JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 14:208-217 (19901
Three-Dimensional Fine Structure of the Biliary Tract: Scanning Electron Microscopy of Biliary Casts KAZUHIDE YAMAMOTO, TATSUYA ITOSHIMA, TAKA0 TSUJI, AND TAKURO MURAKAMI First Department of Internal Medicine and Second Department of Anatomy, Okayama University Medical School, Okayama 700, Japan
KEY WORDS vascular plexus
Hilar biliary plexus, Periductal sacculi, Bile ductular plexus, Peribiliary
ABSTRACT
The three-dimensional structure of the biliary tract was studied by scanning electron microscopy (SEM) of biliary casts. The replica of the biliary tract was successfully prepared by retrograde injection of low viscosity resin into the common bile duct. Bile canaliculi are intricate networks in which hexagonal and pentagonal meshworks are interconnected. Each hexagonal or pentagonal meshwork is on a plane, but adjoining meshworks are on different planes. Bile canalicular networks connect with bile ductules a t the periphery of the portal tract. The intrahepatic bile duct showed considerable interspecies variation. The human bile duct has plexiform side branches and periductal sacculi, which are most numerous near the liver hilum and fewest in the smaller portal tracts. The hilar plexus and sacculi are present on opposite sides of the bile duct. The plexus formed a t the bifurcation of the bile ducts exhibits a plane. Periductal sacculi were also observed in the monkey and pig bile ducts, particularly the latter, while rat bile ducts possess a peculiar portal bile ductular plexus situated between the portal tract and the surrounding liver parenchyma. No such structures were observed in either the dog or rabbit bile ducts. SEM of the biliary casts showed that the biliary tract was not a simple draining tube but had additional structures, such as periductal sacculi and plexiform side branches. These structures, together with the peribiliary vascular plexus, may be implicated in the modification of bile.
INTRODUCTION The biliary tract is the bile drainage route from the liver parenchyma to the duodenum and consists of bile canaliculi, bile ductules, and intra- and extrahepatic bile ducts. The direction of bile flow is countercurrent to the blood stream. An important question is whether or not the biliary tract is a simple drainage tube. The presence of rich vascular networks around the bile duct, the peribiliary plexus (Kiernan, 1833; Murakami et al., 1974),suggests that it is not merely for drainage, but may participate in the modification of bile (Jones et al., 1980). Certain functional data support this possibility (Erlinger, 1987). Morphological and functional studies of the biliary tract have been hampered due to its inaccessibility. As a result, there have been few structural and functional correlations in the biliary tract. The three-dimensional structure of the biliary tract is difficult to study. Current methods include reconstruction studies using serial sections or stereoscopic observations of biliary tracts injected with dyes. Bile canaliculi can be observed under the light microscope following histochemical staining or the injection of dyes secreted into the biliary space (Elias, 1949; Elias and Sherrick, 1969). However, these methods are insufficient due to limited resolution and depth of field. We recently applied scanning electron microscopic analysis (SEM) of resin casts to study the biliary tract with successful results (Murakami et al., 1984; Yamamoto and Phillips, 1984; Yamamoto et al., 1985). The method was originally developed for the study of
B 1990 WILEY-LISS, INC.
the vascular system (Murakami, 19711, but can be applied to the study of other channel structures. Furthermore, the development of less viscous resins permitted injection to the depth of the bile canaliculi which are 1 pm in diameter (Murakami et al., 1984). In the present paper, we introduce this method and present results concerning the finite morphology of the biliary tract.
MATERIALS AND METHODS Injection medium Two types of injection medium, resins A and B, were used in the present study. Resin A was a mixture of 50-60% (VIV)methyl methacrylate monomer (Nakarai Chemicals Ltd., Kyoto, Japan) and 4 0 4 0 % (VIV) 2-hydroxy methacrylate monomer supplemented with 1.5% (WIV) benzol peroxide (catalyst) (Katayama Chemicals Ltd.) and 1.5%N, N-dimethylaniline (accelerator) (Katayama Chemicals Ltd.) just prior to injection. This was used to make minute biliary casts such as bile ductules and bile canaliculi (Murakami et al., 1984). Resin B was prepared by diluting commercially available resin, Mercox (Dai Nippon Ink C.C., Japan),
Received January 30, 1988; accepted in revised form April 8, 1988. Address reprint requests to Dr. Kazuhide Yamamoto, First Department of Internal Medicine, Okayama University Medical School, Shikata-cho.2-5.1, Okayama, 700, Japan
THREE-DIMENSIONAL STRUCTURE OF BILIARY TRACT
with monomeric methyl methacrylate (Nakarai Chemicals Ltd., Kyoto, Japan) a t ratios ranging from 1:l to 1:3 (Yamamoto and Phillips, 1984). Various amounts of catalyst were added to the mixture in order to control the polymerization time.
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RESULTS Intrahepatic biliary tract Bile canaliculi and canaliculoductular junction. Resin A was successfully injected into the periportal and perivenular bile canaliculi without any leakage (Fig. 1). The three-dimensional arrangement Human and animal livers of bile canaliculi appeared as a chickenwire meshwork Human livers were obtained a t autopsy within 5 composed of interconnecting hexagonal and pentagonal hours after death from patients without liver diseases. frameworks (Figs. 1 , 2 ) . Each hexagonal or pentagonal Livers from a variety of animal species, were studied, framework was on a plane, but adjoining frameworks e.g., monkey, pig, guinea pig, rabbit, and rat. Prior to were on different planes sharing only one side in injecting the resin into the common bile duct, the liver common. This spatial arrangement was easily diswas perfused with Ringer’s solution via the portal vein, cerned by observing stereomicrographs. Each side of hepatic artery, or both. the bile canalicular framework consisted of straight channels, which exhibited some narrowing and dilataInjection into bile ductules and canaliculi tion during their course. The rat liver was perfused with Ringer’s solution or Bile canaliculi connected with bile ductules at the physiological saline followed by buffered 2% glutaral- periphery of the portal tract. The transition from bile dehyde. Immediately after glutaraldehyde fixation, canaliculi to bile ductules was not uniform. Some resin A was infused into the common bile duct at a canalicular networks drained into a wider channel, i.e., pressure of 70-80 mmHg. This injection was contin- canal of Hering, which ultimately connected with a bile ued, without decrease in pressure, until the medium ductule (Figs. 2, 3). Ampullary dilatation a t the junchardened in the syringe. The resin was polymerized in tion was sometimes observed, whereas others abruptly a water bath at 60°C and the liver tissue was macer- connected with bile ductules. ated in a 30% NaOH solution. In some cases, a small Bile ducts amount of Mercox was infused into the portal vein after Human. Large intrahepatic bile ducts near the biliary injection in order to visualize the relationship hilum exhibited many irregular side branches and between the biliary tract and the portal vein. After pouches, 150-270 pm in diameter (Fig. 4). Some side rinsing in water, the biliary casts were frozen and branches ended blindly, while others anastomosed to freeze-dried. form a plexus. Smaller blind pouches extending from side branches were also observed. These side branches Injection in the intrahepatic bile duct and pouches were situated on opposite sides of the bile After vascular perfusion, a mixture of Mercox and ducts within the same plane. At the bifurcation of the monomeric methyl methacrylate (resin B) was injected bile duct, side branches from two or three main bile through a catheter inserted into the common bile duct ducts communicated with each other via the plexus. a t a pressure of 14-16 mmHg. Polymerization and These structures correspond to the periductal glands maceration were a s described above. After rinsing observed by light microscopy (Fig. 5). Side branches thoroughly in water, the biliary casts were observed and pouches were sparse in the smaller bile ducts. under a stereoscope and trimmed into small pieces. Animals. Numerous species differences were obvious in the intrahepatic bile ducts of animal livers. The Vascular casts monkey liver possessed both side branches and sacculi Hepatic vascular casts were prepared in human, similar to those observed in humans, but fewer in monkey, and rat livers a s described by Murakami et al. number. The pig liver expressed numerous sacculi up (1974). Special attention was paid to the portal and to and including the smaller bile ducts, but no side peribiliary blood supply. branches. The rat liver had a plexiform bile ductular network surrounding the portal tract (Fig. 6). No Injection into portal lymphatics analogous structures were observed in the livers of In rat and rabbit livers, resin A was injected in rabbits or dogs. excess into the common bile duct a t a pressure of 14-16 mmHg (Yamamoto et al., 1986a). The injection was Vascular supply of the portal or biliary tract continued a t this pressure until the resin polymerized The proper hepatic artery divides into the terminal in the syringe. The lymphatic casts were treated simbranches in the portal canal. The main terminal ilar to the biliary casts. branches of this artery form the peribiliary plexus Scanning electron microscopy surrounding the biliary tract and emit the efferent Biliary and lymphatic casts were carefully trimmed vessels which continue either into the liver sinusoids or into small pieces under a stereomicroscope, mounted on portal terminal branches (Fig. 7). The vascular pathmetal stubs, conductively stained by vaporized osmium way via the peribiliary plexus is called the peribiliary teroxide-hydrazine hydrate, or sputter-coated with or intrahepatic portal system (Murakami et al., 1974). gold and observed in a scanning electron microscope Other terminals continue into the sinusoids or supply with a n accelerating voltage of 10-15 kV. Electron the portal canal. The portal plexus is coarse and drains micrographs were taken in stereopairs for three- into the sinusoids, peribiliary efferent vessels or portal terminal branches (Fig. 8). dimensional analysis.
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Fig. 1. Scanning electron micrograph of bile canalicular casts. Note bile canalicular networks (c) drain into the canal of Hering (b) and then into bile ductules (B). x 1,100.
THREE-DIMENSIONAL STRUCTURE OF BILIARY TRACT
~ i2, ~~ , i magnification ~ h of c~a n a ~~~ c u ~ o d u c tjunctions u~ar (arrowheads). b, bile ductule; c, bile canaliculus. x 2,700.
Portal lymphatics Casts of lymphatic channels were prepared by injecting excessive amounts of resin into the bile duct. Resin B did not enter the lumen of bile canaliculi and excess resin leaked after filling the biliary tract. The resin leaked into the portal interstitial spaces near the terminal portal tract and drained into the lymphatic channels (Fig. 9a). Lymphatic casts demonstrated that the terminal portal lymphatics end blindly (Fig. 9b). The lymphatics found in the portal tract were composed of long, straight channels and interconnecting short branches. Valvelike constrictions were sometimes observed (Fig. 10). Lymphatic networks were especially rich around the bile duct and the bifurcation of the portal tract and these ultimately drained into the lymphatics in the hilum of the liver.
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Fig. 3. SEM of fractured liver surface. Note ampullary dilatation at canaliculo(c)-ductular junction (arrowhead). The other bile canaliculus terminates directly into the bile ductule (arrow). x 900.
Steiner and Carruthers, 1961). On the other hand, the three-dimensional morphology of the liver is still incomplete. The recent application of SEM to the study of hepatic tissues (Itoshima et al., 1980; Motta et al., 1978) had contributed t o the understanding of its three-dimensional structure. However, the threedimensional architecture of hepatic channel structures, such as the vascular and biliary systems, has been difficult to demonstrate. Injection of various materials into the channels has been a popular method since the late 19th century (Beale, 1889; Hering, 1872). However, the method has seen only limited use since both the resolution and depth of field are insufficient. SEM of resin casts to study hepatic microvasculature (Murakami, 1971) introduced a new era for the study of this sytem (Gannon, 1978; Hodde and Nowell, 1980). This method has been applied to the study of other DISCUSSION channel structures, S U C -as ~ the biliary &act (Murakami et al., 1984; Yamamoto and Phillips, 1984; The two-dimensional fine structure of the hepatobil- Yamamoto et al., 1985) and lymphatics (Yamamoto iary system has been studied by light and transmission and Phillips, 1986a). In the present study, the threeelectron microscopy and the detail is well established, dimensional arrangement of the biliary tract has been even at the level of subcellular structure (Biava, 1964; examined using this method.
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Fig. 4. Survey SEM of human hilar biliary tract. Note the rich plexiform side branches and sacculi (arrowheads) on the opposite sides of the bile duct (D). x 12.
THREE-DIMENSIONAL STRUCTURE OF BILIARY TRACT
Fig. 5. Light micrograph of bile ductules (arrowheads) in the portal connective tissue near the hilum corresponding to side branches in Figure 4. x 50.
Intrahepatic biliary tract Bile canuliculi and canaliculoductular junction. Bile canaliculi are the smallest biliary channels
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Fig. 6. SEM of rat bile ductular plexus. Bile ductules (d) surround portal interstitial space (P). x 50.
ing complexes may represent a structural basis for maintaining intralobular gradients for the secretion of various materials into bile canaliculi (Erlinger, 1987). formed by two or three adjacent hepatocytes. The Periportal hepatocytes (zone 1) are bathed in plasma two-dimensional fine structure of these channels has containing higher concentrations of constituents than been studied by transmission electron microscopy (Bi- those cells at the hepatic venous end (zone 3). This ava, 1964; Steiner and Carruthers, 1961).Bile canalic- concentration gradient in the blood is reflected in the uli are situated in the center of the muralium (Elias solute content of the bile. Partial loss of tight junction and Sherrick, 1969) of one-cell-thick plates and are integrity may facilitate the movement of ions and equidistant from the sinusoids on both sides. Their water between the intercellular space and the bile arrangement has been clearly demonstrated by scan- canaliculi. The bile canalicular networks drain into the bile ning electron microscopy of fractured liver surfaces (Fig. 3) (Itoshima et al., 1980; Motta et al., 1978). The ductules at the periphery of the portal tract. The bile canalicular lumen is defined by grooves in adjacent nature of this connection has been a subject of considhepatocytes and the wall is composed of a specialized erable debate (Itoshima et al., 1980; McIndoe, 1928; membrane with microvilli. The lumen is separated Steiner and Carruthers, 1961). A recent SEM study of from the intercellular space by junctional complexes, fractured liver surfaces demonstrated the presence of including tight junctions, although molecules, such as two types of termination (Itoshima et al., 19801, and water and small ions, are thought to permeate this this has been confirmed in the present study using bile barrier (Boyer, 1980; Coleman, 1987; Friend and Di- canalicular casts. In the first type, several bile canaliculi coalesce into a thick channel corresponding to a lula, 1972). SEM of bile canalicular casts revealed their three- canaliculoductular junction, i.e., a canal of Hering, in dimensional arrangement for the first time (Figs. 1,2). which the channel is formed partly by hepatocytes and They form intricate networks composed of a hexagonal partly by ductular cells (Fig. 1). In the rat liver, or pentagonal meshwork. Each straight side of a hexa- ampullary dilatation is observed at this type of termigon or pentagon is formed by the neighboring two or nation (Itoshima et al., 1980). The alternative type three hepatocytes. However, there is little geometric consists of bile canaliculi terminating directly into uniformity in this network. More sophisticated meth- portal bile ductules without ampullary dilatation. Bile ducts. The present study demonstrated that ods, such as computer analysis, are necessary for human intrahepatic bile ducts possess additional strucanalyzing this complicated spatial architecture. The bile canalicular networks are intercalated with tures, such as periductal sacculi and plexiform side vascular networks, i.e., sinusoids. These interdigitat- branches. These structures may correspond to the
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Fig. 7. SEM of vascular casts forming peribiliary Plexus in the monkey liver. An efferent vessel (e) drains into sinusoids (s). x 100.
l?ig. 8. SEM of vascular casts of plexus supplying the portal canal and which drains into sinusoids (s). 60,
parietal sacculi and vasa aberantia reported by Henle (1873) and Beale (1889). Similar structures have been termed tubuloalveolar glands or periductal glands (Burden, 1925; McMinn and Kugler, 1961; Spitz and Petropoulos, 1979). Numerous periductal glands are also present in the extrahepatic bile duct (Andrews and Andrews, 1979) and contain mucinous materials secreted into the bile (Chou and Gibson, 1979). Similar mucous-type glycoproteins have been demonstrated in human bile (Bouchier et al., 1965; Lee et al., 1979). Recent histochemical data suggest the presence of secretary component and immunoglobulins in the periductal glands (Terada et al., 1987). Furthermore, these glands increase during certain infections and conditions of stagnated bile, suggesting that periductal glands may contribute to local immunity (Chou and Gibson, 1970; Yamamoto, 1982). Plexiform side branches or vasa aberantia may also function a s collaterals for bile drainage (Yamamoto et al., 1985). Many species differences were demonstrated in the structure of the intrahepatic bile ducts which may explain, in part, concomitant differences in bile compo-
sition (Erlinger, 1987). For example, in the rat, which lacks a gall bladder, the ductular plexus may function as a reservoir for bile.
Vascular supply of portal and biliary tracts The bile ducts are surrounded by a thick vascular network called the peribiliary vascular plexus (Kiernan, 1833; Murakami et al., 1974). The plexus is composed of inner afferent networks and outer efferent networks, the latter draining directly into liver sinusoids, peribiliary efferent vessels or terminal portal branches. The drainage into efferent vessels and terminal portal branches is termed the peribiliary portal system (Murakami et al., 1974). The exact function(s) of the plexus remains unresolved, but may include the exchange of solute(s)between bile and blood, secretion into or reabsorption from the bile. It is interesting to note that even in the lamprey liver, the bile ducts exhibit a n intimate relationship with the vascular system (Yamamoto et al., 1986131, suggesting that this biliovascular relationship is a well-conserved trait. The portal system may function as a type of feedback
THREE-DIMENSIONAL STRUCTURE OF BILIARY TRACT
Fig. 9 a: SEM of lymphatic casts in the large portal tract. Note rich anastomosis at the bifurcation of the portal tract. x 30. b: Leaked resin near the terminal portal tract drains into lymphatics (L). D, bile duct. ~ 8 0 .
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of bile composition a t the various levels of the biliary tract is essential to this issue.
REFERENCES
Fig. 10. Higher magnification of lymphatic casts showing a valvelike constriction (arrowhead). X 350.
system, wherein some bile constituents may be reabsorbed into the blood and, eventually back to the hepatocytes (Jones et al., 1980). Thus, some biliary signals may be transferred to hepatocytes in order to modulate bile secretion.
Portal lymphatics The lymphatics represent another drainage route from the liver. The excess resin injected into the biliary system leaked into the interstitial spaces of the portal tract and, thus, into the lymphatics. The implication of this observation is that biliary materials may easily enter the lymphatics and drain back into the blood during increased biliary pressure. This may explain the early appearance of bilirubin in the thoracic lymphatics of patients with biliary obstruction (Dumont, 1973). CONCLUSIONS The present study summarizes the threedimensional fine structure of the biliary tract as visualized by scanning electron microscopy of biliary tract casts. The most important morphological feature of the biliary tract is its intimate relationship with the vascular system throughout its course. This biliovascular relationship suggests that the biliary tract may not be a totally autonomous structure but, rather, exhibits a dynamic exchange of constituents with the tissue space and blood. However, the properties corresponding to the various morphological components of the biliary tract remain to be resolved. A comprehensive analysis
Andrews, C.J.H., and Andrews, W.H.H. (1979) The relation between structure and function of bile ducts in man, some laboratory animals and the Adelie Penguin. J . Exp. Physiol., 64:61-67. Beale, L.S. (1889)The Liver. Lecture on the Principles and Practice of Medicine. Churchill, London, pp. 47-52. Biava, C.G. (1964) Studies on cholestasis. A re-evaluation of the fine structure of normal human bile canaliculi. Lab. Invest., 13340864. Bouchier, I.A.D., Cooperband, S.R., and ElKodsi, B.M. (1965) Mucous substances and viscosity of normal and pathological human bile. Gastroenterology, 49:343-353. Boyer, J.L. (1980) New concepts of mechanisms of hepatocyte bile formation. Physiol. Rev., 36:303-326. Burden, V.G. (1925) Observations on the histologic and pathologic anatomy of the hepatic, cyctic, and common bile ducts. Ann. Surg., 82584-587. Chou, S.T., and Gibson, J.B. (1970) The histochemistry of biliary mucins and the changes caused by infestation with Clonorchis Sinensis. J. Pathol., 101:185-197. Coleman, R. (1987) Biochemistry of bile secretion. Biochem. J., 244249-261. Dumont, A.E. (1973) Icteric thoracic duct lymph: Significance in patients with manifestations of obstructive jaundice. Ann. Surg., 178:53-58. Elias, H. (1949) A re-examination of the structure of the mammalian liver. 11. The hepatic lobule and its relation to the vascular and biliary system. Am. J. Anat., 85379-456. Elias, H., and Sherrick, J.C. (1969) Morphology of the Liver. Academic Press, Inc., New York and London, pp, 137-185. Erlinger, S. (1987) Bile secretion. In: Diseases of the Liver. L. Schiff and E.R. Schiff, eds. Lippincott, Philadelphia, pp. 77-101. Friend, D.S., and Dilula N.B. (1972) Variations in tight and gap junctions in mammalian tissues. J . Cell Biol., 53:758-776. Gannon, B.J. (1978)Vascular casting. In: Principles and techniques of scanning electron microscopy. Vol. 6. M.A. Hayat, ed. Van Nostrand Reinhold, New York, pp. 170-190. Henle, J . (1873) Handbuch der Systematis den Anatomie des Menschen. I1 Band, Eingeweidelehre. Friedrich Vieweg und Sohn, Braunschwirg, pp. 197-225. Hering, E. (1872) The Liver. Manual of Human Comparative Histology. Vol. 2. The New Sydenham Society, London, pp. 1-33. Hodde, K.C., and Nowell, J.A. (1980) SEM of microcorrosion casts. In: Scanning Electron Microscopyll980/II, R.P. Becker and 0. Johari, eds. SEM Inc., AMF OHare, IL, pp. 88-106. Itoshima, T., Kiyotoshi, S., Kawaguchi, K., Yoshino, K., Munetomo, F., Ohta, W., Shimada, Y., and Nagashima, H. (1980) Scanning electron microscopy of the rat bile canaliculo-ductularjunction. In: Scanning Electron Microscopy/l980/III,R.P. Becker and 0. Johari, eds. SEM Inc., AMF O’Hare, IL, pp. 373-378. Jones, A.L., Schmucker, D.L., Renston, R.H., and Murakami, T. (1980) The architecture of bile secretion. A morphological perspective of physiology. Dig. Dis. Sci., 25:609-629. Kiernan, (1833) The anatomy and physiology of the liver. Philos. Trans. R. SOC.Lond. [Biol.], 123:711-770. Lee, S.P., Lim, T.H., and Scott, A.J. (1979) Carbohydrate moieties of glycoproteins in human hepatic and gall-bladder bile, gall-bladder mucosa and gall stones. Clin. Sci., 56533-538. McIndoe, A.H. (1928) The structure and arrangement of the bile canaliculi. Arch. Pathol. Lab. Med., 6598-614. McMinn, R.M.H., and Kugler, J.H. (1961) The glands of the bile and pancreatic ducts: Autoradiographic and histochemical studies. J. Anat., 95:l-11. Motta, P., Muto, M., and Fujita, T. (1978) The Liver. An Atlas of Scanning Electron Microscopy. Igaku-shoin, Tokyo, New York. Murakami, T. (1971) Application of the scanning electron microscope to the study of the fine distribution of the blood vessels. Arch. Histol. Jpn., 32445-454. Murakami, T., Itoshima, T., and Shimada, Y. (1974)Peribiliary portal system in the monkey liver as evidenced by the injection replica scanning electron microscope method. Arch. Histol. Jpn., 37:245260. Murakami, T., Itoshima, T., Hitomi, K., Ohtsuka, A,, and Jones, A.L. (1984) A monomeric methyl and hydroxypropyl methacrylate injec-
THREE-DIMENSIONAL STRUCTURE OF BILIARY TRACT tion medium and its utility in casting blood capillaries and liver bile canaliculi for scanning electron microscopy. Arch. Histol. Jpn., 47:223-237. Spitz, L., and Petropoulos, A. (1979) The development of the glands of the common bile duct. J. Pathol., 128:213-220. Steiner, J.W., and Carruthers, J.S. (1961) Studies on the fine structure of the terminal branches of the biliary tree. I. The morphology of normal bile canaliculi, bile pre-ductule (Ducts of Hering) and bile ductules. Am. J. Pathol., 38:639-661. Terada, T., Nakanuma, Y., and Ohta, G. 11987) Glandular elements around the intrahepatic bile ducts in man; their morphology and distribution in normal livers. Liver, 7:l-8. Yamamoto, K. (1982) Intrahepatic periductal glands and their signif-
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icance in primary intrahepatic lithiasis. Jpn. J. Surg., 12163170. Yamamoto, K., and Phillips, M.J. (1984) A hitherto unrecognized bile ductular plexus in normal rat liver. Hepatology, 4:381-385. Yamamoto, K., Fisher, M.M., and Phillips, M.J. (1985) Hilar biliary plexus in human liver. A comparative study of the intrahepatic bile ducts in man and animals. Lab. Invest., 52:103-106. Yamamoto, K., and Phillips, M.J. (1986a) Three-dimensional observation of the intrahepatic lymphatics by scanning electron microscopy of corrosion casts. Anat. Rec., 214:67-70. Yamamoto, K., Sargent P.A., Fisher, M.M., and Youson, J.H. (1986b3 Convoluted bile ducts in the liver of the larval lamprey, Petromyzon marinus L. Anat. Embryol., 173:355-359.
Three-Dimensional Fine Structure of the Biliary Tract: Scanning Electron Microscopy of Biliary Casts KAZUHIDE YAMAMOTO, TATSUYA ITOSHIMA, TAKA0 TSUJI, AND TAKURO MURAKAMI First Department of Internal Medicine and Second Department of Anatomy, Okayama University Medical School, Okayama 700, Japan
KEY WORDS vascular plexus
Hilar biliary plexus, Periductal sacculi, Bile ductular plexus, Peribiliary
ABSTRACT
The three-dimensional structure of the biliary tract was studied by scanning electron microscopy (SEM) of biliary casts. The replica of the biliary tract was successfully prepared by retrograde injection of low viscosity resin into the common bile duct. Bile canaliculi are intricate networks in which hexagonal and pentagonal meshworks are interconnected. Each hexagonal or pentagonal meshwork is on a plane, but adjoining meshworks are on different planes. Bile canalicular networks connect with bile ductules a t the periphery of the portal tract. The intrahepatic bile duct showed considerable interspecies variation. The human bile duct has plexiform side branches and periductal sacculi, which are most numerous near the liver hilum and fewest in the smaller portal tracts. The hilar plexus and sacculi are present on opposite sides of the bile duct. The plexus formed a t the bifurcation of the bile ducts exhibits a plane. Periductal sacculi were also observed in the monkey and pig bile ducts, particularly the latter, while rat bile ducts possess a peculiar portal bile ductular plexus situated between the portal tract and the surrounding liver parenchyma. No such structures were observed in either the dog or rabbit bile ducts. SEM of the biliary casts showed that the biliary tract was not a simple draining tube but had additional structures, such as periductal sacculi and plexiform side branches. These structures, together with the peribiliary vascular plexus, may be implicated in the modification of bile.
INTRODUCTION The biliary tract is the bile drainage route from the liver parenchyma to the duodenum and consists of bile canaliculi, bile ductules, and intra- and extrahepatic bile ducts. The direction of bile flow is countercurrent to the blood stream. An important question is whether or not the biliary tract is a simple drainage tube. The presence of rich vascular networks around the bile duct, the peribiliary plexus (Kiernan, 1833; Murakami et al., 1974),suggests that it is not merely for drainage, but may participate in the modification of bile (Jones et al., 1980). Certain functional data support this possibility (Erlinger, 1987). Morphological and functional studies of the biliary tract have been hampered due to its inaccessibility. As a result, there have been few structural and functional correlations in the biliary tract. The three-dimensional structure of the biliary tract is difficult to study. Current methods include reconstruction studies using serial sections or stereoscopic observations of biliary tracts injected with dyes. Bile canaliculi can be observed under the light microscope following histochemical staining or the injection of dyes secreted into the biliary space (Elias, 1949; Elias and Sherrick, 1969). However, these methods are insufficient due to limited resolution and depth of field. We recently applied scanning electron microscopic analysis (SEM) of resin casts to study the biliary tract with successful results (Murakami et al., 1984; Yamamoto and Phillips, 1984; Yamamoto et al., 1985). The method was originally developed for the study of
B 1990 WILEY-LISS, INC.
the vascular system (Murakami, 19711, but can be applied to the study of other channel structures. Furthermore, the development of less viscous resins permitted injection to the depth of the bile canaliculi which are 1 pm in diameter (Murakami et al., 1984). In the present paper, we introduce this method and present results concerning the finite morphology of the biliary tract.
MATERIALS AND METHODS Injection medium Two types of injection medium, resins A and B, were used in the present study. Resin A was a mixture of 50-60% (VIV)methyl methacrylate monomer (Nakarai Chemicals Ltd., Kyoto, Japan) and 4 0 4 0 % (VIV) 2-hydroxy methacrylate monomer supplemented with 1.5% (WIV) benzol peroxide (catalyst) (Katayama Chemicals Ltd.) and 1.5%N, N-dimethylaniline (accelerator) (Katayama Chemicals Ltd.) just prior to injection. This was used to make minute biliary casts such as bile ductules and bile canaliculi (Murakami et al., 1984). Resin B was prepared by diluting commercially available resin, Mercox (Dai Nippon Ink C.C., Japan),
Received January 30, 1988; accepted in revised form April 8, 1988. Address reprint requests to Dr. Kazuhide Yamamoto, First Department of Internal Medicine, Okayama University Medical School, Shikata-cho.2-5.1, Okayama, 700, Japan
THREE-DIMENSIONAL STRUCTURE OF BILIARY TRACT
with monomeric methyl methacrylate (Nakarai Chemicals Ltd., Kyoto, Japan) a t ratios ranging from 1:l to 1:3 (Yamamoto and Phillips, 1984). Various amounts of catalyst were added to the mixture in order to control the polymerization time.
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RESULTS Intrahepatic biliary tract Bile canaliculi and canaliculoductular junction. Resin A was successfully injected into the periportal and perivenular bile canaliculi without any leakage (Fig. 1). The three-dimensional arrangement Human and animal livers of bile canaliculi appeared as a chickenwire meshwork Human livers were obtained a t autopsy within 5 composed of interconnecting hexagonal and pentagonal hours after death from patients without liver diseases. frameworks (Figs. 1 , 2 ) . Each hexagonal or pentagonal Livers from a variety of animal species, were studied, framework was on a plane, but adjoining frameworks e.g., monkey, pig, guinea pig, rabbit, and rat. Prior to were on different planes sharing only one side in injecting the resin into the common bile duct, the liver common. This spatial arrangement was easily diswas perfused with Ringer’s solution via the portal vein, cerned by observing stereomicrographs. Each side of hepatic artery, or both. the bile canalicular framework consisted of straight channels, which exhibited some narrowing and dilataInjection into bile ductules and canaliculi tion during their course. The rat liver was perfused with Ringer’s solution or Bile canaliculi connected with bile ductules at the physiological saline followed by buffered 2% glutaral- periphery of the portal tract. The transition from bile dehyde. Immediately after glutaraldehyde fixation, canaliculi to bile ductules was not uniform. Some resin A was infused into the common bile duct at a canalicular networks drained into a wider channel, i.e., pressure of 70-80 mmHg. This injection was contin- canal of Hering, which ultimately connected with a bile ued, without decrease in pressure, until the medium ductule (Figs. 2, 3). Ampullary dilatation a t the junchardened in the syringe. The resin was polymerized in tion was sometimes observed, whereas others abruptly a water bath at 60°C and the liver tissue was macer- connected with bile ductules. ated in a 30% NaOH solution. In some cases, a small Bile ducts amount of Mercox was infused into the portal vein after Human. Large intrahepatic bile ducts near the biliary injection in order to visualize the relationship hilum exhibited many irregular side branches and between the biliary tract and the portal vein. After pouches, 150-270 pm in diameter (Fig. 4). Some side rinsing in water, the biliary casts were frozen and branches ended blindly, while others anastomosed to freeze-dried. form a plexus. Smaller blind pouches extending from side branches were also observed. These side branches Injection in the intrahepatic bile duct and pouches were situated on opposite sides of the bile After vascular perfusion, a mixture of Mercox and ducts within the same plane. At the bifurcation of the monomeric methyl methacrylate (resin B) was injected bile duct, side branches from two or three main bile through a catheter inserted into the common bile duct ducts communicated with each other via the plexus. a t a pressure of 14-16 mmHg. Polymerization and These structures correspond to the periductal glands maceration were a s described above. After rinsing observed by light microscopy (Fig. 5). Side branches thoroughly in water, the biliary casts were observed and pouches were sparse in the smaller bile ducts. under a stereoscope and trimmed into small pieces. Animals. Numerous species differences were obvious in the intrahepatic bile ducts of animal livers. The Vascular casts monkey liver possessed both side branches and sacculi Hepatic vascular casts were prepared in human, similar to those observed in humans, but fewer in monkey, and rat livers a s described by Murakami et al. number. The pig liver expressed numerous sacculi up (1974). Special attention was paid to the portal and to and including the smaller bile ducts, but no side peribiliary blood supply. branches. The rat liver had a plexiform bile ductular network surrounding the portal tract (Fig. 6). No Injection into portal lymphatics analogous structures were observed in the livers of In rat and rabbit livers, resin A was injected in rabbits or dogs. excess into the common bile duct a t a pressure of 14-16 mmHg (Yamamoto et al., 1986a). The injection was Vascular supply of the portal or biliary tract continued a t this pressure until the resin polymerized The proper hepatic artery divides into the terminal in the syringe. The lymphatic casts were treated simbranches in the portal canal. The main terminal ilar to the biliary casts. branches of this artery form the peribiliary plexus Scanning electron microscopy surrounding the biliary tract and emit the efferent Biliary and lymphatic casts were carefully trimmed vessels which continue either into the liver sinusoids or into small pieces under a stereomicroscope, mounted on portal terminal branches (Fig. 7). The vascular pathmetal stubs, conductively stained by vaporized osmium way via the peribiliary plexus is called the peribiliary teroxide-hydrazine hydrate, or sputter-coated with or intrahepatic portal system (Murakami et al., 1974). gold and observed in a scanning electron microscope Other terminals continue into the sinusoids or supply with a n accelerating voltage of 10-15 kV. Electron the portal canal. The portal plexus is coarse and drains micrographs were taken in stereopairs for three- into the sinusoids, peribiliary efferent vessels or portal terminal branches (Fig. 8). dimensional analysis.
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Fig. 1. Scanning electron micrograph of bile canalicular casts. Note bile canalicular networks (c) drain into the canal of Hering (b) and then into bile ductules (B). x 1,100.
THREE-DIMENSIONAL STRUCTURE OF BILIARY TRACT
~ i2, ~~ , i magnification ~ h of c~a n a ~~~ c u ~ o d u c tjunctions u~ar (arrowheads). b, bile ductule; c, bile canaliculus. x 2,700.
Portal lymphatics Casts of lymphatic channels were prepared by injecting excessive amounts of resin into the bile duct. Resin B did not enter the lumen of bile canaliculi and excess resin leaked after filling the biliary tract. The resin leaked into the portal interstitial spaces near the terminal portal tract and drained into the lymphatic channels (Fig. 9a). Lymphatic casts demonstrated that the terminal portal lymphatics end blindly (Fig. 9b). The lymphatics found in the portal tract were composed of long, straight channels and interconnecting short branches. Valvelike constrictions were sometimes observed (Fig. 10). Lymphatic networks were especially rich around the bile duct and the bifurcation of the portal tract and these ultimately drained into the lymphatics in the hilum of the liver.
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Fig. 3. SEM of fractured liver surface. Note ampullary dilatation at canaliculo(c)-ductular junction (arrowhead). The other bile canaliculus terminates directly into the bile ductule (arrow). x 900.
Steiner and Carruthers, 1961). On the other hand, the three-dimensional morphology of the liver is still incomplete. The recent application of SEM to the study of hepatic tissues (Itoshima et al., 1980; Motta et al., 1978) had contributed t o the understanding of its three-dimensional structure. However, the threedimensional architecture of hepatic channel structures, such as the vascular and biliary systems, has been difficult to demonstrate. Injection of various materials into the channels has been a popular method since the late 19th century (Beale, 1889; Hering, 1872). However, the method has seen only limited use since both the resolution and depth of field are insufficient. SEM of resin casts to study hepatic microvasculature (Murakami, 1971) introduced a new era for the study of this sytem (Gannon, 1978; Hodde and Nowell, 1980). This method has been applied to the study of other DISCUSSION channel structures, S U C -as ~ the biliary &act (Murakami et al., 1984; Yamamoto and Phillips, 1984; The two-dimensional fine structure of the hepatobil- Yamamoto et al., 1985) and lymphatics (Yamamoto iary system has been studied by light and transmission and Phillips, 1986a). In the present study, the threeelectron microscopy and the detail is well established, dimensional arrangement of the biliary tract has been even at the level of subcellular structure (Biava, 1964; examined using this method.
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Fig. 4. Survey SEM of human hilar biliary tract. Note the rich plexiform side branches and sacculi (arrowheads) on the opposite sides of the bile duct (D). x 12.
THREE-DIMENSIONAL STRUCTURE OF BILIARY TRACT
Fig. 5. Light micrograph of bile ductules (arrowheads) in the portal connective tissue near the hilum corresponding to side branches in Figure 4. x 50.
Intrahepatic biliary tract Bile canuliculi and canaliculoductular junction. Bile canaliculi are the smallest biliary channels
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Fig. 6. SEM of rat bile ductular plexus. Bile ductules (d) surround portal interstitial space (P). x 50.
ing complexes may represent a structural basis for maintaining intralobular gradients for the secretion of various materials into bile canaliculi (Erlinger, 1987). formed by two or three adjacent hepatocytes. The Periportal hepatocytes (zone 1) are bathed in plasma two-dimensional fine structure of these channels has containing higher concentrations of constituents than been studied by transmission electron microscopy (Bi- those cells at the hepatic venous end (zone 3). This ava, 1964; Steiner and Carruthers, 1961).Bile canalic- concentration gradient in the blood is reflected in the uli are situated in the center of the muralium (Elias solute content of the bile. Partial loss of tight junction and Sherrick, 1969) of one-cell-thick plates and are integrity may facilitate the movement of ions and equidistant from the sinusoids on both sides. Their water between the intercellular space and the bile arrangement has been clearly demonstrated by scan- canaliculi. The bile canalicular networks drain into the bile ning electron microscopy of fractured liver surfaces (Fig. 3) (Itoshima et al., 1980; Motta et al., 1978). The ductules at the periphery of the portal tract. The bile canalicular lumen is defined by grooves in adjacent nature of this connection has been a subject of considhepatocytes and the wall is composed of a specialized erable debate (Itoshima et al., 1980; McIndoe, 1928; membrane with microvilli. The lumen is separated Steiner and Carruthers, 1961). A recent SEM study of from the intercellular space by junctional complexes, fractured liver surfaces demonstrated the presence of including tight junctions, although molecules, such as two types of termination (Itoshima et al., 19801, and water and small ions, are thought to permeate this this has been confirmed in the present study using bile barrier (Boyer, 1980; Coleman, 1987; Friend and Di- canalicular casts. In the first type, several bile canaliculi coalesce into a thick channel corresponding to a lula, 1972). SEM of bile canalicular casts revealed their three- canaliculoductular junction, i.e., a canal of Hering, in dimensional arrangement for the first time (Figs. 1,2). which the channel is formed partly by hepatocytes and They form intricate networks composed of a hexagonal partly by ductular cells (Fig. 1). In the rat liver, or pentagonal meshwork. Each straight side of a hexa- ampullary dilatation is observed at this type of termigon or pentagon is formed by the neighboring two or nation (Itoshima et al., 1980). The alternative type three hepatocytes. However, there is little geometric consists of bile canaliculi terminating directly into uniformity in this network. More sophisticated meth- portal bile ductules without ampullary dilatation. Bile ducts. The present study demonstrated that ods, such as computer analysis, are necessary for human intrahepatic bile ducts possess additional strucanalyzing this complicated spatial architecture. The bile canalicular networks are intercalated with tures, such as periductal sacculi and plexiform side vascular networks, i.e., sinusoids. These interdigitat- branches. These structures may correspond to the
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Fig. 7. SEM of vascular casts forming peribiliary Plexus in the monkey liver. An efferent vessel (e) drains into sinusoids (s). x 100.
l?ig. 8. SEM of vascular casts of plexus supplying the portal canal and which drains into sinusoids (s). 60,
parietal sacculi and vasa aberantia reported by Henle (1873) and Beale (1889). Similar structures have been termed tubuloalveolar glands or periductal glands (Burden, 1925; McMinn and Kugler, 1961; Spitz and Petropoulos, 1979). Numerous periductal glands are also present in the extrahepatic bile duct (Andrews and Andrews, 1979) and contain mucinous materials secreted into the bile (Chou and Gibson, 1979). Similar mucous-type glycoproteins have been demonstrated in human bile (Bouchier et al., 1965; Lee et al., 1979). Recent histochemical data suggest the presence of secretary component and immunoglobulins in the periductal glands (Terada et al., 1987). Furthermore, these glands increase during certain infections and conditions of stagnated bile, suggesting that periductal glands may contribute to local immunity (Chou and Gibson, 1970; Yamamoto, 1982). Plexiform side branches or vasa aberantia may also function a s collaterals for bile drainage (Yamamoto et al., 1985). Many species differences were demonstrated in the structure of the intrahepatic bile ducts which may explain, in part, concomitant differences in bile compo-
sition (Erlinger, 1987). For example, in the rat, which lacks a gall bladder, the ductular plexus may function as a reservoir for bile.
Vascular supply of portal and biliary tracts The bile ducts are surrounded by a thick vascular network called the peribiliary vascular plexus (Kiernan, 1833; Murakami et al., 1974). The plexus is composed of inner afferent networks and outer efferent networks, the latter draining directly into liver sinusoids, peribiliary efferent vessels or terminal portal branches. The drainage into efferent vessels and terminal portal branches is termed the peribiliary portal system (Murakami et al., 1974). The exact function(s) of the plexus remains unresolved, but may include the exchange of solute(s)between bile and blood, secretion into or reabsorption from the bile. It is interesting to note that even in the lamprey liver, the bile ducts exhibit a n intimate relationship with the vascular system (Yamamoto et al., 1986131, suggesting that this biliovascular relationship is a well-conserved trait. The portal system may function as a type of feedback
THREE-DIMENSIONAL STRUCTURE OF BILIARY TRACT
Fig. 9 a: SEM of lymphatic casts in the large portal tract. Note rich anastomosis at the bifurcation of the portal tract. x 30. b: Leaked resin near the terminal portal tract drains into lymphatics (L). D, bile duct. ~ 8 0 .
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of bile composition a t the various levels of the biliary tract is essential to this issue.
REFERENCES
Fig. 10. Higher magnification of lymphatic casts showing a valvelike constriction (arrowhead). X 350.
system, wherein some bile constituents may be reabsorbed into the blood and, eventually back to the hepatocytes (Jones et al., 1980). Thus, some biliary signals may be transferred to hepatocytes in order to modulate bile secretion.
Portal lymphatics The lymphatics represent another drainage route from the liver. The excess resin injected into the biliary system leaked into the interstitial spaces of the portal tract and, thus, into the lymphatics. The implication of this observation is that biliary materials may easily enter the lymphatics and drain back into the blood during increased biliary pressure. This may explain the early appearance of bilirubin in the thoracic lymphatics of patients with biliary obstruction (Dumont, 1973). CONCLUSIONS The present study summarizes the threedimensional fine structure of the biliary tract as visualized by scanning electron microscopy of biliary tract casts. The most important morphological feature of the biliary tract is its intimate relationship with the vascular system throughout its course. This biliovascular relationship suggests that the biliary tract may not be a totally autonomous structure but, rather, exhibits a dynamic exchange of constituents with the tissue space and blood. However, the properties corresponding to the various morphological components of the biliary tract remain to be resolved. A comprehensive analysis
Andrews, C.J.H., and Andrews, W.H.H. (1979) The relation between structure and function of bile ducts in man, some laboratory animals and the Adelie Penguin. J . Exp. Physiol., 64:61-67. Beale, L.S. (1889)The Liver. Lecture on the Principles and Practice of Medicine. Churchill, London, pp. 47-52. Biava, C.G. (1964) Studies on cholestasis. A re-evaluation of the fine structure of normal human bile canaliculi. Lab. Invest., 13340864. Bouchier, I.A.D., Cooperband, S.R., and ElKodsi, B.M. (1965) Mucous substances and viscosity of normal and pathological human bile. Gastroenterology, 49:343-353. Boyer, J.L. (1980) New concepts of mechanisms of hepatocyte bile formation. Physiol. Rev., 36:303-326. Burden, V.G. (1925) Observations on the histologic and pathologic anatomy of the hepatic, cyctic, and common bile ducts. Ann. Surg., 82584-587. Chou, S.T., and Gibson, J.B. (1970) The histochemistry of biliary mucins and the changes caused by infestation with Clonorchis Sinensis. J. Pathol., 101:185-197. Coleman, R. (1987) Biochemistry of bile secretion. Biochem. J., 244249-261. Dumont, A.E. (1973) Icteric thoracic duct lymph: Significance in patients with manifestations of obstructive jaundice. Ann. Surg., 178:53-58. Elias, H. (1949) A re-examination of the structure of the mammalian liver. 11. The hepatic lobule and its relation to the vascular and biliary system. Am. J. Anat., 85379-456. Elias, H., and Sherrick, J.C. (1969) Morphology of the Liver. Academic Press, Inc., New York and London, pp, 137-185. Erlinger, S. (1987) Bile secretion. In: Diseases of the Liver. L. Schiff and E.R. Schiff, eds. Lippincott, Philadelphia, pp. 77-101. Friend, D.S., and Dilula N.B. (1972) Variations in tight and gap junctions in mammalian tissues. J . Cell Biol., 53:758-776. Gannon, B.J. (1978)Vascular casting. In: Principles and techniques of scanning electron microscopy. Vol. 6. M.A. Hayat, ed. Van Nostrand Reinhold, New York, pp. 170-190. Henle, J . (1873) Handbuch der Systematis den Anatomie des Menschen. I1 Band, Eingeweidelehre. Friedrich Vieweg und Sohn, Braunschwirg, pp. 197-225. Hering, E. (1872) The Liver. Manual of Human Comparative Histology. Vol. 2. The New Sydenham Society, London, pp. 1-33. Hodde, K.C., and Nowell, J.A. (1980) SEM of microcorrosion casts. In: Scanning Electron Microscopyll980/II, R.P. Becker and 0. Johari, eds. SEM Inc., AMF OHare, IL, pp. 88-106. Itoshima, T., Kiyotoshi, S., Kawaguchi, K., Yoshino, K., Munetomo, F., Ohta, W., Shimada, Y., and Nagashima, H. (1980) Scanning electron microscopy of the rat bile canaliculo-ductularjunction. In: Scanning Electron Microscopy/l980/III,R.P. Becker and 0. Johari, eds. SEM Inc., AMF O’Hare, IL, pp. 373-378. Jones, A.L., Schmucker, D.L., Renston, R.H., and Murakami, T. (1980) The architecture of bile secretion. A morphological perspective of physiology. Dig. Dis. Sci., 25:609-629. Kiernan, (1833) The anatomy and physiology of the liver. Philos. Trans. R. SOC.Lond. [Biol.], 123:711-770. Lee, S.P., Lim, T.H., and Scott, A.J. (1979) Carbohydrate moieties of glycoproteins in human hepatic and gall-bladder bile, gall-bladder mucosa and gall stones. Clin. Sci., 56533-538. McIndoe, A.H. (1928) The structure and arrangement of the bile canaliculi. Arch. Pathol. Lab. Med., 6598-614. McMinn, R.M.H., and Kugler, J.H. (1961) The glands of the bile and pancreatic ducts: Autoradiographic and histochemical studies. J. Anat., 95:l-11. Motta, P., Muto, M., and Fujita, T. (1978) The Liver. An Atlas of Scanning Electron Microscopy. Igaku-shoin, Tokyo, New York. Murakami, T. (1971) Application of the scanning electron microscope to the study of the fine distribution of the blood vessels. Arch. Histol. Jpn., 32445-454. Murakami, T., Itoshima, T., and Shimada, Y. (1974)Peribiliary portal system in the monkey liver as evidenced by the injection replica scanning electron microscope method. Arch. Histol. Jpn., 37:245260. Murakami, T., Itoshima, T., Hitomi, K., Ohtsuka, A,, and Jones, A.L. (1984) A monomeric methyl and hydroxypropyl methacrylate injec-
THREE-DIMENSIONAL STRUCTURE OF BILIARY TRACT tion medium and its utility in casting blood capillaries and liver bile canaliculi for scanning electron microscopy. Arch. Histol. Jpn., 47:223-237. Spitz, L., and Petropoulos, A. (1979) The development of the glands of the common bile duct. J. Pathol., 128:213-220. Steiner, J.W., and Carruthers, J.S. (1961) Studies on the fine structure of the terminal branches of the biliary tree. I. The morphology of normal bile canaliculi, bile pre-ductule (Ducts of Hering) and bile ductules. Am. J. Pathol., 38:639-661. Terada, T., Nakanuma, Y., and Ohta, G. 11987) Glandular elements around the intrahepatic bile ducts in man; their morphology and distribution in normal livers. Liver, 7:l-8. Yamamoto, K. (1982) Intrahepatic periductal glands and their signif-
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