Computerised morphometry was used to define the distribution of aortic atheroma in 10 normal White. Carneau pigeons (age 42 months) and in groups of 10 ...
Acta path. microbiol. immunol. scand. Sect. A, 94: 285-290, 1986.
LOCALIZATION OF ATHEROMATOUS LESIONS IN NORMAL AND HYPERCHOLESTEROLAEMIC PIGEONS AUD D. SVINDLAND, PATRICK J. GALLAGHER* and LARS WALL0E Department of Pathology and Informatics. University of Oslo, Norway and Department of Pathology, University of Southampton*, Great Britain
Svindland, A,, Gallagher, P. J. & Wallrae, L. Localisation of atheromatous lesions in normal and hypercholesterolaemic pigeons. Acta path. microbiol. immunol. scand. Sect. A, 94: 285-290, 1986. Computerised morphometry was used to define the distribution of aortic atheroma in 10 normal White Carneau pigeons (age 42 months) and in groups of 10 weanling or mature birds given a cholesterol coated diet for 24 weeks. Mean serum total and HDL-cholesterol was substantially higher in cholesterol fed animals (15 v 8 mmol/l). The spatial distribution of disease was identical in each group. Lesions developed just proximal to the first major abdominal branch and were more plentiful on the right hand side. The major histological component of the lesions was fibrous intimal thickening and when studied separately its distribution closely matched that of the overall lesion. Foam cell aggregates were more plentiful on the right. This distribution pattern is very similar to that seen in early human atheroma and supports the hypothesis that lesions form in regions of low flow velocities. Key words: White Carneau Pigeons; hypercholesterolaemia; computerised morphometry; shear stress, flow velocity. A. Svindland, Department of Pathology, Ullevaal Hospital, 0407 Oslo 4, Norway.
Received 15.xii.85
Accepted 5 3 . 8 6
There is compelling clinical, experimental and epidemiological evidence that hypercholesterolaemia is a powerful, even the major, risk factor for atheroma in both man (Shekelle et al. 1981) and laboratory animals ( Wissler 1980). Equally physiological or pathological haemodynamic forces play some role in the genesis of atheromatous lesions (Stehbens 1975). For example the larger the calibre of a vessel, the aorta for instance, the greater the severity of disease. Furthermore hypertension aggravates atheroma in both the systemic and pulmonary circulations and it has been shown in a number of detailed topographical studies that early lesions have a predilection for particular sites around arterial branchings and curvatures
few spontaneous lesions. Furthermore the histological appearances of both the naturally occurring and diet-induced lesions are rather different from human atheroma. White Carneau pigeons, however, develop significant spontaneous aortic atheroma at “normal” human serum cholesterol concentrations (c 5-7 mmol/l). In addition the serum cholesterol levels of these birds can be quite accurately adjusted by modest dietary cholesterol supplementation to produce premature disease (Jerome & Lewis 1985). In the current work we have compared the spatial distribution of naturally occurring and cholesterol induced aortic lesions in these pigeons. Our particular objectives were to relate the posi(Kjaernes et al. 1981, Grottum et al. 1983, Svind- tioning of lesions to the major abdominal bifurland 1984). cations, to compare the pattern of disease to that Experimental studies in this field have been seen in man and to determine whether the distrihampered by the lack of a satisfactory animal bution of atheroma vaned at different serum chomodel. Rabbits have been used by some groups lesterol concentrations. (Cornhill & Roach 1976) but they develop very 285
MATERIALS AND METHODS Animals and Dietary Conditions Three groups of 10 White Carneau pigeons were studied (see Table 1). Group 1 was composed of birds that had been fed a mixed grain diet of flaked wheat, maize, New Zealand maple peas and milo throughout life. They were killed at between 171-198 weeks of age. The birds in group 2 were killed at between 44 and 85 weeks of age, having been fed a cholesterol supplemented diet for the last 24 weeks of life. To make this diet 1 kg batches of maple peas in shallow plastic trays were treated with 15 g of Analar grade cholesterol in 150 ml of petroleum ether. The peas were gently mixed in the solution and the ether was then evaporated off in a ventilated cupboard for a minimum of 4 hours. The dried peas acquired a fine white coating of cholesterol, 0.5% by weight. They were eaten normally by the birds along with the remaining 3 grains. Animals in group 3 were fed cholesterol from weaning (age 6 weeks) and killed about 24 weeks later. In this group both the maple peas and milo were treated with 26 g of cholesterol per kg as described above. Serial Observations Blood samples were taken from the wing veins of group 1 birds (normal diet) 8 weeks before killing and at the time of death. Cholesterol fed animals were bled before the coated diet was started and at 6 weekly intervals subsequently. Total serum cholesterol and triglycerides were measured by a colourimetric cholesterol oxidase reaction (Boehringer) or by enzymatic triglyceride hydrolysis and pyruvate induced NADH oxidation (Beckman). HDL cholesterol levels were estimated in representative serum samples taken at termination, and stored at -20 "C for less than 8 weeks. The precipitation reaction (Lopes Virella et al. 1977) used 0.5 M Mg C1, and 4% sodium phosphotungstate. The specimens were centrifuged at 1500 x g for 30 minutes. The cholesterol content of the supernatant was estimated in the usual way by the cholesterol oxidase reaction. Pathology The heart, aorta, common carotid and brachial (wing) arteries were dissected en bloc. Care was taken to preserve the attachment of the first two abdominal aortic branches and to remove the proximal 20 mm of these vessels and the adjacent mesenteric tissues and organs. After overnight formalin fixation connective and other tissues were dissected from the aorta which was then opened longitudinally from the dorsal aspect with fine ophthalmic scissors. The arteries were stained in a saturated solution of Oil red 0 in 70% ethanol for 1 hour and differentiated in 70% ethanol for 3 hours. Aortic plaques in the pigeon are usually well demarcated and the staining procedure did not appear to alter either the size or the clarity of the lesion. The area around the first (coeliac) abdominal branch was flattened under a glass slide, the intimal surface was photographed and black and white prints prepared at x4 magnification.
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A 6 mm segment of aorta just above the orifice of the coeliac artery was then dissected and divided transversely. The proximal half was frozen, cryostat sections cut at 8 pm and stained with Oil red 0. The remainder was processed into paraffin, cut at multiple levels and stained with haematoxylin and eosin and by Gomori's aldehyde fuchsin method. Samples of liver, kidneys spleen and gonads were processed from all cholesterol fed birds (groups 2 and 3) and from 5 representative animals in group 1 (mature, normal diet).
Computer Studies Using a digitising pad a drawing was made of the outline of 17 mm of the opened aorta, the position of the 2 major abdominal branches and the location of all atheromatous lesions. This information was then fed to an Altos microcomputer. The programme, developed in the Department of Informatics, University of Oslo, summarises the results of the drawings as maps whose contour lines connect points with an equal frequency of involvement by disease (maernes et al. 198 I , Gr0ttirm et al. 1983). For greater clarity the contour lines were smoothed by an ancillary programme. Photomicrographs (x80) of the haematoxylin and eosin or Gomori stained arteries were also digitised. The information stored included the outline of the endothelial surface, the position of the innermost elastic lamella (IEL), the distribution of areas of intimal fibrosis and the position of significant foam cell aggregates. The computer programme then transformed the digitised drawing of the opened artery to an idealised closed circle with its centre of gravity at the same point as in the original drawing and with a radius appropriate to the smoothed internal elastic lamella. The thickness of the intima was taken to be the distance between the IEL and the endothelial surface. This was measured by the computer at 360 degree intervals around the circumference of the transformed artery. In group 1 animals, the thickness of specific areas of obvious intimal fibrosis and foam cell accumulation were assessed separately in the same way. The information for each group was then pooled and summarised diagramatically.
RESULTS
Macroscopic Distribution of Lesions Some atheroma was present in all birds, but there were no statistically significant intergroup differences in surface area involvement (Table 1). As shown in Fig. 1 the spatial distribution of atheroma was virtually identical in each group. Lesions were centred on the area just above and lateral to the orifice of the coeliac axis. In each case contour lines converged to two distinct peaks, the right slightly more proximal than the left. Occasional cholesterol fed birds had lesions close to the second abdominal branch but these were far less extensive than around the coeliac artery.
TABLE 1. Serum Cholesterol Values and Aortic Atherosclerosis in Dietary Groups NORMAL DIET
Group 2
Group 3
10 (5MSF)
10 (5MSF)
10 (6M,4F)
30 (28-31)
Group 1 n
,
CHOLESTEROL FED
Median age (range) at termination, weeks
182
61
( 171-189)
(44-85)
Mean serum total cholesterol (range), mmol/l*
8.8 f 0.7 (7.2-16.8)
15.2 3.2 (8.4-28.0)
15.6 f 0.8 (8.6-28.8)
% surface area involvement
0.7-25.4" (17.5)
1.9-29Sb (7.0)
3.7- 19.1' (11.4)
of digitised segment of aorta by atheroma (median value)+
*
* 2 estimates each animal in group 1 4 estimates groups 2 and 3.
+ No significant intergroup differences Mann Whitney test: a vs b U = 28 a vs c U = 40 (5% critical value U ~ V S C= U32
=
27)
Microscopic Analysis of Lesions The histological features of both the naturally occurring and cholesterol induced lesions were broadly similar (Fig. 2). Fibrous intimal thickening was the most prominent feature but aggregates of foam cells, areas of endothelial ulceration and
foci of thrombus formation were identified in some animals. In Fig. 3 the comparative thickness of the intima in the three groups is contrasted. The spontaneous lesions in the 42 month old normally fed pigeons were considerably thicker than in the younger cholesterol fed birds. However the pattern
d Fig. 1. Frequency distribution of aortic atheroma in left group 1 birds, normal diet; centre: group 2, cholesterol diet; right: group 3, cholesterol diet from weaning. The distribution of disease is similar in.all groups. Contour lines converge to peaks proximal to the coeliac artery (marked with a U).
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b Fig. 2. Transverse sections of aorta proximal to coeliac bifurcation. a) Group 1 (normal diet, age 182 weeks). Marked intimal thickening. The clear spaces (arrowheads) are foam cell aggregates. (Elastic-van Gieson x 55). b) Group 2 (cholesterol diet, age 68 weeks). Less pronounced fibrous intimal thickening. Foam cell aggregates are not visible at this magnification (Elastic-van Gieson x 55).
of distribution around the periphery of the aorta was broadly similar in each group. Fig. 4 shows that localisation of areas of fibrosis and foam cell accumulation in normally fed pigeons (group 1). The distribution pattern of fibrous intimal thickening was similar to that of the overall atheromatous lesion. Curiously foam cell accumulation was virtually restricted to the right hand side of the coeliac orifice. As foam cell aggregates were so sparsely scattered in the younger cholesterol fed birds it was not feasible to analyse their thickness separately.
Other Observations Total serum cholesterol levels increased rapidly in the groups given cholesterol coated grains. There was moderate variation in values, not only between birds but in individual animals from estimation to estimation. There was no significant correlation between the percent of surface area involvement by atheroma and the serum cholesterol level in either normally or cholesterol fed birds (Kendall’s coefficient of rank correlation group 1 : 0.18, p>0.3; group 2 : 0.22, p>0.3; group 3 : 0.31, p>O.l). In the terminal samples
Fig. 3. lntimal thickness expressed as a % of the mean aortic diameter. Left: group 1 (normal diet); centre: group 2 (cholesterol diet); right: group 3 (cholesterol diet from weaning). The lines, which are only clearly separated in group 1, correspond to 75%, 50% (solid line) and 25% of the group mean intimal thickness. In all groups atherosclerosis is more common on the right hand side but intimal thickness is clearly greatest in group 1 birds. The arrow indicates the posterior wall of the aorta.
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Fig. 4. Fibrous intimaLthickening (left) and foam cell accumulation (right) in normally fed group I birds (42 months old). The lines indicate 75%, 50% (solid line) and 25% of the mean thickeness of areas of fibrosis or foam cell accumulation. The pattern of fibrous intimal thickening corresponds well to the overall distribution of atherosclerosis (Fig. 3 left) but foam cell accumulation is largely restricted to the right hand side.
taken from group 1 birds serum HDL cholesterol was estimated as 2.4 If: 0.7 (SEM) of a total of 8.9 +_ 0.7 mmol/l, in group 2, (mature, cholesterol fed) 5.4 f 1.8 of a total of 17.4 & 2.3 mmol/l, group 3, 7.2 0.9 of a total of 18.2 -t 1.7 mmol/l. Although total and HDL cholesterol Values were slightly greater in group 3 (fed two coated grains) than in gruop 2 birds (fed one coated grain) the difference was not statistically significant. The serum triglyceride concentration showed marked individual variation with values ranging from 1.3 to >25 mmol/l. The cause of this variation, which was most pronounced in cholesterol fed animals, is largely uncertain. Because these animals have large crops (gizzards) they were not starved before venepuncture, and this may account for some of the wide range in triglyceride values. The majority of the paraffin processed sections of liver from cholesterol fed animals showed fine cytoplasmic vaculation and there was abundant fat in appropriately stained frozen section. Only small amounts of lipid were identified in normally fed animals. There was no evidence of myocardial fibrosis or systemic lipid deposition in any group. The density and distribution of splenic lymphoid tissue was similar in all animals. No significant histological abnormalities were detected in the kidneys.
DISCUSSION This study, and a previous report by Cornhill et al. ( 1980), indicate that early pigeon atherosclerosis
develops just proximal to the orifice of the coeliac artery. In this respect there is a close similarity
between the human and pigeon disease. A series of post mortem studies of different arteries performed in the Oslo laboratories have shown that early lesions in humans are concentrated on the inner curvature of arterial bends, the outer wall of bifurcations and proximal to major branches (Kjaernes et al. 1981, Gr0ttum et al. 1983, Svindland 1984). The fluid dynamics of blood flow in major human arteries is complex since the flow itself is pulsatile, commonly with a reversing phase, and passes through vessels of variable geometry. From a series of experimental studies Stehbens (1975) has suggested that vibrational injury may be an important factor in the induction of both atheromatous lesions and their secondary complications. Another viewpoint is that the shearing or dragging forces which blood flow applies to the vessel wall is of major importance in this respect (Roach & Smith 1983). The direct measurement of shearing stress in small vessels is technically difficult but from a variety of model systems it appears that it is lowest on the inner carvature of arterial bends and just proximal to major arterial branches and highest in the area immediately distal to bifucations. Fry (1972) originally postulated that high shear stress caused direct endothelial injury, thereby favouring the development of atherosclerosis. However Car0 et al. (1 969) provided evidence that atheromatous lesions were commonest at the proximal lip of the larger abdominal branches of the aorta, thereby relating these lesions to areas of low wall shear stress. The results of the current study, along with recent computerised analyses of early human atheroma (Kjaernes et al. 1981 , Gr0ttum et al. 1983, Svindland 1984), strongly support Caro’s findings. 289
Histochemical and radiochemical experiments have shown that the cholesterol present in the intima and atheromatous plaques is not only derived from the plasma but in equilibrium with it (Scott & Hurle 1970, Smith & Slater 1972). The normal concentration gradient is from intima to plasma because the intimal cholesterol concentration is substantially greater than that of the circulation. The precise reason that atherosclerosis develops so predictably in White Carneau pigeons is uncertain. Although genetic factors are of undoubted importance as yet no singularly important biochemical or morphological abnormality has been detected in this species (St. Clair 1983). Because early lesions develop in such specific sites in both man and birds we suspect that haemodynamic factors influence the normal equilibrium that exists between intimal and plasma lipoprotein. This work was supported by the Norwegian Research Councilfor Science and the Humanities and The British Heart Foundation.
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