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Spontaneous arteriosclerosis in repeatedly bred male and female rats
J O U R N A L OF ATHEROSCLEROSIS RESEARCH
57
S P O N T A N E O U S A R T E R I O S C L E R O S I S IN R E P E A T E D L Y B R E D M A L E AND F E M A L E RATS 13. C. W E X L E R
The NIav Institute for Medical Research, Jewish Hospital, and the Department of Pathology, University of Cincinnati College of Medicine, Cincinnati, Ohio (U.S.A.) ( R e c e i v e d J u n e 20th, 1963)
INTRODUCTION
Several investigators have described the resistance of the rat to the development of arteriosclerosis and the various manipulations required to induce arterial lesions in this species 1-3. These procedures unfortunately require rather stringent and timeconsuming measures. In an initial report we described the development of arteriosclerosis in the repeatedly bred female Sprague-Dawley rat 4. The arterial lesions in these female breeders were grossly visible and readily detectable. L a t e r it was found t h a t male breeder rats also developed spontaneous arteriosclerosis 5. In the case of the male breeders, however, the arterial lesions could not be seen b y gross inspection and were visible only when the arteries were examined microscopically. I t should be emphasized t h a t arterial lesions have been detected, microscopically, as early as 3 weeks after the first breeding in both males and females a. These early lesions consist of focal subintimal accumulations of m u c o p r o t e i n a c e o u s material. These same foci later become fibrosed. There is little or no lipid present in the lesions. Arteriosclerosis develops in these breeder rats on a regular rat diet which is low in fat. Serum cholesterol levels are normal. The pathogenesis of the arterial lesions can best be correlated with the n u m b e r and frequency of breedings 5. I t is felt t h a t the spontaneous development of the arterial lesions in these breeder rats and the relatively short space of time required for its production should be brought to the attention of those interested in the investigation of experimental arteriosclerosis. I n this p a p e r we shall compare the morphological nature and anatomical distribution of the arterial lesions in several strains of rats. MATERIALS AND METHODS
Male and female breeder rats of the following strains were studied: Sprague-Dawley, Holtzman, Mead-Johnson, Long-Evans, Wistar and Lewis. Our most extensive experience has been with the Sprague-Dawley rat of the Sprague-Dawley Farms, Madison, Wisc. This strain of r a t is highly inbred and was established forty years ago from litters delivered b y cesarean section. For the past 5 years we have received regular hi-weekly shipments of discarded male and female breeders from the SpragueJ. Atheroseler. Res., 4 (1964) 5 7 ~ 0
58
B.C.
WEXLER
Dawley Farms. No exact record has been kept b y these commercial breeders of the number of breedings or the size of the litter of each rat. Breeders are discarded if they cannibalize, refuse to suckle their young, or if they begin to show signs of outward deterioration. Deterioration becomes marked when animals have been bred repeatedly for an extended period of time. The hi-weekly shipments of discarded breeders have not been uniform in that the female breeders m a y have been pregnant once or several times. The majority of females have been bred 3-4 times. It is assumed that the male breeders have been used as studs on several occasions before they are discarded. In order to learn whether the phenomenon of spontaneous arteriosclerosis would occur in breeder rats other than the Sprague-Dawley strain, rats from other sources were examined. For each strain, approximately 100 male and 100 female breeders were autopsied. None of the commercial breeders could provide exact information regarding the breeding histories of their discarded breeders. In all of the strains examined breeder rats were used which had been bred approximately 3-5 times. In searching for the presence of spontaneous arteriosclerosis in breeder rats, all animals were autopsied as soon as possible after arrival. At autopsy, the aorta was exposed so that the heart, carotids, mesenteric branches, iliacs and proximal portions of the femoral arteries were visualized. In the case of the female breeder, the arterial lesions could often be seen on gross inspection. Animals with aortas having one or more sclerotic plaques were classified as "arteriosclerotic". Those with no grossly detectable plaques were called "clear" or "non-arteriosclerotic". For microscopic confirmation of the presence or absence of arteriosclerosis representative samples were taken routinely in prescribed segments of the systemic aorta. The arch of the aorta was taken so that it included the base of the innominate and common carotid arteries. The thoracic aorta was removed just caudal to where the azygos vein crosses the aorta to where the aorta leaves the thorax between the folia of the diaphragm. The abdominal artery was taken from the level of the diaphragm down to and including portions of the iliacs. The aortas of male breeder rats were examined and sampled in the same manner. The iliac arteries of males occasionally showed sclerotic plaques which could be detected b y gross examination. All segments of the aorta were fixed in 10% buffered neutral formalin (Lillie) for histological study. The brains, hearts, kidneys and carotid arteries of these animals were also taken so t h a t the contained cerebral, coronary, renal and the main carotid artery and its branches could be examined microscopically. The tissue was embedded in paraffin and sectioned at 3/~. Frozen sections were cut at 5-10 #. Adjacent sections were stained with hematoxylin-eosin for routine analysis; VERHOEFF, VAN GIESON and GOMORI'Saldehyde fuchsin stains for elastic tissue; alcian blue and toluidine blue for metachromasia; the HALE stain for mucopolysaccharides; the voN KOSSA method for calcium; and Sudans I I and Ill, Oil Red O, and Sudan Black B were used to demonstrate lipids on both frozen and paraffin sections. The word "arteriosclerosis" is used t h r o u g h o u t this paper to describe the variety of degenerative arterial changes. The word "atherosclerosis" was not used because these lesions do not contain significant deposits of lipid.
J. dtheroscler. Res., 4 (1964) 57-80
SPONTANEOUS
ARTERIOSCLEROSIS
IN REPEATEDLY
BRED
MALE AND FEMALE
RATS
59
RESULTS
Incidence
The results of these investigations were largely based on bi-weekly autopsies conducted during the years 1957-1959 in order to determine whether there was any seasonal variation in the incidence of spontaneous arteriosclerosis in Sprague-Dawley breeder rats. A total of about 3500 female and 500 male breeders were sacrificed during this period. The routine bi-weekly autopsies were abandoned in 1959 because it was found that there was no apparent seasonal variation in the incidence of arteriosclerosis (see Fig. 1). From 1959 until the present, autopsies of a similar nature were performed at r a n d o m intervals as a continuous check on the incidence of spontaneous arteriosclerosis in breeder rats of the Sprague-Dawley strain. During this 5-year period of observation we have found that the average incidence of grossly detectable arteriosclerosis in the female breeder of this strain is approximately 40 %. 5O
~40
°so t.
to
1(2
January to March
April to June
July to October to September December
Fig. 1. Incidence of gross arteriosclerosis in female breeder rats of the Sprague-Dawley strain. Results of bi-weekly autopsies performed throughout the year of 1958.
There have been occasions when this average has run as high as 60-70 %. However the conditions to which these particular breeders were exposed indicated to us that stressful conditions can alter the pathogenesis of the arteriosclerotic process in breeder rats (to be published). At no time have gross lesions been found in the systemic aorta of male breeder Sprague-Dawley rats. However, it should be emphasized that although breeder rats m a y show no gross evidence of arterial lesions, virtually 80-90 % of the female aortas contain microscopic lesions, and 70-80 % of the male aortas which all appear to be "clear" on gross inspection have arteriosclerosis when examined microscopically. If time and facilities had permitted routine serial sections of the aorta, it is felt that the incidence of microscopic lesions in male breeders would be higher than the observed 70~80 %. Arterial lesions in the male breeder aortas are small and focal and can be easily missed in random sections. The female breeder rats of the Holtzman strain which were autopsied were about one year old and h a d been bred 5-6 times. These animals were a few months older and had been bred more than the average Sprague-Dawley breeder. The female Holtzman breeder rats showed advanced arteriosclerosis which was grossly visible f . Atheroscler. Res., 4 (1964) 57~0
60
B . C . WEXLER
in 45 of the 100 animals examined. None of the 50 male breeders of this strain showed gross evidence of arterial damage. The gross anatomical distribution of the lesions was the same in this strain as in the Sprague-Dawley strain. The microscopic morphology of the lesions was also identical to those seen in the Sprague-Dawley strain. The incidence of microscopic lesions also increased to a value of 80 %. Female breeders of the Mead-Johnson strain* were autopsied. None of these animals showed gross evidence of arteriosclerosis. On microscopic examination, however, 60 % of the animals showed histological alterations of the aortic wall which were identical to the early lesions seen in the other strains of breeder rats (see below). It should be noted t h a t the Mead-Johnson breeding practices differ from those used b y the Sprague-Dawley F a r m s in the following respects: (J) these animals are not bred until they are fully mature; (2) they are allowed to rest between litters; and (3) they are not bred more t h a n 3-4 times. In a single sample (100 rats) of female breeder rats of the Long-Evans strain only 10% were found to have grossly evident arteriosclerosis. These animals had been bred 5-6 times. On microscopic examination 7 8 % of these animals were found to have arterial lesions identical morphologically to t h a t observed in previous strains of breeder rats. In this case, however, 4 0 % of the animals h a d microscopic lesions which were quite advanced and considered to be of a severe degree. TABLE I GROSS
AND
MICROSCOPIC
INCIDENCE
OF ARTERIOSCLEROSIS
IN
MALE
AND
FEMALE
BREEDER
RATS
oF SEVERALS~RAINS(%) Strain
Sprague-Dawley Holtzman Mead- J o h n s o n Long-Evans Wistar Lewis
Number of rats
3500 500 100 50 1O0 100 100 6 3
Sex
Female Male Female Male Female Female Female Female Male
Gross lesions
Microscopic lesions
(%)
(%)
40 0 45 0 0 10 0 67 0
80-90 70 80 80 80 60 78 65 67 100
Discarded breeder female rats of the Wistar strain, a p p r o x i m a t e l y 12-13 m o n t h s of age and bred 5 times, were autopsied. None of these animals showed arteriosclerosis grossly but 65 % of these displayed arterial lesions microscopically. Another strain of breeder females known as the Lewis strain** was found to have * P r o v i d e d t h r o u g h the c o u r t e s y of DRS. TRUE AND McCoRMACK, Mead J o h n s o n Co., Evansville, Ind. ** This special s t r a i n of h i g h l y i n b r e d r a t s was p r o v i d e d t h r o u g h t h e c o u r t e s y of DRS. "W. K. SILVERS AND R. E. BILLINGHAM, W i s t a r I n s t i t u t e , P h i l a d e l p h i a , Pa.
J. Atheroscler. Res., 4 (1964) 5 7 - 8 0
SPONTANEOUS ARTERIOSCLEROSIS IN REPEATEDLY BRED MALE AND FEMALE RATS 61
grossly detectable arteriosclerosis. This was a small sample of 6 breeder female rats. Four of the 6 female rats had moderately advanced arteriosclerosis grossly and histologically. These females had had 5 litters. All of the three male breeders which were available to us had tile typical microscopic lesions as seen in other strains. Table I is a s u m m a r y of the incidence of gross and microscopic arteriosclerosis in several strains of breeder rats. At no time has arteriosclerosis been observed, grossly or microscopically, in virgin rats of comparable age to the breeder rats. In summation, breeder rats of several strains of diverse breeding history have been examined for the presence of spontaneously developing arteriosclerosis either grossly or histologically. Grossly detectable arteriosclerosis was not evident in all strains. However, breeder rats of all of the strains examined manifested arterial lesions histologically. Similarly, male breeder rats which do not show gross arteriosclerosis of the systemic aorta were found to have microscopic arterial lesions. Virgin rats of the same age do not develop arterial lesions.
Fig. 2. Dissection of a r c h a n d t h o r a c i c a o r t a of a female breeder ( S p r a g u e - D a w l e y ) gross arteriosclerosis. T h e a r r o w s p o i n t to t w o raised plaques in t h e a s c e n d i n g aortic arch. T h e a r c h s h o w s b e g i n n i n g ectasia a n d t e n d e n c y t o w a r d s o u t p o u c h i n g f o r m a t i o n . A: a close-up of t h e t w o circumscribed p e a r l y w h i t e p l a q u e s
with moderate p o r t i o n of t h e and aneurysm shown.
J. Atheroscler. Res., 4 (1964) 57-80
62
B . C . WEXLER
Anatomical distribution There is a characteristic anatomical p a t t e r n in the appearance of the arteriosclerotic lesions. The first grossly visible lesions a p p e a r as thin pearl-white annular streaks in the lower portion of the abdominal aorta between the renal artery and the bifurcation of the iliacs. I t should be kept in mind first, t h a t the gross arteriosclerosis described applies to the female breeder only, and secondly, t h a t when the arteriosclerosis can be detected grossly the lesions have become considerably advanced histologically. Small p u n c t a t e foci, which are also white and raised appear in a r a n d o m pattern along the abdominal aorta extending from the bifurcation of the iliacs to the origin of the renal and superior and inferior mesenteric arteries. At this time, irregularly shaped plaques begin to appear in the ascending portion of the arch of the aorta (Fig. 2). These plaques broaden and coalesce until all of the arch of the aorta is involved and seldom do they extend beyond the crossing of the azygos vein over the ventral surface of the aorta. In the more advanced cases of arteriosclerosis, thin white annular streaks begin to appear in the thoracic aorta at rather wide intervals. As the process progresses the abdominal aorta shows coalescence of the annular plaques and progression of the punctate lesions u p into the celiac artery and out into the ramifications of the intestinal arteries. At the same time, the entire systemic aorta shows increasing ectasia, occasionally saccular aneurysms appear in the arch of the aorta, and punctate white plaques a p p e a r on all surfaces of the thoracic aorta.
Fig. 3. Severe gross arteriosclerosis in repeatedly bred female r a t (Sprague-Dawley) showing m a r k e d ectasia a n d multiple calcific loci t h r o u g h o u t t h e arch a n d thoracic portions of t h e aorta a n d e x t e n d i n g up into the i n n o m i n a t e a n d c o m m o n carotid arteries.
J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOSCLEROSIS IN R E P E A T E D L Y BRED MALE AND FEMALE RATS 6 3
Of special import, is the extension of the arteriosclerosis up into and along the entire length of the carotid arteries (Fig. 3), along the subclavian and brachial arteries, and distally, at r a n d o m intervals into the iliacs, hypogastric, femoral, popliteal, and tibial arteries. The male breeder does not display lesions grossly in the systemic aorta. However, the aorta does show progressive ectasia. In some cases, white annular streaks similar to those found in the female breeder will appear in the iliac arteries of male breeders. In summary, the grossly detectable arteriosclerosis which develops in the breeder female of all of the strains examined follows a characteristic anatomical pattern of distribution. The earliest gross lesions appear in the abdominal aorta followed b y the arch of the a o r t a and later the thoracic aorta. In the m o s t severe cases of arteriosclerosis the arterial plaques m a y be found in the carotid and peripheral arteries. Male breeders do not show gross lesions in the systemic aorta.
Histological development The earliest microscopic change which can be detected in the a o r t a of breeder rats occurs in the lower portions of the abdominal aorta m i d w a y between the renal artery and the bifurcation of the iliacs. This first change is usually confined to a single segment of the medial tissue of the aorta. Within this focus there is a generalized distention of the tunica m e d i a accompanied b y swelling and stretching of elastic fibers (Fig. 4). The nuclei of the smooth muscle cells become intensely basophilic in this area accompanied b y a h e a v y infiltration of deeply basophilic mesenchymal cells. This basophilia is evanescent and subsides. As the medial swelling and basophilia diminish there is a focal thickening of the tunica intima just central to the medial segment mentioned above. A clear mucoproteinaceous material accumulates in these
Fig. 4.
Fig. S.
Fig. 4. F e m a l e b r e e d e r ( S p r a g u e - D a w l e y ) s h o w i n g t h e earliest c h a n g e s in t h e d e v e l o p m e n t of t h e a r t e r i a l lesions in t h e a b d o m i n a l a o r t a . T h e i n t i m a s h o w s focal s w e l l i n g d u e to a m u c o p r o t e i n a c e o u s m a t e r i a l . T h e r e is t h i c k e n i n g of elastic fibers a n d g r o u n d s u b s t a n c e swelling, w h i c h is a c c o m p a n i e d b y t h e a p p e a r a n c e of i n t e r l u m i n a r elastic fibrils. VERHOEFF-VAN GIESON elastic t i s s u e stain; × 150. Fig. 5. F e m a l e b r e e d e r ( M e a d - J o h n s o n ) s h o w i n g s i m i l a r m e d i a l elastic t i s s u e c h a n g e s d e s c r i b e d in Fig. 4 a n d focal s u b i n t i m a l s w e l l i n g a n d a c c u m u l a t i o n of a m u c o p r o t e i n a c e o u s m a t e r i a l , H e m a t o x y l i n - e o s i n ; × 150.
J. Atheroscler. Res,, 4 (1964) 5 7 - 8 0
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B.C. WEXLER
subintimal loci (Fig. 5). This substance becomes increasingly metachromatic and HALE positive. Within this pool of mucopolysaccharide, strands of collagen are laid down further contributing to the subintimal involvement (Fig. 6). In the more extensive intimal lesions the edges of the degenerative sites are usually demarcated b y clear, vacuolated cells which resemble foam cells. Occasionally, these early lesions contain scattered droplets of lipid. .......ii!!ii!i!i!iiii:~i]
Fig. 6.
Fig. 7.
Fig. 6. Female breeder (Lewis). Intimal swelling characteristic of early arterial degeneration beginning in the abdominal aorta of breeder rats. The mucoproteinaceous material described in Fig. 5 becomes increasingly metachromatic and positive to the HALE stain (deep black material in photo) especially at the base of the atheroma. Collagen (light grey material in photo) fills in beneath the HALE positive material. HALE stain; × 150. Fig. 7. Male breeder (Holtzman). Raised cartilaginous plaque in the arch of the aorta. This is the typical lesion found in male breeders and consists of deeply basophilic large rounded mesenchymal cells bathed b y mucoproteinaceous material (light grey). The deeply basophilic material (black in photo) is metachromatic and is probably mucopolysaccharide. Hematoxylin-eosin; × 75.
Fig. 8.
Fig. 9.
Fig. 8. Male breeder (Sprague-Dawley). Multiplication of the basophilic mesenchymal elements giving rise to a raised intimal plaque (arch of aorta). These cells are cartilaginous in appearance. Hematoxylin-eosin; X 150. Fig. 9. Male breeder (Long-Evans). Advanced d e v e l o p m e n t of lesion shown in Fig. 8. Cartilaginous cells are intensely metachromatic and positive to the HALE stain (black material in photo). Material, giving a positive reaction with the HALE stain, begins to pool about elastic fibers in media. HALE stain; × 150. J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUSARTERIOSCLEROSISIN REPEATEDLYBRED MALEAND FEMALE RATS 65 At this point there is an interesting dichotomy in the manner in which the lesions continue to develop in the two sexes. In the female breeder, the early lesions are characterized b y endothelial proliferation accompanied b y increased mucopolysaccharide and scarring. The scarring is most prominent at the base of the d a m a g e d site. In the male breeder, however, the intimal swellings remain rather self-contained and are invaded b y heavily basophilic mesenchymal cells which become rounded and quite large. These giant round cells have a vacuolated cytoplasm and are surrounded b y a deeply basophilic halo (Fig. 7). These cells often coalesce and take on the appearance of cartilage ceils (Fig. 8). These clumps of cells begin to exude an intensely basophilic and metachromatic material which is believed to be mucopolysaccharide. At this time the focal subintimal swelling begins to increase in scope (Fig. 9). These lesions cannot be detected macroscopically. It should be emphasized that although the early lesions described above are the type most commonly found, they are not the only variety encountered. In some cases, lesions are found in the main branches of the abdominal aorta which are specifically characterized b y h e a v y scarring leading to occlusion of the lumen (Fig. 10). These sites of intense fibrosis do not contain lipid. This type o f lesion also occurs in the muscular arteries of the intestinal arcades (Fig. 11). Many of the main branches of the abdominal aorta display thromboses and rechannelization of the lumen (Fig. 12).
Fig. 10.
Fig. 11.
Fig. 10. Female breeder (Wistar). Marked fibrosis leading to occlusion of lumen of abdominal artery. Note broken and contorted elastica. This lesion contains little or no lipid. These changes are found in breeder rats with and without high blood pressure. Hematoxylin-eosin; × 150. Fig. 11. Female breeder (Holtzman). Large branch of abdominal aorta with thrombus in lumen, intensive scarring, medial swelling and elastosis. Hematoxylin-eosin; × 100. J. Atheroscler. Res., 4 (1964) 57-80
66
B. C, W E X L E R
A n o t h e r lesion found in t h e a b d o m i n a l a o r t a a n d t h e m e s e n t e r i c b r a n c h e s shows s u b i n t i m a l a c c u m u l a t i o n of a d e e p l y b a s o p h i l i c m a t e r i a l w h i c h encircles t h e entire c i r c u m f e r e n c e of the a r t e r y . This is u s u a l l y a c c o m p a n i e d b y f r a g m e n t a t i o n of t h e i n n e r elastic m e m b r a n e . W h e n t h e a b d o m i n a l i n t i m a l lesions are f o u n d e n c r o a c h i n g on t h e m e d i a , t h e a r c h a n d o t h e r p o r t i o n s of t h e a o r t a also b e g i n to s h o w a r t e r i o s c l e r o s i s (Fig. 13). T h e m e d i a in t h e s e sites shows an i n c r e a s e d p o p u l a t i o n of m e s e n c h y m a l cells a n d g r e a t e r a c c u m u l a t i o n of m u c o p o l y s a c c h a r i d e . T h e i n d i v i d u a l elastic fibers of t h e m e d i a begin to swell. T h e g r o u n d s u b s t a n c e b e t w e e n elastic l a m e l l a e also swells so t h a t i n t e r l a m e l l a r fibrils s t a n d o u t in b o l d relief (Figs. 4, 13-15). A t first, t h e g r o u n d s u b s t a n c e swelling h a s an a m o r p h o u s a p p e a r a n c e a n d r e s e m b l e s t h e m u c o p r o t e i n a c e o u s m a t e r i a l w h i c h a c c u m u l a t e s in t h e s u b i n t i m a l space. L i k e t h e i n t i m a l lesion t h e m e d i a l a l t e r a t i o n in g r o u n d s u b s t a n c e b e c o m e s p r o g r e s s i v e l y m e t a c h r o m a t i c a n d i n c r e a s i n g l y p o s i t i v e to t h e alcian blue, t o l u i d i n e blue, a n d HALE stains. T h i s m u c o p o l y s a c c h a r i d e m a t e r i a l s o m e t i m e s occurs in g r a n u l a r or b e a d e d a r r a n g e m e n t a r o u n d i n d i v i d u a l e l a s t i c fibers or occurs as an e x u d a t e or pool. I n all cases, t h e elastic fibers which a r e a s s o c i a t e d w i t h these m u c o p o l y s a c c h a r i d e pools b e c o m e c o n v o l u t e d a n d d i s t o r t e d , s h o w r e d u p l i c a t i o n a n d s p l i t t i n g , a n d finally r u p t u r e (Fig. 15). This p a r t i c u l a r t y p e of lesion occurs in all of t h e m a j o r elastic a r t e r i e s i n c l u d i n g t h e c a r o t i d a n d m e s e n t e r i c
Fig. 12.
Fig. 13.
Fig. 12. Female breeder (Sprague-Dawley). Large branch of abdominal aorta with thrombosis and vacuolization, infimal and medial growth and elastosis. Hematoxylin-eosin; × 75. Fig. 13. Male breeder (Sprague-Dawley). Aortic arch showing advanced intimal outgrowth encroaching upon lumen. This intimal lesion is deeply positive to the HAL~ stain (black material in photo) and at the base of the intimal cushion there is heavy collagen deposition (crescent-shaped area at center of lesion). HALE stain; × 35. J. Atheroscler. Res., 4 (1964) fi7-80
SPONTANEOUS ARTERIOSCLEROSIS IN REPEATEDLY BRED MALE AND FEMALE RATS 6"]
arteries. I t is this t y p e of lesion which is prone to the d e v e l o p m e n t of the saccular aneurysms which occur in the arch and abdominal portions of the aorta (Fig. 14). If the arteriosclerotic process progresses even further, these sites become engulfed with intensely m e t a c h r o m a t i c pools of the mucopolysaccharide material which inundates
Fig. 14. F e m a l e b r e e d e r ( S p r a g u e - D a w l e y ) . R u p t u r e of elastic fibers leading to a n e u r y s m of t h e arch of the aorta. T h e sites of h e r n i a t i o n are d e m a r c a t e d in t h e p h o t o b y arrows. I n t h e wall of the a o r t a (right side) one c a n see t h e b r o k e n elastic fibers w i t h i n pools of acid m u c o p o l y s a c c h a r i d e (black-colored material) w h e r e t h e arterial wall b e c a m e weakened. Clotted blood fills the a n e u r y s m . HALE stain; × 35.
the entire media from intima to adventitia (Fig. 16). Elastic elements are destroyed or lost within these sites (Fig. 17). These sites often show dystrophic calcification. This process of elastic tissue degeneration and mucopolysaccharide accumulation is much more prevalent in the female breeder. The ground substance lesions in the male breeders seldom reach this stage of complexity. This m a y be a consequence of the shorter life span of male breeder rats. However, the male breeder does show swelling and m a r k e d mucopolysaccharide alterations around the ostia of the intercostal arteries. This occurs in r e m a r k a b l e regularity throughout the length of the thoracic aorta at each intercostal level. Another v a r i e t y of lesion which has a dual association with the arch of the a o r t a and its main arterial branches appears in both male and female. This lesion, as described earlier, begins in the intima with infiltration of heavily basophilic mesenJ. Atheroscler. Res., 4 (1964) 57-80
68
B . C . WEXLER
chymal cells. Within such lesions the mesenchymal elements round-up and take on the appearance of cartilaginous cells. In some cases these cells accumulate within the intima and extend out toward the lumen or else their proliferation is short-lived and the endothelial cells immediately associated with these cells undergo hyperplasia. The growth of these cartilaginous appearing cells can best be described, perhaps, as cartilaginous metaplasia (Figs. 7-9, 18). The above changes are most common in the
Fig. 15. Male breeder (Sprague-Dawley). Pooling of HALE positive material in media of carotid artery. Note the contortion and disappearance of elastic elements within the mucopolysaccharide pools (black-colored areas). HALE stain; × 75.
male breeder a n d seldom reach the proportions of its p r o t o t y p e in the female breeder. In the female breeder, this same process occurs in the intima and the media as well (Figs. 18, 19). This process encompasses b o t h intima and media and encroaches upon the adventitia so t h a t it can be detected grossly in the female breeder. In advanced cases of arteriosclerosis, cartilaginous metaplasia in the female breeder is frequent. There is a v e r y definite anatomical area in which change occurs. I t appears in the arch of the aorta, innominate and carotid arteries. The sites of cartilaginous m e t a plasia often become transformed into bone (Fig. 20). Dystrophic calcification of the media is another form of arterial degeneration which occurs only in the female breeder. Unlike M6NCKEBERG'S sclerosis in the human, this dystrophic calcification is found in the m e d i a of elastic arteries only, e.g., carotid, thoracic, abdominal and mesenteric branches (Fig. 21). J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOSCLEROSIS IN REPEATEDLY BRED MALE AND FEMALE RATS 69
After the arteriosclerosis has become well established in the systemic aorta the coronary, renal and cerebral arteries begin to show some evidence of degenerative change. Again, the morphology of the renal, coronary and cerebral lesions is also varied. For example, some of the coronary arteries show intimal growth (Fig. 22) which m a y eventually occlude the lumen of the artery. These intimal lesions m a y or m a y not contain foam cells. Some coronary arteries show endothelial proliferation, elastic membrane rupture, and medial swelling. Other coronary arteries show distention and the deposition of an intensely basophilic substance within the intima. In
Fig. 16. F e m a l e b r e e d e r ( L o n g - E v a n s ) . I n t e n s e HALE positive m a t e r i a l d i s t e n d i n g entire m e d i a in one focus. T h e light g r e y m a t e r i a l in the i n t i m a i m m e d i a t e l y a b o v e t h e medial lesion is collagen. HALE stain; × 75.
the kidney, the arcuate arteries develop intimal cushions of endothelial cells, the larger hilar arteries show accumulation of a basophilic material in the intima accompanied b y elastosis, swelling and palisade arrangement of the smooth muscle elements in the media (Fig. 23). The cerebral arteries never show changes except in animals with the most severe arteriosclerosis. The most frequent cerebral arterial lesion consists of multiple invaginations of the internal elastic membrane into the lumen (Fig. 24). The visceral (Fig. 25), intestinal (Fig. 26) and more peripheral arteries of the extremities (Fig. 27) characteristically show intimal and medial thickening, endothelial proliferation, scarring, and disruption of the normal contour of the elastica. The arterial lesions found in the carotid and cerebral arteries have been described in detail 8. The coronary, renal, intestinal and peripheral arterial changes will be described separately in greater detail in subsequent publications. J. Atheroscler. Res., 4 (1964) 5 7 - 8 0
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In summation, the abdominal aorta is the first site for the appearance of histological as well as grossly detectable lesions. The earliest event that can be detected is swelling of the media and infiltration of heavily basophilic mesenchymal elements. In such areas there is focal subintimal accumulation of an amorphous mucoproteinaceous
Fig. 17. Female breeder (Sprague-Dawley). Advanced arterial lesion of long standing in abdominal aorta. Note eccentric heavy endothelial proliferation of intima with marked elastosis and dystrophic calcification of the media. This type of lesion is easily detected on gross examination. Hematoxylin-eosin; × 10O. material which later becomes metachromatic and is believed to be made up of mucopolysaccharides. These sites contain a few foam cells and soon develop fibrosis. In the male breeder these lesions seldom become more developed or complex. In addition, in the male breeder this type of lesion develops a distinctive type of morphology characterized by intimal invasion with deeply basophilic mesenchymal cells resembling cartilage cells. These sites either show cartilaginous metaplasia or endothelial hyperplasia immediately adjacent to the cartilaginous cells. I n the female breeder there is a spectrum of lesions which appear to develop in specific anatomical locations. Endothelial proliferation, thromboses, scarring, complex degeneration of mucopolysaccharides within the medial ground substance and elastic tissue destruction all occur within the abdominal aorta. Thromboses and endothelial proliferation are rare in the arch and thoracic portions of the aorta. Instead, these J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOSCLEROSIS IN REPEATEDLY BRED MALE AND FEMALE RATS
71
sites show intense mucopolysaccharide changes, elastosis, medial dystrophic calcification and cartilaginous metaplasia of a much more foliating t y p e than the male breeder. In advanced cases of arteriosclerosis, there is an unusual predilection for calcification and bone formation in the arch, innominate and carotid arteries. The peripheral arteries show early changes characterized b y intimal hyperplasia,
Fig. 18. F e m a l e b r e e d e r ( S p r a g u e - D a w l e y ) . I n t e n s e cartilaginous m e t a p l a s i a in o p p o s i t e sides of t h e aorta. B o t h i n t i m a a n d m e d i a are involved. These lesions are i n t e n s e l y m e t a c h r o m a t i c a n d p o s i t i v e to t h e HALE stain. H e m a t o x y l i n - e o s i n ; × 60.
fibrosis and elastosis. The coronary, renal and cerebral arteries do not show signs of any arterial d a m a g e until arteriosclerosis is well established in the systemic aorta. The microscopic m o r p h o l o g y of the arteriosclerotic lesions is essentially identical in breeder rats of all of the strains examined. DISCUSSION
The spontaneous d e v e l o p m e n t of arteriosclerosis in male a n d female breeder rats is a strong argument for using this type of animal in the s t u d y of experimental arteriosclerosis. The finding of vascular disease in all six strains of breeder rats e x a m i n e d demonstrates t h a t this is not an isolated phenomenon confined to a special strain of rat and t h a t it is p r o b a b l y characteristic of all strains of breeder rats. I t is likely t h a t J. Atheroscler. Res., 4 (1964) 5 7 - 8 0
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the hormonal alterations which take place with the reproductive effort are contributory, in some way, toward the development of this disease in rats. Virgin rats of comparable age to breeders do not develop arteriosclerosis spontaneously. Senile, virgin rats (2 3 years), however, do develop arterial lesionsL We have found arteriosclerosis in repeatedly bred Sprague-Dawley rats which were purchased as weanlings
Fig. 19. F e m a l e breeder (HoltzmanL Cartilaginous m e t a p l a s i a i n v o l v i n g the entire p e r i m e t e r of t h e carotid artery. T h e deep black material is positive to t h e HALE stain. HALE stain; × 50.
and raised and bred, under controlled conditions, in our own laboratory (vide infra) 5. INGLE AND BAKER8 also found t h a t discarded breeder female rats of the SpragueDawley strain developed arteriosclerosis. Further, GILLMAN AND HATHORN9 found spontaneous arteriosclerosis in discarded breeder females (3-7 litters) of the Glaxo strain. All of these findings would underscore the role of breeding in the development of the arteriosclerosis. J u s t how breeding constributes to the d e v e l o p m e n t of this arterial disease in rats is an intriguing question. For example, we have found t h a t the n u m b e r and frequency of breedings can be directly correlated with severity of arteriosclerosis which develops in the Sprague-Dawley rat. During the past 5 years, we have conducted several controlled breeding studies in our laboratory in which we sacrificed animals after 1, 2, 3, 4, 5 and 6 breedings. The arterial lesions begin to a p p e a r grossly in the female J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOSCLEROSIS IN R E P E A T E D L Y BRED MALE AND F E M A L E RATS 7 3
by the third or fourth breeding and become increasingly more pronounced after each successive litter. I t was found that if aninmls were allowed to rest between litters the severity of the arteriosclerosis was lessened. The findings could explain w h y certain strains of rat show more or less intense arteriosclerosis, i.e., an actively bred rat would show more arteriosclerosis than a less actively bred rat. Possibly superimposed upon this are factors such as genetic differences between strains in susceptibility toward arteriosclerosis. Also to be considered is the fact t h a t m a n y of these
Fig. 20. F e m a l e b r e e d e r ( S p r a g u e - D a w l e y ) . C a r t i l a g i n o u s m e t a p l a s i a a n d b o n e f o r m a t i o n ( u p p e r right) in c a r o t i d a r t e r y . H e m a t o x y l i n - e o s i n ; > 75.
animals develop hypertension and renal damage, such as glomerulosclerosis, t u b u l a r dilatation and cast formation. However, these changes in blood pressure and in the kidney do not become manifest until well after m a n y of the arterial lesions described have become well-established. Therefore, the author feels that the rapidity and frequency with which the animals are bred seems to him to be the most essential, although not the only factor in the development of arteriosclerosis*. I t is interesting that diet appears to play a secondary role in the development of * I n p r e v i o u s y e a r s , t h e d i s c a r d e d b r e e d e r r a t s f r o m c o m m e r c i a l s o u r c e s w e r e b r e d as m a n y a s 10 12 t i m e s . T h e s e a n i m a l s d e v e l o p e d , in a d d i t i o n to v e r y s e v e r e arteriosclerosis, h y p e r t e n s i o n , ulcers, p o l y a r t e r i t i c lesions, r e n a l d a m a g e a n d g e n e r a l i z e d a c c e l e r a t e d d e b i l i t a t i o n . D u r i n g r e c e n t y e a r s t h e i n t e n s i t y of t h e b r e e d i n g h a s b e e n r e d u c e d a n d c o n c o m i t a n t l y t h e a r t e r i o s c l e r o s i s a n d t h e a f o r e m e n t i o n e d d e g e n e r a t i v e c h a n g e s h a v e b e c o m e less severe. C o n s e q u e n t l y , t h e u l c e r s a n d t h e p o l y a r t e r i t i s so c o m m o n l y s e e n in b r e e d e r r a t s (bred 6 - 1 2 t i m e s ) h a v e b e c o m e less f r e q u e n t .
J. Atheroscler. Res., 4 (1964) 5 7 - 8 0
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arteriosclerosis in these breeder rats. Although m a n y commercial breeders are unwilling to divulge the exact content of their diets, it is least likely t h a t the differences in development of arteriosclerosis can be ascribed to diet. The breeder rats which developed arteriosclerosis in our laboratory were fed a commercial rat diet (Rockland) and tho microscopic detail of their lesions was in no w a y dissimilar to the lesions observed in other strains of rats. I t should also be mentioned t h a t the serum chol-
Fig. 21. Female breeder (Long-Evans). Abdominal aorta showing medial dystrophic calcification, elastosis, and intimal proliferation. Despite the medial calcification this is not equivalent to M6NCKEBERG'S sclerosis. Hematoxylin-eosin; × 50.
esterol levels are normal in all of the strains of rats examined. In addition, the p a u c i t y of histologically demonstrable lipid in the lesions of different strains of breeders militates against the participation of diet in the production of the disease (to be published). Particularly striking is the m a r k e d h y p e r t r o p h y of the adrenal glands in these breeders. The adrenal h y p e r t r o p h y is invariably accompanied b y involution of the t h y m u s gland. Biochemical and histological studies indicate t h a t the adrenal glands become more active with each breeding and t h a t the profound alterations in arterial ground substance m a y be related to changes in adrenal steroid production 10. The adrenal glands of the female breeder are consistently larger t h a n those of the male breeder. Adrenal h y p e r t r o p h y in connection with the reproductive effort is not an isolated phenomenon peculiar to rats. During the period of gestation in the h u m a n J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOCSLEROSIS IN REPEATEDLY BRED MALE AND FEMALE RATS 75
Fig. 22.
Fig. 23.
Fig. 22. Female breeder (Sprague-Dawley). Advanced coronary lesion displaying marked reduction of lumen by intimal ingrowth. Note large, vacuolated foam cells. Hematoxylin-eosin; x 250. Fig. 23. Female breeder (Sprague-Dawley). Hilar renal artery showing endothelial proliferation, fragmentation and reduplication of intimal elastic membrane, swelling and pallisade arrangement of the smooth muscle cells of the media. Hematoxylin-eosin; x 75.
Fig. 24. Female breeder (Sprague-Dawley). Cerebral artery with multiple aneurysms of the internal elastica. Hematoxylin-eosin; x 150.
j . Atheroscler. Res., 4 (1964) 57-80
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it is well k n o w n t h a t the a d r e n a l g l a n d u n d e r g o e s h y p e r t r o p h y a n d t h a t it s u b s e q u e n t l y b e c o m e s r e d u c e d in size following p a r t u r i t i o n 11. F u r t h e r , in fish we h a v e f o u n d h y p e r a d r e n o c o r t i c i s m a n d arteriosclerosis a s s o c i a t e d w i t h spawningl2, is. T h e r e is a
Fig. 25. Male breeder (Wistar). Splenic artery showing basophilia and disruption of the regular convolutions of the internal elastic membrane. This type of lesion is found frequently in visceral arteries. Hematoxylin-eosin; × 150.
Fig. 26. Male breeder (Sprague-Dawley). Mesenteric artery with intimal hyperplasia, basophilia and disruption of elastica which is also frequentlyfoundinmesentericarteries. Hematoxylin-eosin; x 200. J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOSCLEROSIS IN R E P E A T E D L Y BRED MALE AND FEMALE RATS 77
remarkable similarity between the degenerative changes in spawning fish, breeder rats, and those found in CUSHING'S syndrome in man 14. The sex difference in the morphology of arteriosclerosis in male and female breeders
Fig. 27. F e m a l e b r e e d e r ( S p r a g u e - D a w l e y ) . P o p l i t e a l a r t e r y s h o w i n g i n t i m a l p r o l i f e r a t i o n , elastosis, m e d i a l swelling a n d h y p e r t r o p h y . H e m a t o x y l i n - e o s i n ; × 150.
raises the question of the participation of sex hormones in the pathogenesis of arterial degeneration. I n all strains examined b y us to date, the male breeders do not show gross arteriosclerosis of the systemic aorta except for occasional grossly visible plaques in the iliac arteries. In addition to our own experience, neither INGLE AND BAKER s nor GILLMAN AND HATHORN9 found grossly visible arterial lesions in male breeders. In the author's opinion the male breeder seldom exhibits gross arteriosclerosis for two major reasons: (1) the male breeder, despite having a less florid arteriosclerosis than the female, has an exceptionally high mortality rate and dies at an earlier age than the female breeder; (2) the arteriosclerosis in the male develops in a centripetal direction, i.e., is confined to the intimal layer almost exclusively, while in the female the lesions are centrifugal and more fulminating. In the latter case, where the arteriosclerotic process involves media and also impinges on the adventitia, conditions favor gross recognition of lesions. ACKNOWLEDGEMENTS
This work was supported by grants from the JOHN A. HARTFORD Foundation, the GUSTAVUS AND LOUISE PFEIFFER Foundation, American Heart Association, the Cincinnati Heart Association and the U.S. Public Health Service (Grant H-3874). The investigation was supported (in part) b y a Public Health Service research career program award (HE-K3-1734) from the National Heart Institute. J. Atheroscler. Res., 4 (1964) 5 7 - 8 0
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The author is indebted to the following technical personnel for their participation in this project: N. BLAMER, M. BICKERS, D. CONATSER,J. DORON, K. KUCHENBUCH, R. LEBLOND, D. MANTHY, C. NEIMANN, M. SPRINGS, L. STAPLES, L. THOMAS, M. TORBECK, J. TRACY, R. VAUGHN AND A. WHITE. The photomicrographs were taken by J. HUTZELMAN, Medical Photography, Jewish Hospital, Cincinnati, Ohio. SUMMARY
Repeatedly bred male and female rats of several strains develop arteriosclerosis spontaneously. The arterial lesions in the male breeder rats remain microscopic while the arteriosclerosis becomes grossly recognizable in the female breeder. The incidence and severity of the arterial damage is enhanced with each breeding. The lesions appear first in the abdominal aorta and spread to the arch and thoracic portions. Later, as arteriosclerosis becomes well established the coronary, renal and cerebral arteries also develop arterial lesions. A large variety of degenerative arterial changes have been found which are believed to represent early and advanced lesions as well as various stages of repair and degeneration. It has also been found that there are specific morphological reactions peculiar to certain segments of the aorta. The most frequent lesion encountered, in both sexes, is a focal subintimal accumulation of mucopolysaccharide followed b y scarring. In these same sites the media shows pooling of mucopolysaccharide and swelling and eventual fracture of elastic fibers. This t y p e of lesion is also found in male breeders. However, the male breeder frequently develops an additional lesion consisting of intense focal mesenchymal cell infiltration of the intima. These ceils become enlarged and intensely basophilic, develop a resemblance to cartilage cells, and ultimately m a y undergo cartilaginous metaplasia. The gross and histological aspects of the pathogenesis of this type of arteriosclerosis is identical in breeder rats of all of the strains examined thus far. Virgin rats of comparable age do not develop arterial lesions. Repeated breeding is the one outstanding factor which appears to be best correlated with the development of the arterial disease. RI~SUMI~
Des rats reproducteurs males et femelles, provenant de plusieurs souches, d6veloppent une art6rioscl6rose spontan6e. Les l~sions art6rielles chez les rats reproducteurs males restent microscopiques, tandis que l'art6rioscl~rose est bien reconnaissable chez les femelles. La fr6quence et la s6v6rit6 des dommages art6riels augmentent ~ chaque ~levage. Les 16sions apparaissent d'abord dans l'aorte abdominale et s'~tendent A la crosse et aux portions thoraciques. Plus tard, quand l'art6rioscl6rose est bien install6e, les art~res coronaires, r6nales et c6r6brales d6veloppent aussi des 16sions art6rielles. I1 a 6t6 trouv6 une grande vari~t6 de changements art~riels d6g~n6ratifs suppos6s J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS
ARTERIOSCLEROSIS
IN REPEATEDLY
BRED MALE AND FEMALE RATS
79
repr6senter des 16sions pr6coces et avanc6es ainsi que diff6rents stades de r6tablissem e n t et de ddg6ndration. On a aussi d6couvert des r6actions morphologiques sp6cifiques propres ~ certains segments de l'aorte. La ldsion la plus fr6quemment observ6e dans les deux sexes est une accumulation subintimale focale de mucopolysaccharides suivi de cicatrisation. Aux mSmes endroits, la m~dia montre un a m a s de mucopolysaccharides, une dilatation et une 6ventuelle fracture de fibres 6lastiques. Ce t y p e de 16sion a 6t6 aussi trouv6e chez les reproducteurs mMes. Toutefois, ces derniers d6veloppent fr6quemment une ldsion additionnelle composde d'une intense infiltration focale de l'intima par des cellules m6senchymales. Ces cellules s'agrandissent et deviennent intens6ment basophiles, prennent une ressemblance avec les cellules cartilagineuses et peuvent finalement subir une m6taplasie cartilagineuse. Les aspect g6n6raux et histologiques de la pathog6nie de ce t y p e d'art6rioscl6rose est identique chez les rats reproducteurs de toutes les souches examindes jusqu'A prdsent. Les rates vierges d'~ge comparable ne d~veloppent pas de 16sions art6rielles. L'61evage r6p6td est le seul facteur qui semble le mieux se r a p p o r t e r au d6veloppement de la maladie art6rielle. ZUSAMMENFASSUNG
Mehrmalig geziichteten m~innlichen und weiblichen Ratten verschiedener A b s t a m m u n g zeigen spontan Arteriosklerose. Die arteriellen Sch~idigungen bei m~innlichen R a t t e n bleiben n u t mikroskopisch, w/ihrend die Arteriosklerose sehr deutlich wahrn e h m b a r ist bei den Weibchen. Frequenz und Ausmass der arteriellen SchAdigung v e r m e h r t sich nach jeder neuen Ztichtung. Die Sch~idigungen erscheinen zuerst in der B a u c h a o r t a und breiten sich auf den Bogen und die B r u s t p a r t i e aus. Spiiter, wenn die Arteriosklerose deutlich ausgepr~igt ist, zeigen auch die Koronar-, Nierenund Hirnarterien arterielle Defekte. Es wurde eine grosse Verschiedenheit arterieller Ver~inderungen degenerativer N a t u r aufgefunden, die sowohl als friihe und auch ausgesprochene Sch~tdigungen wie aueh als den unterschiedlichen Wiederherstellungs- und Degenerationsphasen bet r a c h t e t werden k6nnen. Es wurde zugleich festgestellt, dass b e s t i m m t e morphologische Reaktionen charakteristisch sind ffir bestimmte Aortasegmente. Als die a m meisten v o r k o m m e n d e Sch~idigung - - bei beiden Geschlechtern - - k a n n die fokale subintimale Anh~iufung von Mukopolysacchariden betrachtet werden, mit danach auftretender Vernarbung. An gerade diesen Stellen zeigt die Media Pooling der Mukopolysacchariden und Schwellung und m a n c h m a l Einreissen der elastischen Fibrillen. Obwohl dieser Sch/idigungstyp auch bei den M/innchen aufgefunden wurde, entwickelt sich in diesen doch 6fters auch ein Defekt, bestehend aus einer intensiven fokalen Mesenchymzellen-Infiltration der Intima. Diese Zellen vergr6ssern sich, werden basophil, entwickeln eine Ahnlichkeit zu Knorpelzellen, und k6nnen schliesslich eine kartilagin6se Metaplasie erleiden. Die allgemeinen und histologischen Aspekte der Pathogenese dieser Art der Arteriosklerose scheint in den geziichteten R a t t e n aller bis jetzt untersuchten Stiimme J. Atheroscler. Res., 4 ( 1 9 6 4 ) 5 7 - 8 0
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identisch zu sein. Jungfr~uliche Ratten vergleichbaren Alters zeigen keine arteriellen Sch~digungen. Wiederholte Zt~chtung scheint der einzige Faktor zu sein, der eine deutliche Korrelation zeigt mit der Entwicklung tier Arterienerkrankung. REFERENCES 1 L. N. KATZ AND J. STAMLER, Experimental A therosclerosis, Charles C. T h o m a s , Springfield, Ill., 1952. 2 W. A. THOMAS AND W. S. HARTROFT, Circulation, 19 (1959) 6S. 3 L. C. FILLIOS, S. B. ANI)RUS, G. W. MANN AND F. B. STONE, J. Exptl. Med., 104 (1956) 539. 4 B. C. WEXLER AND B. F. MILLER, Science, 127 (1958) 590. 5 B. C. WEXLER, T. E. BROWN AND B. F. MILLER, Circulation Res., 8 (1960) 278. 6 B. C. WEXLER AND C. W. TRUE, Circulation Res., 12 (1963) 659. 7 S. L. WILENS AND E. E. SPROUL, Am. jr. Pathol., 14 (1938) 177. 8 D. J. INGLE AND B. L. BAKER, Recent Progr. Hormone Res., 8 (1953) 143. 9 T. GILLMAN AND M. HATHORN, Nature, 183 (1959) 1139. 10 B. C. WEXLER AND G. W. KITTINGER, u n p u b l i s h e d o b s e r v a t i o n s . 11 E. H. VENNING, Endocrinology, 39 (1946) 203. 12 O. S . ROBERTSON AND B. C. WEXLER, Endocrinology, 66 (1960) 222. 13 O. H. ROBERTSON, B. C. WEXLER AND B. V. MILLER, Circulation Res., 9 (1961) 826. 14 C. M. PLOTZ, A. T. KNOWLTON AND C. RAGAN, Am. J. Med., 13 (1952) 597.
j . Atheroscler. Res., 4 (1964) 57-80
57
S P O N T A N E O U S A R T E R I O S C L E R O S I S IN R E P E A T E D L Y B R E D M A L E AND F E M A L E RATS 13. C. W E X L E R
The NIav Institute for Medical Research, Jewish Hospital, and the Department of Pathology, University of Cincinnati College of Medicine, Cincinnati, Ohio (U.S.A.) ( R e c e i v e d J u n e 20th, 1963)
INTRODUCTION
Several investigators have described the resistance of the rat to the development of arteriosclerosis and the various manipulations required to induce arterial lesions in this species 1-3. These procedures unfortunately require rather stringent and timeconsuming measures. In an initial report we described the development of arteriosclerosis in the repeatedly bred female Sprague-Dawley rat 4. The arterial lesions in these female breeders were grossly visible and readily detectable. L a t e r it was found t h a t male breeder rats also developed spontaneous arteriosclerosis 5. In the case of the male breeders, however, the arterial lesions could not be seen b y gross inspection and were visible only when the arteries were examined microscopically. I t should be emphasized t h a t arterial lesions have been detected, microscopically, as early as 3 weeks after the first breeding in both males and females a. These early lesions consist of focal subintimal accumulations of m u c o p r o t e i n a c e o u s material. These same foci later become fibrosed. There is little or no lipid present in the lesions. Arteriosclerosis develops in these breeder rats on a regular rat diet which is low in fat. Serum cholesterol levels are normal. The pathogenesis of the arterial lesions can best be correlated with the n u m b e r and frequency of breedings 5. I t is felt t h a t the spontaneous development of the arterial lesions in these breeder rats and the relatively short space of time required for its production should be brought to the attention of those interested in the investigation of experimental arteriosclerosis. I n this p a p e r we shall compare the morphological nature and anatomical distribution of the arterial lesions in several strains of rats. MATERIALS AND METHODS
Male and female breeder rats of the following strains were studied: Sprague-Dawley, Holtzman, Mead-Johnson, Long-Evans, Wistar and Lewis. Our most extensive experience has been with the Sprague-Dawley rat of the Sprague-Dawley Farms, Madison, Wisc. This strain of r a t is highly inbred and was established forty years ago from litters delivered b y cesarean section. For the past 5 years we have received regular hi-weekly shipments of discarded male and female breeders from the SpragueJ. Atheroseler. Res., 4 (1964) 5 7 ~ 0
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WEXLER
Dawley Farms. No exact record has been kept b y these commercial breeders of the number of breedings or the size of the litter of each rat. Breeders are discarded if they cannibalize, refuse to suckle their young, or if they begin to show signs of outward deterioration. Deterioration becomes marked when animals have been bred repeatedly for an extended period of time. The hi-weekly shipments of discarded breeders have not been uniform in that the female breeders m a y have been pregnant once or several times. The majority of females have been bred 3-4 times. It is assumed that the male breeders have been used as studs on several occasions before they are discarded. In order to learn whether the phenomenon of spontaneous arteriosclerosis would occur in breeder rats other than the Sprague-Dawley strain, rats from other sources were examined. For each strain, approximately 100 male and 100 female breeders were autopsied. None of the commercial breeders could provide exact information regarding the breeding histories of their discarded breeders. In all of the strains examined breeder rats were used which had been bred approximately 3-5 times. In searching for the presence of spontaneous arteriosclerosis in breeder rats, all animals were autopsied as soon as possible after arrival. At autopsy, the aorta was exposed so that the heart, carotids, mesenteric branches, iliacs and proximal portions of the femoral arteries were visualized. In the case of the female breeder, the arterial lesions could often be seen on gross inspection. Animals with aortas having one or more sclerotic plaques were classified as "arteriosclerotic". Those with no grossly detectable plaques were called "clear" or "non-arteriosclerotic". For microscopic confirmation of the presence or absence of arteriosclerosis representative samples were taken routinely in prescribed segments of the systemic aorta. The arch of the aorta was taken so that it included the base of the innominate and common carotid arteries. The thoracic aorta was removed just caudal to where the azygos vein crosses the aorta to where the aorta leaves the thorax between the folia of the diaphragm. The abdominal artery was taken from the level of the diaphragm down to and including portions of the iliacs. The aortas of male breeder rats were examined and sampled in the same manner. The iliac arteries of males occasionally showed sclerotic plaques which could be detected b y gross examination. All segments of the aorta were fixed in 10% buffered neutral formalin (Lillie) for histological study. The brains, hearts, kidneys and carotid arteries of these animals were also taken so t h a t the contained cerebral, coronary, renal and the main carotid artery and its branches could be examined microscopically. The tissue was embedded in paraffin and sectioned at 3/~. Frozen sections were cut at 5-10 #. Adjacent sections were stained with hematoxylin-eosin for routine analysis; VERHOEFF, VAN GIESON and GOMORI'Saldehyde fuchsin stains for elastic tissue; alcian blue and toluidine blue for metachromasia; the HALE stain for mucopolysaccharides; the voN KOSSA method for calcium; and Sudans I I and Ill, Oil Red O, and Sudan Black B were used to demonstrate lipids on both frozen and paraffin sections. The word "arteriosclerosis" is used t h r o u g h o u t this paper to describe the variety of degenerative arterial changes. The word "atherosclerosis" was not used because these lesions do not contain significant deposits of lipid.
J. dtheroscler. Res., 4 (1964) 57-80
SPONTANEOUS
ARTERIOSCLEROSIS
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MALE AND FEMALE
RATS
59
RESULTS
Incidence
The results of these investigations were largely based on bi-weekly autopsies conducted during the years 1957-1959 in order to determine whether there was any seasonal variation in the incidence of spontaneous arteriosclerosis in Sprague-Dawley breeder rats. A total of about 3500 female and 500 male breeders were sacrificed during this period. The routine bi-weekly autopsies were abandoned in 1959 because it was found that there was no apparent seasonal variation in the incidence of arteriosclerosis (see Fig. 1). From 1959 until the present, autopsies of a similar nature were performed at r a n d o m intervals as a continuous check on the incidence of spontaneous arteriosclerosis in breeder rats of the Sprague-Dawley strain. During this 5-year period of observation we have found that the average incidence of grossly detectable arteriosclerosis in the female breeder of this strain is approximately 40 %. 5O
~40
°so t.
to
1(2
January to March
April to June
July to October to September December
Fig. 1. Incidence of gross arteriosclerosis in female breeder rats of the Sprague-Dawley strain. Results of bi-weekly autopsies performed throughout the year of 1958.
There have been occasions when this average has run as high as 60-70 %. However the conditions to which these particular breeders were exposed indicated to us that stressful conditions can alter the pathogenesis of the arteriosclerotic process in breeder rats (to be published). At no time have gross lesions been found in the systemic aorta of male breeder Sprague-Dawley rats. However, it should be emphasized that although breeder rats m a y show no gross evidence of arterial lesions, virtually 80-90 % of the female aortas contain microscopic lesions, and 70-80 % of the male aortas which all appear to be "clear" on gross inspection have arteriosclerosis when examined microscopically. If time and facilities had permitted routine serial sections of the aorta, it is felt that the incidence of microscopic lesions in male breeders would be higher than the observed 70~80 %. Arterial lesions in the male breeder aortas are small and focal and can be easily missed in random sections. The female breeder rats of the Holtzman strain which were autopsied were about one year old and h a d been bred 5-6 times. These animals were a few months older and had been bred more than the average Sprague-Dawley breeder. The female Holtzman breeder rats showed advanced arteriosclerosis which was grossly visible f . Atheroscler. Res., 4 (1964) 57~0
60
B . C . WEXLER
in 45 of the 100 animals examined. None of the 50 male breeders of this strain showed gross evidence of arterial damage. The gross anatomical distribution of the lesions was the same in this strain as in the Sprague-Dawley strain. The microscopic morphology of the lesions was also identical to those seen in the Sprague-Dawley strain. The incidence of microscopic lesions also increased to a value of 80 %. Female breeders of the Mead-Johnson strain* were autopsied. None of these animals showed gross evidence of arteriosclerosis. On microscopic examination, however, 60 % of the animals showed histological alterations of the aortic wall which were identical to the early lesions seen in the other strains of breeder rats (see below). It should be noted t h a t the Mead-Johnson breeding practices differ from those used b y the Sprague-Dawley F a r m s in the following respects: (J) these animals are not bred until they are fully mature; (2) they are allowed to rest between litters; and (3) they are not bred more t h a n 3-4 times. In a single sample (100 rats) of female breeder rats of the Long-Evans strain only 10% were found to have grossly evident arteriosclerosis. These animals had been bred 5-6 times. On microscopic examination 7 8 % of these animals were found to have arterial lesions identical morphologically to t h a t observed in previous strains of breeder rats. In this case, however, 4 0 % of the animals h a d microscopic lesions which were quite advanced and considered to be of a severe degree. TABLE I GROSS
AND
MICROSCOPIC
INCIDENCE
OF ARTERIOSCLEROSIS
IN
MALE
AND
FEMALE
BREEDER
RATS
oF SEVERALS~RAINS(%) Strain
Sprague-Dawley Holtzman Mead- J o h n s o n Long-Evans Wistar Lewis
Number of rats
3500 500 100 50 1O0 100 100 6 3
Sex
Female Male Female Male Female Female Female Female Male
Gross lesions
Microscopic lesions
(%)
(%)
40 0 45 0 0 10 0 67 0
80-90 70 80 80 80 60 78 65 67 100
Discarded breeder female rats of the Wistar strain, a p p r o x i m a t e l y 12-13 m o n t h s of age and bred 5 times, were autopsied. None of these animals showed arteriosclerosis grossly but 65 % of these displayed arterial lesions microscopically. Another strain of breeder females known as the Lewis strain** was found to have * P r o v i d e d t h r o u g h the c o u r t e s y of DRS. TRUE AND McCoRMACK, Mead J o h n s o n Co., Evansville, Ind. ** This special s t r a i n of h i g h l y i n b r e d r a t s was p r o v i d e d t h r o u g h t h e c o u r t e s y of DRS. "W. K. SILVERS AND R. E. BILLINGHAM, W i s t a r I n s t i t u t e , P h i l a d e l p h i a , Pa.
J. Atheroscler. Res., 4 (1964) 5 7 - 8 0
SPONTANEOUS ARTERIOSCLEROSIS IN REPEATEDLY BRED MALE AND FEMALE RATS 61
grossly detectable arteriosclerosis. This was a small sample of 6 breeder female rats. Four of the 6 female rats had moderately advanced arteriosclerosis grossly and histologically. These females had had 5 litters. All of the three male breeders which were available to us had tile typical microscopic lesions as seen in other strains. Table I is a s u m m a r y of the incidence of gross and microscopic arteriosclerosis in several strains of breeder rats. At no time has arteriosclerosis been observed, grossly or microscopically, in virgin rats of comparable age to the breeder rats. In summation, breeder rats of several strains of diverse breeding history have been examined for the presence of spontaneously developing arteriosclerosis either grossly or histologically. Grossly detectable arteriosclerosis was not evident in all strains. However, breeder rats of all of the strains examined manifested arterial lesions histologically. Similarly, male breeder rats which do not show gross arteriosclerosis of the systemic aorta were found to have microscopic arterial lesions. Virgin rats of the same age do not develop arterial lesions.
Fig. 2. Dissection of a r c h a n d t h o r a c i c a o r t a of a female breeder ( S p r a g u e - D a w l e y ) gross arteriosclerosis. T h e a r r o w s p o i n t to t w o raised plaques in t h e a s c e n d i n g aortic arch. T h e a r c h s h o w s b e g i n n i n g ectasia a n d t e n d e n c y t o w a r d s o u t p o u c h i n g f o r m a t i o n . A: a close-up of t h e t w o circumscribed p e a r l y w h i t e p l a q u e s
with moderate p o r t i o n of t h e and aneurysm shown.
J. Atheroscler. Res., 4 (1964) 57-80
62
B . C . WEXLER
Anatomical distribution There is a characteristic anatomical p a t t e r n in the appearance of the arteriosclerotic lesions. The first grossly visible lesions a p p e a r as thin pearl-white annular streaks in the lower portion of the abdominal aorta between the renal artery and the bifurcation of the iliacs. I t should be kept in mind first, t h a t the gross arteriosclerosis described applies to the female breeder only, and secondly, t h a t when the arteriosclerosis can be detected grossly the lesions have become considerably advanced histologically. Small p u n c t a t e foci, which are also white and raised appear in a r a n d o m pattern along the abdominal aorta extending from the bifurcation of the iliacs to the origin of the renal and superior and inferior mesenteric arteries. At this time, irregularly shaped plaques begin to appear in the ascending portion of the arch of the aorta (Fig. 2). These plaques broaden and coalesce until all of the arch of the aorta is involved and seldom do they extend beyond the crossing of the azygos vein over the ventral surface of the aorta. In the more advanced cases of arteriosclerosis, thin white annular streaks begin to appear in the thoracic aorta at rather wide intervals. As the process progresses the abdominal aorta shows coalescence of the annular plaques and progression of the punctate lesions u p into the celiac artery and out into the ramifications of the intestinal arteries. At the same time, the entire systemic aorta shows increasing ectasia, occasionally saccular aneurysms appear in the arch of the aorta, and punctate white plaques a p p e a r on all surfaces of the thoracic aorta.
Fig. 3. Severe gross arteriosclerosis in repeatedly bred female r a t (Sprague-Dawley) showing m a r k e d ectasia a n d multiple calcific loci t h r o u g h o u t t h e arch a n d thoracic portions of t h e aorta a n d e x t e n d i n g up into the i n n o m i n a t e a n d c o m m o n carotid arteries.
J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOSCLEROSIS IN R E P E A T E D L Y BRED MALE AND FEMALE RATS 6 3
Of special import, is the extension of the arteriosclerosis up into and along the entire length of the carotid arteries (Fig. 3), along the subclavian and brachial arteries, and distally, at r a n d o m intervals into the iliacs, hypogastric, femoral, popliteal, and tibial arteries. The male breeder does not display lesions grossly in the systemic aorta. However, the aorta does show progressive ectasia. In some cases, white annular streaks similar to those found in the female breeder will appear in the iliac arteries of male breeders. In summary, the grossly detectable arteriosclerosis which develops in the breeder female of all of the strains examined follows a characteristic anatomical pattern of distribution. The earliest gross lesions appear in the abdominal aorta followed b y the arch of the a o r t a and later the thoracic aorta. In the m o s t severe cases of arteriosclerosis the arterial plaques m a y be found in the carotid and peripheral arteries. Male breeders do not show gross lesions in the systemic aorta.
Histological development The earliest microscopic change which can be detected in the a o r t a of breeder rats occurs in the lower portions of the abdominal aorta m i d w a y between the renal artery and the bifurcation of the iliacs. This first change is usually confined to a single segment of the medial tissue of the aorta. Within this focus there is a generalized distention of the tunica m e d i a accompanied b y swelling and stretching of elastic fibers (Fig. 4). The nuclei of the smooth muscle cells become intensely basophilic in this area accompanied b y a h e a v y infiltration of deeply basophilic mesenchymal cells. This basophilia is evanescent and subsides. As the medial swelling and basophilia diminish there is a focal thickening of the tunica intima just central to the medial segment mentioned above. A clear mucoproteinaceous material accumulates in these
Fig. 4.
Fig. S.
Fig. 4. F e m a l e b r e e d e r ( S p r a g u e - D a w l e y ) s h o w i n g t h e earliest c h a n g e s in t h e d e v e l o p m e n t of t h e a r t e r i a l lesions in t h e a b d o m i n a l a o r t a . T h e i n t i m a s h o w s focal s w e l l i n g d u e to a m u c o p r o t e i n a c e o u s m a t e r i a l . T h e r e is t h i c k e n i n g of elastic fibers a n d g r o u n d s u b s t a n c e swelling, w h i c h is a c c o m p a n i e d b y t h e a p p e a r a n c e of i n t e r l u m i n a r elastic fibrils. VERHOEFF-VAN GIESON elastic t i s s u e stain; × 150. Fig. 5. F e m a l e b r e e d e r ( M e a d - J o h n s o n ) s h o w i n g s i m i l a r m e d i a l elastic t i s s u e c h a n g e s d e s c r i b e d in Fig. 4 a n d focal s u b i n t i m a l s w e l l i n g a n d a c c u m u l a t i o n of a m u c o p r o t e i n a c e o u s m a t e r i a l , H e m a t o x y l i n - e o s i n ; × 150.
J. Atheroscler. Res,, 4 (1964) 5 7 - 8 0
64
B.C. WEXLER
subintimal loci (Fig. 5). This substance becomes increasingly metachromatic and HALE positive. Within this pool of mucopolysaccharide, strands of collagen are laid down further contributing to the subintimal involvement (Fig. 6). In the more extensive intimal lesions the edges of the degenerative sites are usually demarcated b y clear, vacuolated cells which resemble foam cells. Occasionally, these early lesions contain scattered droplets of lipid. .......ii!!ii!i!i!iiii:~i]
Fig. 6.
Fig. 7.
Fig. 6. Female breeder (Lewis). Intimal swelling characteristic of early arterial degeneration beginning in the abdominal aorta of breeder rats. The mucoproteinaceous material described in Fig. 5 becomes increasingly metachromatic and positive to the HALE stain (deep black material in photo) especially at the base of the atheroma. Collagen (light grey material in photo) fills in beneath the HALE positive material. HALE stain; × 150. Fig. 7. Male breeder (Holtzman). Raised cartilaginous plaque in the arch of the aorta. This is the typical lesion found in male breeders and consists of deeply basophilic large rounded mesenchymal cells bathed b y mucoproteinaceous material (light grey). The deeply basophilic material (black in photo) is metachromatic and is probably mucopolysaccharide. Hematoxylin-eosin; × 75.
Fig. 8.
Fig. 9.
Fig. 8. Male breeder (Sprague-Dawley). Multiplication of the basophilic mesenchymal elements giving rise to a raised intimal plaque (arch of aorta). These cells are cartilaginous in appearance. Hematoxylin-eosin; X 150. Fig. 9. Male breeder (Long-Evans). Advanced d e v e l o p m e n t of lesion shown in Fig. 8. Cartilaginous cells are intensely metachromatic and positive to the HALE stain (black material in photo). Material, giving a positive reaction with the HALE stain, begins to pool about elastic fibers in media. HALE stain; × 150. J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUSARTERIOSCLEROSISIN REPEATEDLYBRED MALEAND FEMALE RATS 65 At this point there is an interesting dichotomy in the manner in which the lesions continue to develop in the two sexes. In the female breeder, the early lesions are characterized b y endothelial proliferation accompanied b y increased mucopolysaccharide and scarring. The scarring is most prominent at the base of the d a m a g e d site. In the male breeder, however, the intimal swellings remain rather self-contained and are invaded b y heavily basophilic mesenchymal cells which become rounded and quite large. These giant round cells have a vacuolated cytoplasm and are surrounded b y a deeply basophilic halo (Fig. 7). These cells often coalesce and take on the appearance of cartilage ceils (Fig. 8). These clumps of cells begin to exude an intensely basophilic and metachromatic material which is believed to be mucopolysaccharide. At this time the focal subintimal swelling begins to increase in scope (Fig. 9). These lesions cannot be detected macroscopically. It should be emphasized that although the early lesions described above are the type most commonly found, they are not the only variety encountered. In some cases, lesions are found in the main branches of the abdominal aorta which are specifically characterized b y h e a v y scarring leading to occlusion of the lumen (Fig. 10). These sites of intense fibrosis do not contain lipid. This type o f lesion also occurs in the muscular arteries of the intestinal arcades (Fig. 11). Many of the main branches of the abdominal aorta display thromboses and rechannelization of the lumen (Fig. 12).
Fig. 10.
Fig. 11.
Fig. 10. Female breeder (Wistar). Marked fibrosis leading to occlusion of lumen of abdominal artery. Note broken and contorted elastica. This lesion contains little or no lipid. These changes are found in breeder rats with and without high blood pressure. Hematoxylin-eosin; × 150. Fig. 11. Female breeder (Holtzman). Large branch of abdominal aorta with thrombus in lumen, intensive scarring, medial swelling and elastosis. Hematoxylin-eosin; × 100. J. Atheroscler. Res., 4 (1964) 57-80
66
B. C, W E X L E R
A n o t h e r lesion found in t h e a b d o m i n a l a o r t a a n d t h e m e s e n t e r i c b r a n c h e s shows s u b i n t i m a l a c c u m u l a t i o n of a d e e p l y b a s o p h i l i c m a t e r i a l w h i c h encircles t h e entire c i r c u m f e r e n c e of the a r t e r y . This is u s u a l l y a c c o m p a n i e d b y f r a g m e n t a t i o n of t h e i n n e r elastic m e m b r a n e . W h e n t h e a b d o m i n a l i n t i m a l lesions are f o u n d e n c r o a c h i n g on t h e m e d i a , t h e a r c h a n d o t h e r p o r t i o n s of t h e a o r t a also b e g i n to s h o w a r t e r i o s c l e r o s i s (Fig. 13). T h e m e d i a in t h e s e sites shows an i n c r e a s e d p o p u l a t i o n of m e s e n c h y m a l cells a n d g r e a t e r a c c u m u l a t i o n of m u c o p o l y s a c c h a r i d e . T h e i n d i v i d u a l elastic fibers of t h e m e d i a begin to swell. T h e g r o u n d s u b s t a n c e b e t w e e n elastic l a m e l l a e also swells so t h a t i n t e r l a m e l l a r fibrils s t a n d o u t in b o l d relief (Figs. 4, 13-15). A t first, t h e g r o u n d s u b s t a n c e swelling h a s an a m o r p h o u s a p p e a r a n c e a n d r e s e m b l e s t h e m u c o p r o t e i n a c e o u s m a t e r i a l w h i c h a c c u m u l a t e s in t h e s u b i n t i m a l space. L i k e t h e i n t i m a l lesion t h e m e d i a l a l t e r a t i o n in g r o u n d s u b s t a n c e b e c o m e s p r o g r e s s i v e l y m e t a c h r o m a t i c a n d i n c r e a s i n g l y p o s i t i v e to t h e alcian blue, t o l u i d i n e blue, a n d HALE stains. T h i s m u c o p o l y s a c c h a r i d e m a t e r i a l s o m e t i m e s occurs in g r a n u l a r or b e a d e d a r r a n g e m e n t a r o u n d i n d i v i d u a l e l a s t i c fibers or occurs as an e x u d a t e or pool. I n all cases, t h e elastic fibers which a r e a s s o c i a t e d w i t h these m u c o p o l y s a c c h a r i d e pools b e c o m e c o n v o l u t e d a n d d i s t o r t e d , s h o w r e d u p l i c a t i o n a n d s p l i t t i n g , a n d finally r u p t u r e (Fig. 15). This p a r t i c u l a r t y p e of lesion occurs in all of t h e m a j o r elastic a r t e r i e s i n c l u d i n g t h e c a r o t i d a n d m e s e n t e r i c
Fig. 12.
Fig. 13.
Fig. 12. Female breeder (Sprague-Dawley). Large branch of abdominal aorta with thrombosis and vacuolization, infimal and medial growth and elastosis. Hematoxylin-eosin; × 75. Fig. 13. Male breeder (Sprague-Dawley). Aortic arch showing advanced intimal outgrowth encroaching upon lumen. This intimal lesion is deeply positive to the HAL~ stain (black material in photo) and at the base of the intimal cushion there is heavy collagen deposition (crescent-shaped area at center of lesion). HALE stain; × 35. J. Atheroscler. Res., 4 (1964) fi7-80
SPONTANEOUS ARTERIOSCLEROSIS IN REPEATEDLY BRED MALE AND FEMALE RATS 6"]
arteries. I t is this t y p e of lesion which is prone to the d e v e l o p m e n t of the saccular aneurysms which occur in the arch and abdominal portions of the aorta (Fig. 14). If the arteriosclerotic process progresses even further, these sites become engulfed with intensely m e t a c h r o m a t i c pools of the mucopolysaccharide material which inundates
Fig. 14. F e m a l e b r e e d e r ( S p r a g u e - D a w l e y ) . R u p t u r e of elastic fibers leading to a n e u r y s m of t h e arch of the aorta. T h e sites of h e r n i a t i o n are d e m a r c a t e d in t h e p h o t o b y arrows. I n t h e wall of the a o r t a (right side) one c a n see t h e b r o k e n elastic fibers w i t h i n pools of acid m u c o p o l y s a c c h a r i d e (black-colored material) w h e r e t h e arterial wall b e c a m e weakened. Clotted blood fills the a n e u r y s m . HALE stain; × 35.
the entire media from intima to adventitia (Fig. 16). Elastic elements are destroyed or lost within these sites (Fig. 17). These sites often show dystrophic calcification. This process of elastic tissue degeneration and mucopolysaccharide accumulation is much more prevalent in the female breeder. The ground substance lesions in the male breeders seldom reach this stage of complexity. This m a y be a consequence of the shorter life span of male breeder rats. However, the male breeder does show swelling and m a r k e d mucopolysaccharide alterations around the ostia of the intercostal arteries. This occurs in r e m a r k a b l e regularity throughout the length of the thoracic aorta at each intercostal level. Another v a r i e t y of lesion which has a dual association with the arch of the a o r t a and its main arterial branches appears in both male and female. This lesion, as described earlier, begins in the intima with infiltration of heavily basophilic mesenJ. Atheroscler. Res., 4 (1964) 57-80
68
B . C . WEXLER
chymal cells. Within such lesions the mesenchymal elements round-up and take on the appearance of cartilaginous cells. In some cases these cells accumulate within the intima and extend out toward the lumen or else their proliferation is short-lived and the endothelial cells immediately associated with these cells undergo hyperplasia. The growth of these cartilaginous appearing cells can best be described, perhaps, as cartilaginous metaplasia (Figs. 7-9, 18). The above changes are most common in the
Fig. 15. Male breeder (Sprague-Dawley). Pooling of HALE positive material in media of carotid artery. Note the contortion and disappearance of elastic elements within the mucopolysaccharide pools (black-colored areas). HALE stain; × 75.
male breeder a n d seldom reach the proportions of its p r o t o t y p e in the female breeder. In the female breeder, this same process occurs in the intima and the media as well (Figs. 18, 19). This process encompasses b o t h intima and media and encroaches upon the adventitia so t h a t it can be detected grossly in the female breeder. In advanced cases of arteriosclerosis, cartilaginous metaplasia in the female breeder is frequent. There is a v e r y definite anatomical area in which change occurs. I t appears in the arch of the aorta, innominate and carotid arteries. The sites of cartilaginous m e t a plasia often become transformed into bone (Fig. 20). Dystrophic calcification of the media is another form of arterial degeneration which occurs only in the female breeder. Unlike M6NCKEBERG'S sclerosis in the human, this dystrophic calcification is found in the m e d i a of elastic arteries only, e.g., carotid, thoracic, abdominal and mesenteric branches (Fig. 21). J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOSCLEROSIS IN REPEATEDLY BRED MALE AND FEMALE RATS 69
After the arteriosclerosis has become well established in the systemic aorta the coronary, renal and cerebral arteries begin to show some evidence of degenerative change. Again, the morphology of the renal, coronary and cerebral lesions is also varied. For example, some of the coronary arteries show intimal growth (Fig. 22) which m a y eventually occlude the lumen of the artery. These intimal lesions m a y or m a y not contain foam cells. Some coronary arteries show endothelial proliferation, elastic membrane rupture, and medial swelling. Other coronary arteries show distention and the deposition of an intensely basophilic substance within the intima. In
Fig. 16. F e m a l e b r e e d e r ( L o n g - E v a n s ) . I n t e n s e HALE positive m a t e r i a l d i s t e n d i n g entire m e d i a in one focus. T h e light g r e y m a t e r i a l in the i n t i m a i m m e d i a t e l y a b o v e t h e medial lesion is collagen. HALE stain; × 75.
the kidney, the arcuate arteries develop intimal cushions of endothelial cells, the larger hilar arteries show accumulation of a basophilic material in the intima accompanied b y elastosis, swelling and palisade arrangement of the smooth muscle elements in the media (Fig. 23). The cerebral arteries never show changes except in animals with the most severe arteriosclerosis. The most frequent cerebral arterial lesion consists of multiple invaginations of the internal elastic membrane into the lumen (Fig. 24). The visceral (Fig. 25), intestinal (Fig. 26) and more peripheral arteries of the extremities (Fig. 27) characteristically show intimal and medial thickening, endothelial proliferation, scarring, and disruption of the normal contour of the elastica. The arterial lesions found in the carotid and cerebral arteries have been described in detail 8. The coronary, renal, intestinal and peripheral arterial changes will be described separately in greater detail in subsequent publications. J. Atheroscler. Res., 4 (1964) 5 7 - 8 0
70
B.c. WEXLER
In summation, the abdominal aorta is the first site for the appearance of histological as well as grossly detectable lesions. The earliest event that can be detected is swelling of the media and infiltration of heavily basophilic mesenchymal elements. In such areas there is focal subintimal accumulation of an amorphous mucoproteinaceous
Fig. 17. Female breeder (Sprague-Dawley). Advanced arterial lesion of long standing in abdominal aorta. Note eccentric heavy endothelial proliferation of intima with marked elastosis and dystrophic calcification of the media. This type of lesion is easily detected on gross examination. Hematoxylin-eosin; × 10O. material which later becomes metachromatic and is believed to be made up of mucopolysaccharides. These sites contain a few foam cells and soon develop fibrosis. In the male breeder these lesions seldom become more developed or complex. In addition, in the male breeder this type of lesion develops a distinctive type of morphology characterized by intimal invasion with deeply basophilic mesenchymal cells resembling cartilage cells. These sites either show cartilaginous metaplasia or endothelial hyperplasia immediately adjacent to the cartilaginous cells. I n the female breeder there is a spectrum of lesions which appear to develop in specific anatomical locations. Endothelial proliferation, thromboses, scarring, complex degeneration of mucopolysaccharides within the medial ground substance and elastic tissue destruction all occur within the abdominal aorta. Thromboses and endothelial proliferation are rare in the arch and thoracic portions of the aorta. Instead, these J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOSCLEROSIS IN REPEATEDLY BRED MALE AND FEMALE RATS
71
sites show intense mucopolysaccharide changes, elastosis, medial dystrophic calcification and cartilaginous metaplasia of a much more foliating t y p e than the male breeder. In advanced cases of arteriosclerosis, there is an unusual predilection for calcification and bone formation in the arch, innominate and carotid arteries. The peripheral arteries show early changes characterized b y intimal hyperplasia,
Fig. 18. F e m a l e b r e e d e r ( S p r a g u e - D a w l e y ) . I n t e n s e cartilaginous m e t a p l a s i a in o p p o s i t e sides of t h e aorta. B o t h i n t i m a a n d m e d i a are involved. These lesions are i n t e n s e l y m e t a c h r o m a t i c a n d p o s i t i v e to t h e HALE stain. H e m a t o x y l i n - e o s i n ; × 60.
fibrosis and elastosis. The coronary, renal and cerebral arteries do not show signs of any arterial d a m a g e until arteriosclerosis is well established in the systemic aorta. The microscopic m o r p h o l o g y of the arteriosclerotic lesions is essentially identical in breeder rats of all of the strains examined. DISCUSSION
The spontaneous d e v e l o p m e n t of arteriosclerosis in male a n d female breeder rats is a strong argument for using this type of animal in the s t u d y of experimental arteriosclerosis. The finding of vascular disease in all six strains of breeder rats e x a m i n e d demonstrates t h a t this is not an isolated phenomenon confined to a special strain of rat and t h a t it is p r o b a b l y characteristic of all strains of breeder rats. I t is likely t h a t J. Atheroscler. Res., 4 (1964) 5 7 - 8 0
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B . C . WEXLER
the hormonal alterations which take place with the reproductive effort are contributory, in some way, toward the development of this disease in rats. Virgin rats of comparable age to breeders do not develop arteriosclerosis spontaneously. Senile, virgin rats (2 3 years), however, do develop arterial lesionsL We have found arteriosclerosis in repeatedly bred Sprague-Dawley rats which were purchased as weanlings
Fig. 19. F e m a l e breeder (HoltzmanL Cartilaginous m e t a p l a s i a i n v o l v i n g the entire p e r i m e t e r of t h e carotid artery. T h e deep black material is positive to t h e HALE stain. HALE stain; × 50.
and raised and bred, under controlled conditions, in our own laboratory (vide infra) 5. INGLE AND BAKER8 also found t h a t discarded breeder female rats of the SpragueDawley strain developed arteriosclerosis. Further, GILLMAN AND HATHORN9 found spontaneous arteriosclerosis in discarded breeder females (3-7 litters) of the Glaxo strain. All of these findings would underscore the role of breeding in the development of the arteriosclerosis. J u s t how breeding constributes to the d e v e l o p m e n t of this arterial disease in rats is an intriguing question. For example, we have found t h a t the n u m b e r and frequency of breedings can be directly correlated with severity of arteriosclerosis which develops in the Sprague-Dawley rat. During the past 5 years, we have conducted several controlled breeding studies in our laboratory in which we sacrificed animals after 1, 2, 3, 4, 5 and 6 breedings. The arterial lesions begin to a p p e a r grossly in the female J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOSCLEROSIS IN R E P E A T E D L Y BRED MALE AND F E M A L E RATS 7 3
by the third or fourth breeding and become increasingly more pronounced after each successive litter. I t was found that if aninmls were allowed to rest between litters the severity of the arteriosclerosis was lessened. The findings could explain w h y certain strains of rat show more or less intense arteriosclerosis, i.e., an actively bred rat would show more arteriosclerosis than a less actively bred rat. Possibly superimposed upon this are factors such as genetic differences between strains in susceptibility toward arteriosclerosis. Also to be considered is the fact t h a t m a n y of these
Fig. 20. F e m a l e b r e e d e r ( S p r a g u e - D a w l e y ) . C a r t i l a g i n o u s m e t a p l a s i a a n d b o n e f o r m a t i o n ( u p p e r right) in c a r o t i d a r t e r y . H e m a t o x y l i n - e o s i n ; > 75.
animals develop hypertension and renal damage, such as glomerulosclerosis, t u b u l a r dilatation and cast formation. However, these changes in blood pressure and in the kidney do not become manifest until well after m a n y of the arterial lesions described have become well-established. Therefore, the author feels that the rapidity and frequency with which the animals are bred seems to him to be the most essential, although not the only factor in the development of arteriosclerosis*. I t is interesting that diet appears to play a secondary role in the development of * I n p r e v i o u s y e a r s , t h e d i s c a r d e d b r e e d e r r a t s f r o m c o m m e r c i a l s o u r c e s w e r e b r e d as m a n y a s 10 12 t i m e s . T h e s e a n i m a l s d e v e l o p e d , in a d d i t i o n to v e r y s e v e r e arteriosclerosis, h y p e r t e n s i o n , ulcers, p o l y a r t e r i t i c lesions, r e n a l d a m a g e a n d g e n e r a l i z e d a c c e l e r a t e d d e b i l i t a t i o n . D u r i n g r e c e n t y e a r s t h e i n t e n s i t y of t h e b r e e d i n g h a s b e e n r e d u c e d a n d c o n c o m i t a n t l y t h e a r t e r i o s c l e r o s i s a n d t h e a f o r e m e n t i o n e d d e g e n e r a t i v e c h a n g e s h a v e b e c o m e less severe. C o n s e q u e n t l y , t h e u l c e r s a n d t h e p o l y a r t e r i t i s so c o m m o n l y s e e n in b r e e d e r r a t s (bred 6 - 1 2 t i m e s ) h a v e b e c o m e less f r e q u e n t .
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arteriosclerosis in these breeder rats. Although m a n y commercial breeders are unwilling to divulge the exact content of their diets, it is least likely t h a t the differences in development of arteriosclerosis can be ascribed to diet. The breeder rats which developed arteriosclerosis in our laboratory were fed a commercial rat diet (Rockland) and tho microscopic detail of their lesions was in no w a y dissimilar to the lesions observed in other strains of rats. I t should also be mentioned t h a t the serum chol-
Fig. 21. Female breeder (Long-Evans). Abdominal aorta showing medial dystrophic calcification, elastosis, and intimal proliferation. Despite the medial calcification this is not equivalent to M6NCKEBERG'S sclerosis. Hematoxylin-eosin; × 50.
esterol levels are normal in all of the strains of rats examined. In addition, the p a u c i t y of histologically demonstrable lipid in the lesions of different strains of breeders militates against the participation of diet in the production of the disease (to be published). Particularly striking is the m a r k e d h y p e r t r o p h y of the adrenal glands in these breeders. The adrenal h y p e r t r o p h y is invariably accompanied b y involution of the t h y m u s gland. Biochemical and histological studies indicate t h a t the adrenal glands become more active with each breeding and t h a t the profound alterations in arterial ground substance m a y be related to changes in adrenal steroid production 10. The adrenal glands of the female breeder are consistently larger t h a n those of the male breeder. Adrenal h y p e r t r o p h y in connection with the reproductive effort is not an isolated phenomenon peculiar to rats. During the period of gestation in the h u m a n J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOCSLEROSIS IN REPEATEDLY BRED MALE AND FEMALE RATS 75
Fig. 22.
Fig. 23.
Fig. 22. Female breeder (Sprague-Dawley). Advanced coronary lesion displaying marked reduction of lumen by intimal ingrowth. Note large, vacuolated foam cells. Hematoxylin-eosin; x 250. Fig. 23. Female breeder (Sprague-Dawley). Hilar renal artery showing endothelial proliferation, fragmentation and reduplication of intimal elastic membrane, swelling and pallisade arrangement of the smooth muscle cells of the media. Hematoxylin-eosin; x 75.
Fig. 24. Female breeder (Sprague-Dawley). Cerebral artery with multiple aneurysms of the internal elastica. Hematoxylin-eosin; x 150.
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it is well k n o w n t h a t the a d r e n a l g l a n d u n d e r g o e s h y p e r t r o p h y a n d t h a t it s u b s e q u e n t l y b e c o m e s r e d u c e d in size following p a r t u r i t i o n 11. F u r t h e r , in fish we h a v e f o u n d h y p e r a d r e n o c o r t i c i s m a n d arteriosclerosis a s s o c i a t e d w i t h spawningl2, is. T h e r e is a
Fig. 25. Male breeder (Wistar). Splenic artery showing basophilia and disruption of the regular convolutions of the internal elastic membrane. This type of lesion is found frequently in visceral arteries. Hematoxylin-eosin; × 150.
Fig. 26. Male breeder (Sprague-Dawley). Mesenteric artery with intimal hyperplasia, basophilia and disruption of elastica which is also frequentlyfoundinmesentericarteries. Hematoxylin-eosin; x 200. J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS ARTERIOSCLEROSIS IN R E P E A T E D L Y BRED MALE AND FEMALE RATS 77
remarkable similarity between the degenerative changes in spawning fish, breeder rats, and those found in CUSHING'S syndrome in man 14. The sex difference in the morphology of arteriosclerosis in male and female breeders
Fig. 27. F e m a l e b r e e d e r ( S p r a g u e - D a w l e y ) . P o p l i t e a l a r t e r y s h o w i n g i n t i m a l p r o l i f e r a t i o n , elastosis, m e d i a l swelling a n d h y p e r t r o p h y . H e m a t o x y l i n - e o s i n ; × 150.
raises the question of the participation of sex hormones in the pathogenesis of arterial degeneration. I n all strains examined b y us to date, the male breeders do not show gross arteriosclerosis of the systemic aorta except for occasional grossly visible plaques in the iliac arteries. In addition to our own experience, neither INGLE AND BAKER s nor GILLMAN AND HATHORN9 found grossly visible arterial lesions in male breeders. In the author's opinion the male breeder seldom exhibits gross arteriosclerosis for two major reasons: (1) the male breeder, despite having a less florid arteriosclerosis than the female, has an exceptionally high mortality rate and dies at an earlier age than the female breeder; (2) the arteriosclerosis in the male develops in a centripetal direction, i.e., is confined to the intimal layer almost exclusively, while in the female the lesions are centrifugal and more fulminating. In the latter case, where the arteriosclerotic process involves media and also impinges on the adventitia, conditions favor gross recognition of lesions. ACKNOWLEDGEMENTS
This work was supported by grants from the JOHN A. HARTFORD Foundation, the GUSTAVUS AND LOUISE PFEIFFER Foundation, American Heart Association, the Cincinnati Heart Association and the U.S. Public Health Service (Grant H-3874). The investigation was supported (in part) b y a Public Health Service research career program award (HE-K3-1734) from the National Heart Institute. J. Atheroscler. Res., 4 (1964) 5 7 - 8 0
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The author is indebted to the following technical personnel for their participation in this project: N. BLAMER, M. BICKERS, D. CONATSER,J. DORON, K. KUCHENBUCH, R. LEBLOND, D. MANTHY, C. NEIMANN, M. SPRINGS, L. STAPLES, L. THOMAS, M. TORBECK, J. TRACY, R. VAUGHN AND A. WHITE. The photomicrographs were taken by J. HUTZELMAN, Medical Photography, Jewish Hospital, Cincinnati, Ohio. SUMMARY
Repeatedly bred male and female rats of several strains develop arteriosclerosis spontaneously. The arterial lesions in the male breeder rats remain microscopic while the arteriosclerosis becomes grossly recognizable in the female breeder. The incidence and severity of the arterial damage is enhanced with each breeding. The lesions appear first in the abdominal aorta and spread to the arch and thoracic portions. Later, as arteriosclerosis becomes well established the coronary, renal and cerebral arteries also develop arterial lesions. A large variety of degenerative arterial changes have been found which are believed to represent early and advanced lesions as well as various stages of repair and degeneration. It has also been found that there are specific morphological reactions peculiar to certain segments of the aorta. The most frequent lesion encountered, in both sexes, is a focal subintimal accumulation of mucopolysaccharide followed b y scarring. In these same sites the media shows pooling of mucopolysaccharide and swelling and eventual fracture of elastic fibers. This t y p e of lesion is also found in male breeders. However, the male breeder frequently develops an additional lesion consisting of intense focal mesenchymal cell infiltration of the intima. These ceils become enlarged and intensely basophilic, develop a resemblance to cartilage cells, and ultimately m a y undergo cartilaginous metaplasia. The gross and histological aspects of the pathogenesis of this type of arteriosclerosis is identical in breeder rats of all of the strains examined thus far. Virgin rats of comparable age do not develop arterial lesions. Repeated breeding is the one outstanding factor which appears to be best correlated with the development of the arterial disease. RI~SUMI~
Des rats reproducteurs males et femelles, provenant de plusieurs souches, d6veloppent une art6rioscl6rose spontan6e. Les l~sions art6rielles chez les rats reproducteurs males restent microscopiques, tandis que l'art6rioscl~rose est bien reconnaissable chez les femelles. La fr6quence et la s6v6rit6 des dommages art6riels augmentent ~ chaque ~levage. Les 16sions apparaissent d'abord dans l'aorte abdominale et s'~tendent A la crosse et aux portions thoraciques. Plus tard, quand l'art6rioscl6rose est bien install6e, les art~res coronaires, r6nales et c6r6brales d6veloppent aussi des 16sions art6rielles. I1 a 6t6 trouv6 une grande vari~t6 de changements art~riels d6g~n6ratifs suppos6s J. Atheroscler. Res., 4 (1964) 57-80
SPONTANEOUS
ARTERIOSCLEROSIS
IN REPEATEDLY
BRED MALE AND FEMALE RATS
79
repr6senter des 16sions pr6coces et avanc6es ainsi que diff6rents stades de r6tablissem e n t et de ddg6ndration. On a aussi d6couvert des r6actions morphologiques sp6cifiques propres ~ certains segments de l'aorte. La ldsion la plus fr6quemment observ6e dans les deux sexes est une accumulation subintimale focale de mucopolysaccharides suivi de cicatrisation. Aux mSmes endroits, la m~dia montre un a m a s de mucopolysaccharides, une dilatation et une 6ventuelle fracture de fibres 6lastiques. Ce t y p e de 16sion a 6t6 aussi trouv6e chez les reproducteurs mMes. Toutefois, ces derniers d6veloppent fr6quemment une ldsion additionnelle composde d'une intense infiltration focale de l'intima par des cellules m6senchymales. Ces cellules s'agrandissent et deviennent intens6ment basophiles, prennent une ressemblance avec les cellules cartilagineuses et peuvent finalement subir une m6taplasie cartilagineuse. Les aspect g6n6raux et histologiques de la pathog6nie de ce t y p e d'art6rioscl6rose est identique chez les rats reproducteurs de toutes les souches examindes jusqu'A prdsent. Les rates vierges d'~ge comparable ne d~veloppent pas de 16sions art6rielles. L'61evage r6p6td est le seul facteur qui semble le mieux se r a p p o r t e r au d6veloppement de la maladie art6rielle. ZUSAMMENFASSUNG
Mehrmalig geziichteten m~innlichen und weiblichen Ratten verschiedener A b s t a m m u n g zeigen spontan Arteriosklerose. Die arteriellen Sch~idigungen bei m~innlichen R a t t e n bleiben n u t mikroskopisch, w/ihrend die Arteriosklerose sehr deutlich wahrn e h m b a r ist bei den Weibchen. Frequenz und Ausmass der arteriellen SchAdigung v e r m e h r t sich nach jeder neuen Ztichtung. Die Sch~idigungen erscheinen zuerst in der B a u c h a o r t a und breiten sich auf den Bogen und die B r u s t p a r t i e aus. Spiiter, wenn die Arteriosklerose deutlich ausgepr~igt ist, zeigen auch die Koronar-, Nierenund Hirnarterien arterielle Defekte. Es wurde eine grosse Verschiedenheit arterieller Ver~inderungen degenerativer N a t u r aufgefunden, die sowohl als friihe und auch ausgesprochene Sch~tdigungen wie aueh als den unterschiedlichen Wiederherstellungs- und Degenerationsphasen bet r a c h t e t werden k6nnen. Es wurde zugleich festgestellt, dass b e s t i m m t e morphologische Reaktionen charakteristisch sind ffir bestimmte Aortasegmente. Als die a m meisten v o r k o m m e n d e Sch~idigung - - bei beiden Geschlechtern - - k a n n die fokale subintimale Anh~iufung von Mukopolysacchariden betrachtet werden, mit danach auftretender Vernarbung. An gerade diesen Stellen zeigt die Media Pooling der Mukopolysacchariden und Schwellung und m a n c h m a l Einreissen der elastischen Fibrillen. Obwohl dieser Sch/idigungstyp auch bei den M/innchen aufgefunden wurde, entwickelt sich in diesen doch 6fters auch ein Defekt, bestehend aus einer intensiven fokalen Mesenchymzellen-Infiltration der Intima. Diese Zellen vergr6ssern sich, werden basophil, entwickeln eine Ahnlichkeit zu Knorpelzellen, und k6nnen schliesslich eine kartilagin6se Metaplasie erleiden. Die allgemeinen und histologischen Aspekte der Pathogenese dieser Art der Arteriosklerose scheint in den geziichteten R a t t e n aller bis jetzt untersuchten Stiimme J. Atheroscler. Res., 4 ( 1 9 6 4 ) 5 7 - 8 0
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identisch zu sein. Jungfr~uliche Ratten vergleichbaren Alters zeigen keine arteriellen Sch~digungen. Wiederholte Zt~chtung scheint der einzige Faktor zu sein, der eine deutliche Korrelation zeigt mit der Entwicklung tier Arterienerkrankung. REFERENCES 1 L. N. KATZ AND J. STAMLER, Experimental A therosclerosis, Charles C. T h o m a s , Springfield, Ill., 1952. 2 W. A. THOMAS AND W. S. HARTROFT, Circulation, 19 (1959) 6S. 3 L. C. FILLIOS, S. B. ANI)RUS, G. W. MANN AND F. B. STONE, J. Exptl. Med., 104 (1956) 539. 4 B. C. WEXLER AND B. F. MILLER, Science, 127 (1958) 590. 5 B. C. WEXLER, T. E. BROWN AND B. F. MILLER, Circulation Res., 8 (1960) 278. 6 B. C. WEXLER AND C. W. TRUE, Circulation Res., 12 (1963) 659. 7 S. L. WILENS AND E. E. SPROUL, Am. jr. Pathol., 14 (1938) 177. 8 D. J. INGLE AND B. L. BAKER, Recent Progr. Hormone Res., 8 (1953) 143. 9 T. GILLMAN AND M. HATHORN, Nature, 183 (1959) 1139. 10 B. C. WEXLER AND G. W. KITTINGER, u n p u b l i s h e d o b s e r v a t i o n s . 11 E. H. VENNING, Endocrinology, 39 (1946) 203. 12 O. S . ROBERTSON AND B. C. WEXLER, Endocrinology, 66 (1960) 222. 13 O. H. ROBERTSON, B. C. WEXLER AND B. V. MILLER, Circulation Res., 9 (1961) 826. 14 C. M. PLOTZ, A. T. KNOWLTON AND C. RAGAN, Am. J. Med., 13 (1952) 597.
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