein in the plaque. Whether or not the changes observed in the elastic tissue in the present report are due to a direct effect of thiouracil upon the elastic fibers or alternatively represent changes induced by thyroid suppression caused by this drug remains to be elucidated. It is generally accepted that the antithyroid action of thiouracil results from the inhibition of the formation of thyroid hormone, primarily at the step of the protein binding of iodine. However, it has been suggested that thiouracil may have extrathyroidal actions not yet determineds. The rarefication and ultimate dissolution of the elastic tissues in the arteries of dogs treated with thiouracil suggests that this drug may exert a damaging action

376

A. TRILL0

upon the arterial elastic tissue, thus either initiating or aggravating the arterial lesions. The fragmentation of the elastic elements, possibly associated with an altered amino acid composition, could favor the deposition of lipids administered exogenously as in the case of cholesterol feeding.

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EFFECT OF THIOURACIL

UPON CANINE ARTERIAL

ELASTIC TISSUE

377

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