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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.

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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.
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morphology, nematoda, bilet, nematodacucullanidae, 1910, duct, sueur, brooks, truttaedacnitis, stelmioides, vessichelli, lamottenii, lamprey, infectious, lampetra
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