Act&Path. Jap. 27(6): 809-822, 1977
PATHOLOGICAL STUDY ON AMYLOIDOSIS -
Scanning Electron Microscopic Observation of Amyloid-Laden Mouse Liver Mutsuo TAKAHASHI First Department of Pathology, Yamaguchi University School of Medicine, Ube
(Received on Feb. 2, 1977)
Amyloidosis of liver in mice induced by casein injection was studied by scanning electron microscopy (SEM). In the fractured-surfaces of the central portion of amyloid nodules, they appeared a s chestnut-bur-like structures and were composed of numerous thread-like structures of fasciculated amyloid fibrils and of stick-like structures of amyloid bundles in three dimensional figure. In the cytoplasm of the Kupffer cell, amyloid bundles were revealed a s band-like bulges. These bulges were projected from the Kupffer cell into the Disse’s space and continued with thread-like structures outside the cytoplasm of Kupffer cells. ACTA PATH. JAP. 27: 809-822, 1977.
Introduction
Recent studies on amyloidosis have advanced since the demonstration of amyloid fibrils in various tissues by COHEN and CALKINS4 using the transmission electron microscope (TEM). GLENNERet a1.8 have succeeded in producing fibrils closely resembling amyloid fibrils from Bence Jones proteins in vitro, and they have suggested that immunoglobulins in the serum phagocytized by the macrophages are converted to amyloid fibrils after lysosomal digestion in the cells. Today, it is clear that the main component of amyloid is amyloid fibril proteins. Biochemically, this fibril protein can be classified into three groups; (1) the protein, amino acid sequence of which is derived from the light chain of immunoglobulin, (2) products of so-called APUD cells, and (3) amyloid fibril protein in which amino acid sequence is different from that of any known proteins nowadays. Morphological ~tudies~,10-12,14,17,~~,~0 of amyloidosis are mainly done by the use of transmission electron microscope and an intimate relationship between amyloid fibrils and reticuloendothelial cells has supported the concept that amyloid is produced by these cells. Recently, ISHIHARA et a l l 3 observed amyloid-laden mouse liver by using freeze-etching method of TEM in three dimensional image. However, it is still controversial whether the reticuloendothelial cells synthesize amyloid de now0 within ___.~
&@
EZEA Director: Prof. Fumiya UOHINO Presented a t the 63th Annual Meeting of the Japanese Pathological Society held in Tokyo, in Nagoya, April, 1974. Research Grant for Specific Disease from the Ministry of Health and Welfare. 809
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their cytoplasm or not. More recently, ESHUN-WILSON et aL5 reported on pulmonary alveolar septa1 amyloidosis of a human autopsy case by using SEM. The present author will describe detailed three dimensional structures of amyloid in experimental amyloidosis in mice for the purpose of making clear the relationship between amyloid and Kupffer cells.
Materials and Methods AKR mice (approximately 6 weeks of age and weighing about 20 g) received subcutaneous injections of 0.5 ml of 5% sodium caseineit six times weekly. The mice were killed a t 12 weeks after the start of casein injection and untreated mice were used as control. The sections of the liver were stained with hematoxylin and eosin, periodic-acid-Schiff and Puchtler’s alkaline Congo red for light microscopic observation. For scanning electron microscopic study, amyloid-laden mice were sacrificed under ether anesthesia without perfusion and small tissue blocks (1 x 1 x 5 mm) from the liver were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer a t 4°C for 3 hrs and postfixed in 1% buffered osmium tetroxide for 1 hr, dehydrated by graded concentrations of alcohol and propylene oxide. The tissues were placed in gelatin capsules (No 2) which were filled with Cemedine 1500. The capsules were left overnight a t 37°C t o get sufficient soaking of Cemedine into the tissue, and then the Cemedine in the capsules was hardened in a cold chamber a t 3OoC for 3 hrs. After adequate hardening of the Cemedine, the capsules were cracked into two pieces with a chisel and hammer according to Tanaka’s freezed resin cracking method’*. The cracked pieces of the capsules were put into propylene oxide to remove the Cemedine from the tissue a t 37°C for overnight. After the Cemedine were sufficiently removed, the tissues were transfered t o isoamyl acetate and then dried by Anderson’s critical point drying method’. The specimens were vacuum-coated with a 100 to 200 A thin layer of carbon and a 200 to 300 p\ thin layer of gold, and photographed in a Hitachi HHS2R or JSM-S1 scanning electron microscope. After observation of SEM, the same specimens were provided for TEM by the methods described below. For transmission electron microscopic study, small tissue blocks from the liver were fixed in 4.15% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) a t 4°C for 2 hrs and postfixed in 1% buffered osmium tetroxide, dehydrated by graded concentrations of alcohol and finally embedded in Epon 812 according to the method of Luftlj. Thin sections cut with Ivan Sorvall I I b ultramicrotome were doubly stained with uranyl acetate and lead citrate and photographed in a Hitachi HS-8 transmission electron microscope.
Results Light microscopically, several hyaline-like nodules were detected in the Disse’s space. These nodules revealed homogenous and pale pink color with hematoxylin-eosin stain. At a high magnification, the nodules showed radiating fine fibrillar structures (amyloid stars). And the nodules demonstrated green polarization color under polarizing microscope stained with Congo red. Transmission electron microscopy of the liver revealed amyloid bundles composed of numerous amyloid fibrils and of projections from the cytoplasm of Kupffer cells into the Disse’s space, and randomly arranged fibrils showing a felt-like structure a t the Fig. 1. A typical amyloid nodule in TEM. A characteristic felt-like structure is seen in the central portion, but a t the periphery in the vicinity of the Kupffer cell, amyloid bundles are arranged in a radial fashion. x 12,000. Fig. 2. Polyhedral, angulated hepatocytes (H) form hepatic plates and sinusoids (S) are round or oval on cross-section. Dome-shaped nuclei (n) of the hepatocytes are seen (control liver). x 1,200.
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central portion of the nodule. I n the peripheral cytoplasm of Kupffer cell, the amyloid bundles were observed in the cytoplasmic invagination and arranged in a radial fashion (Fig. 1). I n the samples of TEM provided from those of SEM, amyloid fibrils were clearly depicted as fine fibrillar profiles in spite of mild artifacts i.e. slightly widened Disse’s space. Scanning electron microscopy revealed the followings; I n the control liver, polyhedral, angulated hepatocytes formed hepatic plates which were one cell thick. Sinusoids were round or oval in cross-section and their greatest diameter were about 20 to 40 ,u. In favorably fractured specimens, hepatic plates were generally straight and were separated from neighboring plates by sinusoids. The nuclei of hepatocytes were occasionally seen as hemispherical convex or concave structures in the center of the cells (Fig. 2). Small spherical or oval bodies varying in size from 2 to 4 ,u in diameter were detected in the cytoplasm of the hepatocytes and they were presumed to represent the mitochondria. However, cristae of the mitochondria could not be seen (Fig. 3). Several vacuoles ranging from 1 to 3 p in diameter were seen a t the periphery of the cytoplasm of some hepatocytes (Fig. 4). I n the Disse’s space, numerous microvilli of the hepatocytes were found protruding and some of them seemed to intrude into the cytoplasm of the sinusoidal endothelial cells. Thin cytoplasmic processes of the sinusoidal endothelial cells extended outward in all lateral directions and formed thin cytoplasmic veils, which composed the major extent of
Fig. 3. In the cytoplasm of hepatocytes (H), numerous mitochondria (m) are seen, but the cristae of the mitochondria can not be seen. S: Sinusoid x 12,000.
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Fig. 4. Several vacuoles (v) are seen a t the periphery of the cytoplasm of some hepatocytes (H). A Kupffer cell (K) is demonstrated in the sinusoid (S) (control liver). x 6,000. Fig. 5. In the fractured surface of Kupffer cells (K), the cells are attached cloeely to the sinusoidal walls (control liver). H: Hepatocyte S: Sinusoid n: nucleus x 6,000.
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Figs. 6, 7 . Several amyloid nodules (amy) are seen between the hepatocytes (H). Some of them are shown as chestnut-bur-like bodies. A typical amyloid star (arrow) is also seen. S: Sinusoid x 2,000, x 2,000.
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sinusoidal walls. The cytoplasm of the sinusoidal endothelial cell formed small, round fenestrations measuring about 0.1 p in diameter. A few larger fenestrations ranging from 1 to 3 p in diameter were occasionally seen. Another type of sinusoidal cell with plump cytoplasm was rarely seen in the sinusoids. The surface of these cells were ridged or ruffled and occupied by scattered, long and filopodia-like protrusions (Fig. 4). On the fractured surface of the cells, it was demonstrated that the cells were attached closely to the sinusoidal walls (Fig. 5). I n the amyloid-laden liver, chestnut-bur-like bodies measuring up t o 20 p in diameter were noted between the hepatic plates (Figs. 6, 7). A high magnification of the bodies showed that they were composed of numerous thread-like and stick-like structures measuring from about 50 t o 100 mp and 200 to 500 mp in width respectively. The thread-like structures were generally located a t the central portion of the body displaying a compactly or intertwingly oriented lace-like pattern. The stick-like structures were located a t the periphery of the body and they were usually arranged in a radial fashion. They branched out and connected with other branches not only a t the peripheral portion but also a t the central portion of the body. Among these structures many pores or slits were detected and they were increased in size a t the peripheral area of the body (Fig. 8). In some areas, an accumulation of thread-like structures was discernible in the Disse’s space, compressing the sinusoidal space
Fig. 8. A high magnification of amyloid nodule (amy). It is composed of thread-like structures and stick-like structures. A Kupffer cell (K) is seen at the lower side. H: Hepatocyte x 12,000.
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Fig. 9. An amyloid nodule (amy) compressing the sinusoidal space (arrows) is seen in thc Disse’s space. H: Hepatocyte S: Sinusoid x 5,000.
almost conipletely (Fig. 9). I n the fractured surface, an accumulation of these structures appeared to be encircled by sinusoidal endothelial cells (Fig. 10). As also shown in the control liver, the cells with plump cytoplasm which were attached closely to the sinusoidal walls were seen in the amyloid-laden liver. The surfaces of the cell that faced the sinusoid were somewhat ridged or folded, and a t the surface facing the Disse’s space, several filopodia-like protrusions were seen. In some parts of the cytoplasm of the cell, band-like bulges were detected with several rows. These bulges were connected with the thread-like structures outside of the cytoplasm (Fig. 1 1). Occasionally, these cells with plump cytoplasms were detected exclusively in the vicinity of the chestnut-bur-like body. A high magnification showed that flattened cytoplasm of the cell extending toward the sinusoid contained some fenestrations or vacuoles, and some parts of the cytoplasm possessed several stick-like protrusions. These protrusions stood nearly perpendicularly to the cytoplasm of the cell revealing a wood-like appearance. They Erequently branched out, fused with other branches and finally transfering into the chestnut-bur-like body (Figs. 12, 13). On other fractured surface, the different processes of accumulations of the thread-like Fig. 10. An amyloid nodule (amy) appears to be encircled by sinusoidal endothelial cells. H: Hepatocyte S: Sinusoid x 6,000. Fig. 11. A Kupffer cell (K) containing band-like bulges with several rows (arrow). The bulges are connected with thread-like structures outside of the cytoplasm. amy: amyloid H: Hepatocyte S: Sinusoid x 10,000.
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Fig. 12. Stick-like protrusions (arrows) stand nearly perpendicularly to the cytoplasm of the Kupffer cell (K). amy: amyloid H: Hepatocyte x 20,000. Fig. 13. A higher magnification of figure 10. Stick-like structures are closely connected with the cytoplasm of the Kupffer cell (K) and they branch out and fuse with other branches. amy: arnyloid x 60.000.
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Fig. 14. The thread-like structures appear to intrude into the cytoplasm of hepatocyte (H). amy: amyloid x 20,000. Fig. 15. The stick-like structures appear to push up the wall of the vein (V). amy: amyloid x 37,000.
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and stick-like structures were seen. These structures appeared to intrude into the cytoplasm of the hepatocyte (Fig. 14). Rarely, in the neighborhood of the vein, the structures appeared to be pushing up the wall of the vein (Fig. 15).
Discussion
In the amyloid-laden liver, SEM demonstrates various types of accumulations of the thread-like and stick-like structures. The reasons why these accumulations are identified with amyloid are as follows; (1) such figures are never found in the control livers, (2) they are located in the Disse's space, (3) their fractured surfaces are similar to amyloid nodules in TEM, (4)in the samples of TEM provided from those of SEM, amyloid fibrils are clearly depicted as fine fibrillar profiles, (5) the images of amyloid in et d 6 . SEM are almost identical with those reported by ESHUN-WILSON The chestnut-bur-like bodies and the stick-like or the band-like structures in SEhi are consistent with the amyloid nodules (amyloid stars) and the amyloid bundles in TEM, respectively. However, the thread-like structures are different from the amyloid fibril in width. These thread-like structures may represent the fasciculus of several amyloid fibrils which have been coated together presumably a t the time of vacuum evaporation. In three dimensional image, it is clear that the fasciculated amyloid fibrils are generally placed at the central portion of the amyloid nodules and are compactly and intertwingly oriented revealing a lace-like pattern. The amyloid bundles are located at the periphery of the nodules and these bundles are generally arranged in a radial fashion. These fasciculated amyloid fibrils and amyloid bundles branch out and connect with other branches. In SEM, many pores or slits are clearly seen among the thread-like structures. And it is conceivable that these pores or slits have occurred by draining off the tissue fluid during sampling of the specimens, as reported by E'uRuTAN17and ISHIHARA~~ using the TEM. Unfortunately, the amyloid fibril cannot be elucidated because of the limitation of resolution power in SEM. As to the Kupffer cell, there are several r e p 0 r t s ~ 1 ~as P to the three dimensional figures of the Kupffer cell in normal condition. According to NOPANITAYA and GRISHAM~~, sinusoids contain an additional type of cell and these cells are plump and their surfaces are ridged or folded and sparsely populated with stubby microvilli. They did not identify the cells with the Kupffer cells but presumed that they might represent the Kupffer cells. However, they described only the surface structure of the cells and did not clarify the relationship between these cells and sinusoidal endothelial cells. The author observed similar cells with plump cytoplasm both in control liver and amyloid-laden liver. Although it is sometimes difficult to differentiate a Kupffer cell from lymphocyte or leucocyte only in surface structure, as shown in Pig. 4, the Kupffer cells are easily recognized on cross-fractured surfce, because the cells are plump, the surface are ridged or ruffled and they are attached closely to the sinusoidal walls.
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Based on the observations by TEM, UCHINO’’ reported that, in amyloidosis in mice, the Kupffer cells contained an increased number of granular endoplasmic reticulum and lysosome and well-developed Golgi complex, and these morphological evidences suggested the functional abnormality of the reticuloendothelial cells during amyloid production. In SEM, it is difficult t o demonstrate these changes of the organella of Kupffer cells because of the limited resolution ability of SEM. As shown in Fig. 11, some portions of the cytoplasm of Kupffer cells contain band-like bulges and these bulges are usually continuous with the thread-like structures of amyloid bundles outside of the cytoplasm. The band-like bulges correspond with the stone-column-like structures reported by ISHIHARA and co-workers13 who observed amyloid-laden mouse liver by using freeze-etching method of TEM. According to their report, the stone-column-like structures of the amyloid bundles were ensheathed by the cytoplasmic membrane of Kupffer cells. However, in SEM, it is not clear whether the band-like bulges of the amyloid bundles are ensheathed or exposed directly in the sinusoidal space. Although the image obtained in this study cannot deny the possibility that the Kupffer cell may phagocytize the band-like bulges, the author considers that the bulges are projected from the Kupffer cell because of their radial arrangement and width, and this image may represent the early stage of amyloid production by Kupffer cells. As shown in Figs. 12 and 13 the stick-like structures of amyloid bundles stand nearly perpendicularly to the cytoplasm of the Kupffer cell. These structures of amyloid bundles frequently branch out, fuse with other branches and finally form the amyloid nodule. This image can be interpreted to represent the progressive stages of amyloid formation. I n conclusion, the amyloid bundles are in close contact to the cytoplasm of the Kupffer cell and are projected from the cell into the Disse’s space when observed in three dimensional figures by using SEM. These figures cannot deny the possibility that Kupffer cells may phagocytize the amyloid bundles. I n regard to this point, further studies may be required. Acknowledgement: The author is gratefully indebted to Prof. F. UCHMOfoq his encouragements throughout this study. Thanks are also due to Dr. N. MATSUMOTO,Dr. T. ISHIHARA, Dr. M. SHIBATA, Mr. M. YAMASHITA, and Mr. Y. MURAKAMIfor their technical assistances.
References 1. ANDERSON, T.F. : Technique for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N.Y. Acad. Sci., Ser. I11 13: 130-134, 1951. 2. BROOKS, S.E.H. and HAGGIS, G.H.: Scanning electron microscopy of rat’s liver. Application of freeze-fracture and freeze-drying techniques. Lab. Invest. 29: 60-64, 1973. 3. CAESAR,R.: Die Feinstruktur von Milz und Leber bei experimenteller Amyloidoses. Z. Zellforsch. 52: 653-673, 1960. 4. COHEN,A.S. and CALRINS, E.: Electron microscopic observations on a fibrous component in amyloid of diverse origins. Nature 183: 1202-1203, 1959. 5. COHEN, A.S., GROSS, E., and SEIRAHAMA, T.: The light and electron microscopic autoradiographic demonstration of local amyloid formation in spleen explants. Am. J. Path. 47: 1079-1112, 1965. 6. ESHUN-WILSON, K., FRANDIEN, N.E., and CHRISTENSEN, H.E.: Pulmonary alveolar septa1
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9.
10. 11. 12.
13.
14. 15.
16. 17. 18. 19. 20.
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amyloidosis. A scanning and transmission electron microscopy study. Virchow Arch. -4 Path. Anat. and Histolo. 371 : 89-99, 1976. FURUTANI, H. : Pathological study on amyloidosis. The behavior of exogenous peroxidase in experimental amyloidosis of liver. Jap. Soc. R.E.S. 13: 149-162, 1974 (in Japanese). GLENNER, G.G., EIN, D., EANES,E.D., BLADEN,H A . , TERRY,W., and PAGE, D.L.: Creation of “Amyloid” fibrils from Bence Jones proteins in vitro. Science 174: 712-714, 1971. GRISHAM,J.W., NOPANITAYA, W., COMPAGNO, J., and NAQEL,A.E.H. : Scanning electron microscopy of normal r a t liver. The surface structure of its cells and tissue components. Am. J. Anat. 144: 295422, 1975. GUEFT, B. and CHIDONI,J.J.: The site of formation and ultrastructure of amyloid. Am. J. Path. 43: 837-854, 1963. HOSOKAWA, 8.: Pathological study on amyloidosis. Trans. Soc. Path. Jap. 61: 5-31, 1972 (in Japanese). ISHIHARA, T. : Experimental amyloidosis using silver nitrate. Electron microscopic study on the relationship between silver granules, amyloid fibrils and reticuloendothelial system. Acta Path. Jap. 23(3): 439464, 1973. ISHLHARA, T., UOHINO,F., and TAKAHASHI, M.: Pathological study on amyloidosis. The fine structure of amyloid-laden liver as revealed by freeze-etching. Beta Path. Jap. 26(3): 357-366, 1976. ISHIHARA, T. and UCHINO,F. : Pathological study on amyloidosis. Amyloid formation and resorption in Kupffer cell. Recent Advances in RES Resarch. 15: 14S171, 1975. LUFT, J.H.: Improvement in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9 : 409414, 1961. NOPANLTAYA, W. and ORISHAM, H. W. : Scanning electron microscopy of mouse intrahepatic structures. Exper. Mol. Path. 23: 441458, 1975. J.P. : Experimental amyloidosis. 11. SORENSON, G.D., HEEFNER,W.B., and KIRKPATRICK, Light and electron microscopic observations of liver. Am. J. Path. 44: 629444, 1964. TANAKA, K. : Preened resin cracking method for scanning electron microscopy of biological materials. Naturwiss. 59: 77, 1972. UCHINO,F. : Pathological study on amyloidosis. Role of reticuloendothelial cells in inducing amyloidosis. Acta Path. Jap. 17: 49-82, 1967. UCHINO, F. : Amyloidosis. Histopathological iderpretation of lichen amyloidosis and study on producing mechanism of amyloid substance in experimental cases. J. Jap. Derm atol. 82: 704-715, 1972 (in Japanese).
PATHOLOGICAL STUDY ON AMYLOIDOSIS -
Scanning Electron Microscopic Observation of Amyloid-Laden Mouse Liver Mutsuo TAKAHASHI First Department of Pathology, Yamaguchi University School of Medicine, Ube
(Received on Feb. 2, 1977)
Amyloidosis of liver in mice induced by casein injection was studied by scanning electron microscopy (SEM). In the fractured-surfaces of the central portion of amyloid nodules, they appeared a s chestnut-bur-like structures and were composed of numerous thread-like structures of fasciculated amyloid fibrils and of stick-like structures of amyloid bundles in three dimensional figure. In the cytoplasm of the Kupffer cell, amyloid bundles were revealed a s band-like bulges. These bulges were projected from the Kupffer cell into the Disse’s space and continued with thread-like structures outside the cytoplasm of Kupffer cells. ACTA PATH. JAP. 27: 809-822, 1977.
Introduction
Recent studies on amyloidosis have advanced since the demonstration of amyloid fibrils in various tissues by COHEN and CALKINS4 using the transmission electron microscope (TEM). GLENNERet a1.8 have succeeded in producing fibrils closely resembling amyloid fibrils from Bence Jones proteins in vitro, and they have suggested that immunoglobulins in the serum phagocytized by the macrophages are converted to amyloid fibrils after lysosomal digestion in the cells. Today, it is clear that the main component of amyloid is amyloid fibril proteins. Biochemically, this fibril protein can be classified into three groups; (1) the protein, amino acid sequence of which is derived from the light chain of immunoglobulin, (2) products of so-called APUD cells, and (3) amyloid fibril protein in which amino acid sequence is different from that of any known proteins nowadays. Morphological ~tudies~,10-12,14,17,~~,~0 of amyloidosis are mainly done by the use of transmission electron microscope and an intimate relationship between amyloid fibrils and reticuloendothelial cells has supported the concept that amyloid is produced by these cells. Recently, ISHIHARA et a l l 3 observed amyloid-laden mouse liver by using freeze-etching method of TEM in three dimensional image. However, it is still controversial whether the reticuloendothelial cells synthesize amyloid de now0 within ___.~
&@
EZEA Director: Prof. Fumiya UOHINO Presented a t the 63th Annual Meeting of the Japanese Pathological Society held in Tokyo, in Nagoya, April, 1974. Research Grant for Specific Disease from the Ministry of Health and Welfare. 809
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their cytoplasm or not. More recently, ESHUN-WILSON et aL5 reported on pulmonary alveolar septa1 amyloidosis of a human autopsy case by using SEM. The present author will describe detailed three dimensional structures of amyloid in experimental amyloidosis in mice for the purpose of making clear the relationship between amyloid and Kupffer cells.
Materials and Methods AKR mice (approximately 6 weeks of age and weighing about 20 g) received subcutaneous injections of 0.5 ml of 5% sodium caseineit six times weekly. The mice were killed a t 12 weeks after the start of casein injection and untreated mice were used as control. The sections of the liver were stained with hematoxylin and eosin, periodic-acid-Schiff and Puchtler’s alkaline Congo red for light microscopic observation. For scanning electron microscopic study, amyloid-laden mice were sacrificed under ether anesthesia without perfusion and small tissue blocks (1 x 1 x 5 mm) from the liver were fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer a t 4°C for 3 hrs and postfixed in 1% buffered osmium tetroxide for 1 hr, dehydrated by graded concentrations of alcohol and propylene oxide. The tissues were placed in gelatin capsules (No 2) which were filled with Cemedine 1500. The capsules were left overnight a t 37°C t o get sufficient soaking of Cemedine into the tissue, and then the Cemedine in the capsules was hardened in a cold chamber a t 3OoC for 3 hrs. After adequate hardening of the Cemedine, the capsules were cracked into two pieces with a chisel and hammer according to Tanaka’s freezed resin cracking method’*. The cracked pieces of the capsules were put into propylene oxide to remove the Cemedine from the tissue a t 37°C for overnight. After the Cemedine were sufficiently removed, the tissues were transfered t o isoamyl acetate and then dried by Anderson’s critical point drying method’. The specimens were vacuum-coated with a 100 to 200 A thin layer of carbon and a 200 to 300 p\ thin layer of gold, and photographed in a Hitachi HHS2R or JSM-S1 scanning electron microscope. After observation of SEM, the same specimens were provided for TEM by the methods described below. For transmission electron microscopic study, small tissue blocks from the liver were fixed in 4.15% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) a t 4°C for 2 hrs and postfixed in 1% buffered osmium tetroxide, dehydrated by graded concentrations of alcohol and finally embedded in Epon 812 according to the method of Luftlj. Thin sections cut with Ivan Sorvall I I b ultramicrotome were doubly stained with uranyl acetate and lead citrate and photographed in a Hitachi HS-8 transmission electron microscope.
Results Light microscopically, several hyaline-like nodules were detected in the Disse’s space. These nodules revealed homogenous and pale pink color with hematoxylin-eosin stain. At a high magnification, the nodules showed radiating fine fibrillar structures (amyloid stars). And the nodules demonstrated green polarization color under polarizing microscope stained with Congo red. Transmission electron microscopy of the liver revealed amyloid bundles composed of numerous amyloid fibrils and of projections from the cytoplasm of Kupffer cells into the Disse’s space, and randomly arranged fibrils showing a felt-like structure a t the Fig. 1. A typical amyloid nodule in TEM. A characteristic felt-like structure is seen in the central portion, but a t the periphery in the vicinity of the Kupffer cell, amyloid bundles are arranged in a radial fashion. x 12,000. Fig. 2. Polyhedral, angulated hepatocytes (H) form hepatic plates and sinusoids (S) are round or oval on cross-section. Dome-shaped nuclei (n) of the hepatocytes are seen (control liver). x 1,200.
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central portion of the nodule. I n the peripheral cytoplasm of Kupffer cell, the amyloid bundles were observed in the cytoplasmic invagination and arranged in a radial fashion (Fig. 1). I n the samples of TEM provided from those of SEM, amyloid fibrils were clearly depicted as fine fibrillar profiles in spite of mild artifacts i.e. slightly widened Disse’s space. Scanning electron microscopy revealed the followings; I n the control liver, polyhedral, angulated hepatocytes formed hepatic plates which were one cell thick. Sinusoids were round or oval in cross-section and their greatest diameter were about 20 to 40 ,u. In favorably fractured specimens, hepatic plates were generally straight and were separated from neighboring plates by sinusoids. The nuclei of hepatocytes were occasionally seen as hemispherical convex or concave structures in the center of the cells (Fig. 2). Small spherical or oval bodies varying in size from 2 to 4 ,u in diameter were detected in the cytoplasm of the hepatocytes and they were presumed to represent the mitochondria. However, cristae of the mitochondria could not be seen (Fig. 3). Several vacuoles ranging from 1 to 3 p in diameter were seen a t the periphery of the cytoplasm of some hepatocytes (Fig. 4). I n the Disse’s space, numerous microvilli of the hepatocytes were found protruding and some of them seemed to intrude into the cytoplasm of the sinusoidal endothelial cells. Thin cytoplasmic processes of the sinusoidal endothelial cells extended outward in all lateral directions and formed thin cytoplasmic veils, which composed the major extent of
Fig. 3. In the cytoplasm of hepatocytes (H), numerous mitochondria (m) are seen, but the cristae of the mitochondria can not be seen. S: Sinusoid x 12,000.
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Fig. 4. Several vacuoles (v) are seen a t the periphery of the cytoplasm of some hepatocytes (H). A Kupffer cell (K) is demonstrated in the sinusoid (S) (control liver). x 6,000. Fig. 5. In the fractured surface of Kupffer cells (K), the cells are attached cloeely to the sinusoidal walls (control liver). H: Hepatocyte S: Sinusoid n: nucleus x 6,000.
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Figs. 6, 7 . Several amyloid nodules (amy) are seen between the hepatocytes (H). Some of them are shown as chestnut-bur-like bodies. A typical amyloid star (arrow) is also seen. S: Sinusoid x 2,000, x 2,000.
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sinusoidal walls. The cytoplasm of the sinusoidal endothelial cell formed small, round fenestrations measuring about 0.1 p in diameter. A few larger fenestrations ranging from 1 to 3 p in diameter were occasionally seen. Another type of sinusoidal cell with plump cytoplasm was rarely seen in the sinusoids. The surface of these cells were ridged or ruffled and occupied by scattered, long and filopodia-like protrusions (Fig. 4). On the fractured surface of the cells, it was demonstrated that the cells were attached closely to the sinusoidal walls (Fig. 5). I n the amyloid-laden liver, chestnut-bur-like bodies measuring up t o 20 p in diameter were noted between the hepatic plates (Figs. 6, 7). A high magnification of the bodies showed that they were composed of numerous thread-like and stick-like structures measuring from about 50 t o 100 mp and 200 to 500 mp in width respectively. The thread-like structures were generally located a t the central portion of the body displaying a compactly or intertwingly oriented lace-like pattern. The stick-like structures were located a t the periphery of the body and they were usually arranged in a radial fashion. They branched out and connected with other branches not only a t the peripheral portion but also a t the central portion of the body. Among these structures many pores or slits were detected and they were increased in size a t the peripheral area of the body (Fig. 8). In some areas, an accumulation of thread-like structures was discernible in the Disse’s space, compressing the sinusoidal space
Fig. 8. A high magnification of amyloid nodule (amy). It is composed of thread-like structures and stick-like structures. A Kupffer cell (K) is seen at the lower side. H: Hepatocyte x 12,000.
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Fig. 9. An amyloid nodule (amy) compressing the sinusoidal space (arrows) is seen in thc Disse’s space. H: Hepatocyte S: Sinusoid x 5,000.
almost conipletely (Fig. 9). I n the fractured surface, an accumulation of these structures appeared to be encircled by sinusoidal endothelial cells (Fig. 10). As also shown in the control liver, the cells with plump cytoplasm which were attached closely to the sinusoidal walls were seen in the amyloid-laden liver. The surfaces of the cell that faced the sinusoid were somewhat ridged or folded, and a t the surface facing the Disse’s space, several filopodia-like protrusions were seen. In some parts of the cytoplasm of the cell, band-like bulges were detected with several rows. These bulges were connected with the thread-like structures outside of the cytoplasm (Fig. 1 1). Occasionally, these cells with plump cytoplasms were detected exclusively in the vicinity of the chestnut-bur-like body. A high magnification showed that flattened cytoplasm of the cell extending toward the sinusoid contained some fenestrations or vacuoles, and some parts of the cytoplasm possessed several stick-like protrusions. These protrusions stood nearly perpendicularly to the cytoplasm of the cell revealing a wood-like appearance. They Erequently branched out, fused with other branches and finally transfering into the chestnut-bur-like body (Figs. 12, 13). On other fractured surface, the different processes of accumulations of the thread-like Fig. 10. An amyloid nodule (amy) appears to be encircled by sinusoidal endothelial cells. H: Hepatocyte S: Sinusoid x 6,000. Fig. 11. A Kupffer cell (K) containing band-like bulges with several rows (arrow). The bulges are connected with thread-like structures outside of the cytoplasm. amy: amyloid H: Hepatocyte S: Sinusoid x 10,000.
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Fig. 12. Stick-like protrusions (arrows) stand nearly perpendicularly to the cytoplasm of the Kupffer cell (K). amy: amyloid H: Hepatocyte x 20,000. Fig. 13. A higher magnification of figure 10. Stick-like structures are closely connected with the cytoplasm of the Kupffer cell (K) and they branch out and fuse with other branches. amy: arnyloid x 60.000.
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Fig. 14. The thread-like structures appear to intrude into the cytoplasm of hepatocyte (H). amy: amyloid x 20,000. Fig. 15. The stick-like structures appear to push up the wall of the vein (V). amy: amyloid x 37,000.
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and stick-like structures were seen. These structures appeared to intrude into the cytoplasm of the hepatocyte (Fig. 14). Rarely, in the neighborhood of the vein, the structures appeared to be pushing up the wall of the vein (Fig. 15).
Discussion
In the amyloid-laden liver, SEM demonstrates various types of accumulations of the thread-like and stick-like structures. The reasons why these accumulations are identified with amyloid are as follows; (1) such figures are never found in the control livers, (2) they are located in the Disse's space, (3) their fractured surfaces are similar to amyloid nodules in TEM, (4)in the samples of TEM provided from those of SEM, amyloid fibrils are clearly depicted as fine fibrillar profiles, (5) the images of amyloid in et d 6 . SEM are almost identical with those reported by ESHUN-WILSON The chestnut-bur-like bodies and the stick-like or the band-like structures in SEhi are consistent with the amyloid nodules (amyloid stars) and the amyloid bundles in TEM, respectively. However, the thread-like structures are different from the amyloid fibril in width. These thread-like structures may represent the fasciculus of several amyloid fibrils which have been coated together presumably a t the time of vacuum evaporation. In three dimensional image, it is clear that the fasciculated amyloid fibrils are generally placed at the central portion of the amyloid nodules and are compactly and intertwingly oriented revealing a lace-like pattern. The amyloid bundles are located at the periphery of the nodules and these bundles are generally arranged in a radial fashion. These fasciculated amyloid fibrils and amyloid bundles branch out and connect with other branches. In SEM, many pores or slits are clearly seen among the thread-like structures. And it is conceivable that these pores or slits have occurred by draining off the tissue fluid during sampling of the specimens, as reported by E'uRuTAN17and ISHIHARA~~ using the TEM. Unfortunately, the amyloid fibril cannot be elucidated because of the limitation of resolution power in SEM. As to the Kupffer cell, there are several r e p 0 r t s ~ 1 ~as P to the three dimensional figures of the Kupffer cell in normal condition. According to NOPANITAYA and GRISHAM~~, sinusoids contain an additional type of cell and these cells are plump and their surfaces are ridged or folded and sparsely populated with stubby microvilli. They did not identify the cells with the Kupffer cells but presumed that they might represent the Kupffer cells. However, they described only the surface structure of the cells and did not clarify the relationship between these cells and sinusoidal endothelial cells. The author observed similar cells with plump cytoplasm both in control liver and amyloid-laden liver. Although it is sometimes difficult to differentiate a Kupffer cell from lymphocyte or leucocyte only in surface structure, as shown in Pig. 4, the Kupffer cells are easily recognized on cross-fractured surfce, because the cells are plump, the surface are ridged or ruffled and they are attached closely to the sinusoidal walls.
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Based on the observations by TEM, UCHINO’’ reported that, in amyloidosis in mice, the Kupffer cells contained an increased number of granular endoplasmic reticulum and lysosome and well-developed Golgi complex, and these morphological evidences suggested the functional abnormality of the reticuloendothelial cells during amyloid production. In SEM, it is difficult t o demonstrate these changes of the organella of Kupffer cells because of the limited resolution ability of SEM. As shown in Fig. 11, some portions of the cytoplasm of Kupffer cells contain band-like bulges and these bulges are usually continuous with the thread-like structures of amyloid bundles outside of the cytoplasm. The band-like bulges correspond with the stone-column-like structures reported by ISHIHARA and co-workers13 who observed amyloid-laden mouse liver by using freeze-etching method of TEM. According to their report, the stone-column-like structures of the amyloid bundles were ensheathed by the cytoplasmic membrane of Kupffer cells. However, in SEM, it is not clear whether the band-like bulges of the amyloid bundles are ensheathed or exposed directly in the sinusoidal space. Although the image obtained in this study cannot deny the possibility that the Kupffer cell may phagocytize the band-like bulges, the author considers that the bulges are projected from the Kupffer cell because of their radial arrangement and width, and this image may represent the early stage of amyloid production by Kupffer cells. As shown in Figs. 12 and 13 the stick-like structures of amyloid bundles stand nearly perpendicularly to the cytoplasm of the Kupffer cell. These structures of amyloid bundles frequently branch out, fuse with other branches and finally form the amyloid nodule. This image can be interpreted to represent the progressive stages of amyloid formation. I n conclusion, the amyloid bundles are in close contact to the cytoplasm of the Kupffer cell and are projected from the cell into the Disse’s space when observed in three dimensional figures by using SEM. These figures cannot deny the possibility that Kupffer cells may phagocytize the amyloid bundles. I n regard to this point, further studies may be required. Acknowledgement: The author is gratefully indebted to Prof. F. UCHMOfoq his encouragements throughout this study. Thanks are also due to Dr. N. MATSUMOTO,Dr. T. ISHIHARA, Dr. M. SHIBATA, Mr. M. YAMASHITA, and Mr. Y. MURAKAMIfor their technical assistances.
References 1. ANDERSON, T.F. : Technique for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N.Y. Acad. Sci., Ser. I11 13: 130-134, 1951. 2. BROOKS, S.E.H. and HAGGIS, G.H.: Scanning electron microscopy of rat’s liver. Application of freeze-fracture and freeze-drying techniques. Lab. Invest. 29: 60-64, 1973. 3. CAESAR,R.: Die Feinstruktur von Milz und Leber bei experimenteller Amyloidoses. Z. Zellforsch. 52: 653-673, 1960. 4. COHEN,A.S. and CALRINS, E.: Electron microscopic observations on a fibrous component in amyloid of diverse origins. Nature 183: 1202-1203, 1959. 5. COHEN, A.S., GROSS, E., and SEIRAHAMA, T.: The light and electron microscopic autoradiographic demonstration of local amyloid formation in spleen explants. Am. J. Path. 47: 1079-1112, 1965. 6. ESHUN-WILSON, K., FRANDIEN, N.E., and CHRISTENSEN, H.E.: Pulmonary alveolar septa1
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amyloidosis. A scanning and transmission electron microscopy study. Virchow Arch. -4 Path. Anat. and Histolo. 371 : 89-99, 1976. FURUTANI, H. : Pathological study on amyloidosis. The behavior of exogenous peroxidase in experimental amyloidosis of liver. Jap. Soc. R.E.S. 13: 149-162, 1974 (in Japanese). GLENNER, G.G., EIN, D., EANES,E.D., BLADEN,H A . , TERRY,W., and PAGE, D.L.: Creation of “Amyloid” fibrils from Bence Jones proteins in vitro. Science 174: 712-714, 1971. GRISHAM,J.W., NOPANITAYA, W., COMPAGNO, J., and NAQEL,A.E.H. : Scanning electron microscopy of normal r a t liver. The surface structure of its cells and tissue components. Am. J. Anat. 144: 295422, 1975. GUEFT, B. and CHIDONI,J.J.: The site of formation and ultrastructure of amyloid. Am. J. Path. 43: 837-854, 1963. HOSOKAWA, 8.: Pathological study on amyloidosis. Trans. Soc. Path. Jap. 61: 5-31, 1972 (in Japanese). ISHIHARA, T. : Experimental amyloidosis using silver nitrate. Electron microscopic study on the relationship between silver granules, amyloid fibrils and reticuloendothelial system. Acta Path. Jap. 23(3): 439464, 1973. ISHLHARA, T., UOHINO,F., and TAKAHASHI, M.: Pathological study on amyloidosis. The fine structure of amyloid-laden liver as revealed by freeze-etching. Beta Path. Jap. 26(3): 357-366, 1976. ISHIHARA, T. and UCHINO,F. : Pathological study on amyloidosis. Amyloid formation and resorption in Kupffer cell. Recent Advances in RES Resarch. 15: 14S171, 1975. LUFT, J.H.: Improvement in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9 : 409414, 1961. NOPANLTAYA, W. and ORISHAM, H. W. : Scanning electron microscopy of mouse intrahepatic structures. Exper. Mol. Path. 23: 441458, 1975. J.P. : Experimental amyloidosis. 11. SORENSON, G.D., HEEFNER,W.B., and KIRKPATRICK, Light and electron microscopic observations of liver. Am. J. Path. 44: 629444, 1964. TANAKA, K. : Preened resin cracking method for scanning electron microscopy of biological materials. Naturwiss. 59: 77, 1972. UCHINO,F. : Pathological study on amyloidosis. Role of reticuloendothelial cells in inducing amyloidosis. Acta Path. Jap. 17: 49-82, 1967. UCHINO, F. : Amyloidosis. Histopathological iderpretation of lichen amyloidosis and study on producing mechanism of amyloid substance in experimental cases. J. Jap. Derm atol. 82: 704-715, 1972 (in Japanese).
