Scanning Electron Microscopy of Normal R a t Liver : The Surface Structure of Its Cells and Tissue Components ' J. W. GRISHAM,2 W. NOPANITAYA, J. COMPAGN03
AND
A . E. H . NAGEL Departments of Pathology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 2751 4 and Washington University School of Medicine, S t . Louis, Missouri 63110
ABSTRACT Scanning electron microscopy (SEM) allows the surface ultrastructure of intrahepatic cells and other tissue components of liver to be delineated. Excellent depth of focus of the SEM makes it possible to visualize surfaces of intact cells in their native configurations. This report details the surface characteristics and inter-relationships of hepatocytes and hepatic plates, sinusoidal endothelial cells and sinusoids, presumed Kupffer cells, vessels, bile ducts, connective tissue, and the capsule of rat liver. Hepatocytes present three structurally distinctive faces - the intercellular face containing flat surfaces and bile canaliculus, the sinusoidal face, and the connective tissue face which abuts portal tracts and hepatic veins. Sinusoidal endothelium is penetrated by large (1 to 3 cLm) and small (0.1 cLm) fenestrae, the latter occurring in clusters of up to 50. The width of bile canaliculi and distribution of large fenestrae vary proximodistally along hepatic plate or sinusoid. The cells of portal bile ductules contain microvilli located in linear rows and sparse cilia. Endothelium of hepatic artery and of portal vein is sparsely fenestrated.
Scanning electron microscopy (SEM) has afforded new information on the surface structure of cells and their supracellular organization in a variety of tissues. The liver is only now receiving detailed examination by this technique, mainly because of the difficulty i n adequately preparing the interior of this solid tissue for SEM study (Miyai et al., '74). Illustrations in early studies showed severe artifacts of cellular surface structure caused by uncontrolled drying of tissues (Fugita et al., '71; Bierring and Skaaring, '73). Brooks and Haggis ('73) demonstrated the utility of freeze-drying combined with fracturing of tissue to reveal hepatocyte surfaces. Compagno and Grisham ('74) introduced a simple, rapid method of tissue preparation which combines fixation by aldehyde perfusion, manual fracture by simply breaking hepatic lobes, post-osmication, and critical-point drying. Other methods for preparing liver for SEM examination have since been elaborated which also AM. J. ANAT.,144: 295-322.
yield satisfactory preservation of cellular surface structure (Miyai et al., '74; Motta and Porter, '74). Reports by Brooks and Haggis ('73), Compagno and Grisham ('74), Motta and Porter ('74), Motta and Fumagalli ('74, '75), Miyai et al. ('74), Grisham et al. ('75), and Andrews and Porter ('73) have illustrated certain features of the surfaces of normal and altered liver cells. In this report we extend and refine these earlier findings and detail new observations on hepatocellular structure and hepatic tissue organization. METHODS
Rats employed were adult males and females of Wistar-derived strains, weighing 200 to 300 gm. Livers were fixed by Accepted June 24, '75. 1 Support by grants AM 07595, A,M 17595, GM 092, and GM 097 from the National Institutes of Health. ZReprint requests should be directed to the North Carolina address. 3 Present address: Armed Forces Institute of Pathology, Washington, D.C. 20306.
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intracardiac perfusion of 2.5% glutaraldehyde solution (Fahimi, '67) or 4% paraformaldehyde solution (Carson et al., '72) in phosphate buffer at pH 7.4, using techniques described previously (Compagno and Grisham, '74). I n order to disclose internal hepatic cells and structures for examination by SEM, aldehyde-fixed liver lobes were fractured manually or they were incised with a sharp blade. The latter procedure was effected either by slicing fixed, unembedded liver with a razor blade or by cutting slices of liver, previously embedded in paraffin, with a microtome. Paraffin was removed from slices before they were processed further. Specimens were trimmed into blocks of varying sizes, one surface of which was protected for ultimate examination. After osmication in a 1% or 2% solution of osmium tetroxide in phosphate buffer, tissue blocks were dehydrated in ethanolic solutions, dried using a critical-point method (Anderson, '51), coated with metal (chromium or gold-palladium) and examined in a Cambridge Stereoscan Mark I1 or a Coates and Welter Cwiksc an-104 scanning electron microscope. RESULTS
I. Parenchymal segmentation The segmentation of hepatic parenchyma by the regular interdigitation of hepatic veins and portal tracts was clearly seen in fractured specimens; hepatic veins and portal tracts branched as trees; portal tracts ran in a roughly perpendicular direction about midway between adjacent hepatic venous "limbs" and vice versa (fig. 1 ). Favorably fractured specimens clearly demonstrated a microvascular segment of hepatic parenchyma centered about a terminal portal venule and adjoined at its periphery by one or more hepatic veins (fig. 2). 11. Portal tracts and contents Portal tracts were readily identified on surfaces of tissue blocks prepared by slicing fixed liver (fig. 3 ) , and demonstrated the well-known vascular and biliary ductal components and connective tissue. In favorably sliced specimens the interiors of these structures were exposed.
A. Bile ducts On their lumenal surface, ductal epithelial cells contained frequent microvilli (fig. 4 ) and occasional cilia (fig. 5 ) . Microvilli, about 0.5 pm long by 0.1 wide, were often concentrated in rows along the circumference of the ductal lumen (fig. 4 ) . Randomly located cilia measured about 0.2 to 0.3 @mi n width and more than 5 pm in length (fig. 5 ) . B. Hepatic artery Hepatic arterial branches could be identified by their small size and relatively thick wall (fig. 3 ) . Their endothelium, which was sparsely studded with short, stubby microvilli, typically formed longitudinal ridges (fig. 6 ) , presumably due to contraction of underlying smooth muscle. C. Portal vein Endothelium of portal veins was generally smoother than that of hepatic arteries (fig. 7 ) , although it also contained a few stubby microvilli (fig. 8). Many areas of portal venous endothelium contained scattered holes or fenestrae which varied in size (fig. 8). I n addition, endothelial surfaces of portal veins had many tiny pits or holes near the limits of resolution of the SEM.
D. Connective tissue Connective tissue fibers were poorly seen in cleanly incised portal tracts (fig. 3 ) . However, portal tracts uncovered longitudinally by either fracturing or cutting (fig. 9), contained a mass of twisted, rope-like fibers 0.5 to 1.0 pm in diameter. In some foci these were frayed, forming aggregations of smaller fibers. Intertwined with these large fibers were small (0.05 pm or less) highly branched fibrils. 111. Parenchymal elements
A. Hepatocellular plates Polyhedral, multifaceted hepatocytes formed distinct plates (laminae) which were one cell thick (fig. 10). Plates appeared to be continuous, except for holes where sinusoids penetrated them (lacunae). I n favorably fractured specimens plates were generally straight (fig. 1 0 ) and were oriented directly between adja-
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cent terminal portal and hepatic veins. Neighboring plates were separated by sinusoids (figs. 10-12). 1. Canalicular surface. Opened canaliculi (figs. 11-15) measured from 0.5 Pm to a little over 1.0 @min width; canaliculi adjacent to portal tracts were wider (fig. 1 4 ) than were those at opposite ends of hepatocellular plates (fig. 15). The interiors of narrow canaliculi were obscured by the microvilli crowding their margins (fig. 1 5 ) . Where it was possible to see into the depths of wide canaliculi, microvilli were clearly concentrated at their lateral margins, where adjacent hepatocytes joined (fig. 14). The canalicular surface facing the hepatic cell body was smooth, containing only a few short microvilli and occasional small pits or holes (figs. 13-15). Canaliculi were typically located in the centers of hepatocellular plates, and in many areas they were straight and unbranched (fig. 11). In other situations, canaliculi meandered over the face of hepatocytes (fig. 12) or branched blindly on the surface of single hepatocytes (figs. 13, 20). Blindly ending canalicular branches sometimes extended to within 0.1 pm of the sinusoidal surfaces of hepatocytes (fig. 13), but direct connections between canaliculi and perisinusoidal spaces were not seen. 2. Intercellular cleft. Cell surfaces laterally bordering canaliculi were the smoothest areas of the hepatocyte membrane (figs. 13-15). Although these areas contained attachment complexes, the latter were not visible at the resolution attainable with the SEM. However, even at SEM resolution the so-called flat surface was not free of microvilli, but contained more sparse, stubbier structures than did other surfaces (fig. 14). The flat surface also contained a few processes that were wider than microvilli (about 0.5 +,.m wide by 1.0 pm long), together with about the same number of holes of about equal diameter (figs. 13, 14). The membranes of the flat surface also contained many small pits, the diameters of which were near the limits of resolution of the SEM. 5. Sinusoidal smface. Hepatocellular surf aces facing sinusoids were densely covered by microvilli which averaged about 0.5 Pm long by 0.1 pm wide (fig.
12). Sinusoidal surfaces of rat hepatocytes contained 25 to 50 microvilli/pm2 of cell surface, which largely filled the perisinusoidal space of Disse. Intermixed with microvilli were occasional fibers and cells, which have not yet been clearly identified from their surface characteristics. At the sharply angled corners of hepatocytes, microvillus-studded membranes contiguous with sinusoidal surfaces approached to within 0.1 Pm of canaliculi (fig. 15).
B. Sinusoids In cross-section sinusoids were round or oval, averaging about 20-40 pm in greatest diameter (fig. 21). 1. Sinusoidal endothelium. Sinusoidal endothelium was widely fenestrated (figs. 16-19). Fenestrae differed greatly i n size, but fell into two general size categories, large and small (figs. 18, 19). Large fenestrae measured 1.0 to 3.0 pm in diameter; small fenestrae were typically near 0.1 p in diameter and occurred in clusters of 10 to 50 (figs. 18, 19). Microvilli of the sinusoidal surface of hepatocytes were visible through many fenestrae (fig. 1 9 ) , indicating the lack of a covering membrane. Large fenestrae were more numerous and larger in sinusoidal endothelium near portal tracts (fig. 16), whereas sinusoidal endothelium near terminal hepatic veins contained almost entirely small fenestrae (fig. 17). Thin sinusoidal endothelial cytoplasm extended outward from a plump cell body, which probably contained the nucleus (fig. 21). 2. Kupffer cells. Kupffer cells were not identified unequivocally on the basis of their phagocytic behavior. Sinusoidal cells with plump nonfestrated cytoplasm were suspected to be Kupffer cells (fig. 20). The surfaces of these cells, which projected into sinusoidal lumens, contained small ridged folds, intermingled with stubby microvilli (fig. 20). IV. Hepatic veins
A. Terminal hepatic veins Terminal hepatic veins were usually two to three times the diameter of the sinusoids that drained directly into them, and their walls consisted of little more
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than endothelium overlying a delicate reticular stroma (fig. 22). Endothelium with small fenestrations, typical of that found in the distal end of sinusoids, extended into terminal hepatic veins for short distances around sinusoidal entrances (fig. 23). Surfaces of endothelial cells contained sparse stubby microvilli (fig. 23).
B. Larger hepatic veins Larger hepatic veins had walls with progressively heavier connective tissue reinforcement (figs. 24, 25). Some sinusoids emptied directly into second order hepatic veins (fig. 24), but larger branches of the hepatic veins did not receive sinusoidal openings (fig. 25). Fibers, presumably collagen, i n the walls of tertiary and larger hepatic veins formed thick, ropelike bundles (fig. 25). Continuous endothelium was disposed in gentle undulations, presumably reflecting the location of cell nuclei (fig. 25).
V. Portal and hepatic canals Fracturing fixed hepatic parenchyma avulsed the collagen-rich contents of some portal and hepatic canals, which left these channels through the parenchymal substance empty. Large portal and hepatic canals were lined by a nearly continuous layer of hepatocytes (fig. 26), unlike terminal structures which were penetrated frequently by capillary-sized openings. The surfaces of hepatocytes forming the walls of portal and hepatic canals contained flattened or leaf-shaped microvilli (fig. 27). This hepatocyte surface also contained characteristic smooth linear indentations (fig. 27), which appeared to represent molding of cells around adjacent connective tissue fibers. VI. Capsular surface The hepatic capsule was covered by a continuous layer of serosal cells which were studded with large numbers of microvilli of various lengths (figs. 28, 29). Many microvilli were long and filamentous, measuring up to 2 pm long by 0.1 pm thick. Areas of serosa covered by such tall microvilli alternated with foci covered by stubby structures (about 0.5 p long) (figs. 28, 29).
DISCUSSION
SEM clearly demonstrates the laminar structure of hepatic plates (Brooks and Haggis, '73; Compagno and Grisham ,'74; Miyai et al., '74; Motta and Porter, '74; Motta and Fumagali, '75), first forcefully described by Elias ('49) from his studies on wax reconstructions of serially-sectioned liver. This SEM study clearly shows that channels which carry larger portal tracts and hepatic veins through the parenchymal substance (portal and hepatic canals) are lined by a nearly continuous layer of hepatocytes. These more-or-less continuous plates of hepatocytes that surround larger portal and hepatic canals were noted in LM studies and termed the periportal and perihepatic "limiting plates" (Elias, '49). The surfaces of hepatocytes which form the connective tissue face of portal and hepatic limiting plates are covered with unusual, flattened microvilli, except for smooth linear indentions presumed to be caused by pressure from closely applied connective tissue fibers. Individual hepatocytes which form interlobular plates are rhomboid and sharply angulated, each having several surf ace facets (Elias and Sherrick, '69). Three distinct types of cell surfaces, readily seen with the SEM, occur among these several facets: the canalicular surface, the flat or smooth intercellular surface, and the sinusoidal surface (Heath and Wissig, '66). Fracturing of fixed liver for SEM separates adjacent hepatocytes by breaking junctional complexes and exposes hemicanaliculi (Compagno and Grisham, '74; Miyai et al., '74; Motta and Porter, '74). Canaliculi typically occupy the centers of the intercellular faces of hepatocytes, and usually are as straight as the plates of which they are a part. However, in many situations canaliculi meander over the face of individual hepatocytes and they sometimes have branches that end blindly near the sinusoidal surf aces (Compagno and Grisham, '74; Motta and Fumagalli, '75). Similar branches have been discerned by Matter et al. ('69) from studies utilizing reconstructions of TEM photographs, obtained from serial thin sections. Tips of canalicular branches come close to the space of Disse, without apparently directly connecting with that space
SEM OF RAT LIVER
(Compagno and Grisham, '74; Motta and Porter, '75). This study has also disclosed that the largest bile canaliculi are located in the portal ends of the hepatocellular plates. Microvilli appear to fill small canaliculi but they are clearly clustered at the lateral margins of large canaliculi (Motta and Porter, '74; Motta and Fumagalli, '75). I n large canaliculi the surface facing the cell body is almost free of microvilli and it is smooth except for a few tiny pits or holes. The intercellular face of hepatocytes laterally bordering biliary canaliculi is relatively smooth, but still it contains a variety of pits, holes, and stubby protrusions (Brooks and Haggis, '73; Compagno and Grisham, '74; Motta and Fumagalli, '75). The largest holes and protrusions appear to correspond to the so-called stud or collar-button processes, which have been noted by TEM and are hypothesized to have a role i n hepatocyte attachment (Fawcett, '55; Bruni and Porter, '65; Heath and Wissig, '66; Tandler and Hoppel, '74). Sinusoidal endothelial cells contain numerous cytoplasmic fenestrae (Brooks and Haggis, '73; Miyai et al., '74; Motta and Porter, '74). Two general size classes of fenestrae are present in rat liver endothelium, i.e., greater than 1 pm and less than 0.2 pm in diameter (Motta and Porter, '74). Small fenestrae are frequently clustered into groups of 50 or more and are located in endothelium throughout sinusoids, and this study shows that they even extend for short distances into terminal portal and hepatic venules around sinusoidal openings. Large fenestrae are more numerous and/or larger in endothelium at the portal ends of sinusoids and they are uncommon adjacent to terminal hepatic veins. Some fenestrae penetrate sinusoidal endothelial cells completely, since microvilli of the sinusoidal surface of hepatocytes are visible through them. The question of gaps or fenestrae i n sinusoidal endothelium has been the subject of some disagreement in the past. TEM studies have consistently shown discontinuities in profiles of sinusoidal endothelium (Fawcett, '55; Bruni and Porter, '65). However, many investigators have believed that these gaps were the result
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of preparative artifact (Aterman, '64). Wisse ('70), Orci et al. ('71), and Ogawa e t al. ('73) have conclusively demonstrated small fenestrae by the use of freeze-etching combined with surf ace replication and TEM, and Ogawa et al. ('73) also noted large fenestrae with this technique. The functional distinction between large and small fenestrae and the physiological meaning of the differential proximodistal distribution of large fenestrae along sinusoids remains to be shown. It is evident that the plasmatic portion, but not cells, of sinusoidal blood has free access to the perisinusoidal space and the sinusoidal surface of hepatocytes. Sinusoidal endothelial cells are morphologically distinct from the cells which are presumed to be Kupffer cells. Presumptive Kupffer cells face sinusoidal lumens interspersed with endothelial cells. However, unlike the latter they are plump and their surface is wrinkled by numerous small cerebroid ridges and by occasional microvilli. Previous studies combining LM and TEM with various tracer techniques have indicated the cytologic a1 and function a1 distinctiveness of Kupffer cells and sinusoidal lining cells (Wisse, '70; Widman et al., '72). This SEM study is compatible with these views, but definitive identification of Kupffer cells on the basis of their surface structure awaits the combination OF SEM with cellular uptake of particulate material. Endothelium of larger portal and hepatic veins appears similar to that already noted in SEM studies of various endothelia (Shimamato et al., '69). A curious difference pertaining to portal veins is that the endothelium of some is infrequently fenestrated. The possibility that these holes represent an artifactitious opening of tight junctions resulting from mild damage has not been rigorously excluded, but they are regularly seen only in portal veins. Terminal portal and hepatic venules in the rat differ little in structure from large sinusoids, fenestrated endothelium even extends for short distances into both types of terminal hepatic vessels about sinusoidal openings. Terminal venules have very weakly supported walls, consisting only of a few reticulin or collagen fibers overlaid by endothelium, and both types of
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vessels communicate widely with sinusoids. Study of advantageous fracture planes by SEM corroborates the existence of a microscopic unit of parenchyma which surrounds a terminal portal venule and at its periphery merges with one or more terminal hepatic venules. This unit of tissue corresponds to the acinus, delineated by Rappaport ('73) from studies combining LM with vascular injections of various dyes. TEM studies have demonstrated that intrahepatic bile ducts are composed of cells whose lumenal surface contains numerous microvilli and infrequent long cilia (Grisham, '63). However, SEM examination shows that cilia are much more numerous than the isolated TEM observations have suggested (Motta and Fumagalli, '75). This study discloses that ductal microvilli are concentrated in longitudinal rows, the reason for which is not known. Surface microvilli on capsular mesothelial cells of the liver are similar to those covering other viscera (Andrews and Porter, '73). Mesothelial microvilli hypothetically are involved in exchange of materials between liver and peritoneal cavity (Odor, '54) or in maintaining, with mucins, surface lubricity (Andrews and Porter, '73). LITERATURE CITED Anderson, T. F. 1951 Technique for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N. Y. Acad. Sci. Ser. 111, 13: 130-134. 1973 The Andrews, P. M., and K. R. Porter ultrastructural morphology and possible significance of mesothelial microvilli. Anat. Rec., 177: 409426. Aterman, K. 1964 The structure of liver sinusoids and the sinusoidal cells. In: The Liver. C. Rouiller, ed. Academic Press, Inc. New York, pp. 61-136. Bierring, F., and P. Skaaring 1973 Scanning electron microscopy of the liver. JEOL News, I l e : 16-17. Brooks, S. E. H., and G. H. Haggis 1973 Scanning electron microscopy of rat's liver. Application of freeze-fracture and freeze-drying techniques. Lab. Invest., 29: 60-64. Bruni, C., and K. R. Porter 1965 The fine structure of the parenchymal cell of the normal rat liver. I. General observations. Am. J. Path., 56: 691-775. Carson, F., J. A. Lynn and J. H. Martin 1972 Ultrastructural effects of various buffers, osmolarity, and temperature on paraformaldehyde fixation of the formed elements of blood and bone marrow. Texas Rep. Biol. Med., 30: 125142.
Compagno, J., and J. W. Grisham 1974 Scanning electron microscopy of extrahepatic biliary obstruction. Arch. Path., 97: 348-351. Elias, H. 1949 A re-examination of the structure of mammalian liver. I. Parenchymal architecture. Am. J. Anat., 84: 311-334. Elias, H., and J. L. Sherrick 1969 Morphology of the liver. Academic Press, Inc., New York, 389 pp. Fahimi, H. D. 1967 Perfusion and immersion h a t i o n of rat liver with glutaraldehyde. Lab. Invest., 16: 736-750. Fawcett, D. W. 1955 Observations o n the cytology and electron microscopy of hepatic cells. J. Nat. Cancer Inst., 15: 1457-1502. Fugita, T., J. Tokumaga and H. Inoue 1971 Atlas of scanning electron microscopy, Igaku Shoin, Tokyo, pp. 20-21. Grisham, J. W. 1963 Ciliated cells i n normal murine intrahepatic bile ducts. Proc. SOC.Exp. Biol. Med., 144: 318-320. Grisham, J. W., R. L. Tillman, A. E. H. Nagel and J. Compagno 1975 Ultrastructure of the proliferating hepatocyte: Sinusoidal surfaces and endoplasmic reticulum. In: Liver Regeneration after Experimental Injury. R. Lesch and W. Reuttner, eds. Intern. Med. Book Corp., New York, pp. 6-23. Heath, T., and S. L. Wissig 1966 Fine structure of the surface of mouse hepatic cells. Am. J. Anat., 119: 97-128. Matter, A., L. Orci and C. Rouiller 1969 A study on the permeability barriers between Disse's space and the bile canaliculus. J. Ultrastruct. Res. (Suppl.), 11: 1-71. Miyai, K., R. M. Wagner and A. L. Richardson 1974 Preparation of liver for combined SEM and TEM study. In: Scanning Electron Microscopy/l974. 0. Johari, ed. IIT Research Inst., Chicago, pp. 283-290. Motta, P., and G. Fumagalli 1974 Scanning electron microscopy demonstration of cilia i n rat intrahepatic bile ducts. Z. Anat. Entwickl. Gesch., 145: 223-226. 1975 Structure of rat bile canaliculi as revealed by scanning electron microscopy. Anat. Rec., 182: 499-513. Motta, P., and K. R. Porter 1974 Structure of rat liver sinusoids and associated tissue spaces as revealed by scanning electron microscopy. Cell Tiss. Res., 148: 111-125. Odor, D. L. 1954 Observation on rat mesothelium with the electron and phase microscopes. Am. J. Anat., 95: 433-465. Ogawa, K., T. Minase, K. Enomoto and T. Onoe 1973 Ultrastructure of fenestrated cells in the sinusoidal wall of rat liver after perfusion fixation. Tohoku J. Exp. Med., 110: 89-101. Orci, L., A. Matter and C. Rouiller 1971 A comparative study of freeze-etch replicas and thin sections of rat liver. J. Ultrastruct. Res., 35: 1-19. Rappaport, A. M. 1973 The microcirculatory hepatic unit. Microvasc. Res., 6: 212-228. Shimamoto, T., Y. Yamashita and T. Sunaga 1969 Scanning electron microscope observations of the endothelial surface of heart and blood vessels. Proc. Japan Acad., 45: 507-512.
SEM O F RAT LIVER Tandler, B., and C. L. Hoppel 1974 Subsurface cisterns in mouse hepatocytes. Anat. Rec., 179: 273-284. Wisse, E. 1970 An electron microscopic study of the fenestrated lining of rat liver sinusoids. J. Ultrastruct. Res., 31: 125-150.
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Widman, J. J., R. S. Cotran and H . D. Fahimi 1972 Mononuclear phagocytes (Kupffer cells) and endothelial cells. Identification of two functional cell types i n rat liver sinusoids by endogenous peroxidase activity. J. Cell Biol., 52: 159-170.
PLATE 1 EXPLANATION O F FIGURES
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1
Fractured liver surface showing the tree-like branching of a portal canal (P). x 50.
2
Microscopic segmentation of hepatic parenchyma. Hepatic plates run from a terminal portal canal ( P ) toward a terminal hepatic venule (arrow). x 110.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 1
303
PLATE 2 EXPLANATION OF FIGURES
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3
Sliced liver surface showing a transected portal tract. Portal vein ( V ) and hepatic artery ( A ) are indicated. Two sectioned bile ducts are located to the left of the vein and the right of the artery. X 525.
4
Lumenal surface of a n intrahepatic bile duct showing focally concentrated microvilli. x 13,650.
5
A cilium protruding from the lumenal surface of a n intrahepatic bile duct. Microvilli are much shorter. x 17,650.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE a
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PLATE 3 EXPLANATION O F FIGUEES
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6
Endothelial ( E ) surface of hepatic artery i n the liver. Ridges of endothelium are presumed to be caused by contraction of subjacent smooth muscle cells. Sparse, short microvilli occupy the surface of endothelial cells. X 6,825.
7
Interior of a portal vein with a sinusoidal opening ( S ) containing a leukocyte (WBC). Shingle-like endothelial cells contain sparse microvilli on their surfaces. ~ 4 , 9 0 0 .
8
Detail of endothelial surface of portal vein showing microvilli (arrow heads) and fenestrae (arrows). x 7,840.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 3
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PLATE 4 EXPLANATION OF FIGURES
9 10
Rope-like bundles and filaments of connective tissue fibers on exterior of a portal tract. x 1,470. Interdigitation of hepatic plates and sinusoids ( S ) .
x 1,270.
11 Detail of one-cell thick hepatocytic plate showing a n unroofed bile canaliculus (arrows) i n its center. Sinusoids ( S ) on either side of the hepatocytic plate contain erythrocytes. X 5,250.
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SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 4
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PLATE 5 EXPLANATION O F FIGURES
12
A similar hepatocytic plate i n which the canaliculus is not so precisely centered. Sinusoidal endothelium rests on microvilli forming the hepatocytic sinusoidal surface. (Sinusoids, S ) . x 5,250.
13 Intercellular surface of a single hepatocyte containing a highly branched bile canaliculus (BC). Several branches end blindly near the sinusoidal surface. The flat intercellular surface contains several holes (arrows) and protrusions. X 7,350.
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PLATE 5
Grisham, Nopanitaya, Compagno and Nagel
31 1
PLATE 6 EXPLANATION OF FIGURES
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14
Surface of hepatocyte located at the portal end of a n hepatocytic plate. The bile canaliculus ( B C ) is broad and its microvilli are concentrated on its lateral margins. Holes (arrows) and studs (arrow heads) occupy the flat intercellular space. Dense populations of microvilli on the sinusoidal surface of this cell are seen at the left and right sides of the photograph. x 13,000.
15
Surface of hepatocyte located at the hepatic venous end of a n hepatocytic plate. The bile canaliculus ( B C ) is much narrower than at the portal end (above). At the acute margins of the cell, the microvilluscovered sinusoidal surface extends close to the canaliculus. x 13,000.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 6
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PLATE 7 EXPLANATION OF FIGURES
16 The portal end of a sinusoid is lined with endothelium containing many large fenestrae. Clusters of small fenestrae are also present. X 4,200.
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17
The hepatic venous end of a sinusoid contains endothelium with only small fenestrae. x 5,250.
18
Sinusoidal endothelium showing both large and small fenestrae. x 15,680.
19
Microvilli o n the sinusoidal surface of underlying hepatocytes (arrows) are visible through some fenestrae. x 19,600.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 7
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PLATE 8 EXPLANATION OF FIGURES
316
20
Portion of a sinusoid ( S ) and hepatocytic plate. A presumed Kupffer cell ( K ) is continuous with the sinusoidal endothelium. x 3,190.
21
Oval cross-section of a sinusoid ( S ) showing a n endothelial cell body (E ), which gradually thins peripherally to form typically fenestrated cytoplasm. x 4,550.
22
Cross-section of a terminal hepatic vein (THV) which is about three times the diameter of sinusoids draining into it. X 800.
23
Endothelial surface of a terminal hepatic vein with a sinusoidal opening ( S ) . Endothelium about the sinusoidal opening is fenestrated although elsewhere it is continuous and contains sparse microvilli. X 6,300.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 8
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PLATE 9 EXPLANATION OF FIGURES
318
24
Endothelial surface and transected wall of a second order hepatic vein, penetrated by sinusoidal openings (arrows) and containing sparse connective tissue fibers i n its walls. x 1,000.
25
A large hepatic vein whose wall is composed of thick bundles of connective tissue fibers. The endothelium (upper right) is continuous and is disposed i n gentle undulations. Sinusoids do not open into this vein. X 2,500.
26
A portal canal from which the portal tract has been torn away. The portal canal is lined by a nearly continuous plate of hepatocytes (limiting plate), penetrated only occasionally by openings (arrows). Canals containing hepatic veins are structurally similar. x 525.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 9
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PLATE 10 EXPLANATION O F FIGURES
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27
Detail of the surface of a n hepatocyte in the limiting plate. This surface faces the connective tissue of the portal tract or hepatic vein and is distinguished by irregular, flattened microvilli and smooth linear indentions (I). The latter are presumed to reflect molding of the hepatocyte around connective tissue fibers.
28
Peritoneal surface of mesothelial cells ( M ) on the capsule of the liver. Patches of filamentous microvilli alternate with areas devoid of these structures. x 3,180.
29
Both long and short microvilli are present on the peritoneal surface of mesothelial cells of the hepatic capsule. x 10,920.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 10
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AND
A . E. H . NAGEL Departments of Pathology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 2751 4 and Washington University School of Medicine, S t . Louis, Missouri 63110
ABSTRACT Scanning electron microscopy (SEM) allows the surface ultrastructure of intrahepatic cells and other tissue components of liver to be delineated. Excellent depth of focus of the SEM makes it possible to visualize surfaces of intact cells in their native configurations. This report details the surface characteristics and inter-relationships of hepatocytes and hepatic plates, sinusoidal endothelial cells and sinusoids, presumed Kupffer cells, vessels, bile ducts, connective tissue, and the capsule of rat liver. Hepatocytes present three structurally distinctive faces - the intercellular face containing flat surfaces and bile canaliculus, the sinusoidal face, and the connective tissue face which abuts portal tracts and hepatic veins. Sinusoidal endothelium is penetrated by large (1 to 3 cLm) and small (0.1 cLm) fenestrae, the latter occurring in clusters of up to 50. The width of bile canaliculi and distribution of large fenestrae vary proximodistally along hepatic plate or sinusoid. The cells of portal bile ductules contain microvilli located in linear rows and sparse cilia. Endothelium of hepatic artery and of portal vein is sparsely fenestrated.
Scanning electron microscopy (SEM) has afforded new information on the surface structure of cells and their supracellular organization in a variety of tissues. The liver is only now receiving detailed examination by this technique, mainly because of the difficulty i n adequately preparing the interior of this solid tissue for SEM study (Miyai et al., '74). Illustrations in early studies showed severe artifacts of cellular surface structure caused by uncontrolled drying of tissues (Fugita et al., '71; Bierring and Skaaring, '73). Brooks and Haggis ('73) demonstrated the utility of freeze-drying combined with fracturing of tissue to reveal hepatocyte surfaces. Compagno and Grisham ('74) introduced a simple, rapid method of tissue preparation which combines fixation by aldehyde perfusion, manual fracture by simply breaking hepatic lobes, post-osmication, and critical-point drying. Other methods for preparing liver for SEM examination have since been elaborated which also AM. J. ANAT.,144: 295-322.
yield satisfactory preservation of cellular surface structure (Miyai et al., '74; Motta and Porter, '74). Reports by Brooks and Haggis ('73), Compagno and Grisham ('74), Motta and Porter ('74), Motta and Fumagalli ('74, '75), Miyai et al. ('74), Grisham et al. ('75), and Andrews and Porter ('73) have illustrated certain features of the surfaces of normal and altered liver cells. In this report we extend and refine these earlier findings and detail new observations on hepatocellular structure and hepatic tissue organization. METHODS
Rats employed were adult males and females of Wistar-derived strains, weighing 200 to 300 gm. Livers were fixed by Accepted June 24, '75. 1 Support by grants AM 07595, A,M 17595, GM 092, and GM 097 from the National Institutes of Health. ZReprint requests should be directed to the North Carolina address. 3 Present address: Armed Forces Institute of Pathology, Washington, D.C. 20306.
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intracardiac perfusion of 2.5% glutaraldehyde solution (Fahimi, '67) or 4% paraformaldehyde solution (Carson et al., '72) in phosphate buffer at pH 7.4, using techniques described previously (Compagno and Grisham, '74). I n order to disclose internal hepatic cells and structures for examination by SEM, aldehyde-fixed liver lobes were fractured manually or they were incised with a sharp blade. The latter procedure was effected either by slicing fixed, unembedded liver with a razor blade or by cutting slices of liver, previously embedded in paraffin, with a microtome. Paraffin was removed from slices before they were processed further. Specimens were trimmed into blocks of varying sizes, one surface of which was protected for ultimate examination. After osmication in a 1% or 2% solution of osmium tetroxide in phosphate buffer, tissue blocks were dehydrated in ethanolic solutions, dried using a critical-point method (Anderson, '51), coated with metal (chromium or gold-palladium) and examined in a Cambridge Stereoscan Mark I1 or a Coates and Welter Cwiksc an-104 scanning electron microscope. RESULTS
I. Parenchymal segmentation The segmentation of hepatic parenchyma by the regular interdigitation of hepatic veins and portal tracts was clearly seen in fractured specimens; hepatic veins and portal tracts branched as trees; portal tracts ran in a roughly perpendicular direction about midway between adjacent hepatic venous "limbs" and vice versa (fig. 1 ). Favorably fractured specimens clearly demonstrated a microvascular segment of hepatic parenchyma centered about a terminal portal venule and adjoined at its periphery by one or more hepatic veins (fig. 2). 11. Portal tracts and contents Portal tracts were readily identified on surfaces of tissue blocks prepared by slicing fixed liver (fig. 3 ) , and demonstrated the well-known vascular and biliary ductal components and connective tissue. In favorably sliced specimens the interiors of these structures were exposed.
A. Bile ducts On their lumenal surface, ductal epithelial cells contained frequent microvilli (fig. 4 ) and occasional cilia (fig. 5 ) . Microvilli, about 0.5 pm long by 0.1 wide, were often concentrated in rows along the circumference of the ductal lumen (fig. 4 ) . Randomly located cilia measured about 0.2 to 0.3 @mi n width and more than 5 pm in length (fig. 5 ) . B. Hepatic artery Hepatic arterial branches could be identified by their small size and relatively thick wall (fig. 3 ) . Their endothelium, which was sparsely studded with short, stubby microvilli, typically formed longitudinal ridges (fig. 6 ) , presumably due to contraction of underlying smooth muscle. C. Portal vein Endothelium of portal veins was generally smoother than that of hepatic arteries (fig. 7 ) , although it also contained a few stubby microvilli (fig. 8). Many areas of portal venous endothelium contained scattered holes or fenestrae which varied in size (fig. 8). I n addition, endothelial surfaces of portal veins had many tiny pits or holes near the limits of resolution of the SEM.
D. Connective tissue Connective tissue fibers were poorly seen in cleanly incised portal tracts (fig. 3 ) . However, portal tracts uncovered longitudinally by either fracturing or cutting (fig. 9), contained a mass of twisted, rope-like fibers 0.5 to 1.0 pm in diameter. In some foci these were frayed, forming aggregations of smaller fibers. Intertwined with these large fibers were small (0.05 pm or less) highly branched fibrils. 111. Parenchymal elements
A. Hepatocellular plates Polyhedral, multifaceted hepatocytes formed distinct plates (laminae) which were one cell thick (fig. 10). Plates appeared to be continuous, except for holes where sinusoids penetrated them (lacunae). I n favorably fractured specimens plates were generally straight (fig. 1 0 ) and were oriented directly between adja-
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cent terminal portal and hepatic veins. Neighboring plates were separated by sinusoids (figs. 10-12). 1. Canalicular surface. Opened canaliculi (figs. 11-15) measured from 0.5 Pm to a little over 1.0 @min width; canaliculi adjacent to portal tracts were wider (fig. 1 4 ) than were those at opposite ends of hepatocellular plates (fig. 15). The interiors of narrow canaliculi were obscured by the microvilli crowding their margins (fig. 1 5 ) . Where it was possible to see into the depths of wide canaliculi, microvilli were clearly concentrated at their lateral margins, where adjacent hepatocytes joined (fig. 14). The canalicular surface facing the hepatic cell body was smooth, containing only a few short microvilli and occasional small pits or holes (figs. 13-15). Canaliculi were typically located in the centers of hepatocellular plates, and in many areas they were straight and unbranched (fig. 11). In other situations, canaliculi meandered over the face of hepatocytes (fig. 12) or branched blindly on the surface of single hepatocytes (figs. 13, 20). Blindly ending canalicular branches sometimes extended to within 0.1 pm of the sinusoidal surfaces of hepatocytes (fig. 13), but direct connections between canaliculi and perisinusoidal spaces were not seen. 2. Intercellular cleft. Cell surfaces laterally bordering canaliculi were the smoothest areas of the hepatocyte membrane (figs. 13-15). Although these areas contained attachment complexes, the latter were not visible at the resolution attainable with the SEM. However, even at SEM resolution the so-called flat surface was not free of microvilli, but contained more sparse, stubbier structures than did other surfaces (fig. 14). The flat surface also contained a few processes that were wider than microvilli (about 0.5 +,.m wide by 1.0 pm long), together with about the same number of holes of about equal diameter (figs. 13, 14). The membranes of the flat surface also contained many small pits, the diameters of which were near the limits of resolution of the SEM. 5. Sinusoidal smface. Hepatocellular surf aces facing sinusoids were densely covered by microvilli which averaged about 0.5 Pm long by 0.1 pm wide (fig.
12). Sinusoidal surfaces of rat hepatocytes contained 25 to 50 microvilli/pm2 of cell surface, which largely filled the perisinusoidal space of Disse. Intermixed with microvilli were occasional fibers and cells, which have not yet been clearly identified from their surface characteristics. At the sharply angled corners of hepatocytes, microvillus-studded membranes contiguous with sinusoidal surfaces approached to within 0.1 Pm of canaliculi (fig. 15).
B. Sinusoids In cross-section sinusoids were round or oval, averaging about 20-40 pm in greatest diameter (fig. 21). 1. Sinusoidal endothelium. Sinusoidal endothelium was widely fenestrated (figs. 16-19). Fenestrae differed greatly i n size, but fell into two general size categories, large and small (figs. 18, 19). Large fenestrae measured 1.0 to 3.0 pm in diameter; small fenestrae were typically near 0.1 p in diameter and occurred in clusters of 10 to 50 (figs. 18, 19). Microvilli of the sinusoidal surface of hepatocytes were visible through many fenestrae (fig. 1 9 ) , indicating the lack of a covering membrane. Large fenestrae were more numerous and larger in sinusoidal endothelium near portal tracts (fig. 16), whereas sinusoidal endothelium near terminal hepatic veins contained almost entirely small fenestrae (fig. 17). Thin sinusoidal endothelial cytoplasm extended outward from a plump cell body, which probably contained the nucleus (fig. 21). 2. Kupffer cells. Kupffer cells were not identified unequivocally on the basis of their phagocytic behavior. Sinusoidal cells with plump nonfestrated cytoplasm were suspected to be Kupffer cells (fig. 20). The surfaces of these cells, which projected into sinusoidal lumens, contained small ridged folds, intermingled with stubby microvilli (fig. 20). IV. Hepatic veins
A. Terminal hepatic veins Terminal hepatic veins were usually two to three times the diameter of the sinusoids that drained directly into them, and their walls consisted of little more
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than endothelium overlying a delicate reticular stroma (fig. 22). Endothelium with small fenestrations, typical of that found in the distal end of sinusoids, extended into terminal hepatic veins for short distances around sinusoidal entrances (fig. 23). Surfaces of endothelial cells contained sparse stubby microvilli (fig. 23).
B. Larger hepatic veins Larger hepatic veins had walls with progressively heavier connective tissue reinforcement (figs. 24, 25). Some sinusoids emptied directly into second order hepatic veins (fig. 24), but larger branches of the hepatic veins did not receive sinusoidal openings (fig. 25). Fibers, presumably collagen, i n the walls of tertiary and larger hepatic veins formed thick, ropelike bundles (fig. 25). Continuous endothelium was disposed in gentle undulations, presumably reflecting the location of cell nuclei (fig. 25).
V. Portal and hepatic canals Fracturing fixed hepatic parenchyma avulsed the collagen-rich contents of some portal and hepatic canals, which left these channels through the parenchymal substance empty. Large portal and hepatic canals were lined by a nearly continuous layer of hepatocytes (fig. 26), unlike terminal structures which were penetrated frequently by capillary-sized openings. The surfaces of hepatocytes forming the walls of portal and hepatic canals contained flattened or leaf-shaped microvilli (fig. 27). This hepatocyte surface also contained characteristic smooth linear indentations (fig. 27), which appeared to represent molding of cells around adjacent connective tissue fibers. VI. Capsular surface The hepatic capsule was covered by a continuous layer of serosal cells which were studded with large numbers of microvilli of various lengths (figs. 28, 29). Many microvilli were long and filamentous, measuring up to 2 pm long by 0.1 pm thick. Areas of serosa covered by such tall microvilli alternated with foci covered by stubby structures (about 0.5 p long) (figs. 28, 29).
DISCUSSION
SEM clearly demonstrates the laminar structure of hepatic plates (Brooks and Haggis, '73; Compagno and Grisham ,'74; Miyai et al., '74; Motta and Porter, '74; Motta and Fumagali, '75), first forcefully described by Elias ('49) from his studies on wax reconstructions of serially-sectioned liver. This SEM study clearly shows that channels which carry larger portal tracts and hepatic veins through the parenchymal substance (portal and hepatic canals) are lined by a nearly continuous layer of hepatocytes. These more-or-less continuous plates of hepatocytes that surround larger portal and hepatic canals were noted in LM studies and termed the periportal and perihepatic "limiting plates" (Elias, '49). The surfaces of hepatocytes which form the connective tissue face of portal and hepatic limiting plates are covered with unusual, flattened microvilli, except for smooth linear indentions presumed to be caused by pressure from closely applied connective tissue fibers. Individual hepatocytes which form interlobular plates are rhomboid and sharply angulated, each having several surf ace facets (Elias and Sherrick, '69). Three distinct types of cell surfaces, readily seen with the SEM, occur among these several facets: the canalicular surface, the flat or smooth intercellular surface, and the sinusoidal surface (Heath and Wissig, '66). Fracturing of fixed liver for SEM separates adjacent hepatocytes by breaking junctional complexes and exposes hemicanaliculi (Compagno and Grisham, '74; Miyai et al., '74; Motta and Porter, '74). Canaliculi typically occupy the centers of the intercellular faces of hepatocytes, and usually are as straight as the plates of which they are a part. However, in many situations canaliculi meander over the face of individual hepatocytes and they sometimes have branches that end blindly near the sinusoidal surf aces (Compagno and Grisham, '74; Motta and Fumagalli, '75). Similar branches have been discerned by Matter et al. ('69) from studies utilizing reconstructions of TEM photographs, obtained from serial thin sections. Tips of canalicular branches come close to the space of Disse, without apparently directly connecting with that space
SEM OF RAT LIVER
(Compagno and Grisham, '74; Motta and Porter, '75). This study has also disclosed that the largest bile canaliculi are located in the portal ends of the hepatocellular plates. Microvilli appear to fill small canaliculi but they are clearly clustered at the lateral margins of large canaliculi (Motta and Porter, '74; Motta and Fumagalli, '75). I n large canaliculi the surface facing the cell body is almost free of microvilli and it is smooth except for a few tiny pits or holes. The intercellular face of hepatocytes laterally bordering biliary canaliculi is relatively smooth, but still it contains a variety of pits, holes, and stubby protrusions (Brooks and Haggis, '73; Compagno and Grisham, '74; Motta and Fumagalli, '75). The largest holes and protrusions appear to correspond to the so-called stud or collar-button processes, which have been noted by TEM and are hypothesized to have a role i n hepatocyte attachment (Fawcett, '55; Bruni and Porter, '65; Heath and Wissig, '66; Tandler and Hoppel, '74). Sinusoidal endothelial cells contain numerous cytoplasmic fenestrae (Brooks and Haggis, '73; Miyai et al., '74; Motta and Porter, '74). Two general size classes of fenestrae are present in rat liver endothelium, i.e., greater than 1 pm and less than 0.2 pm in diameter (Motta and Porter, '74). Small fenestrae are frequently clustered into groups of 50 or more and are located in endothelium throughout sinusoids, and this study shows that they even extend for short distances into terminal portal and hepatic venules around sinusoidal openings. Large fenestrae are more numerous and/or larger in endothelium at the portal ends of sinusoids and they are uncommon adjacent to terminal hepatic veins. Some fenestrae penetrate sinusoidal endothelial cells completely, since microvilli of the sinusoidal surface of hepatocytes are visible through them. The question of gaps or fenestrae i n sinusoidal endothelium has been the subject of some disagreement in the past. TEM studies have consistently shown discontinuities in profiles of sinusoidal endothelium (Fawcett, '55; Bruni and Porter, '65). However, many investigators have believed that these gaps were the result
299
of preparative artifact (Aterman, '64). Wisse ('70), Orci et al. ('71), and Ogawa e t al. ('73) have conclusively demonstrated small fenestrae by the use of freeze-etching combined with surf ace replication and TEM, and Ogawa et al. ('73) also noted large fenestrae with this technique. The functional distinction between large and small fenestrae and the physiological meaning of the differential proximodistal distribution of large fenestrae along sinusoids remains to be shown. It is evident that the plasmatic portion, but not cells, of sinusoidal blood has free access to the perisinusoidal space and the sinusoidal surface of hepatocytes. Sinusoidal endothelial cells are morphologically distinct from the cells which are presumed to be Kupffer cells. Presumptive Kupffer cells face sinusoidal lumens interspersed with endothelial cells. However, unlike the latter they are plump and their surface is wrinkled by numerous small cerebroid ridges and by occasional microvilli. Previous studies combining LM and TEM with various tracer techniques have indicated the cytologic a1 and function a1 distinctiveness of Kupffer cells and sinusoidal lining cells (Wisse, '70; Widman et al., '72). This SEM study is compatible with these views, but definitive identification of Kupffer cells on the basis of their surface structure awaits the combination OF SEM with cellular uptake of particulate material. Endothelium of larger portal and hepatic veins appears similar to that already noted in SEM studies of various endothelia (Shimamato et al., '69). A curious difference pertaining to portal veins is that the endothelium of some is infrequently fenestrated. The possibility that these holes represent an artifactitious opening of tight junctions resulting from mild damage has not been rigorously excluded, but they are regularly seen only in portal veins. Terminal portal and hepatic venules in the rat differ little in structure from large sinusoids, fenestrated endothelium even extends for short distances into both types of terminal hepatic vessels about sinusoidal openings. Terminal venules have very weakly supported walls, consisting only of a few reticulin or collagen fibers overlaid by endothelium, and both types of
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vessels communicate widely with sinusoids. Study of advantageous fracture planes by SEM corroborates the existence of a microscopic unit of parenchyma which surrounds a terminal portal venule and at its periphery merges with one or more terminal hepatic venules. This unit of tissue corresponds to the acinus, delineated by Rappaport ('73) from studies combining LM with vascular injections of various dyes. TEM studies have demonstrated that intrahepatic bile ducts are composed of cells whose lumenal surface contains numerous microvilli and infrequent long cilia (Grisham, '63). However, SEM examination shows that cilia are much more numerous than the isolated TEM observations have suggested (Motta and Fumagalli, '75). This study discloses that ductal microvilli are concentrated in longitudinal rows, the reason for which is not known. Surface microvilli on capsular mesothelial cells of the liver are similar to those covering other viscera (Andrews and Porter, '73). Mesothelial microvilli hypothetically are involved in exchange of materials between liver and peritoneal cavity (Odor, '54) or in maintaining, with mucins, surface lubricity (Andrews and Porter, '73). LITERATURE CITED Anderson, T. F. 1951 Technique for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N. Y. Acad. Sci. Ser. 111, 13: 130-134. 1973 The Andrews, P. M., and K. R. Porter ultrastructural morphology and possible significance of mesothelial microvilli. Anat. Rec., 177: 409426. Aterman, K. 1964 The structure of liver sinusoids and the sinusoidal cells. In: The Liver. C. Rouiller, ed. Academic Press, Inc. New York, pp. 61-136. Bierring, F., and P. Skaaring 1973 Scanning electron microscopy of the liver. JEOL News, I l e : 16-17. Brooks, S. E. H., and G. H. Haggis 1973 Scanning electron microscopy of rat's liver. Application of freeze-fracture and freeze-drying techniques. Lab. Invest., 29: 60-64. Bruni, C., and K. R. Porter 1965 The fine structure of the parenchymal cell of the normal rat liver. I. General observations. Am. J. Path., 56: 691-775. Carson, F., J. A. Lynn and J. H. Martin 1972 Ultrastructural effects of various buffers, osmolarity, and temperature on paraformaldehyde fixation of the formed elements of blood and bone marrow. Texas Rep. Biol. Med., 30: 125142.
Compagno, J., and J. W. Grisham 1974 Scanning electron microscopy of extrahepatic biliary obstruction. Arch. Path., 97: 348-351. Elias, H. 1949 A re-examination of the structure of mammalian liver. I. Parenchymal architecture. Am. J. Anat., 84: 311-334. Elias, H., and J. L. Sherrick 1969 Morphology of the liver. Academic Press, Inc., New York, 389 pp. Fahimi, H. D. 1967 Perfusion and immersion h a t i o n of rat liver with glutaraldehyde. Lab. Invest., 16: 736-750. Fawcett, D. W. 1955 Observations o n the cytology and electron microscopy of hepatic cells. J. Nat. Cancer Inst., 15: 1457-1502. Fugita, T., J. Tokumaga and H. Inoue 1971 Atlas of scanning electron microscopy, Igaku Shoin, Tokyo, pp. 20-21. Grisham, J. W. 1963 Ciliated cells i n normal murine intrahepatic bile ducts. Proc. SOC.Exp. Biol. Med., 144: 318-320. Grisham, J. W., R. L. Tillman, A. E. H. Nagel and J. Compagno 1975 Ultrastructure of the proliferating hepatocyte: Sinusoidal surfaces and endoplasmic reticulum. In: Liver Regeneration after Experimental Injury. R. Lesch and W. Reuttner, eds. Intern. Med. Book Corp., New York, pp. 6-23. Heath, T., and S. L. Wissig 1966 Fine structure of the surface of mouse hepatic cells. Am. J. Anat., 119: 97-128. Matter, A., L. Orci and C. Rouiller 1969 A study on the permeability barriers between Disse's space and the bile canaliculus. J. Ultrastruct. Res. (Suppl.), 11: 1-71. Miyai, K., R. M. Wagner and A. L. Richardson 1974 Preparation of liver for combined SEM and TEM study. In: Scanning Electron Microscopy/l974. 0. Johari, ed. IIT Research Inst., Chicago, pp. 283-290. Motta, P., and G. Fumagalli 1974 Scanning electron microscopy demonstration of cilia i n rat intrahepatic bile ducts. Z. Anat. Entwickl. Gesch., 145: 223-226. 1975 Structure of rat bile canaliculi as revealed by scanning electron microscopy. Anat. Rec., 182: 499-513. Motta, P., and K. R. Porter 1974 Structure of rat liver sinusoids and associated tissue spaces as revealed by scanning electron microscopy. Cell Tiss. Res., 148: 111-125. Odor, D. L. 1954 Observation on rat mesothelium with the electron and phase microscopes. Am. J. Anat., 95: 433-465. Ogawa, K., T. Minase, K. Enomoto and T. Onoe 1973 Ultrastructure of fenestrated cells in the sinusoidal wall of rat liver after perfusion fixation. Tohoku J. Exp. Med., 110: 89-101. Orci, L., A. Matter and C. Rouiller 1971 A comparative study of freeze-etch replicas and thin sections of rat liver. J. Ultrastruct. Res., 35: 1-19. Rappaport, A. M. 1973 The microcirculatory hepatic unit. Microvasc. Res., 6: 212-228. Shimamoto, T., Y. Yamashita and T. Sunaga 1969 Scanning electron microscope observations of the endothelial surface of heart and blood vessels. Proc. Japan Acad., 45: 507-512.
SEM O F RAT LIVER Tandler, B., and C. L. Hoppel 1974 Subsurface cisterns in mouse hepatocytes. Anat. Rec., 179: 273-284. Wisse, E. 1970 An electron microscopic study of the fenestrated lining of rat liver sinusoids. J. Ultrastruct. Res., 31: 125-150.
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Widman, J. J., R. S. Cotran and H . D. Fahimi 1972 Mononuclear phagocytes (Kupffer cells) and endothelial cells. Identification of two functional cell types i n rat liver sinusoids by endogenous peroxidase activity. J. Cell Biol., 52: 159-170.
PLATE 1 EXPLANATION O F FIGURES
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1
Fractured liver surface showing the tree-like branching of a portal canal (P). x 50.
2
Microscopic segmentation of hepatic parenchyma. Hepatic plates run from a terminal portal canal ( P ) toward a terminal hepatic venule (arrow). x 110.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 1
303
PLATE 2 EXPLANATION OF FIGURES
304
3
Sliced liver surface showing a transected portal tract. Portal vein ( V ) and hepatic artery ( A ) are indicated. Two sectioned bile ducts are located to the left of the vein and the right of the artery. X 525.
4
Lumenal surface of a n intrahepatic bile duct showing focally concentrated microvilli. x 13,650.
5
A cilium protruding from the lumenal surface of a n intrahepatic bile duct. Microvilli are much shorter. x 17,650.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE a
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PLATE 3 EXPLANATION O F FIGUEES
306
6
Endothelial ( E ) surface of hepatic artery i n the liver. Ridges of endothelium are presumed to be caused by contraction of subjacent smooth muscle cells. Sparse, short microvilli occupy the surface of endothelial cells. X 6,825.
7
Interior of a portal vein with a sinusoidal opening ( S ) containing a leukocyte (WBC). Shingle-like endothelial cells contain sparse microvilli on their surfaces. ~ 4 , 9 0 0 .
8
Detail of endothelial surface of portal vein showing microvilli (arrow heads) and fenestrae (arrows). x 7,840.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 3
307
PLATE 4 EXPLANATION OF FIGURES
9 10
Rope-like bundles and filaments of connective tissue fibers on exterior of a portal tract. x 1,470. Interdigitation of hepatic plates and sinusoids ( S ) .
x 1,270.
11 Detail of one-cell thick hepatocytic plate showing a n unroofed bile canaliculus (arrows) i n its center. Sinusoids ( S ) on either side of the hepatocytic plate contain erythrocytes. X 5,250.
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SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 4
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PLATE 5 EXPLANATION O F FIGURES
12
A similar hepatocytic plate i n which the canaliculus is not so precisely centered. Sinusoidal endothelium rests on microvilli forming the hepatocytic sinusoidal surface. (Sinusoids, S ) . x 5,250.
13 Intercellular surface of a single hepatocyte containing a highly branched bile canaliculus (BC). Several branches end blindly near the sinusoidal surface. The flat intercellular surface contains several holes (arrows) and protrusions. X 7,350.
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SEM OF RAT LIVER
PLATE 5
Grisham, Nopanitaya, Compagno and Nagel
31 1
PLATE 6 EXPLANATION OF FIGURES
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14
Surface of hepatocyte located at the portal end of a n hepatocytic plate. The bile canaliculus ( B C ) is broad and its microvilli are concentrated on its lateral margins. Holes (arrows) and studs (arrow heads) occupy the flat intercellular space. Dense populations of microvilli on the sinusoidal surface of this cell are seen at the left and right sides of the photograph. x 13,000.
15
Surface of hepatocyte located at the hepatic venous end of a n hepatocytic plate. The bile canaliculus ( B C ) is much narrower than at the portal end (above). At the acute margins of the cell, the microvilluscovered sinusoidal surface extends close to the canaliculus. x 13,000.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 6
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PLATE 7 EXPLANATION OF FIGURES
16 The portal end of a sinusoid is lined with endothelium containing many large fenestrae. Clusters of small fenestrae are also present. X 4,200.
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17
The hepatic venous end of a sinusoid contains endothelium with only small fenestrae. x 5,250.
18
Sinusoidal endothelium showing both large and small fenestrae. x 15,680.
19
Microvilli o n the sinusoidal surface of underlying hepatocytes (arrows) are visible through some fenestrae. x 19,600.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 7
315
PLATE 8 EXPLANATION OF FIGURES
316
20
Portion of a sinusoid ( S ) and hepatocytic plate. A presumed Kupffer cell ( K ) is continuous with the sinusoidal endothelium. x 3,190.
21
Oval cross-section of a sinusoid ( S ) showing a n endothelial cell body (E ), which gradually thins peripherally to form typically fenestrated cytoplasm. x 4,550.
22
Cross-section of a terminal hepatic vein (THV) which is about three times the diameter of sinusoids draining into it. X 800.
23
Endothelial surface of a terminal hepatic vein with a sinusoidal opening ( S ) . Endothelium about the sinusoidal opening is fenestrated although elsewhere it is continuous and contains sparse microvilli. X 6,300.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 8
317
PLATE 9 EXPLANATION OF FIGURES
318
24
Endothelial surface and transected wall of a second order hepatic vein, penetrated by sinusoidal openings (arrows) and containing sparse connective tissue fibers i n its walls. x 1,000.
25
A large hepatic vein whose wall is composed of thick bundles of connective tissue fibers. The endothelium (upper right) is continuous and is disposed i n gentle undulations. Sinusoids do not open into this vein. X 2,500.
26
A portal canal from which the portal tract has been torn away. The portal canal is lined by a nearly continuous plate of hepatocytes (limiting plate), penetrated only occasionally by openings (arrows). Canals containing hepatic veins are structurally similar. x 525.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 9
319
PLATE 10 EXPLANATION O F FIGURES
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27
Detail of the surface of a n hepatocyte in the limiting plate. This surface faces the connective tissue of the portal tract or hepatic vein and is distinguished by irregular, flattened microvilli and smooth linear indentions (I). The latter are presumed to reflect molding of the hepatocyte around connective tissue fibers.
28
Peritoneal surface of mesothelial cells ( M ) on the capsule of the liver. Patches of filamentous microvilli alternate with areas devoid of these structures. x 3,180.
29
Both long and short microvilli are present on the peritoneal surface of mesothelial cells of the hepatic capsule. x 10,920.
SEM OF RAT LIVER Grisham, Nopanitaya, Compagno and Nagel
PLATE 10
321
