MICROSCOPY RESEARCH AND TECHNIQUE 38:547–551 (1997) Introduction to The Biliary Tract, The Gallbladder, and Gallstones JACQUES GILLOTEAUX* Summa Health System Foundation, Akron, Ohio 44304 KEY WORDS biliary tract; gallbladder; nomenclature; microscopy; bile acids ABSTRACT This paper serves to introduce a topical section of fifteen invited original research contributions dealing with normal and pathological development of the human biliary tract. This section also includes comparative anatomy of the gallbladder and the cystic duct as well as, the formation of gallstone. This series of reports have used advanced microscopic and ancillary techniques to study adaptative changes in gallbladder epithelial cell changes regarding permeability, renewal, mucous secretion as well as cholesterol uptake and nucleation. Several contributions deal with the blood and lymphatic drainage of the gallbladder. The gallbladder contractility is clarified by recent findings about its innervation, elegantly demonstrated and supported by complementary immunohistochemical and neurophysiological techniques. In vivo models for production of cholelithiasis in the ground squirrel and the Syrian hamster are introduced. Recent in vitro cellular and molecular models have substantially increased the understanding of biliary tract calculi formation. Finally, a survey and new data about progesterone gene regulation of both cholesterol metabolism and gallstone formation obtained in the Syrian hamster model are compared with cholelithogenesis in human. Microsc. Res. Tech. 38:547–551, 1997. r 1997 Wiley-Liss, Inc. OVERVIEW OF THE BILIARY SYSTEM The biliary system includes the secretory units of the liver, the bile canaliculi, bile ductules (canals of Hering), intrahepatic bile ducts, extrahepatic bile ducts, the common hepatic duct, the cystic duct, the gallbladder, and the common bile duct. Some confusion remains as to the terminology of the components of the biliary system (i.e., interlobular 5 intrahepatic). All vertebrates possess a biliary tract but not all of them possess a gallbladder whose function as an accessory organ of the digestive system is to participate in the enterohepatic circulation of bile (Hofmann, 1968, 1990, 1994) by storing and concentrating biliary compounds through the removal of water and the exchange of electrolytes (Diamond, 1962; Dietschy and Moore, 1964; Wheeler, 1968). The biliary system in man includes all of the aforementioned components, although this is not true of all vertebrates. Pfuhl (1932) reviewed the literature and histology of the biliary system in selected vertebrates, including humans. Morphological aspects of the biliary system during development, differentiation (Desmet, 1994; Phillips, 1994), and histopathology (Sirica, 1995) have been reviewed recently. In this monograph, the paper of Nakanuma et al. (1996a) describes the normal morphology from early development to a differentiated stage of the human biliary tract and its pathology. It has been reported that the histochemical characterization of biliary hepatic cells could play an important diagnostic role in pathology (Strain et al., 1995). Nakanuma et al. (1997a) have also described the extra- and intrahepatic peribiliary glands (development, differentiation, apoptotic growth, remodeling and differentiation, immunologic functions, etc.). These glands constitute intramural and extramural r 1997 WILEY-LISS, INC. mixed mucous and serous acini with the serous parts resembling exocrine pancreatic acini (Terada et al., 1990) that do not contain islets of Langerhans’s tissue. This observation is particularly interesting because Gilloteaux et al. (1995) and Oldham-Ott and Gilloteaux (1997) illustrate that in lower vertebrates (fish) pancreatic tissue is commonly detected in the vicinity of the gallbladder. The bile canaliculi are formed by the adjacent plasma membranes of the hepatocytes. This was seen by light microscopy in 1911, and one of the first TEM studies of bile canaliculi demonstrated this feature (Rouiller, 1956). Previously, these channels were thought to be intercellular tubules with their own walls. Even Rouiller once believed that the bile canaliculi constituted an incomplete barrier between the space of Disse and the lumen of the canaliculi (Steiner and Carruthers, 1961). It is now known that tight junctions and other specialized junctions (gap junctions and desmosomes) separate the canaliculi lumen from the hepatocytes. In an interesting monograph of Matter et al. (1969) it is shown that these tight junctions are efficient permeability barriers. Moreover, bile canaliculi are able to have a coordinated contractile activity by pericanalicular, actomyosinic interactions controlled by calcium. This action was observed in pathology (Oda et al., 1974; Oda and Phillips, 1977) and in vitro (Oshio and Phillips, 1981, in review by Phillips, 1994; Phillips et al., 1983; Smith et al., 1985; Watanabe et al., 1983; Watanabe and Phillips, 1984). In addition, abundant microvilli, with varied *Correspondence to: Dr. J. Gilloteaux, Summa Health System Foundation, Arch Street 41, Suite 506, Akron, Ohio 44304. Accepted 1 August, 1995 548 GILLOTEAUX shapes and sizes, due to actin filaments remodeling (Forscher et al., 1992) protrude into the lumen of the canaliculi (Matter et al., 1969; Rouiller and Jezequel, 1963). The bile ductules are circumscribed by two to four epithelial cells, eventually becoming cuboidal, with blunt microvilli, a basal lamina, a large spherical nucleus, poorly developed rough endoplasmic reticulum, many free ribosomes, and few mitochondria. The cells contact one another through complex interdigitations and have extensive tight junctions (Gabe, 1973). The microvilli-coated intrahepatic bile duct cells progress from a cuboidal to a columnar epithelium on a basal lamina and they often bear a single extended (perhaps motile) flexuous cilium ranging between 2 to 4 µm in length. An overview of the embryology, anatomy, histochemical typing, and functioning of the epithelial cells of the intrahepatic bile ducts (cholangiocytes) has been performed by Alpini et al. (1994). The extrahepatic bile ducts are composed of tall, columnar epithelial cells, surrounded by a sheet of connective tissue. Elastic and collagen fibers have been observed in the basement membrane; tubular or tubuloalveolar glands, varying according to species, have also been observed. The material secreted by the extrahepatic duct has been identified as mucin with acid mucopolysaccharides (Gabe, 1973). A submucosa, muscularis, and adventitia are present. The extrahepatic bile ducts join to form the common hepatic duct. This duct connects with the cystic duct from the gallbladder to form the common bile duct (ductus choledochus). The common bile duct has been observed to contain tubular glands (Spitz and Petropoulos, 1979) and PASpositive cells. The glands are more frequent in animals without a gallbladder, such as the rat (Jones and Spring-Mills, 1983). We refer the reader to Nakanuma et al. (1997a) for a more detailed and recent account of these structures. During bile formation and all along the pathway to the gallbladder many of the cells involved show evidence of active and passive transport of water and electrolytes. The anatomy of the various bile ducts differs widely among vertebrate groups. The Gallbladder A review of the morphological components of the biliary tract, the gallbladder, and the formation of gallstones across the vertebrates is offered by OldhamOtt and Gilloteaux (1997). This review shows that the gallbladder’s microscopic architecture displays a homologic histological pattern throughout the vertebrates in terms of layering. A lamina propria, fibromuscular tunic, subserosal, and serosal tunics are normally present. Small nerve ganglia and adipose tissue are present between the fibromuscular and subserosal layers. The thickness of the gallbladder wall also varies in vertebrates. It may be quite thick, as in many carnivores and primates, or very thin, as in the lower vertebrates (fish, amphibians), mouse, and guinea pig. The gallbladder’s mucosal epithelium is usually composed of tall columnar cells, displays a PAS-positive apical aspect (in carnivores it contains lipidic inclusions), and is coated with microvilli and other characteristics of absorptive cells, including basolateral spaces and digitations. Prominent junctional complexes are present (Gabe, 1973). Bulging epithelial apices and decapita- tions of excrescent apical structures can be found occasionally. These are more abundant in cholesterolosis (Gilloteaux et al., 1996b), in male, in ovariectomized, and in intact female Syrian hamster where cholelithiasis was induced by steroid hormones (Gilloteaux et al., 1992, 1993a–c; Karkare et al., 1995; Karkare and Gilloteaux, 1995). The same apical excrescences are morphologically similar to the secretory endometrial epithelial cell apices during the postovulatory phase (Wessel, 1960; Wetzstein and Wagner, 1960; Wynn and Wooley, 1967). This phenomenon has also been observed in fetal gallbladders (Laitio and Nevalainen, 1972) and in fish gallbladder (Gilloteaux et al., 1995; Oldham-Ott and Gilloteaux, 1997; Viehberger, 1982). In the surface epithelium of the mouse gallbladder, columnar brush cells, which are possibly sensory cells, were discovered in 1969 by Luciano and Reale and are elegantly illustrated in this monograph, as they have included a freeze-fracture technique (Luciano and Reale, 1997). Interestingly enough, similar brush cells appear to be present in some amphibians and reptilian gallbladders (Oldham-Ott and Gilloteaux, 1997). Lamote and Willems (1997) demonstrate that the normal gallbladder epithelium has a very low rate of cell renewal, while mechanical distension by ligation of the common bile duct or by neoplastic obstruction in humans increases the mitotic activity of the tissue. Other influences, such as hormones (caerulein, cholecystokinin (CCK)-octapeptide, CCK analogs), or a diet rich in cholesterol and cholic acid, can stimulate high DNA synthesis in the gallbladder epithelium. Fundusectomy or the presence of gallstones stimulates proliferative activity. These authors suggest that this proliferative activity could be the result of hypergastrinemia. Rokitansky-Aschoff crypts reaching the fibromuscular layer are present in humans. Canals of Luschka, in which the epithelium invaginates through the fibromuscular layer and may contact blood vessels, have been observed in humans, are well defined in the rabbit, and almost absent in the cat (Gabe, 1973). Gallbladder tubular and tubulo-alveolar glands, whose presence and abundance is variable in the lamina propria, are similar to those found in the extrahepatic ducts. In primates, these glands are located near the infundibulum of the gallbladder, and in ruminants they form a subepithelial layer (equivalent to Brunner’s glands). These glands are rare in carnivores (Gabe, 1973). Permeability Many of the regulatory mechanisms of ion transport across the gallbladder epithelium have been resolved physiologically (Reuss, 1989; Reuss et al., 1991; Wheeler, 1971). Hopwood and Ross (1997) survey the permeability ‘‘barriers’’ of the gallbladder epithelium in terms of water and ion removal. Using the mouse, they demonstrate that the gallbladder wall is able to take up significant amounts of lipids and that inflammatory reactions can alter membrane composition and functions. Finally, using a cationic, cytochemical probe (cuprolinic blue; reviewed by Van Kuppevelt and Veerkamp, 1994), Hopwood and Ross demonstrate a significant increase of glycosaminoglycans in the basement membrane in the cholecystic gallbladder. BILIARY TRACT, GALLBLADDER, AND GALLSTONES Mucus Using histo- and cytochemical lectin markers, Madrid et al. (1989, 1994) surveyed and investigated characteristics of the glycoproteinaceous secretory products and glycocalyx of the gallbladder epithelium across several species, including humans. In their contribution, Madrid et al. (1997) verify that most of the mucus is neutral, heavily sulfated, and contains scarce sialic residues. They also discuss the presence of core proteins which are known molecular markers for mucus types (Audie et al., 1993; Campion et al., 1995). Our ultrastructural description of the human cholecystitic (Gilloteaux et al., 1989) and cholelithiatic gallbladder shows anionic mucus secretory granules (Gilloteaux et al., 1997c). Koga (1985) and Satoh and Koga (1997) review the pathological gallbladder storage of cholesterol in cholesterolosis. In the case of the cystic duct, Gilloteaux et al. (1997a) confirm the differential morphology of the gallbladder in cholesterolosis and cholelithiasis. It is interesting to note that LaMont (1989) has hypothesized that oxygen radicals can stimulate gallbladder glycoprotein secretion. Blood and Lymphatic Supply The complex vasculature of the biliary tract and its development is surveyed by Nakanuma et al. (1997a). Blood supply to the mammalian gallbladder is by way of small and medium arteries which cross the fibromuscular layer and form a fenestrated subepithelial plexus in the lamina propria. There, blood flow could be controlled by neuropeptides, such as NPY (Gilloteaux et al., 1990–1991). Using SEM on corrosion casts and on KOH-macerated samples, Ohtani et al. (1997) illustrate the blood and lymphatic microvascularization of the guinea pig gallbladder; they demonstrate that, as in the small intestine, and in the liver lymphatics start their trajectory as blind ends. Innervation Gallbladder innervation resembles that of the intestine and has been comprehensively discussed by Cai and Gabella (1983, 1984), Goehler et al. (1988), Mawe and Gershon (1989), Mawe (1990, 1991, 1993), Talmage et al. (1992), etc. As reported in the monograph by Mawe et al. (1997), the musculature and the epithelial tissues of the guinea pig gallbladder are regulated by small clusters of neurons which constitute a ganglionated network containing diverse important neuropeptides and enzyme markers. From a physiological point of view, they also report that this network acts as a complex neuromodulator, and demonstrate that hormonal CCK can access gallbladder ganglia presynaptically (no blood-barrier) to facilitate vagal terminal release in the musculature and the epithelial layer of the gallbladder. Splanchnic mechanoreceptors of the biliary tract and its mesentery have been studied by Crousillat and Ranieri (1980). Gallbladder contraction is mediated by cholecystokinin, the cholecystokinin antagonist pancreatic polypeptide, and others too numerous to list here (Adrian et al., 1982; Conter et al., 1987; Spellman et al., 1979). The presence and degree of development of a sphincter regulating the flow of bile is directly related to the presence or absence of a gallbladder; in those animals 549 lacking a gallbladder, the sphincter appears poorly developed (Dorst, 1973; Gorham and Ivy, 1938). Models In vivo models for producing cholelithiasis have also been developed by using diverse animals (review in Oldham-Ott and Gilloteaux, 1997). Using ground squirrels fed a high-cholesterol diet, MacPherson and Pemsingh (1996) have shown that mucus hypersecretion occurs prior, during, and after stone formation. Gilloteaux et al. (1996c) have shown similar changes and stone formation after perturbing hepatic bile metabolism using female sex steroids in the male, in ovariectomized, and in intact female Syrian hamsters (Gilloteaux et al., 1992; Gilloteaux et al., 1993a–c; Karkare et al., 1995; Karkare and Gilloteaux, 1995). Medroxyprogesterone treatment alone can alter bile and mucus composition, favoring formation of gallstones via a down-regulation of the cholesterol 7a-hydroxylase gene expression (Qiu et al., 1994; Gilloteaux et al., 1997). In vitro models are being developed to comprehend the cell biology and pathology of the gallbladder epithelium. Nakanuma et al. (1997b) demonstrate that mucus secretion characteristics can be maintained and studied morphologically with in vitro-grown epithelium. Bile Acids The concentrated bile secreted into the duodenum via the permissive effect of cholecystokinin (Mawe et al., 1991, 1997) provides a micellar suspension facilitating intestinal absorption of fat soluble compounds, including vitamins (Borgström et al., 1985), as well as scavenging peroxyl radicals (Stocker and Ames, 1987; Stocker et al., 1987a–b). In addition, bile has a laxative property due to its effect on the motility of the large intestine (Hofmann, 1968). The composition of bile acids in the bile varies in the animal kingdom (Haslewood, 1968; Hoshita, 1985; Hofmann, 1990, 1994; etc.): The fish groups produce various compounds (bile alcohols in ancient fish, such as the shark), amphibians generally produce bile alcohols, and the reptiles produce bile acids but no bile alcohols. In birds and mammals, the type of bile acids produced seem dietdependent. Bile alcohols usually occur only in ancient groups of mammals. Omnivores (including humans) generally produce dihydroxy bile acids and carnivores trihydroxy bile acids. Conjugated xenobiotics and bilirubin are other important components of bile (Arias, 1968). In addition to bile acids, bile pigments vary with the animal species and it is thought that gaining an understanding of this diversity of bile pigments will aid in comprehending altered bile metabolism in humans (Cornelius, 1986). In plants, these pigments perform photosynthetic functions. Bile pigments in mammals have no known function; they are, however, potentially toxic (Cornelius, 1986; Troxler, 1986). Further investigations in this area are clearly indicated. Examining artificial and human native bile by cryotransmission electron microscopy, Kaplun et al. (1996) have demonstrated that dilution induces cholesterol supersaturation, resulting in the production of a range of microstructures: spheroidal, uni- and multilamellar micelles, followed by tubular and helical as well as the classical plate-like cholesterol. This model can help to 550 GILLOTEAUX decipher the beneficial role of bile in preventing the cholesterol nucleation process in the gallbladder. In order to comprehend the pathologic etiology of the hepatobiliary system, most investigations have to rely on case studies, or animal models and in vitro studies. It would be interesting to obtain fresh postmortem or surgical human specimens in order to more fully describe certain structures, such as the cystic duct (Gilloteaux et al., 1997b), which are easily damaged during autopsy or following fixation delays after surgical removal. ACKNOWLEDGMENTS I would like to thank Dr. J.E. Johnson Jr., Chief Editor, for inviting me as Guest Editor for this special monograph. I would also like to thank all the contributors for their efforts in providing informative and well-illustrated manuscripts on the topic, and recognize the experts and reviewers for their scientific assistance and for having returned as promptly as possible the manuscripts submitted to them. I wish, therefore, to acknowledge Drs. Ian A.D. Bouchier, V.J. Desmet, Mr. L.C. Gilloteaux, Drs. A. Groom, R. Hakanson, W.S. Hawkins, M.R. Jacyna, E.R. Lacy, L. Luciano, S. Nagamori, Mrs. C. Oldham-Ott, Drs. G. Perry, E. Reale, W. Schemann, P. Scheuer, B.A. Schulte, D.P. Siegel, R.J. Spontak, P. 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