Morphology of the bile ducts of the brook lamprey Lampetra lamottenii Le Sueur before and during infection with the nematode Truttaedacnitis stelmioides Vessichelli 1910 NematodaCucullanidae.код для вставкиСкачать
THE ANATOMICAL RECORD 2341201-214 (1992) Morphology of the Bile Ducts of the Brook Lamprey, Lampetra lamuttenii (Le Sueur) Before and During Infection With the Nematode, Truttaedacnitisstelmiuides (Vessicheili, 1910) (Nematoda: Cucullanidae) F.ENG AND J.H. YOUSON Department of Zoology and Scarborough Campus, University of Toronto, West Hill, Ontario M l C 1A4, Canada ABSTRACT Routine light microscopy and transmission and scanning electron microscopy were used to describe and compare the biliary tree of larval Lumpetru lumottenii before and during infestation of the bile ducts with the nematode, Truttaedacnitis stelmioides. The most prominent changes to the biliary tree following infection by the parasite are the dilation of the bile ducts, alterations to their epithelial cells, and an increase in periductal fibrous tissue. In recently infected animals, the simple epithelium of dilated bile ducts often contains many mitotic figures. In long-term infestations, the epithelium is stratified or pseudostratified. Changes to the fine structure of the biliary epithelial cells include increase and/or dilation of the RER and SER, and increases in microfilaments, intermediate filaments, and microtubules. The abundance of dense bodies may reflect enhance reabsorption of biliary constituents, and their accumulation may ultimately result in cytolysis. There are increased mucous granules in the apical cytoplasm of biliary epithelial cells and an abundance of mucinous material within the bile duct lumen, and the basal lamina appears thickened. The changes to the liver of L. lumottenii following infection are discussed and compared to those reported in small mammals following bile duct ligation, in patients with extrahepatic biliary obstruction, and in parasitic infection of the biliary tree. o 1992 Wiley-Liss, Inc. Key words: Bile ducts, Lamprey, Nematode, Infection, Face structure The microscopic anatomy of the liver in various species of lampreys has been extensively studied (Bertolini, 1965; De Vos et al., 1973; Shin, 1977; Peek et al., 1979), and it has been shown that this organ in larval (ammocoetes)lampreys is similar to that of adult mammals. The biliary tree collects and transports bile to the intestine where it is eliminated from the body (Youson, 1981) and may play a role in the modification of bile in a manner similar to that found in most mammalian livers (Yamamoto et al., 1986a). The cells of the bile duct are specialized for absorption and transport and are similar to the cells of the bile duct in other vertebrates. The gall bladder, however, is less specialized for water transport (Sidon et al., 1980). During metamorphosis in all species of lampreys, there is reorganization of the liver as well as loss of the biliary tree (i.e., biliary atresia) and gall bladder (Youson and Sidon, 1978; Sidon and Youson, 1983a,b; Youson and Ogilvie, 1990). The loss of the biliary tree does not seem to have immediate visible effects on the recently metamorphosed, juvenile adult, but eventually the bile pigments, bilirubin and biliverdin, accumulate in the liver and blood (Makos and Youson 1987, 1988). During the upstream spawning migration, the 0 1992 WILEY-LISS, INC. consequences of the aductular liver are manifested in the fact that the liver and urine are green and the skin is yellow-orange (i.e.,jaundice). According to Bertolini (1965), the huge bile accumulation that follows the loss of bile ducts in lampreys causes hepatic cell damage and subsequent cytolysis, and thus, the elimination of bile is essential in most vertebrates (McDonagh, 1979). Usually no signs of bile stasis are present in larval lampreys (Makos and Youson, 1987); yet it might be expected that, as in other vertebrates, anything that interrupts the normal flow of bile could result in a cholestatic condition. Previously, Pybus et al. (1978) reported the presence of the nematode Truttaedacnitis stelmioides (Vessichelli, 1910) (Nematoda: Cucullanidae) in the bile ducts of the larval American brook lamprey, Lampetra lamottenii. It is possible that the parasite would obstruct the normal flow of bile and Received October 15, 1990; accepted January 24, 1992. Address reprint requests to J.H. Youson, Division of Life Sciences, Scarborough Campus, University of Toronto, West Hill, Ontario M1C 1A4, Canada. 202 F. ENG AND J.H. YOUSON cause morphological alterations to the liver which parallel those occurring during lamprey biliary atresia a t metamorphosis (Sidon and Youson, 1983a,b),in human pathology (Schaffner and Popper, 1959a,b; Hollander and Schaffner, 1986b; Schaff and Lapis, 19791, or following experimental bile duct obstruction in small mammals (Layden et al., 1975; Jones et al., 1976). In a recent study, we reported that the presence of the bile pigment, biliverdin, was responsible for the green coloration of the serum of larval L. Zamottenii (Eng and Youson, 1991) at a time when they were infected. It was the belief that the above was a consequence of the increased biliary pressure brought about by the obstruction of the bile ducts. Biliary parasites have been known to produce mechanical extrahepatic and intrahepatic biliary obstruction resulting in alterations to the biliary tract. These alterations include biliary epithelia1 proliferation and hyperplasia, dilation of the bile ducts, periductal fibrosis, proliferation of bile ducts and ductules, and replacement of hepatic parenchyma with connective tissue (Belding, 1965a,b; Sun and Tung-Ma, 1973; Muller, 1975; Ong, 1985). In this present study, light microscopy and transmission and scanning electron microscopy are used to describe the biliary tree of larval L. lamottenii before and during their infection with the larvae of the nematode T. stelmioides. These changes are discussed and compared with those reported in small mammals following bile duct ligation, in patients with extrahepatic biliary obstruction, and in parasitic infection of the biliary tree. MATERIALS AND METHODS Animals Larval (ammocoetes)Lampetra lamottenii (Le Sueur, 1827) were collected using an electroshocking device from Duffins Creek (Pickering, Ontario, Canada) from spring until late fall. Animals were transported to Scarborough College, University of Toronto, were maintained in constantly aerated, dechlorinated tap water containing 5 to 10 cm of river substrate for up to 6 months, and were fed Liquidfry for Live Bearers (Hagen, Montreal, Quebec) 3 times a week. In all cases, animals were first anesthetized in 0.02% tricaine methanesulfonate, definitively identified using the external characteristics outlined by Vladykov (1950, 1960), and their weights and lengths were recorded. The ammocoetes were killed by decapitation and the entire liver was removed. Light Microscopy A total of 20 uninfected (<60 mm in length, <0.2 g i n weight), 10 infected (60-65 mm, 0.3-0.5 g), and 80 (70-160 mm, 0.6-9.1 g) infected ammocoetes were used for routine light microscopy. The livers were fixed in Bouin’s fluid for 24-48 h, stored in 70% ethanol for variable periods, transferred to a solution of lithium carbonate, dehydrated in ethanol, and embedded in Paraplast X-TRA embedding medium (melting point, 52-54°C; Monoject Scientific). Serial sections of 5-10 pm were placed on glass slides and stained with periodic acidSchiff (PAS), acid hemalum and orange G (Lillie, 1951). Transmission Electron Microscopy A total of 6 uninfected (<60 mm, <0.2 g) and 6 infected (98-155 mm, 1.4-5.7 g) ammocoetes were used for electron microscopy. Livers were exposed by an abdominal incision, and immediately flooded in situ with a solution of ice-cold 2% glutaraldehyde in phosphate buffer (Millonig, 1961) at pH 7.3. Livers were then removed, chopped into 1mm cubes and fixed in the above fixative for 2 h. The tissues were then rinsed in buffer and postfixed for 2 h in 1%OsO, in the same buffer, dehydrated in ethanol and propylene oxide, embedded in Epon-Araldite (Mollenhauer, 1964), and sectioned with glass knives using a Reichert-Jung ultracut microtome. Semithin sections (0.5 pm thickness) were placed on glass slides and stained with 1%toluidine blue in saturated sodium tetraborate (Bencosme et al., 1959). Thin (silver and gold) sections were mounted on uncoated copper grids, stained with saturated uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 19631, and examined using a Zeiss EM-9S electron microscope. Scanning Electron Microscopy A total of 12 uninfected (<60 mm, C0.2 g) and 10 infected (78-189 mm, 1.0-9.1 g) ammocoetes were used. Whole livers were placed in vials containing icecold 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) for 2 h. Tissue was immersed consecutively in 15, 30, and 50% solutions of dimethyl sulfoxide and then freeze-cut on a copper plate chilled by liquid nitrogen. The specimens were dehydrated in a graded series of ethanol, dried in a critical-point dryer, sputter-coated with gold, and observed in a Hitachi S530 scanning electron microscope. RESULTS General Morphology As noted in a previous study (Pybus et al., 19781, uninfected animals were usually <60 mm in length. The gross morphology of the liver of larval Lampetra Zamottenii was similar to that of other lamprey species (Maskell, 1930; Strahan and MacLean, 1969; Peek et al., 1979; Hilliard et al., 1985).The ventral surface was convex, and the esophagus was in intimate association with the concave dorsal surface (Figs. 1,2). The major differences were that the liver of L. lamottenii was a pyriform-shaped organ that tapered to a blunt posterior tip, a larger surface area of the liver was connected to the body wall, the extrahepatic common bile duct entered the alimentary canal at approximately threefifths the length of the liver, and the large gall bladder appeared close to the surface. The gross morphology of the livers of uninfected and infected animals was similar except that white cyst-like areas appeared occasionally on the surface of the liver of infected animals, >60 mm in length. The livers of uninfected (Fig. 3) and Abbreuiations bd BL DB E F m N P bile duct basal lamina dense body epithelium fibrosis mitochondria nucleus parasite NEMATODE, BILE DUCTS OF LAMPREY LIVER infected (Fig. 4) animals were composed of hepatocytes, sinusoids, and centrally located bile ducts. 203 cases, the epithelium appeared highly attenuated. In the latter, the bile ducts usually possessed a nematode larva. Mitotic figures also appeared to be a prominent Light Microscopy feature in the bile ducts that showed epithelial changes Bile ducts which suggested that some proliferation had occurred In uninfected animals, the five types of passageways (Fig. 7). (bile ductule, intrahepatic bile duct, intrahepatic comEven recently infected animals (60-65 mm in mon bile duct, extrahepatic common bile duct, and cys- length) displayed changes to the biliary epithelium and tic duct) were categorized according to their size and dilation of the bile ducts, and these were accompanied location within the biliary tree (Fig. 5). The various by an increase in fibrous tissue around the ducts. The passageways were intimately associated to sinusoids bile ducts of young animals appeared to just barely which possessed wide lumina (Fig. 5). Each duct pos- accommodate one nematode. In comparison, however, sessed an apical brush border which stained with peri- the bile ducts in animals >110 mm were extremely odic acidSchiff (PAS-positive) and a PAS-positive dilated and contained several nematodes. basement membrane, and was surrounded by a layer of In infected animals, light microscope sections revascularized, fibrous connective tissue. The size of the vealed a marked increase both in the number of bile lumina, the type of epithelial cells, and the thickness of ducts staining with PAS-positive material and in the the surrounding connective tissue of the bile ducts var- intensity of the staining. However, no relationship was ied along the course of the biliary tree. The bile found between the type of epithelial lining, the presductules were of small diameter (Fig. 51, measuring 4 ence of the nematode, and the intensity of PAS staining pm where they drained the bile canaliculi, and con- of the apical cytoplasm of the bile duct cells. sisted of a simple cuboidal cells, 2.5 pm tall. These The lumen of the bile ducts and bile ductules in unductules emptied into intrahepatic bile ducts (Fig. 5) infected animals appeared devoid of any material (Fig. consisting of a simple cuboidal to low columnar epithe- 5). Following infection, the lumina of the bile ducts and lium, 2.5-13.2 pm in height, with large, round to oval, occasionally of the bile ductules contained variable basally located nuclei. Occasionally a cilium was seen amounts of PAS-positive material (Fig. 7). Sometimes at the cell surface, and PAS-positive granules were the bile ducts appeared occluded due t o the presence of present in the apical cytoplasm. The bile ducts were the parasite and the extensive amounts of material and surrounded by a thin layer of fibrous connective tissue debris (Figs. €49).The surface and sometimes the cells which increased slightly as the intrahepatic bile ducts of the nematode also showed PAS staining (Fig. 7). united to form the intrahepatic common bile duct. The Fine Structure latter duct was of a large caliber, and was surrounded Bile ducts by moderate amounts of loose, areolar connective tissue (Fig. 5).This duct was connected to the gall bladder Nucleus and mitochondria. The bile ducts and ductules through a short cystic duct; it consisted of simple, cil- in uninfected animals shared similar ultrastructural iated, tall-columnar epithelium, about 25 pm in height features. A large, round-to-oval nucleus with a promi(Fig. 5). As the intrahepatic common bile duct exited nent nucleolus was located near the basal surface of the the liver, some animals displayed a progressive in- bile duct cells. Numerous mitochondria, round to rod crease in the amount of PAS-positive granules in the shape, were arranged in the long axes of the cells. The apical cytoplasm. The extrahepatic common bile duct numerous cristae were transversely orientated and and cystic duct shared similar morphological charac- tended to span the entire width of the mitochondrion teristics with the intrahepatic common bile duct. (Fig. 10). Following infection, the changes to the bile The infected liver showed tortuous, ectatic bile ducts ducts were variable. The nuclei were sometimes elecwith a thickened and intensely PAS-positive basement tron-dense (pyknotic), and frequently the nuclear enmembrane and thick, supporting connective tissue velope was dilated. Mitochondria were sometimes swolwalls. The degree of periductal fibrosis was variable. len (Fig. ll),and their matrices were electron-lucent Most of the bile ducts were surrounded by a thick, and possessed electron-dense intramitochondrial grandense-regular connective tissue (Fig. 6). In some ani- ules (measuring up to 107 nm compared to 18-45 nm mals, the fibrosis was so extensive that bile ducts were in uninfected animals). Cristae seemed to be decreased widely separated by the connective tissue and sinu- in number and of variable configuration (Fig. 11). This soids were inconspicuous (compare Figs. 5 and 6). Due type of mitochondrion was particularly characteristic to the dilated nature of the bile ducts and the peri- of what seemed to be cells preserved in a state of deductal fibrosis, the bile ducts appeared more prominent generation. It was not unusual to find cells in varying (Fig. 6); and generally it was difficult to differentiate degrees of cell death next to supposed healthy cells the lumina of bile ductules from bile canaliculi of he- (Figs. 11, 12). Endoplasmic reticulum. In some bile ducts of infected patocytes and the intrahepatic bile ducts from the intrahepatic common bile duct. Most of the dilated bile animals, these structures were sparsely distributed canaliculi or bile ductules were within the fibrous con- throughout the cytoplasm, as observed in uninfected nective tissue next to the wide-diameter bile ducts (Fig. animals. However, others possessed an increase and/or moderate dilation of the cisternae of RER and SER 6). In infected livers, the epithelia of many of the bile resulting in a vacuolated appearance to the cytoplasm. ducts showed marked changes. The lining epithelium In particular, dilated cisternae of RER were more conwas thrown into folds and ranged from pseudostratified spicuous in cells of bile ductules. Dense bodies. In uninfected animals, most of the columnar to stratified columnar, to festooned (alternate low and tall cells), or to simple cuboidal; in some dense bodies were located in the apical cytoplasm of 204 F. ENG AND J.H. YOUSON Figs. 1-4. NEMATODE, BILE DUCTS OF LAMPREY LIVER 205 bile ductular cells (Fig. 10). The irregularly shaped dense bodies were bounded by either a single or double limiting membrane. The highly electron-dense matrix contained flocculent material of varying electron densities. In infected animals, dense bodies were conspicuous in the apical cytoplasm of the bile ductules and were more commonly found in the larger bile ducts (Fig. 12). However, they were not confined to the supranuclear cytoplasm of the latter. Lipid was sometimes present within the dense bodies. In addition, dense bodies often possessed moderately electron-dense matrices and were bounded by what appeared to be many concentric layers of membranes. Cytoplasmic matrix. Following infection, coated vesicles and free ribosomes were numerous; and glycogen particles (20-30 nm in diameter), now present as aggregates, were dispersed throughout the cytoplasm. The latter were also concentrated at the periphery of lipid droplets. A pronounced difference between the ductal cells of the uninfected and infected animals was the apparent increase in the number of microtubules (20 nm), microfilaments (5-6 nm), intermediate filaments (7.8-10 nm), and cytoplasmic filaments following parasite infection (Fig. 13). There were small numbers of bundles of microfilaments in the cytoplasm, and these were particularly prominent as parallel groups below the apical surface. Numerous cytoplasmic filaments were arranged parallel to the basal plasma membrane in bile duct cells but were not observed in the bile ductules of uninfected animals. In infected animals, thick bundles of microfilaments were located throughout the cytoplasm and were often interconnected with one another (Fig. 13). The basal cytoplasmic filamentous bundles were usually discontinuous, a t times more conspicuous, and now present in the bile ductules. In cells of the smaller ducts in infected animals, large bundles of intermediate filaments were usually observed encircling the nucleus, as well as scattered throughout the cytoplasm (Fig. 13). In the common bile duct of both uninfected and infected animals, large bundles of intermediate filaments and microfilaments were found primarily encircling the nucleus and scattered throughout the cytoplasm, respectively. Mucous granules. Variable numbers of small round granules (160-240 nm), bounded by a single limiting membrane and containing electron-dense matrices, were present in the apical cytoplasm of uninfected animals (Fig. 10). The location of the granules was in the identical region that stained PAS-positive in the light microscope. Therefore, it was with a high degree of confidence that the apical granules were called mucous granules. Following infection, mucous granules appeared to be more abundant and present in the apical cytoplasm of bile duct cells of all types (Fig. 12). In degenerating cells, the granules seemingly were released into the lumen of the bile ducts along with other cellular debris (Fig. 12). Surface specializations. The apical surface of each bile duct and ductular cell in uninfected animals possessed many closely packed microvilli, 1.5-1.9 pm long. The common bile duct possessed alternating groups of microvilli and cilia. The apical surfaces of the bile duct and ductular cells were highly variable in infected animals. Some bile duct cells resembled those in uninfected animals while others displayed dome-shaped or flattened apices (Figs. 11, 12). Fragmentation and/or loss of microvilli at portions of the apical surface were observed. The shortened microvilli appeared distorted, fragmented and club-shaped (Figs. 11,12). Cytoplasmic blebs or extrusions were a common feature a t the apical surface (Figs. 11,121. In those bile ducts which contained nematodes, these features were particularly apparent. In this situation, the microvilli were short and flattened and in some cases absent. Modifications to the microvilli were sometimes accompanied by a disruption and/or an increase in microfilaments in the apical cytoplasm. There were no apparent changes to the junctional complex following infection (Fig. 11). The width of the lateral intercellular spaces extended over a broader range (22 nm-2.9 pm) than seen in uninfected animals (18 nm-1.7 pm). There were some areas where the lateral intercellular space widened and narrowed (Fig. 11). Coated pits and vesicles were present at the lateral surface. The number of microvillus projections ranged from a few (resembling uninfected animals) to many, and the projections interdigitated with those of adjacent cells. This latter feature was particularly pronounced in bile ducts lined with a stratified or pseudostratified epithelium. Frequently, the intercellular space possessed moderately electron-dense, amorphous debris, and vacuolar or membranous structures. The lateral intercellular space of some bile ductules in infected animals was 770 nm wide (in contrast to 95 nm in uninfected animals) and occasionally possessed a few microvillus projections. Following infection, the basal plasma membrane showed slightly more folding; this was accompanied by a more folded, and sometimes multilayered and thicker, basal lamina (Fig. 11).Fragmented portions of the basal lamina were sometimes Fig. 1. SEM of a dorsal view of the liver (L) and part of the alimentary canal showing the gall bladder (GI and esophagus (01,the anterior mesenteric artery (AM), and the extrahepatic common bile duct (EBD). The latter two are eventually enclosed by a cord of connective tissue that enters the anterior intestine (All. X 31. Fig. 3.A transverse section through a posterior region of a n uninfected liver showing the concave dorsal surface (DS) next to the esophagus (0). Also note numerous bile ducts, hepatic parenchyma (HI, and the anterior mesenteric artery (arrowhead). x 340. Fig.2.SEM of a ventral view of the liver showing the location of the gall bladder (G) and the pyriform shape of the liver with its blunt posterior tip (arrow). x 29. Fig. 4.A transverse section through a posterior region of a n infected liver showing the large, dilated bile ducts containing parasites (arrowhead), the loose arrangement of hepatic parenchyma (H), and the extensive periductal fibrosis. AM, Anterior mesneteric artery; EBD, extrahepatic common bile duct; 0, esophagus; VS, ventral surface. x 85. 206 F. E N G AND J.H. YOUSON present in the surrounding connective tissue. Numerous coated pits were usually found in association with the highly folded membrane while coated vesicles were observed nearby (Fig. 14). Periductal connective tissue The thick periductal connective tissue (Figs. 4,6) in infected animals consisted of large amounts of collagen arranged in bundles, isolated fibrils of collagen, and microfibrils (9-10 nm diameter). Vacuolar or membranous structures, resembling those found in the lumen and lateral intercellular space of the bile ducts, were occasionally present within the connective tissue. Also following infection, numerous fibroblasts (Fig. 9) and lipocytes were observed. Frequently, the RER and the nuclear envelope of these cells were highly dilated, and both cells were associated with collagen fibrils. The cytoplasm of fibroblasts contained bundles of cytoplasmic filaments (3.7-5. 6 nm) running parallel to the plasma membrane; some cells possessed abundant glycogen particles, lipid, and electron-dense bodies. Within the connective tissue were portions of cytoplasm that contained electron-dense bodies, vacuoles with granular and amorphous electron-dense debris, and numerous vesicles. Desmosomes were also observed (Fig. 14). These may be portions of a phagocytic cell, however, the desmosomes suggest that they are portions of extruded epithelial cells. Nerve terminals were abundant throughout the connective tissue surrounding the majority of the bile ducts (Fig. 12) while in the uninfected animals, nerve terminals were confined to the connective tissue of the extrahepatic common bile duct. DISCUSSION The morphology of bile ducts of uninfected L. lamottenii is generally similar to that described in other larval lampreys (De Vos e t al., 1973; Sidon et al., 1980). However, the infected animals show marked differences from the livers of all lampreys so far described. The most prominent alterations to the biliary tree following infection are the dilation of the intrahepatic bile ducts and the accompanying changes in their epithelium. Similar changes have been observed in small mammals following experimental bile duct ligation (Cameron and Oakley, 1932; Carlson et al., 1977) and in patients with extrahepatic bile duct obstruction (Desmet, 1987). More commonly, these are features in mammals infected with Clonorchis sinesis (Hou, 1955, 1965; Viranuvatti and Stitnimankam, 1972; Lee et al., 1988), Opisthorchis felineus, Opisthorchis viverrini (Evans et al., 1971; Muller, 19751, and Fusciolu hepat- Fig. 5. A portion of an uninfected liver showing the intrahepatic common bile duct (CBD), bile ducts, and bile ductules (large arrowhead). The latter ducts are surrounded by a thin layer of fibrous connective tissue (thin arrow), while the common bile duct is encompassed by a loose areolar connective tissue (large arrow). Note a mitotic figure (small arrowhead). S, Sinusoid. x 925. Fig. 6. An infected liver showing extensive periductal fibrosis and a n abundance of dilated structures (arrow) adjacent to the larger, dilated bile ducts. These smaller components may be either dilated ica (Meyers and Neafie, 1976; Sun, 198213). As in these latter cases, the changes to the biliary tree in infected L. lamottenii are probably due to the increase in biliary pressure because of the mechanical obstruction of the bile ducts by the presence of the nematode and the abundance of extraneous material in the lumen. Proliferation of the biliary epithelium is suggested by the mitotic figures and the multilayered epithelium. It is of interest that epithelial proliferation of bile ducts occurs in small mammals following bile duct ligation (Johnstone and Lee, 1976) and experimental infections with C. sinesis (Lee and Lee, 1978).These findings suggest that with increased biliary pressure, there is a n activation and proliferation of the biliary epithelium leading to a multilayered epithelium which ultimately allows for dilation of the bile duct. However, several studies have indicated that proline excretion from a parasite may result in hyperplasia of the bile duct epithelium (Isseroff et al., 1977; Acosta-Ferreira et al., 1979; Vacanti and Folkman, 1979). It is not known for certain that T. stelmioides plays a role in stimulating hyperplasia of bile duct cells, but the similar response of the hosts to the various parasites seen in earlier studies is striking. The observations of the present study would seem to support occlusion of the bile duct lumen as being most important to the pathogenesis of the bile ductular hyperplasia and fibrosis in this animal model. Bile ductular proliferation is a prominent feature of many forms of liver disorders (Buyssens, 1965) including in clonorchiasis (Sun and Tung-Ma, 1973), and in fasciolasis (Acosta-Ferreira et al., 1979). In mammalian cholestasis, ductular proliferation may be a secondary cause of intrahepatic obstruction (Buyssens, 1965; Desmet, 1972). Furthermore, prolonged ductular proliferation may stimulate fibrogenesis since the basement membrane may act a s a scaffold for the development of collagen fibers (Popper and Schaffner, 1961; Buyssens, 1965; Desmet, 1987). Whether there is a similar proliferation of bile ductules in the liver of infected larval L. lamottenii is unclear because we had difficulty in differentiating bile ductules from dilated bile canaliculi at the light microscope level. However, the ultrastructural changes in the ductular epithelial cells of infected L. Zamottenii are strikingly similar to those found in proliferated ductules in mammalian extrahepatic cholestasis (Steiner et al., 1962) and biliary atresia (Hollander and Schaffner, 1962). It would be of interest to determine if ductular proliferation occurred because fibrosis is quite prominent in L. lamottenii following infection. In the meantime, the general impression is that bile ducts are more conspicuous in the livers of infected animals. bile ductules or dilated bile canaliculi of the hepatocytes. Note the presence of PAS-positive material in the bile duct lumen (arrowhead) and parasites. x 370. Fig. 7. Bile ducts from an infected ammocoete possess numerous mitotic figures (arrows), PAS-positive material (arrowhead), and a parasite and are surrounded by a fibrous connective tissue. x 1,360. Fig. 8. A portion of the bile duct lumen showing debris (de), the surfaces of the parasite, and biliary epithelium. x 4,000. NEMATODE, BILE DUCTS O F LAMPREY LIVER Figs. 5-8. 207 Fig. 9. SEM showing a nematode larva in the lumen of a bile duct. Note the epithelium, the other material in the lumen (arrowhead), and the layer of fibrous connective tissue. Inset: High magnification SEM of a fibroblast (f) and the collagen fibers (arrow) of the periductal connective tissue. x 5,900; inset, X 17,500. Fig. 10. A portion of the bile duct epithelium from an uninfected ammocoete showing a few mucous granules (arrow), a dense body, rough endoplasmic reticulum (er), a Golgi apparatus (Ga), mitochondria, nucleus, a vacuole (V), cytoplasmic filaments (arrowhead) and intracytoplasmic cisternae (IC). JC, Junctional complex. x 9,700. NEMATODE, BILE DUCTS OF LAMPREY LIVER Fig. 1 1. A portion of the bile duct epithelium from a n infected ammocoete showing apical cytoplasmic blebs (cy), swollen mitochondria, lateral intercellular spaces of variable width (arrow), cilia (arrowhead), and folding of the basal lamina. d, Desmosome; mv, microvilli. Inset: Enlargement of electron-lucent swollen mitochondria with cristae that are of various configurations. x 9,800; inset, x 28,000. 209 Fig. 12. Bile duct epithelium from an infected ammocoete showing a degenerating cell (D) adjacent to normal looking cells. Note the abundance of mucous granules (mu) and cytoplasmic blebs (cy) at the apical surface and the abundant bundles of collagen fibers (Co) below the epithelium. The arrowhead indicates a dense body. Li, Lipid droplet; NT, nerve terminal. x 4,500. Fig. 13.A portion of the bile duct cell from an infected animal showing abundant intermediate filaments (IF), microfilaments (mf), and a few microtubules (mt). x 29,400. Fig. 14. In a n infected animal, a portion of a cell in the extracellular space between bile ducts contains electron-dense bodies, desmosomes (d), and amorphous electron-dense debris (arrow). Note the presence of coated vesicles along the plasma membrane (arrowhead) of the ductular cell, and a cell process (cp) near the basal lamina. cy, Cytoplasmic filaments. X 16,900. NEMATODE, BILE DUCTS OF LAMPREY LIVER In long-standing infection of L. lamottenii, and hence prolonged increase in biliary pressure, the bile ducts became markedly dilated, and they may reach the surface of the liver where they appear as white cystic areas. This is similar to the situation of mammals infected with biliary parasites (Tansurat, 1971; Simson and Gear, 1987). Difficulty in differentiation of the intrahepatic common bile duct from the intrahepatic bile ducts and bile ductules from the dilated bile canaliculi accompanies prolonged infection by larvae of T. stelmioides. The relative paucity of organelles in the cells of the bile ducts and bile ductules of uninfected larval L. lamottenii indicates that, as in other vertebrates (Sternlieb, 1965; Yamada, 1969) and in larval P. marinus (Sidon et al., 1980), these cells are not as metabolically active as the adjacent hepatocytes. In infected livers, the RER in bile duct cells is prominent and dilated and ribosomes are numerous. Similar observations have been reported in proliferated biliary epithelial cells in extrahepatic biliary obstruction (Carruthers and Steiner, 1961; Steiner et al., 1962), in extrahepatic biliary atresia (Hollander and Schaffner, 1968a), and in clonorchiasis (Sun and Tung-Ma, 1973). It may be that in L. lamottenii, as in mammals, the increase in RER and ribosomes signifies an increase in protein production which is necessary for support of proliferating cells (Carruthers and Steiner, 1961). As in the liver of larval P. marinus (Sidon, 1982), large bundles of intermediate filaments and microfilaments were observed primarily in the biliary cells of the common bile duct of uninfected L. lamottenii. Following infection, the number of microfilaments and intermediate filaments increased in various branches of the biliary tree. A similar increase in microfilaments has been reported in ductular cells of cholestatic livers (Carruthers and Steiner, 1961; Adler et al., 1980), while no significant changes to intermediate filaments have been reported following bile duct ligation in the rat (Okanoue et al., 1988). Since intermediate filaments in hepatocytes can be increased by changes in the environment over an extended period of time in obstructive jaundice (Okanoue et al., 1988) and following experimental bile duct ligation (Hoshino et al., 1989), and the lampreys used in this study have been infected for several years, it seems plausible that the numerous intermediate filaments in bile duct cells ofL. lamottenii are induced by the resultant increased biliary pressure. The role of intermediate filaments in biliary epithelial cells is not known. In hepatocytes, however, the intermediate filaments play an important role in the maintenance of cell shape and stability and act as an anchor for cell organelles (Ohta et al., 1988; Okanoue et al., 1988). Dense bodies were a conspicuous feature in the biliary epithelial cells following infection. The dense bodies are strikingly similar to those found in duct cells during human (Hollander and Schaffner, 1968a) and lamprey biliary atresia (Sidon and Youson, 198313) and in clonorchiasis (Sun and Tung-Ma, 1973). They also resemble those inclusions found in lamprey hepatocytes which were shown to possess various components of bile (Bertolini, 1965; Sidon and Youson, 1983a). The accumulation of these inclusions suggests that, as in a mammalian cholestatic condition (Desmet, 1972; 211 Schaffner and Popper, 19751, there is enhanced reabsorption of biliary constituents by both ductal and ductular cells in L. lamottenii. Such a situation may be due to increased biliary pressure and bile stagnation. Large numbers of dense bodies containing bile products in the bile duct cells may ultimately result in cellular dysfunction and perhaps even cytolysis, for similar structures occur in ductular cells of more extreme cases of bile duct obstruction, such as in lampreys undergoing biliary atresia a t metamorphosis (Sidon and Youson, 1983b) and in hepatocytes of cholestatic livers (Hollander and Schaffner, 1968b; Desmet, 1987). That cytolysis may occur in bile duct cells as a result of infection is evidenced by cells that possess cellular and amorphous debris and swollen mitochondria with unusual cristae. The exfoliation of cells or extrusion of their cellular contents into the bile duct lumen is perhaps the ultimate fate of these cells. The apical cytoplasm of cells of the extrahepatic, and sometimes the intrahepatic, common bile duct of uninfected L. lamottenii possesses small numbers of mucous granules. Although present in cells of the common bile duct of rodents (Yamada, 19691, mucous granules are not common in normal bile duct cells of lampreys. Following infection, there is an increase in the number of mucous granules along most of the branches of the biliary tree, and there is abundant mucinous material within the bile duct lumen. These features have been found in bile ducts of mammals infected with C . sinensis (Chou and Gibson, 1970; Sun, 19841, 0. uiuerrini and 0. felineus (Sun, 1982a1, and F. hepatica (Ong, 1985). It may be that T.stelmioides, as is the case with the other biliary parasites, acts as an irritant to cause elaboration and secretion of mucus by the biliary epithelial cells. The irritation may be chemical (i.e., parasite metabolic products) but not likely mechanical, since T. stelmioides does not migrate once it is in the biliary tree (Pybus et al., 1978). In mammals, the elaboration and release of mucinous material by host epithelial cells are a means of protection from highly concentrated bile which is irritative and injurious to bile duct cells (McMinn and Kugler, 1961; Yamada, 1969). In addition, the mucus may serve as a “glue,” as it is for C . sinensis (Hou, 1955), enabling T . stelmioides to adhere to the bile duct wall. However, the mucus is not likely to be a source of food for T. stelmioides, as it is for C . sinensis (Hou, 1955) and F . hepatica (Ong, 19851, since the nematode is in an arrested state of development (Pybus et al., 1978). Ultimately, the elaboration of excess mucus may contribute to obstruction (Hou, 1955; Gibson and Sun, 1971), as suggested by the occluded appearance of the bile ducts. It has been established that an increase in biliary pressure within the bile canaliculus produces structural alterations to the tight junctions resulting in increased permeability (De Vos and Desmet, 1978; Koga and Toda, 1978). Thin sections show intact tight junctions in infected L. lamottenii in spite of the dilated lumina. Similar observations have been reported in bile ductules (Carruthers and Steiner, 1961; Hollander and Schaffner, 1968a) and in hepatocytes (Metz et al., 1977; Robenek et al., 1981) of cholestatic livers. However, the observation in thin sections of preserved tight junctions does not exclude structural changes inside the junctional area and an increase in permeability of 212 F. ENG AND J.H. YOUSON the canaliculo-sinusoidal barrier (Robenek et al., 1980).To date there has been no report on the structure of the tight junctions between bile duct cells in mammals (Phillips et al., 1986). It is possible that the already “leaky” tight junctions of larval lamprey (Sidon et al., 1980) become more permeable. This view is supported by the presence of material, resembling the biliary material observed in the bile duct lumen and bile canaliculus, in the intracellular space and surrounding connective tissue in infected L. lamottenii. The bile ducts of infected L. lamottenii are surrounded by a prominent PAS-positive basement membrane. Electron micrographs indicate this change was due primarily to thickening and folding of the basal lamina. A similar response was also reported in proliferated ductules from patients with biliary atresia (Hollander and Schaffner, 1968a) or with cholestatic livers (Sasaki et al., 1967), in rats subjected to bile duct ligation (Steiner and Carruthers, 1961; Gay et al., 1976), and in degenerating bile ducts of lampreys during metamorphosis (Sidon and Youson, 1983a). Thickening of the basal lamina is a nonspecific change due to alterations in the biosynthesis or metabolism of basement membrane components which occurs in mammalian cells as a response to sublethal injury (Martinez-Hernandez and Amenta, 1983). In L. lamottenii, the sublethal injury may be in the form of escaped biliary material into the periductal tissue, for this mechanism has been suggested t o be responsible for disruption (i.e., fragmentation) and duplication of the basement membrane in ductules of human cholestatic livers (Sasaki et al., 1967; Desmet, 1987). Accompanying the dilation of the bile ducts and changes to their epithelial cells in infected L. lamottenii is the prominent thickening of the underlying connective tissue with numerous bundles of collagen. Concomitant with the fibrosis, and suggestive of an inflammatory reaction, are numerous mononuclear cells in the surrounding sinusoid. Similar observations have been reported in cholestatic livers (Weinbren, 1953; Carruthers et al., 1962; Kountouras et al., 1984). The implication is that periductal fibrosis may be due to an escape of an irritating material, presumably bile, into the surrounding connective tissue. The fibrosis may initially represent a compensatory response to the rising biliary pressure and serve to provide support for the dilated bile ducts (Johnstone and Lee, 1976; Popper and Martin, 1982). According to Carlson et al. (1977), the increase in biliary pressure may be the initial stimulating force that results in proliferation and activation of a fibrogenic cell population. The extensive fibrosis that encompasses many bile ducts in L. lamottenii has also been observed in the liver following other parasite infections (Dooley and Neafie, 1976; Ong, 1985; Lee et al., 1987). The degree and extent of fibrosis in L. lamottenii appear to be related to the duration of infection and, hence, obstruction. Such is also the case following experimental bile duct obstruction (Johnstone and Lee, 1976; Carlson et al., 1977) and parasitic infections (Belding 1965a,b; Tsutsumi et al., 1980; Lee et al., 1987). As during bile duct regression in lamprey metamorphosis (Yamamoto et al., 1986131, the thick periductal fibrosis separates the bile ducts from the vascular channels and likely, interrupts fluid and nutrient exchange between the two systems, and disturbs the process of bile modification. In contrast to the present situation, however, it has been suggested that periductal fibrosis during lamprey biliary atresia may be a critical event in the process of the destruction of the biliary tree (Yamamoto et al., 198613). Lipocytes have been identified in the perisinusoidal area and interstitial tissue of the liver of larval (Sidon et al., 1980) and transforming lampreys (Sidon and Youson, 1983a; Yamamoto et al., 1986b); they may be equivalent to the vitamin A-containing lipocytes (stellate, fat-storing cells) of higher vertebrates which have been implicated in fibrogenesis (Kawase et al., 1986). Following infection, numerous lipocytes are observed in the periductal connective tissue in close apposition to collagen fibrils suggesting that the lipocytes, in addition to fibroblasts, synthesize collagen and may be responsible for the periductal fibrosis. ACKNOWLEDGMENTS This study was supported by Grant A5945 from the Natural Science and Engineering Research Council of Canada to J.H.Y. The authors would like to thank Rick Raininger for his technical assistance and to Pat Sargent in the collection of animals. LITERATURE CITED Acosta-Ferreira, W., J. Vercelli-Retta, and L.M. 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