Micron, 1977, Vol. 8: 33-36. Pergamon Press. Printed in Great Britain. S H O R T COMMUNICATION
Electron microscopic study of senile cardiac amyloidosis H. LEN TSENG and B. S. LIM
Blackburn Laboratory, Saint Elizabeth Hospital, Washington DC, 20032, U.S.A. Manuscript received December 13, 1976
Using specimensfrom various organs originally processedfor routine histopathologieal study (wax embedding), a total of 2044 autopsy cases were reviewed to identify those with primary amyloidosis of the heart. A total of 7 such cases, involving the heart only, was identified with the aid of appropriate stains and polarized light. The heart tissue was subsequently re-processedfor study by electron microscopy. In all 7 cases, large deposits of amyloid fibrils werefound around the blood capillaries and smaller amounts between individual myocardial fibres. The possible origin of the amyloid fibrils is briefly discussed within the limitations imposed by the source of the material; it is suggested that they may be formed either by the muscle cells or from material reaching the tissue via the capillaries or alternatively, as the result of an interaction between the cells and circulating immunoglobulins.
Since the first report by Wild in 1886, several authors have commented on various aspects of primary amyloidosis of the heart including senile cardiac amyloidosis (Kerwin, 1936; Binford, 1936; King, 1948; Schwartz and Kuruez, 1965; Buja, 1970; Stiller and Katenkamp, 1971). However, in most of the reported cases, the amyloidosis involved other organs in addition to the heart. At present, it is possible to identify amyloidlike substances by three main methods: 1. light microscopy using Congo Red in conjunction with polarized light when the material appears as green fibres (see Luna, 1968). 2. electron microscopy when the amyloid typically appears as fibrils about 10nm dia. (Ghidoni and Guelft, 1962) and 3. X-ray diffraction (Eanes and Glenner, 1968). In this short communication, light microscopy was used to identify cases of primary arnyloidosis involving the heart only and the more precise localization of the amyloid investigated by transmission electron microscopy using material from these cases. Tissue obtained from various organs at autopsy and processed for routine histopathological study (fixed in 10% buffered formaldehyde, dehydrated, cleared, embedded in paraffin wax and stained with haemotoxylin and eosin) was examined to provisionally identify cases of primary amyloidosis. Out of a total of 2,044 33
autopsy cases examined in this way, 12 appeared to contain amyloid-like deposits in the myocardium. This was confirmed by the use of Congo Red and polarized light. It was also established that of these, 2 contained amyloid deposits in the wall of the small arteries of the heart and 3 in one or more organs other than the heart, usually either the lung or kidney but not the spleen or liver. As far as could be determined, the remaining 7 cases only contained amyloid in the myocardium. The age of the 7 cases ranged from 80 to 97 years. In order to make greater use of this unique collection of material, the available paraffin wax blocks of the heart tissue from the above 7 cases were immersed in xylene to remove the wax, passed through descending concentrations of ethanol and finally well-washed in distilled water. The tissue was then re-processed for study by electron microscopy by fixing it in 5% glutaraldehyde in 0.2M phosphate buffer followed by post-fixation in 1% osmium tetroxide. The specimens were then dehydrated with ascending concentrations of ethanol and finally embedded in Epon in the usual way. Sections about 90-120nm thick were cut with a diamond knife using a Porter-Blum MT2ultramicrotome, mounted on 200 mesh copper grids, stained with uranyl acetate and lead citrate and finally examined in a Zeiss EM9S-2.
EM Study of Senile Cardiac Amyloidosis As expected, a number of components had been affected by the rigours of the treatment to which they had been exposed. Thus the mitochondria were very poorly preserved while neither the transverse tubules nor the sarcolemma could be recognized. However, both the structure of the myofibrils and amyloid substance was reasonably well preserved, presumably because of their high protein content (Fig. 1). In the former case, the sarcomeres were readily visible while the amyloid could be identified by the presence of the usual fibrils about 10nm dia (Figs. 1 and 3). In all 7 cases, the amyloid fibres were mainly concentrated around the capillaries where they formed conspicuous large deposits (Fig. 2). Although more detailed observations were limited by the generally low quality of preservation, the peripheral fibres were so close to the wall of the capillaries as to suggest some intimate relationship (Fig. 3). Due to these large deposits, the muscle fibres were much more widely separated from the adjacent capillaries than found in healthy tissue (see, for example, Ebe and Kobayashi, 1972). In some areas, smaller deposits, but still packed with fibrils, were seen between individual myofibrils and these were apparently present within the cardiac muscle cells (Fig. 1). In recent times, and on the basis of both physiological and immunochemical studies, it has been shown that amyloid possesses m a n y of the characteristics of a light chain immunoglobulin with a relatively low molecular weight (Glenner, 1972; Glenner et al., 1973). The same workers have suggested that the known increase in the ' M ' component of immunoglobulin in ageing m a y explain the presence of focal deposits of amyloid in geriatric patients. However,
while this suggests that the deposits observed in the present work involved cells of the immune system, and hence m a y not be formed locally, the evidence from other work is conflicting. Thus in a study on medullary carcinoma of the thyroid, Meyer (1968) observed large deposits which he concluded arose from dense granules produced by the neoplastic cells. Bilbao et al. (1975), in a study of a pituitary adenoma producing an amyloid like-substance, observed precursor material inside cisternae of the granular endoplasmic reticulum and concluded that the amyloid was synthesized de novo by the adenoma cells. The same authors also emphasized that other types of cells which might be expected to be involved (e.g. plasma cells), were too sparse to account for the size of the deposits they observed. On the other hand, Schober and Nelson (1975), again in work on pituitary adenoma, observed amyloid-like material in autophagic vacuoles of histiocytes and felt that these were the source. Yet another type of cell was earlier implicated in a different type of study by Hashimoto et al. (1972). These authors investigated wound healing in cases oflichenoid amyloidosis and believed that the amyloid was formed by the granular endoplasmic reticulum of certain abnormal dermal fibroblasts. Finally it may be mentioned that following injections of casein, Shirahama and Cohen (1975) observed amyloid type fibrils in primary lysosomes of Kupffer cells of the liver and less frequently in the fixed reticular cells and sinus lining ceils of the spleen and occasionally in glomerular mesangial cells of the kidney. It is, of course possible that under certain abnormal conditions, amyloid fibrils m a y be formed by a variety of cells. Theoretically this would seem particularly possible in the case of
Fig. 1. Electron micrograph of heart muscle using tissue obtained at autopsy, processed for routine histopathological study (wax-embedding) and subsequently re-processed for examination by electron microscopy. Although such components as the mitochondria and cytoplasmic matrix are only poorly preserved, structures rich in protein have remained relatively intact. In the upper right hand corner, a dense concentration ofamyloid fibrils can be seen between two myocardial fibrils, x 9000. Fig. 2. Electron micrograph of heart tissue processed as described for Fig. 1 showing a large mass of amyloid fibrils (Am) concentrated around the wall of a blood capillary, x 9000. Fig. 3. Electron micrograph of heart tissue processed as described for Fig. 1 showing a mass of amyloid fibrils (Am) lying between a muscle fibre (MF) and the outer surface of an adjacent blood capillary (Cap). x 19,000.
H. Len Tseng and B. S. 15m
Eanes, E. D. and Glenner, G. t;., 19(i}k X-ra~ diffraction studies on amyloid filaments. 37. littlechem. Cytochern., 16: 673-677. Ebe, T. and Kobayashi, S., 1972. Fine Structures oj Human Cells and Tissues. John Wiley, London and New York. Ghidoni, B. and Gueft, j., 1962. Double nature of amyloid fiber. In: Electron AIicroscopy. Breese, S. S. (ed.), Academic Press, New York, 2 : 1 15. Glenner, G. G., 1972. The pathogenetic and therapeutic implications of the discovery of the immunoglobulin origin of amyloid fibrils. Ituman Path., 3: 157-162. Glenner, G. G. Terry, W. D. and Isersky, C., 1973. Amyloidosis: Its nature and pathogenesis. Seminar in Hematology, 10: 65-86. Hashimoto, K. and Brownstein, M. H., 1972. Amyloidogenesis in healing wounds. A..7. Path., 68: 371-380. Kerwin, A. J., 1936. Idiopathic amyloid diseases ot" the heart. J. Lab. Clin. Med., 22: 255-261. King, L. S., 1948. Atypical amyloid disease with observations on new silver stain for amyloid. Am. J. Path., 24: 1095-1115. Luna, L. G., 1968. Manual of Histological Staining Blethods of Armed Forces Institute of Pathology. 3rd edition, McGraw-Hill, New York. Meyer, J. S., 1968. Fine structure of two amyloidfornfing medullary carcinomas of thyroid. Cancer, 21 : 406-425. Rees, L. H., 1975. The biosynthesis of hormones by non-endocrine tumours--a review. J. Endocr., 67: 143 175. Schober, R. and Nelson, D., 1975. Fine structure and origin of amyloid deposits in pituitary adenoma. Archs. Path., 99: 401-410. Acknowledgements--It is a pleasure to acknowledge the Schwartz, P. and Kuruez, J., 1965. Amyloid deposit technical assistance of Dr. H. C. Chang. in the heart of the aged persons. J. Am. Geriatric. Soc., 27: 711-718. Shirahama, T. and Cohen, A. S., 1975. Intralysosamal REFERENCES formation of amyloid fibrils. Am. 37. Path., 81: Bilbao, J. M. Horvath, E., Hudson, A. R. and 101-116. Kovacs, K., 1975. Pituitary adenoma producing Stiller, Von D. and Katenkamp, D., 1970. Die amyloid-like substance. Archs. Path., 99: 411-415. Amyloidose des Herzens. Deutsche Gesundheitswesen, Binford, H., 1936. Primary amyloid disease of the 26: 2178-2184. myocardium and blood vessels. Archs. Path., 29" Wild, C., 1886. Beitrag zur Kenntnis der Amyloiden 314-320. und der Hyalinen Degeneration des Bindegewebes. Buja, L. M., 1970. Clinical significant cardiac Beitr. zur Path. Anat. und Alle. Path., 1: 174-199. amyloidosis. Am. 37. Cardiol. 26: 394M-05.
t u m o u r cells, some o f w h i c h can e l a b o r a t e substances n o r m a l l y p r o d u c e d by other cells as, for e x a m p l e , in the p r o d u c t i o n of p r o t e i n h o r m o n e s b y t u m o u r cells of n o n - e n d o c r i n e tissue (see Rees, 1975). I t also seems r e a s o n a b l e to consider t h a t in other instances, the deposits m a y be f o r m e d as a result of some i m m u n o l o g i cal r e a c t i o n b e t w e e n the cells found locally a n d c i r c u l a t i n g i m m u n o g l o b u l i n s , an effect which could r e a d i l y be envisaged in senile patients following the suggestion m a d e b y G l e n n e r a n d colleagues (see above). I n the present work, most of the a m y l o i d was present on the outside of the capillaries. I t is, therefore, possible t h a t this was simply the result of m a t e r i a l r e a c h i n g the site via the c a p i l l a r i e s - a l t h o u g h this does not e x p l a i n w h y it should have o c c u r r e d in this o r g a n a n d not elsewhere. A l t e r n a t i v e l y , a n d since smaller deposits were seen w i t h i n the muscle cells, it seems possible t h a t they were the site of origin. H o w e v e r , in view o f the foregoing discussion, a m o r e a t t r a c tive hypothesis is t h a t the a m y l o i d fibrils were f o r m e d as a result of a n i n t e r a c t i o n between the cells a n d i m m u n o g l o b u l i n s of low m o l e c u l a r weight r e a c h i n g the cells via the capillaries as a result of w h i c h most of the a m y l o i d was deposited in the form of fibrils between the two surfaces. I t is h o p e d to be able to investigate these views b y the use of a n i m a l models in w h i c h amyloidosis of the h e a r t has been produced experimentally.