EXPERIMENTAL

AND

Scanning

MOLECULAR

Electron

Microscopy

WAYKIN Department

of Mouse

NOPANITAYA

of Pathology,

AND

University

Chapel Received

23, 441-458

PATHOLOGY

Hill,

April 29, 1975,

JOE

of North

North

Carolina

and in revised

( 1975)

lntrahepatic W.

Structures1

GRISHAM

Carolina, 27514

School

of Medicine,

form &me 12, 1975

Livers of normal mice were prepared for scanning electron microscopic (SEM ) study by fracturing or slicing lobes fixed in situ by perfusion with paraformaldehyde. Fracturing fixed liver exposes surfaces of hepatocytes and sinusoidal endothelial cells, whereas slicing the tissue reveals the internal structures of portal tracts. Earlier studies have delineated the major surface characteristics of hepatocytes and sinusoidal endothelial cells of rats. Surfaces of hepatocytes in the mouse differ from those in the rat by having larger and more numerous peg and hole complexes on the flat intercelhdar surface and less dense populations of perisinusoidal microvilli. Sinusoidal endothelial cells in the mouse have fewer large fenestrations than do similar cells in the rat; chrsters of small fenestrations appear similarly distributed in both species. The surfaces of capsular mesothelial cells, Kupffer cells, bile duct epitheIia1 cells, and endo~e~i~ cells of major vessels are similar in rat and mouse. The methods described for preparing liver for SEM examination are simple, rapid, and reproducible. The SEM is a useful tool with which to study intrahepatic surface structures, and its use may allow further correlations to be made between hepatic structure and function in both health and disease.

INTRODUCTION Several recent studies of hepatic ultrastructure have used the scanning electron microscope (SEM) to elucidate the three-dimensional features of some prominent intrahepatic structures, especially the surface characteristics of hepatocytes and sinusoidal endothelial cells (Bier-ring and Skaaring, 1973; Brooks and Haggis, 1973; Andrews and Porter, 1973; Compagno and Grisham, 1974; Miyai et al., 1974; and Motta and Fumagalli, 1974, 1975). In all of these studies of both normal and pathologically altered livers, the rat is the only species of animal studied. Furthermore, a variety of techniques has been employed to prepare liver for SEM, frequently resulting in artifactual alterations. It is the purpose of this report to detail the intrahepatic surface structure of another species, the mouse, prepared for study by simple, easily reproduced techniques. This study discloses that several surface features of mouse hepatocytes and sinusoidal endothelial cells differ distinctively from those of the rat. MATERIALS

AND METHODS

Male and female Swiss albino mice weighing 25-30 gm were used; mice were anesthesized with ether or they were killed by cervical dislocation prior to open’ Supported

by

Grants

AM

17595

and

GM

92 from 441

Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

the

National

Institutes

of Health.

442

NOPANITAYA

AND GRISHAhf

ing their thoracic and abdominal cavities. A small incision was made in the inferior vena cava just below the renal veins, and intracardiac perfusion was immediately begun. Animals were perfused with chilled (4°C) 0.9% solution of sodium chloride for 2 min, followed by chilled 4% solution of paraformaldehyde in phosphate buffer (pH 7.3) (C arson et al., 1972) for 2 min. The gravity ilow rate was about 45-55 ml/min. The perfused, whole liver, partially hardened by paraformaldehyde, was dissected out and sliced through the anterior-posterior plane into pieces 5 mm or less thick. Slices of liver were immersed in fresh 4% solution of paraformaldehyde in phosphate buffer at 4°C. After fixing for 6-8 hr, blocks of liver were individually fractured by digital pressure according to the method of Compagno and Grisham (1974). In a few instances, whole livers were immediately fractured following in viva perfusion with paraformaldehyde, after which they were fixed for 6-8 hr in fresh fixative. The fracture surface was carefully protected from injury during the second fixation step. Some liver blocks, fixed for 6-8 hr, were sliced into small pieces (about 1 x 5 x 5 mm) with thin, acetone-cleaned razor blades. In this instance a sliced, rather than a fractured, surface was examined. Trimmed, fractured specimens or razor-sliced blocks were rinsed in chilled 0.1 M phosphate buffer solution (pH 7.3) for two changes, 30 min each. Tissues were postfixed in 1% 0~04 solution in phosphate buffer, pH 7.3, (Palade, 1952) for l-2 hr, dehydrated in graded solutions of ethanol, and dried by the critical point method (Anderson, 1951). Some blocks of tissue that had been carried through the osmication step were processed in an Autotechnicon through a graded series of ethanol solutions, three changes of toluene, and three changes of melted paraffin. The total processing time was 2 hr. Paraffin-embedded blocks were faced on a conventional rotary microtome, using an ordinary steel blade, until the surface was smooth. Paraffin was then removed from these blocks by melting at 60°C followed by further deparaffinization in toluene, dehydration in graded solutions of ethanol, and drying by the critical point technique as detailed above. During these subsequent steps, the surface that was microtome-faced in the paraffin block was carefully protected from damage. All specimens were mounted so that the surface for examination was uppermost, coated with gold-palladium, and viewed with a Coates and Welter Cwikscan-104 scanning electron microscope at 15 kV. RESULTS Preparation of Tissue Fixed liver can be exposed for SEM study by either fracturing it (Compagno and Grisham, 1974) or slicing it with a sharp blade with or without paraffin embedment (Miyai et al., 1974). Liver prepared by each method has certain advantages for the study of some structural components. Fracturing adequately fixed liver separates hepatocytes by breaking junctional complexes and provides nearly ideal exposure of the surfaces of hepatocytes and sinusoidal lining cells. Imperfectly fixed liver fractures both through and between hepatocytes, provid-

SEM

FIG. shown.

1. Liver (x180.)

fractured

after

OF

MOUSE

paraformaldehyde

FIG. 2. Liver sliced with a microtome (PV) are exposed. ( X580. )

blade

443

LIVER

fixation.

after

fixation.

An

empty

A portal

portal

triad

canal

and

portal

(P)

is

vein

ing a less adequate view of liver cell surfaces; treatment for 6-8 hr in paraformaldehyde is required to give adequate fixation. However, the fracture process destroys portal tracts and hepatic veins; these connective tissue-rich structures are avulsed, leaving empty portal and hepatic canals (Fig. 1). Fractured surfaces, thus, do not allow the study of hepatic veins or the substructures of portal tracts, but examination of the hepatocytic walls surrounding portal tracts and hepatic veins is facilitated by this process. Slicing fixed liver, either with or without paraffin embedment (Miyai et al., 1974), with a sharp blade cuts portal tracts and hepatic veins cleanly, exposing their contents for examination (Fig. 2) ; sinusoids can also be studied readily in livers prepared by this technique. However, surfaces of hepatocytes are severely distorted by the cutting process, rendering them unsatisfactory for detailed examination. Hepatic

Plates and Hepatocytes

Hepatic plates are uniformly one cell thick and generally straight (Fig. 3). Hepatocytes are polyhedral and sharply angulated (Fig. 4 and 5). Four distinct facets of hepatocyte surfaces are clearly visible with the SEM: The canalicular surface containing an unroofed bile canaliculus (hemicanaliculus) (Figs. 4-6); the flat intercellular surface that laterally borders canaliculi (Figs. 4-6) ; the sinusoidal surface (Fig. 7); and the connective-tissue-facing surface that borders portal tracts and hepatic veins (Figs. 8 and 9).

NOPANITAYA

FIG. 3. One-cell thick centrally located on each

AND

hepatic plates plate. ( x900.)

FIG. 4. Polyhedral, sharply angulated flat intercellular surfaces, which contain canalicuhu (hemicanalicldus). ( x5000.

are

separated

hepatocytes pits, holes, )

GRISHAhl

by

sinusoids

form a one-cell and protrusions,

( S ). Hemicanaliculi

thick hepatic plate. border an unroofed

are

The bile

SEM

FIG. 5. Surface the bile canaliculus ( x8000. )

FIG. cellular

OF

of hepatocyte detailing (BC) at its lateral

6. A high magniikation surface. HoIes (H)

and

MOUSE

the features margins. At

445

LIVER

described in Fig. 4. Microvilli crowd the left the bile canaliculus branches.

view of a bile canaliculus (BC) and the adjacent studlike protrusions (S ) are prominent. (X18,000.)

flat

inter-

446

NOPANITAYA

AND

GRISHAM

FIG. 7. A portion of an hepatocyte showing its sinusoidal microvilli. A fragment of an endothelial cell (E ) lining ( x 10,000. )

surface (SS ) covered by nnmerons the sinusoid is partially lifed off.

The cana~cular surfaces of all hepatocytes have centrally located canaliculi that generally measure from 0.5 to 1.0 pm in width (Figs. 4 and 6). Bile canaliculi adjacent to portal tracts (Fig. 5) are wider (up to 1.5-2 pm) than are those in other parts of hepatic plates. Although canaliculi are generally straight, short branches are occasionally observed (Fig. 5). Canalicular microvilli measure 0X-0.4 pm long by 0.02 pm wide. They are cIearly located both at the lateral margins and on the canalicular surface facing the body of hepatocytes. The flat intercellular surfaces laterally bordering bile canafculi contain a variety of pits, holes, and protrusions (Figs. 4-6). Numerous tiny pits and small protrusions, averaging about 0.1 pm in diameter, are present on the flat surface. Several noticeably larger holes, which measure 0.5-1.5 pm in diameter, and a corresponding number of stubby protrusions of the same diameter and up to l-l.5 ,.m long are present on the intercellular surface of all hepatocytes (Figs. 5 and 6). A sampling of 50 hepatocytes contained 6.3 * 2.7 holes and 6.2 _+ 2.3 protr~lsions on one flat intercelluIar surface, i.e., on one side of a bile hemicanaficulus. Protrusions are often located close to holes, a projection frequently being present on the lip of a hole (Fig. 6). The hepatocytic surfaces that face sinusoids and form the hepatocellular border of the space of Disse are covered with microvilli (25-36 microvilli/mm? of surface). These microvilli measure about 0.15 +n wide by 0.3-0.6 pm long (Fig. 7). Microvillus-studded surfaces extend around the sharply angulated corners of hepatocytes toward the centers of hepatic plates (per~sinusoidal recesses), approaching to within 1-2 pm of bile canaliculi.

SEM OF MOUSE

LIVER

447

FIG, 8. Surfaces of hepatocytes facing connective tissue of a portal tract. Smooth-su~aced, linear indentations (I) are present between groups of microvilli. ( x3500). Fm. 9. A high magni&ation of the hepatocyte surface described in Fig. 8. Micro&% irregularly shaped, varying from fingerlike to leaf-shaped forms. Smooth indentations (I) alsovisible. ( X 14,200. )

are are

Hepatocytes form a nearly continuous layer encircling portal tracts and hepatic veins (portal and hepatic limiting plates). The surfaces of hepatocytes facing connective tissue of portal tracts or hepatic veins are modified from that of any of the other surfaces of hepatocytes located in the interior of p~ench~a; connective tissue-facing hepatocyte surfaces are studded with irregrdarly shaped microvilli, which measure 0.1-0.3 ,CCI-I wide by 0.3-0.6 f*.m long (Figs. 8 and 9). Many microvilli in this location have distinctive leaf-shapes or are fused to form short ridges (Fig. 9). Groups of microvilli are separated by smoothsurfaced, linear indentions that vary in width and form continuous interwoven patterns (Fig. 9).

Sinusoids are round to oval in cross section, appearing in their longitudinal aspect to be tubes (Figs. 10 and If). Their largest diameter is about 20 to 30 e. Sinusoidal endothelial cells have a centrally located cell body (6 to 8 ,m-r in diameter) from which prominent arms of cytoplasm extend outward in all lateral directions, gradually diminishing in thickness to form typical, thin cytoplasmic veiIs, which compose the major extent of sinusoida waIIs (Fig. 12). EndotheIiaI cell bodies contain numerous surface pits, which measure 0.05-0.1 m in diameter (Fig. 12). The thinned endo~elial cytoplasm contains frequent small,

448

NOPANITAYA

FIG. FIG.

AND

GRISHAM

10. Cross section of a sinusoid shows it to be round or oval. (X3000. ) 11. Longitudinal section of a sinusoid discloses its tubular form. (x3200.)

I?IG. 12. A centrally located extends outward from the body ous small pits. ( X7500).

cell body of a sinusoidal endothelial to form the sinusoidal wall. The cell

cell (E ). Its cytoplasm surface contains numer-

SEM OF MOUSE

LIVER

449

FIG. 13. Groups of small, round fenestrations and few larger fenestrations are present on the thinned endohtelial cytoplasm. ( ~12,000.) FK.

14. Hepatocytic

microvilli

are visible through

some fenestrations.

(arrowheads) ( ~13,000.)

round fenestrations about 0.1 pm in diameter, which form clusters of up to 20 fenestrae (Figs. 13 and 14). A few larger fenestrations, measuring up to I e in diameter, are occasionally seen (Fig. 13). Microvilli of the sinusoidal surfaces of hepatocytes can be seen through some fenestrations (Fig. 14). Sinusoids contain an additional type of cell, presumed to represent the cell of Kupffer. These cells are plump, and their surface is ridged or folded and sparsely populated with stubby microvilli (Fig. 15). Presumptive Kupffer cells are clearly located within the lumen of sinusoids and are underlaid by sinusoidal endothelium, or they form part of the sinusoid wall, merging laterally with thin sinusoidal endo~elial cells. The perisinusoidal space of Disse is nearly filled by hepatocytic microvilli. It aIso contains a few fine fibers, presumed to represent connective tissue, which in fractured specimens are occasionally dislodged and strewn over the parenchyma1 surface (Fig. IS). A cell whose surface is smooth with gentIy rounded undulations over underlying spherical outlines (Fig. 16) is frequently found in Disse’s space. Correlated TEM studies demonstrate that these cells are socalled fat-storing cells, Other perisinusoidal cells such as leukocytes or fibroblasts were not observed.

~ntT~~~at~c

Blood Vessels and Bile Ducts

IIepatic arteries can be distin~ished from portal veins by their smaller size and thicker walls (Fig. 17). Openings, which measure 20-30 iurn in diameter,

450

NOPANITAYA

FIG. 15. A Kupffer cell. Fine connective ( x5500. )

AND

cell (K) within a sinusoidal tissue fibers are strewn over

FIG. 16. A fat-storing

cell

(F)

containing

GHISHAM

lumen overlying a sinusoidal the surface of an hepatocyte

internal

spherical

outlines.

endothelial on the left.

(X5500.)

SEM OF MOUSE

LIVER

451

FIG. 17. A longitudinal section of an intrahepatic artery showing its lmnenal long axes of arterial endothleial cells parallel the arterial lbng axis. (x500.)

FIG. 18. A tangentially transected portal vein. Oval-shaped sinusoidal openings (S ) occupy the lumenal surface. ( X 1900. )

endothelial

surface. The

cells (E ) and

452

NOPANITAYA

FIG. 19. A portion of an arterial endothelial microvilli. ( X 10,000.)

AND GRISHAM

cell (E ) sh owing irregular

ridges and stubby

FIG. 20. Venous endothelial cells (E ) also have ridges and stubhy microvilli surfaces. Pits are frequently seen on the cell surfaces. (X6000.)

on their

SEM OF MOUSE

LIVER

453

are frequent in portal veins (Fig. 18) but relatively sparse in hepatic arteries, Arterial endothelial cells are regularly rectangular in shape, with their long axis oriented parallel to the long axis of vessels (Fig. 19). In contrast, venous endothelial cells are more nearly round or oval in shape, and they are randomly oriented on the luminal surface (Fig. 20). Arterial endothelial cell bodies are thicker and more elongated than are venous endothelial cells. Irregular ridges and sparse, stubby microvifli are present on the lumenal surfaces of both arterial and venous endothehal cells (Figs, I9 and 20). Small pits are more commonly seen on the surfaces of venous than of arteria1, endotbelial cells (Fig. 20). The characteristics of the endothelium of hepatic and portal veins are generally similar. Most hepatic veins examined were densely penetrated by openings of sinusoids, which measured 20-30 pm in diameter. Connective tissue fibers surrounding vessels or composing portal tracts consist of a mass of ropelike fibers of various sizes (0.1-2.0 pm in diameter) (Fig. 21). Other known components of connective tissue, smooth muscie of larger vessels, nerves, and lymphatic vessels have not been identified and studied. Lumenal surfaces of bile ducts are occupied by microvilli, which measure 0.1 pm long (Fig. 22), and are often concentrated in longitudinal rows along the duct lumen. Occasional cilia, which are about 0.2 pm wide and S-10 pm long, appear to be randomly located on the lumenal surface.

The hepatic parenchyma is microscopically segmented by the regular interdigitation of portal and hepatic veins, which at the terminal level are oriented more or less perpendicularly. A microvascular segment of parenchyma centering about a terminal portal venule and, at its periphery, touching several terminal hepatic veins is readily seen in favorably fractured specimens (Fig. 23). Hepatic Caps-de The capsule of the mouse liver consists of a uniform, single layer of mesothelial cells, overlying twisted connective tissue fibers (Fig. 24). The outlines of serosal mesothelial nuclei are visible (Fig. 24). Surfaces of mesothelial cells are covered by numerous microvilli; patches of long microvilli, up to 1.5 pm long, alternate with areas con~i~ng short microvilli, less than 0.3 w long (Figs. 24 and 25). Microvilli are occasionally branched (Fig. 25). RegardIess of their length, all microvilli measure approximally 0.15 pm in diameter. DISCUSSION Several methods have now been applied to the preparation of liver for SEM study with variable results (Fugita et aI., 1971; Bierring and Skaaring, 1973; Brooks and Haggis, 1973; Compagno and Grisham, 1974; Motta and Porter, 1974). This study demonstrates a simple and rapid technique for preparing liver for SEM that is cheap and highly reproducible. Phosphate-buffered paraformaldehyde, a cheap and stable fixative that has been recommended for general electron microscope use (Carson et al, 1972)) preserves intrahepatic structure as well as does more expensive and less stable glutaraldehyde, used

454

NOPA~ITA~A

FIG. 21. Connective tissue fibers surrounding vary. ( X5000 ) . FIG. 22. Lumenal ( x 12,000.)

AND

GRISEIAM

an hepatic vein. Sizes of these ropelike

surfirce of bile ducts showing

FKC. 23. A microunit of hepatic venules are shown. ( x 170. f

parenchyma.

many

Terminal

short microvilE

portal

(P)

fibers

and a c&urn.

and Aepatic

(H)

SEM OF MOUSE

LIVER

455

FIG. 21. Mesothelial cells (M) and underlying connective tissue fibers (Cl?) of the hepatic Surfaces of mesothelial cells are studded by both short and long, filamentous microvilli. (X5000.) capsule.

F’IG. 25. Short and long, filamentous microvilli microvilli (arrowheads ) are visible. ( x 12,000. )

on mesothelial

cell surfaces. Branched

4%

NOPAN~TA~A

AND GRISHA~~

by several other investigators (Brooks and Haggis, 19’73; Andrews and Porter, 1973; Compagno and Grisham, 1974; Miyai et al., 1974; Motta and Porter, 1974). In viva perfusion for 2 min. is adequate for m0use liver, probably because this animal has an uncomplicated portal circulation, permitting perfusate to reach all tissue spaces quickly (Elias and Sherrick, 1969; Lee et al., 1960). Livers of larger animals should be perfused for longer periods. Postfixation in osmium tetroxide stabilizes both lipids and proteins in membranes (Hayat, 1970), which may otherwise be altered during dehydration or electron bombardment with resulting artifactual deforn~ation, Critic& point drying of tissues is required to retain the structure of delicate surface membrane specializations (Anderson, 1951). Air drying, used in early SEM studies (Fugita et al., 1971; Bierring and Skaaring, 1973), should be abandoned because of the artifactual flattening of surface structure that results from its use. Many features of mouse intrahepatic structures are closely analogous to similar aspects of rat livers described previously (Brooks and Haggis, 1973; Compagno and Grisham, 1974; Miyai et al., 1974; Motta and Porter, 1974; Grisham et al., 1975a,b,c; Motta and Fumagalli, 1974, 1975). The most striking difference between mouse and rat hepatocytes is in the character of stud processes and holes. In the rat only a few structures are found on the flat ~ntercelluIar surface that conform to the character of these peg and hole processes, as defined by TEM studies (Fawcett, 1955; Bruni and Porter, 1965; Heath and Wissig, 1965; Tandler and Hoppel, 1974). So sparse are these structures on rat hepatocytes that Compagno and Grisham (1974) questioned their existence. However, this study shows that pegs and holes are prominently disposed on mouse hepatocytes, each flat intercelluar surface containing approximately six pegs and an equal number of holes. It has previously been hypothesized that pegs and holes are involved in celluar attachment (Fawcett, 1955; Tandler and Hoppel, 1974). If so, then the quality or mechanism of attachment of adjacent hepatocytes in the rat and mouse must differ. However, it is possibIe that these structures are involved in some other process, such as communication between cells, since they contain concentrations of subcortical cisternae ( Tandler and Hoppel, 1974) . Other ways in which surfaces of mouse and rat hepatocytes vary are in the density of sinusoidal microvilli (rat: 30 to 6O/pm” [Grisham et al., 1975b,c], mouse: 20 to 25/pme), more numerous microvilli (at least less variation in disposition) on the surface of bile canaliculi facing the cell body in mice, and fewer branched bile canaliculi in the rat. The exact functional meaning of these species differences remains to be elucidated. Sinusoidal fenestrae vary distinctively in rats and mice. Sinusoida endothelial ceI1.sin both species have numerous small fenestrations meas~lring about 0.1 pm in diameter disposed evenly throughollt the length of sinusoids. In rats these small fenestrae are prominently grouped into clusters of up to 50 or more (Brooks and Haggis, 1973; Motta and Porter, 1974; Grisham et al., 1975b,c), which Wisse (1970) has termed sieve plates. Clustering of small fenestrae is less marked in the mouse, there seldom being more than lo-20 closely apposed in one locus. SEM shows that sinusoidal endothelium in rats contains prominent large fenestrae that measure 1.0-3.0 pm in diameter (Motta and Porter, 1974; Grisham et al., 1975~). Large fenestrations are less common in mouse liver and rarely measure more than 1.0 pm in diameter. Even in this species, large

SEM

OF

MOUSE

LIVER

4.57

fenestrae appear to be more numerous at the afferent ends of sinusoids. Although fenestrae must be importantly involved in transsinusoidal transport, the roles of fenestrae of different sizes are unknown, as is the meaning of species variation described here. Preliminary observations in our laboratory suggest that fenestrae may be able to close under some physiologic conditions. It is not known whether small fenestrae can enlarge above the relatively uniform 0.1 pm diameter measured almost universally (Wisse, 1970; Orci et al., 1971; Ogawa et al., 1971; Brooks and Haggis, 1973; Motta and Porter, 1974; Grisham et al., 197%). If small fenestrae can enlarge, one cauld explain the occurrence of large fenestrae by the fusion or coalescence of all small fenestrae in a single cluster or sieve plate. This hypothesis would also explain the smaller size of large fenestrae in mice, since clusters or sieve plates in this species contain fewer small fenestrae than are present in rats. However, since we have not seen, and no one else has apparently reported, clusters of small fenetrae that appear to be in the process of fusing, this possibility remains speculative. This study shows that prominent differences in surface structure of hepatocytes and sinusoidal cells distinguish mouse and rat livers. Unreported studies in our laboratory indicate that equahy distinctive differences will be found to occur in livers of other species. It will be interesting and potentially useful to attempt to correlate these species variations with known variations in hepatic function. The utility of SEM as a technique for studying surfaces and interrelationships of hepatic substructures is evident from this report, as well as from previous studies using this technique. REFERENCES ANDERSON, T. F. ( 1951). Technique for the preservation of three-dimensional structure in preparing liver for the electron microscope. Trans. N.Y. Acad. Sci. Ser. III 13, 136-134. ANDREWS, P. M., and PORTER, K. R. (1973). The ultrastructural morphology and possible signiilcance of mesothelial microvilli. An&. Rec. 177, 409425. BIERRDXG, F., and SKAARING, P. ( 1973). Scanning electron microscopy of the liver. IEOL News lie, 16-17. BROOKS, S. E. H., and HAGGIS, G. H. (1973). Scanning electron microscopy of rat’s liver. Application of freeze-fracture and freeze-drying techniques. Lab. Invest. 29, 66-64. BRUNI, C., and PORTER, K. R. ( 1965). The fine structure of the parenchymal cell of the normal rat. I. General observations. Amer. J. Pathol. 56, 691-775. CARSON, F., LYNN, J. A., and MARTIN, J. H. ( 1972). Ultrastrnctural effects of various buffers, osmolarity, and temperature on paraformaldehyde fixation of the formed elements of blood and bone marrow. Tex. Rep. Biol. Med. 30, 125-142. Scanning COMPAGNO, J., and GRISHAM, J. W. (1974). ebctron microscopy of extrahepatic biliary obstruction. Arch. PuthoE. 97, 348-351. EIAS, H., and SHERRICK, J. L. ( 1969). “Morphology of the Liver,” p. 389. Academic Press, New York. FAWCETT, D. W. ( 1955). Observations on the cytology and electron microscopy of hepatic cells. .I. Nut. Cancer Inst. 15, 1485-1502. FXJGITA, T., TOKUMAGA, J., and INOWE, II. (1971). ‘