ATHEROSCLEROSIS IN THE AFRICAN BUFFALO 301

COMBS, J. W. . . . . . . 1966. J. Cell Biol., 31, 563. MILLER, F., DE HARVEN, E., AND 1966. Ibid., 31, 349.

PALAY, S. L., AND KARLIN, L. J. . 1959. J. Biophys. Biochem. Cytol., 5, 363. REYNOLDS, E. S . . . . . . . 1963. J. Cell Biol., 17, 208. ZUCKER-FRANKLIN, DOROTHEA, 1966. J . Exp. Med., 124,521.

ZUCKER-FRANKLIN, DOROTHEA, 1964. Ibid., 120,569.

PALADE, G. E.

DAWDSON, M., AND THOMAS, L.

AND HIRSCH, J. G.

ATHEROSCLEROSIS IN THE AFRICAN BUFFALO (SYNCERUS CAFFER) AND COMPARATIVE STUDIES IN MAN

BRIAN McKI”EY The Royal Free Hospital, London

Fox (1923, 1939), Rigg et ul. (1960) and Finlayson, Symons and T-W-Fiennes (1962) studied naturally occurring arterial lesions in captive wild animals and concluded that some lesions resemble those of atherosclerosis in man. Other workers, notably Keys (1963, penonal communication) and Roser and Magarey (1964), claimed that such lesions are not identical with human atherosclerosis, though they are superficially similar. It was decided to investigate the histochemistry of atherosclerosis in the African buffalo and to compare the findings with those in spontaneous atherosclerosis in man, as such a study has not been made before.

MATERIALS AND METHODS

The aortas of 12 young adult buffaloes from the Queen Elizabeth National Park, Uganda, obtained at necropsy within 6 hr of death, were fixed in 4 per cent. formaldehyde in saline, in which they were kept until used.

Human aortas obtained from routine necropsies at the Royal Free Hospital were obtained within 24 hr of death and similarly fixed and kept; all were from patients aged between 30 and 50 yr.

Blocks were taken from fatty streaks and fibrous plaques. The following techniques were used on 12p frozen sections: the osmium tetroxide alpha-naph- thylamine (OTAN) method for differential staining of phospholipids, hydrophobic fats and cholesterol esters (Adams, 1959): the Schultz reaction for cholesterol and its esters; the nileblue sulphate method for acidic lipids; the periodic acid- Schiff (PAS) reaction for neutral mucopolysaccharides (modified by Pearse, 1953); 2 per cent. alcian blue in ~ N - H ~ S O ~ at pH 0 .245 (Adams and Tuqan, 1961) to stain acid mucopolysaccharides; and oil red 0 and orcein (McKinney and Riley, 1967) to stain lipids and elastic tissue in the same section. On several sections the Verhoeff-Van Gieson stain for elastic tissue or the phosphine3R fluorescent method for lipids was also used. A section of each specimen was defatted in a chloroform- ethanol mixture (Z:l, v/v) for 18 hr at 20°C and then stained with oil red 0 to ensure that non-lipid substances would not take up lipid stains. Other blocks were embedded in paraffin wax; sections were cut at 5 p and stained with haematoxylin and eosin (HE), or phosphotungstic acid-haematoxylin (PTAH) or by Lendrum’s acid picro-Mallory technique.

302 B. McKINNEY

RESULTS Buffalo aortas

In all animals there was infiltration of some areas of the aortic intima with lipid (fig. l), best demonstrated after macroscopic staining of the aorta with oil red 0 (Roser and Magarey). This was often associated with slight elevation and roughening of the intima, was found most commonly at or near to arterial ostia leaving the aorta, and vaned from almost complete involvement of the whole aortic surface to small areas 3 4 mm. in diameter.

In 2 animals, fibrous plaques about 3 cm. in diameter were found in the lower thoracic and upper abdominal aorta (fig. 2). These plaques were firm, smooth, white and shiny and contained only a very small quantity of lipid around their edges.

Microscopic examination Lipid-contcliningplques. HE-stained sections usually show pronounced thicken-

ing of the intima; this is much more marked in areas that are macroscopically elevated or roughened and is caused by proliferation of fibrous and elastic tissue and endothelial cells (fig. 3). These areas vary from 20 to 30 pin thickness where the surface is relatively smooth and regular, to 200-300 p in thickness where the surface is very irregular.

In many other areas the intima (especially where markedly thickened) is acellular or sparsely cellular and appears to consist of hyaline material. There is no evidence of infiltration of the intima or the underlying media with lymphocytes, or of thrombosis or calcification.

In normal areas of the aorta the intima consists of a layer of endothelial cells, and an internal elastic lamina, between which is a little fibrous tissue and an occasional muscle cell. Sections stained with oil red 0 and orcein show that the inner layer of the normal intima consists of an internal elastic lamina consisting of a single continuous layer of elastic tissue; lipids are not present. When there is intimal thickening, lipid is present in or around the internal elastic lamina, often on the intimal side and within macrophages; some lipid is extracellular (fig. 4). Lipid is also present to a lesser degree in the thickened intimal tissue (fig. 5 ) either extracellularly or less commonly in macrophages. In a few areas lipid is seen below the internal elastic lamina (fig. 6), but such deposits are not very extensive. The internal elastic lamina is often abnormal-thickened, irregular or incomplete, especially when it is surrounded by lipid; in other areas it shows reduplication or fragmentation.

The OTAN technique demonstrates red-staining phospholipid in and around the internal elastic lamina (especially where it is fragmented) or in the deeper parts of the thickened intima (fig. 7). Most of this phospholipid appears as fine extra- cellular particles, but some is contained in macrophages. OTAN staining also reveals a small amount of black-staining hydrophobic lipid, mainly on the luminal side of the thickened intima, but little of this reacts as cholesterol with the Schultz technique (fig. 8).

Picro-Mallory and PTAH stains do not demonstrate fibrin in any of the speci- mens, either within the lesions or on the luminal surface of the intima.

Acid mucopolysaccharide is plentiful within the internal elastic lamina where there are alterations in the internal elastic lamina, or other intimal changes, or when lipid is present.

Fibrous plaques. HE stains show compact fibrous tissue, which in a few areas is relatively acellular, the underlying media not being significantly thinned or infiltrated with inflammatory cells; the intima is not ulcerated and there is no sign of haemorrhage.

The orcein and oil red 0 stain shows only a very small amount of lipid, mainly around the edges of the lesions; this lipid is usually extracellular, but in a few small areas is in macrophages. Elastic tissue is present in most fibrous plaques,

MCKINNEY PLATE CXVlI

ATHEROSCLEROSIS IN THE AFRICAN BUFFALO

FIG. ].-Aorta showing intimal deposits of lipid. Macro- scopic specimen. Oil red 0. x 1.9.

FIG. 2.--Intimal fibrous plaque in aorta showing a small deposit of lipid around its edge. Macroscopic specimen. Oil red 0. x25.

FIG. 3.-Thickened intima of fibrous plaque. Haematoxylin and eosin. x 40.

MCKINNEY PLATE CXVIlI

ATHEROSCLEROSIS IN THE AFRICAN BUFFALO

FIG. 4.-Lipid is present in lipophages and also extra- cellularly. Oil red 0. x 450.

FIG. 5.-Thickened internal elastic lamina; the lipid is most plentiful in the deeper intima. Orcein and oil red 0. x30.

FIG. 6.-Lipid extending below the internal elastic lamina and into the media. Orcein and oil red 0. x 36.

MCKINNEY PLATE CXIX

ATHEROSCLEROSIS IN THE AFRICAN BUFFALO

FIG. 7.-Lumen to right. Phospholipids surround the internal elastic lamina; hydro- phobic cholesterol (arrowed) in the superficial intima. Osmium tetroxide alpha- naphthylamine. x 40.

FIG. I.-Lumen to right. Thickened intima, showing only a slight amount of cholesterol (arrowed). Schultz technique. x40.

ATHEROSCLEROSIS IN THE AFRICAN BUFFALO 303

but there are some areas where it is absent; it appears that the elastic tissue of the media often passes directly into some plaques. There are no significant changes in the internal elastic lamina.

OTAN stains show only a small amount of phospholipid and hydrophobic fat around the edges of the fibrous plaques; the Schultz technique shows that some of this is cholesterol.

The PAS reaction shows a few positive areas at or near the edges of the plaques or immediately below the intima.

Human aortas The lesions in human atherosclerosis are similar in some respects. They

contain more lipid. Sometimes the surface of the intima shows small fibrin thrombi. The lesions contain more cholesterol than those of the buffalo, are often

ulcerated and calcified and do not merge so directly with the underlying media. Many of the human lesions are surrounded by chronic inflammatory cells that extend into the underlying media and areas of haemorrhage are sometimes seen within them.

DISCUSSION The arterial lesions in the buffalo aorta are similar in some respects to those of

atherosclerosis in man: there are intimal changes in the internal elastic lamina accompanied by overlying fibro-elastic and endothelial proliferation ; the same types of lipid are present; and there are a few fibrous plaques.

The buffalo lesions differ from those that occur in man: they do not progress beyond the fibrous plaque stage to complicated lesions showing ulceration, or calci- fication, or both; the fibrous plaques are much less frequent and occur mainly in the lower thoracic or upper abdominal aorta and not in the lower abdominal aorta; the fibrous plaques usually contain elastic tissue passing directly into them from the underlying media; fibrin cannot be demonstrated either within or covering them as has been shown in man (Crawford and Levene, 1952); there is no surround- ing intlammatory reaction; and cholesterol, though present, is much less plentiful than in human atherosclerosis.

The distribution of the aortic fibrous plaques in the buffalo may be influenced by posture as a similar distribution has been described in cattle (Giinther, 1958); however, plaques are usually found near the aortic bifurcation in dogs (Lindsay, Chaikoff and Gilmore, 1952) suggesting that the quadrupedal posture is not the sole factor.

S W A R Y

A histochemical study of spontaneous lesions in the aortas of wild African

The types of lipid found resemble those in human atherosclerosis, but histo-

The situation of the fibrous plaques is different in the buffalo. There is absence of cellular infiltration, fibrin deposits, calcification and ulcera-

My thanks are due to the Wellcome Trust for financial support for this investi-

buffaloes is described.

chemical examination shows that considerably less cholesterol is present.

tion.

gation and to Professor K. R. Hill for his helpful criticism and advice.

REFERENCES ADAMS, C. W. M. . . . . . 1959. This Journal, 77,648. ADAMS, C. W. M., AND TUQAN, N. A. 1961. This Journal, 82,131. CRAWFORD, T., AND LEVENE, C. J. . 1952. This Journal, 64,523. FINLAYSON, R., SYMONS, C., AND 1962. Br. Med. J., 1, 501.

T-W-FIENNES, R. N.

304 ANTONIO CAPUTO AND MARIA L. MARCANTE

Fox, H. . . . . . . . . . ,Y . . . . . . . .

GUNTHER, W. . . . . . . LINDSAY, S., CHAIKOFF, 1. L., AND

MCKINNEY, B., AND RILEY, M. . PEARSE, A. G. E. . . . . . .

GILMORE, J. W.

RIGG, K., FINLAYSON, R., SYMONS, C., HILL, K. R., AND T-W- FJENNES, R. N.

ROSER, B. J., AND MAGAREY, F. R. .

1923.

1939. 1958. 1952.

1967. 1953.

1960.

1964.

Disease in captive wild animals and birds, Philadelphia.

Bull. N. Y. Acad. Med., 15, 748. Medsche Klin., 53, 1916. Archs Path., 53,281.

Stain Technol., 42, 245. Histochemistry, theoretical and

applied, London, pp. 415 and 431. Proc. Zool. SOC. Lond., 135, 157.

This Journal., 88, 73.

IMMUNO-ELECTROPHORETIC PATTERNS OF BLOOD SERUM AND ASCITES FLUID IN NORMAL AND ASCITES-TUMOUR-BEARING RATS

ANTONIO CAPUTO AND MARIA L. MARCANTE The Institute of General Pathology, University of Perugia, and the

Regina Elena Institute for Cancer Research, Rome, Italy

PLATES CXX AND CXXI

IT is well known that glycoprotein components undergo significant qualitative and quantitative changes in tumours and other pathological conditions. In the Yoshida ascites tumour, for example, some specific constituents are present; of these an al-glycoprotein has been isolated and the physicochemical (Marcante, 1963) and some of the structural properties (Caputo, 1964; Caputo and Marcante, 1964) have been investigated. As we believe that the most important prerequisite for characterisation of the tumoral glycoproteins is the study of their immunochemical properties, we have undertaken an immuno-electrophoretic investigation of the antigenic relations between normal rat and tumour-bearing rat blood serum proteins and those in Yoshida ascitic fluid.

MATERIALS AND METHODS

Animals and samples. Most of the rats used were of the Wistar strain from the ARSAL colony; a few experiments have also been performed with rats of Long- Evans strain. Preliminary investigations (Gazzaniga, Sonnino and Caputo, 1964) showed that the immuno-electrophoretic pattern of serum proteins is similar in animals from both strains. The blood of normal or amour-bearing rats was obtained by heart puncture under pentobarbital anaesthesia. After clotting and centrifugation, samples of serum from 20 animals of each group were collected, pooled and examined immediately. In a few cases the serum was rapidly frozen and stored at -20°C. In some experiments plasma was used instead of serum, because of the presence of small amounts of fibrinogen in the ascitic fluid. The ascitic fluid was collected 10 days after inoculation of the Yoshida tumour. After centrifugation the samples were collected, pooled and examined immediately.

Antisera. Immune sera were produced in rabbits against normal rat serum or plasma, tumour-bearing-rat serum or plasma and Yoshida ascitic fluid: 1 ml. of serum or 1-5 ml. of ascitic fluid with complete Freund's adjuvant (Difco) were injected intramuscularly on the 1st day and on every 3rd day thereafter till the