The ductular cell reaction of rat liver in extrahepatic cholestasis

EXPERIMENTAL

The

AND

MOLECULAR

Ductular

Cell

PATHOLOGY

1,

162-185 (1962)

Reaction

of

Rat

Liver

in Extrahepatic

C holestasis I. Proliferated

Biliary

Epithelial

Cells’

JAN W. STEINER, JOHN S. CARRUTHERS, AND S. ROBERT KALIFAT~ Department

of Pathology,

Banting

Institute,

Received

University March

of Toronto,

Toronto

2, Canada

8, 1962

INTRODUCTION The ductular cell reaction has been defined as an aggregation of inflammatory cells and a proliferation of organized and disorganized biliary epithelial (ductular) cells in the liver (Popper et al., 1957). The histology of this lesion has presented many difficulties of interpretation particularly when its epithelial components were disorganized and failed to form polarized architectural patterns recognizable as biliary channels.The terms ‘interstitial’ (Popper et aZ., 1957) and ‘oval’ cells (Farber, 1956) attest to this difficulty. There has not even been agreement as to whether they are mesenchymal or epithelial. The “undifferentiated (‘oval’ cells)” (Schaffner and Popper, 1961) have been thought ‘by someto be fibroblasts (Edwards and White, 1941; Fitzhugh and Nelson, 1948; Jaffe et al., 1950; MacDonald and Malloy, 1959), endothelial cells (Davidson, 1935; Edwards and White, 1941; Korpissy and Kovacs, 1949), or histiocytes (Korpassy and Kovacs, 1949)) and others have consideredthem epithelial, either ductular (Banson et al., 1959; Cameron and Oakley, 1932; Cameron and Hou, 1961; Cameron et al., 1960; Edwards and White, 1941; Fitzhugh and Nelson, 1948; Farber, 1956; Lopez and Mazzanti, 1955; McLean and Rees, 1958; Opie, 1944; Popper et al., 1957; Schoental and Magee, 1957) or parenchymal (Leduc, 1959; Wilson and Leduc, 1958) in origin. Two recent investigations have helped to clarify this confusion. Histochemical studies have shown that the ‘oval’ or ‘interstitial’ cells of the ductular reaction are distinct from parenchymal liver cells (Melnick, 1955; Rosenholtz, 1960; Wachstein, 1959), and Grisham and Hartroft (1961) have demonstrated by electron microscopy that in ethionine intoxication and in choline deficiency the ‘oval’ cells are ductular cells which encircle lumina and are surrounded by a basement membrane. Our own unpublished investigations of the ductular cell reaction in ethionine and in a-naphthyl isothiocyanate intoxication (Steiner and Carruthers, unpublished observations) in general confirm Grisham and Hartroft’s findings (1961). However, some of our observations indicate that the reaction is considerably more complex than they have described. In particular, we found that the development of metaplastic changes in the proliferating biliary epithelial cells compounded its intricacy. It, therefore, 1 Supported by a grant-in-aid, MA-785 of the Medical Research Council of Canada, 2 Interne des HBpitaux de Paris and Research Fellow of the Canadian Arthritis and Rheumatism Society. 162

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seemed to us desirable that before attempting to describe the fine structure of this complex lesion, a more simple type of ductular cell reaction should be studied. The reaction which develops in the liver of rats after ligation of the common bile duct seemed suitabIe. All the more recent investigations (Cameron and Oakley, 1932 ; Cameron and Hasan, 1958; McMaster, 1922; Ogata, 1913; Riittner and Stofer, 1960) agree that in this case the epithelial components of the ductular cell reaction can be clearly identified even by the light microscope. The fine structure of the normal intrahepatic biliary passages and the cytoarchitectural features of their lining cells have been examined by a number of investigators (Ashworth and Sanders, 1960; David, 1961; Fawcett, 1955; Grisham and Hartroft, 1961; Popper et al., 1960, 1961; Rouiller, 1956; Schaffner and Popper, 1961; Steiner and Carruthers, 1961b). We have therefore explored some aspects of the changes at an electron microscopic level of observation by studying the alterations which occur in rabbits and rats during the first 14 days following the induction of total extrahepatic cholestasis (Carruthers and Steiner, 1961; Steiner and Carruthers, 1961a, 1962). It is the purpose of the present paper to extend these findings by describing the fine structure of the ductular cell reaction which occurs in rats between the second and sixth weeks after ligation of the common bile duct. MATERIALS

AND

METHODS

=Inimals. White Wistar male rats weighing approximately 250 gm were used. Operative Procedures. The animals were anaesthetized with ether and the abdominal organs exposed by a mid-line incision. The common bile duct was divided immediately adjacent to the duodenum between double ligatures of cotton. The animals were maintained on Purina chow and water ad libitum. Sacrifice. Of the total of forty-one rats operated upon, thirty died spontaneously and they were discarded. The remaining eleven were killed by an overdose of ether on day 14 (two animals), 15, 16, 17, 21 (two animals), 25, 26, 33, and 39 postoperatively. All rats were killed between 9 A.M. and 10 A.M. to reduce as much as possible individual variations caused by diurnal cycles of feeding and other such factors. Histologir techniques. As soon as anaesthesia had been induced in the rats to be killed, the abdomen was opened and tissues taken from the right half of the median lobe of the liver. Tissue for conventional light microscopy was fixed in buffered formalin (pH 7.0) and embedded in paraffin. Sections were stained with hematoxylin and eosin or, where appropriate, with Gomori’s silver impregnation stain for reticulin, or by the periodic acid-Schiff method. Small fragments of tissue for thin-section light microscopy and for electron microscop~f were fixed in Palade’s buffered osmium tetroxide (pH 7.4) containing 0.25 M sucrose (Caufield, 1957). Two milliliters of fixative was used for each sample. After the insertion of the tissue, the vial containing the fixative was maintained at 4’C for 90 minutes, and then for 30 minutes at room temperature. The tissues were next dehydrated in a graded series of ethanol solutions. The fragments were embedded in either a mixture of butyl-methyl methacrylate (8: 1) and polymerized at 6O”C, or were embedded in epoxy resin (Epon 812), by the method of Luft (1961). Thin sections of these blocks were cut on a Porter-Blum ultra-microtome with glass

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knives and were examined by a phase contrast microscope to determine the state of preservation of the tissues. Well preserved samples were stained by flotation on alkaline Azure B, or by the toluidine blue method of Trump et al. (1961). Ultrathin sections were cut on a Porter-Blum ultramicrotome with diamond or glass knives. Some sections were stained by flotation on phosphotungstic acid (PTA), or uranyl acetate (Watson, 1958a). Others were being picked up on Formvar-coated grids, and stained with lead hydroxide by the method of Watson (1958b). The sections were examined in an RCA-EMU-SE electron microscope. Micrographs were taken at initial magnifications of 1400 to 32,000, and were enlarged photographically to the desired size. RESULTS LIGHT

MICROSCOPY

Our tindings in conventiona sections confirm those of other investigators (Cameron and Hasan, 1958; Cameron and Oakley, 1932; Riittner and Stofer, 1960), who examined livers of rats between the second and sixth weeks after ligation of the common bile duct (Fig. 1). The proliferating ductular cells faithfully reconstruct the pattern of smaller and larger ducts. Although this may not be always obvious in hematoxylin and eosinstained sections, it becomesapparent in sections stained for reticulum, in which the contours of the channels are clearly outlined. In contrast to this organized proliferation, the liver cords show evidence of disarray. This is brought about by a process designated as “arkadenfiirmige Einbuchtung” (Riittner and Stofer, 1960). The advancing ductular cells penetrate the lobules of parenchymal cells and leave individual FIG. 1. Conventional Iight microscopic picture of ductular cell reaction following ligation the common bile duct. Proliferated ductules occupy the center of the micrograph and lobules upper and lower margins. Lumina can be recognized in some but not all channels. The ductules surrounded by fairly numerous mesenchymal cells. Hematoxylin and eosin stain, x 186.

of the are

FIG. 2. Thin section of the ductular cell reaction following ligation of the common bile duct, The organized nature of the reaction is easily apparent since the ductular cells show an invariable tendency to line biliary channels. One ductule (lower center) contains edematous microvilli. Capillaries (arrows) are easily apparent. Epoxy embedding, toluidine blue stain, x 522. FIG. 3. Same as Fig. 2. Ductular cell reaction penetrating into a lobule. Isolated parenchymal liver cells find themselves stranded in the midst of the elements of the reaction. Note the marked difference in staining intensity of ductular and parenchymal cell respectively, the discrepancy of their nuclear size and of their nucleoli. The lumina stand out clearly. Mesenchymal cells, occasionally containing phagocytosed material, can be seen though they are difficult to classify. The pale material between mesenchymal cells stains faintly metachromatically. The capillary at the right center margin is probably a sinusoid involved in the reaction (arrow). Epoxy embedding, toluidine blue stain, X 652. Abbreviations used in figures: AP, attachment plate (terminal bar) ; BB, basal body of cilium; BD, bile ductule; BM, basement membrane; C, centriole; CC, zone of periluminal cytoplasmic condensation; CIL, cilium ; CP, cell process; CT, connective tissue; D, desmosome ; EMV, edematous microvillus; EN, capillary endothelium; ER, endoplasmic reticulum (ergastoplasm) ; F, intracytoplasmic filaments; G, Golgi zone; IC, intercalated cell; ID, cell interdigitations; IS, intercellular space; L, lumen of bile ductule; M, mitochondrion; MB, microbody; ME, mesenchymal cel1; MV, microvillus; MVB, microvesicular body; N, nucleus; NE, nuclear envelope; PM, plasma membrane; PR, process of fibroblast ; RBC, erythrocyte; RNP, ribcse nucleoprotein granules; S, shaft of cilium; SV, secretory vacuole; tg, tangentially sectioned area; V, vacuole.

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parenchymal cells or small groups of them stranded in the midst of the proliferating bile duct cells and inflammatory elements of the ductular reaction. A proliferation of fibrous connective tissue always forms part of the ductular cell reaction. Three of its components may be identified in conventional sections; fibroblasts, reticulin fibers, and ground substance. The identification of other mesenchymal cells participating in the reaction is difficult in conventional sections. The thin sections (Figs. 2 and 3) for light microscopy have several advantages. They make clear the organized nature of the biliary proliferation, for the lumina of the newly formed ductules stand out clearly, and the special stains are unnecessary. They also make obvious the isolated parenchymal cells. As seen in thin sections, the ductular cells have vesicular nuclei with indistinct nucleoli and a pale cytoplasm, whereas the parenchymal cells possess a dark staining cytoplasm, usually with round nuclei and distinct large nucleoli. The edematous microvilli of biliary epithelial cells may also be identified. Or again, the better preservation of epoxy-embedded tissues makes recognition of capillaries considerably easier (Fig. 2), and they are found to be more numerous than conventional sections would suggest. However, thin sections add little to our ability to recognize clearly the mesenchymal cells participating in the ductular cell reaction. ELECTRON

MICROSCOPY

Only those observations which are additional, or different to those described previously (Carruthers and Steiner, 1961; Steiner and Carruthers, 1961a, 1962) will be stressed. General aspects The proliferating ductular cells form well-polarized ductules and pre-ductules (Fig. 4) surrounded by a basement membrane (Fig. 5) which is only lacking at the point of contact between ductular and parenchymal cells. The proliferating ductular cells are accompanied by various mesenchymal cells in haphazard distribution and by numerous capillary channels. These will be described in a second separate paper. The ductular

cells

The proliferated biliary epithelial cells deviate from the normal, and from our findings in earlier stages of the reaction, in a number of respects. The loss of luminal microvilli was less marked than in the first 14 days after ligation. Luminal recesses were seen rarely, and “intracellular channels” were almost absent. Edema of microvilli in pre-ductular and ductular lumina was very frequent (Figs. 5 and 6). In many instances, the edematous microvilli nearly occluded the lumen. The edema affected only the microvilli themselves, the main perikaryonic mass of cytoplasm retaining its normal cohesion of organelles. The matrix of the swollen microvilli was less electron-dense than that of the rest of the cytoplasm (Figs. 5 and 6), and contained occasional smooth-surfaced vesicles (Fig. 11) and free RNP granules. Although rupture of edematous microvilli was observed occasionally, we could not assure ourselves that this was not an artifact. There was a marked increase in the rough-surfaced profiles of the endoplasmic reticulum (ER) (Figs. 7, 8, 14) and of free RNP particles (Figs. 8 and 14) which

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FIG. 4. Electron micrograph of normal proliferated ductule adjacent to a parenchymal liver cell. The basement membrane of the ductule is surrounded by processes of fibroblasts arrayed in parallel rows. Note the two main features which distinguish hepatocytes from ductular cells: (a) larger mitochondria, (b) parallel arrays of ER. The lumen of the ductule is provided with an approximately normal complement of microvilli. This is exceptional in proliferated ductules. Lead hydroxide, X 15,200.

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were usually arranged in rosettes. This change was patchy, and though present in one cell, might be absent in its neighbor. Marked variations were also noted in the distribution of the profiles in a given cell. The ergastoplasmic cisternae occurred

FIG. 5. Overall view of a ductular cell in proliferated channel. The nucleus is relatively large. (This is in part caused by the addition of sucrose to the fixative.) A large, edematous microvillus occupies the entire luminai surface. Mitochondria are few and two Golgi zones are seen. Interdigitation of the lateral cell wall with a neighboring cell can be observed at the right border of the electron micrograph. A cell process of an adjacent cell forms an intercalated cell in this plane of section, A continuous basement membrane underlies the antiluminal border of the cell. Uranyl acetate, X 10,140.

FIG. 6. Three ductular cells surround a portion of a lumen. The centrally located cell shows some loss of microvilli and a large, pear-shaped edematous one. Note abrupt cessation of edema at the junction with the main perikaryonic mass of the cell. Numerous smooth-surfaced vesicles are aggregated near the base of the large microvillus. The lateral cell walls are provided with attachment plates (terminal bars), desmosomes and in the right upper corner with interdigitating processes. Uranyl acetate, X 28,080.

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either in short disconnected segments, or as continuous intracytoplasmic canals which communicated with the nuclear envelope (Fig. 16) and in rare instances were seen to end in Golgi zones. The ribosomes attached to the outer surface of the membranes were irregularly distributed, and were lacking in many areas. The mitochondria were not altered in size or number, though occasional distorted corpuscles were observed. The Golgi zones were at times somewhat enlarged, and the cisternae dilated (Figs. 9, 10). In some cells several could be seen randomly distributed throughout the cytoplasm (Fig. 9). Smooth surfaced vesicles were often numerous (Figs. 6 and 14). In many instances their profiles merged with those of similar cisternae in the vicinity of Golgi zones. Most of these appeared empty, but some contained a faintly electron-opaque material. Occasional vesicles were seen in the edematous microvilli, imparting to them a Swiss cheese appearance (Fig. 11). Large intracytoplasmic vacuoles were seen occasionally. Multivesicular bodies were found commonly adjacent to Golgi zones (Fig. 14). A particular feature of the lesion after the day 14 following ligation of the common bile duct, was the appearance of increasing numbers of ‘dark cells’ (Figs. 12 and 13). The cells lining lumina and intercalated cells were both involved in this process. They seemed to gradually decrease in size, and their lateral cell membranes tended to shrink away from their neighbors, leaving between them a space into which their previously interlocking processes project. The cohesion at the location of attachment plates (terminal bars), and desmosomes was unaltered. Both the cytoplasm and nuclei seemed to become progressively more electron-dense, and the organelles, even though unchanged in size and shape, were aggregated more closely around the nucleus. The increased opacity of the matrix of the cytoplasm was heightened by the condensation of RNP particles. The proliferating biliary epithelial cells usually contained prominent centrioles (Fig. 14), the long axes of which were oriented in a random fashion in relation to other organelles. Occasionally, a diplosomal arrangement of centrioles was seen (Fig. 16)) the long axis of one member being often nearly perpendicular to that of the other. Each centriole measured approximately 400 rnp in diameter and 900 mu in length. Some of the proliferating ductular cells showed cilia. These were always single in any one cell in a given plane of section (Fig. 15) and were found in three distinct locations in relation to the luminal cell membrane. The majority projected into the lumen (Figs. 15 and 19) from a basal body which was the only portion of the organelle in direct contact with the cytoplasmic matrix. Some lay entirely within the FIG. 7. The lumen of a ductule is present in the left upper corner. The lateral walls of the biliary epithelial cells are provided with a number of desmosomes. Only one attachment plate (terminal bar) can be seen near the ductular lumen. Note the fairly elaborate ER in the cells. Lead hydroxide, X 27,650. FIG. 7a. (Insert.) A desmosome seen at higher power shows the bilaminar arrangement of each unit cell membrane participating in its formation. Note the somewhat irregular peri-desmosomal density, possibly resulting from tangential sectioning of the area. Uranyl acetate, x 115,360. FIG. 8. A ductular lumen is seen at the right upper corner with its peri-luminal area of condensation of the cell matrix. The ER of the cell is somewhat dilated and forms a communicating network of channels. Numerous rosettes of free RNP granules are scattered between the cisternae of the ER. Lead hydroxide, X 19,980.

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cytoplasm some distance from the lumen, and were surrounded by a continuous membrane except at the point where the basal body opened into the matrix of the cell (Fig. 17). Some were located in a narrow recess formed by an evagination of the cell membrane along the shaft of the cilium to the point of its junction with the basal body (Fig. 18). The average length of the shaft of these cilia was approximately 1.45 p and the length of their basal body approximately 400 rnp. The basal body of cilia is thought to consist of a single modified centriole (Bernhard and De Harven, 1960; Tokuyasu and Yamada, 1959). We are uncertain whether we have observed cross sections of the basal body itself, but transverse sections of centrioles reveal the usual hollow cylindrical array of nine groups of three fibrils (Fig. 15a). Cross sections of the shafts of cilia, as seen in the lumina of ductules, are approximately 650 rnp in widest diameter (Fig. 15b). There are seven double fibril groups peripherally and one of similar configuration centrally. The centrioles thus have a 9 + 0 arrangement, and the shafts of the cilia a 7 + 1 arrangement. The increase in the number of desmosomes which was observed in the earlier phases of the ductular cell reaction was maintained (Fig. 7). Not infrequently, three, four or more of these structures were found along the cell membranes of two adjacent ductular cells in a single section. Their fine structure did not differ from normal. At least two of the layers of the trilaminar cell membrane formed each of the segments (Fig. 7a). The thin dense line which was described by Fawcett (1961a) in the intercellular cleft of desmosomes could not be clearly identified in our material. The surrounding peridesmosomal densities were often irregular rather than rectilinear in shape. DISCUSSION Light microscopy has demonstrated that the ductular cell reaction constitutes the most prominent feature in the response of the liver of rats to ligation of the common bile duct (Cameron and Hasan, 1958; Cameron and Oakley, 1932 ; Koch-Weser et al., 1952; Riittner and Stofer, 1960; Steiner and Martinez, 1961; Trams and Symeonidis, 1957). Dilatation of ductules occurs as early as 1 hour after induction of cholestasis (Cameron and Oakley, 1932), and is followed after 4 hours by the appearance of mitoses in biliary epithelial cells (Cameron and Hasan, 1958). By 8 hours after ligation, the biliary epithelial cells have begun to proliferate (Cameron and Hasan, 1958), and their proliferation is well established by 48 to 72 hours after ligation (Cameron and Hasan, 1958; Cameron and Oakley, 1932; Riittner and Stofer, 1960; Steiner and Martinez, 1961; Trams and Symeonidis, 1957). By day 14 of cholestasis, the liver lobules consist of approximately equal quantities of proliferated ducts and FIG. 9. A portion of a ductular cell is seen. The cell contains four separate zones in a small portion dilated. Uranyl acetate, X 15,960. FIG. 10. are present structure to intracellular FIG. Uranyl

increase of Golgi of the cytoplasm.

complexes is depicted. Some of the cisternae

The are

Higher power view of a Golgi zone. The parallel stacks of elongated cisternal profiles in the center. The peripheral cisternae are distended. The double-membraned round the right of the Golgi area (arrow) is a process of an adjacent cell lying within an tunnel. Uranyl acetate, X 22,680.

11. Swiss-cheese appearance acetate, X 15,960.

of edematous

microvillus

containing

smooth-surfaced

vesicles.

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residual parenchyma (Trams and Symeonidis, 1957). By the third week, adjacent portal areashave becomeconnected by elementsof the ductular cell reaction. By the fourth to sixth week of stasis, ductular sprouts reach central veins (Riittner and Stofer, 1960). At this time, the picture is of diffuse adenomatous hyperplasia of ductular tissue (Cameron and Hasan, 19.58; Riittner and Stofer, 1960). The average survival time of rats with complete obstruction of the common bile duct is approximately 9 to 11 weeks. Our light microscopic findings have shown that the ductular cell reaction following ligation of the common bile duct is a well-organized proliferation. The ductular epithelial cells form regular channels almost invariably aligning themselves around lumina. This regular arrangement is not always obvious in sections, becausethe new ductules are often tortuous and branching. This regular proliferation of ductules is the more important as preliminary observations on ethionine and a-naphthyl isothiocyanate intoxication (Steiner and Carruthers, unpublished observations) indicate that this is by no meansalways the case in that condition. In contrast, parenchymal cells become isolated, singly or in small groups, as the advancing ductular sprouts and their connective tissue envelopes penetrate the lobules. We are not at present able to say whether these isolated cells have any connection with pre-existing or newly formed biliary channels. Serial thin sections may assist in the elucidation of this problem becauseof the easewith which lumina can be followed when this method is employed. Our previous electron microscopic studies (Carruthers and Steiner, 1961; Steiner and Carruthers, 1961a, 1962) of the ductular cell reaction which occurred during the 14 days following ligation of the common bile duct in rabbits and rats demonstrated a number of deviations from normal. The lumina of proliferated ductules were probably dilated. They had a paucity of microvilli, and luminal recesseswere prominent. These outpouchings, which were also noted in pre-ductules, projected into the cytoplasm of the ductular cells and did not passbetween them. By fortuitous sectioning, “intracellular channels” were demonstrated, and were interpreted as further evidence of luminal herniation into cells. An increase of pre-ductular lumina was considered to be the result of hyperplasia of biliary epithelial cells and of abnormal branching of their cytoplasm. Despite the probable increase in intraluminal pressure, the cohesion of ductular cells was maintained by attachment plates (terminal bars) and desmosomes. The cytoarchitecture of proliferated ductular cells was found to change at this time. The microvilli were often sparseor had becomeedematous.The rough-surfaced profiles of the endoplasmic reticulum were increased in number, and intracellular

FIG. 12. Three ductular cells line the lumen of a ductule. early phase of ‘dark’ cell metamorphosis. Both the cytoplasm electron dense. The mitochondria and ER lie closely together Lead hydroxide, X 17,860.

The centrally located one shows the and nucleus are more than usually between the nucleus and the lumen.

FIG. 13. Final phase of ‘dark’ cell metamorphosis involving an intercalated cell near the basement membrane (arrows). The cell has retracted from its neighbors and the cell processes become prominent due to some elongation. No structures can be recognized in the ‘dark’ cell at this magnification. Uranyl acetate, X 20,520.

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vacuoles appeared. The cells had numerous interlocking processes and there was a marked increase in the number of desmosomes. The dilatation of the biliary ductules grows less after day 14 of obstruction, at least if a paucity of microvilli and the presence of luminal outpouchings and recesses (Steiner and Carruthers, 1962) are considered evidence of its presence. The dilatation does not seem due entirely to an increase of intraluminal pressure in the bile ductules. In at least some cases the intraluminal pressure is still rising after day 14 of stasis though the dilatation is less. Also, similar changes in the ductules occur in carbon tetrachloride intoxication (Carruthers and Steiner, 1961) and there is little reason to assume that the ductular pressure is high in this condition. We have noted previously that in the first 14 days following ligation, extrahepatic biliary occlusion may be complicated by an intrahepatic obstruction resulting from edema of microvilli in bile canaliculi (Steiner and Carruthers, 1961c), and in proliferated bile ductules (Carruthers and Steiner, 1961; Steiner and Carruthers, 1961a). This edema is maintained after day 14 of obstruction, and is indeed often more marked than in the earlier phase of the process. Not infrequently ductular lumina are almost completely occluded. It is unlikely that this change plays a major role in impeding bile flow, since it seems likely that the great majority of channels are not occluded, although many are affected to a lesser degree. We are not certain of the cause of this localized edema, though it may be either the result of a disturbance of cell membrane permeability, leading to abnormal imbibition by or abnormal retention of fluid in microvilli, or a disturbance of fluid distribution in the ductular cells. Biliary epithelial cells contain normally only very few lamellae of rough-surfaced profiles of endoplasmic reticulum (ER), and free RNP particles are scanty (Grisham and Hartroft, 1961; Schaffner and Popper, 1961; Steiner and Carruthers, 1961b). This paucity of ergastoplasm is useful in distinguishing ductular from parenchymal liver cells. In proliferated biliary epithelial cells, the ER is increased, and is found either in the form of short disconnected segments or as more complex, communicating networks with prominent, though unevenly distributed, surface ribosomes. The cisternae never show the regular parallel arrays seen in hepatocytes. We were unable to relate the slight but almost uniform basophilia of the proliferated ductular cells to the presence of the ER, since in many cells free ribosomes appear to be far more numerous than those of the membrane system. In this respect the distribution of ribosomes and the basophilia of these cells resembles that found in rapidly growing hepatomas (Howatson and Ham, 1955). It has been stated that the free ribosomes alone may be sufficient for the protein synthesis needed to support rapid cell proliferation, but that their association with the membranes of the reticulum may be necesFIG. 14. Two ductular cells form part of the lining of a ductule. Their lateral cell membranes are provided with an attachment plate (terminal bar) and two desmosomes. (The one nearer the lumen is sectioned tangentially.) Each cell contains a centriole in this plane of section. The ribosomes are prominent on the surface of the vesicles of the ER. Note their uneven distribution. Free cytoplasmic RNP granules are arranged singly or in rosettes in the matrix of the cell. The Golgi zones contain vacuoles whose content is faintly electron dense. Similar, larger, secretory vacuoles are present in the cytoplasm remote from Golgi zones. Others appear to be entirely empty. Note the multivesicular body in the lower center of the micrograph. Lead hydroxide, x 39,950.

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sary for the elaboration of a protein-rich secretory product (Fawcett, 1961b; Howatson and Ham, 1955). There is no evidence that ductular cells elaborate a protein-containing secretion (Schaffner and Popper, 1961). We therefore may assume that in this instance both the free ribosomes and the membrane-bound RNP particles may be primarily concerned with protein synthesis in support of proliferating cells. The development of a fairly elaborate system of ergastoplasmic membranes in some but not all proliferated biliary epithelial cells raises the question of their state of maturity. It has been stated that the ER with its dependence on the nucleus alone, probably exerts an important influence on the development of cell form and surface configuration, as well as on the distribution of the cell organelles which are commonly accepted as expressions of cellular differentiation (Porter, 1961). The endoplasmic reticulum is poorly developed in relatively immature cells during the proliferative phase of any cell line, and becomes more prominent in mature cells which have a distinct function to perform (Porter, 1961). We have noted in our observations that the prominence of the ER varies considerably from cell to cell in proliferated ductules. If Porter’s conclusions (1961) are correct, we may assume that in a relatively rapidly proliferating cell population, ductular cells with little ER may be less mature and possess a less well-defined function than those with an abundant ER. It is of course also possible that the cells with little ER, which closely resemble normal biliary epithelial cells in this respect, are fully mature. Those with an abundant ER may be cells in an interphase generation which will be replaced in successive generations by others possessing a gradually less developed ER until the form of the characteristic mature cell is reached. We are unable to support the concept of Cameron and Hasan (1958) that biliary epithelial cells elaborate mucin as a defense mechanism against toxic products. The fine structure of mucin droplets in goblet cells is now well understood (Florey, 1960). In the penultimate branches of the bile-conducting channels that we examined, cells containing mucin droplets could not be identified. On the other hand, the presence of numerous smooth-surfaced profiles in relation to the Golgi complex, as well as in other parts of the cytoplasm, and particularly within edematous microvilli, may be an indication of secretory and//or absorptive activity. Some of these profiles contain a faintly electron-dense material, and so may be designated as secretory granules, akin to those found in the pancreas (Palade, 1959) or the pituitary (Farquhar, 1961). Schaffner and Popper ( 196 1) suggested that ductular cells secrete a bicarbonatecontaining fluid, and that they may be concerned also with reabsorption of water from ductular lumina, serving to regulate the final water and electrolyte content of bile. The presence of such substances within the secretory granules would well account for their relatively low electron opacity. FIG. 15. A bile ductule is separated by a basement membrane and by an elongated fibroblastic process from a membrane-enclosed blood capillary. The endothelium of this vessel is in one part edematous, A cilium lies in a luminal recess of the ductular lumen, its basal body projecting into the cytoplasm. Uranyl acetate, X 16,720. FIG.

groups. Fro. central

(Insert.) Cross-section of centriole showing A central “hub” is recognizable only faintly. Uranyl

15a.

15b. (Insert.) doublet fiber

Cross-section groups. Uranyl

of cilium in lumen acetate, X 46,980.

nine peripherally placed acetate, X 46,980.

of ductule.

Note

seven

peripheral

triplet fiber and one

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Another observation which supports the theory that biliary epithelial cells may produce a secretory product, is that some proliferated biliary epithelial cells show evidence of a reduction in size and an increase in cytoplasmic and nuclear density. These changes have not been previously noted by light microscopy. They resemble the “schmale Zellen” of Miillendorff (1936) or the ‘Lexhausted” goblet cells of the gut described by Palay (1958) and Florey (1960). In the gut, such cells arise as a result of a massive discharge of mucin. They are regarded by Florey as exhausted rather than resting, since they still contain an ample Golgi network, numerous mitochondria and a few mucin droplets (Florey, 1960). In the ‘dark’ biliary epithelial cells of proliferated ductules, mitochondria are as numerous as in other proliferated ductular cells, although the organelles are crowded together in the limited space available to them. Although the secretory product of ductular cells is not recognizable as clearly as are mucin droplets in intestinal cells, we suggest that the development of ‘dark’ cells is an exhaustion or emptying phenomenon resulting from a more or less sudden discharge of secretion from the cytoplasm. It is possible that the development of ‘dark’ cells is the result of loss of cell water because of an intracellular electrolyte or osmotic imbalance, or because of rupture of edematous microvilli. Whether these cells should be regarded as resting, or as more actively synthesizing secretion than normal cells, cannot be answered on the basis of the evidence available to us. We have described the cilia which develop in proliferated ductular cells. In general, cilia and flagella are divided into those whose basal organization involves one modified centriole, and those in which two centrioles participate. Although we have observed a diplosomal arrangement of centrioles in proliferated ductular cells, and the centrioles are often located in the vicinity of the cell membrane, all the cilia found were of the one-centriole type. Only one cilium was found in any one cell. This observation is of some importance to the study of other forms of the ductular cell reaction, since in ethionine intoxication (Steiner and Carruthers, unpublished observations) two-centriole type cilia are found, and more than one may be present in a single cell. The cilia which we have observed are abnormal in their fiber configuration. Although the centrioles retain the normal 9 + 0 arrangement of triplet fiber units, the shafts of the cilia reveal only seven peripheral and one central doublet fiber groups. In almost all other instances where cilia, flagella or the tails of spermatozoa have FIG. 16. Two centrioles farm a diplosome of the centrioles are oriented at right angles nally and the other (C2) is cross-sectioned. (arrow). Lead hydroxide, X 15,510.

in the cytoplasm of this ductular cell. The long axes to each other so that one (C,) is sectioned longitudiNote continuity of the nuclear envelope with the ER

FIG. 17. A cilium, the shaft enclosed in a membrane, lies within the ductular cell cytop!asm beneath an edematous microvillus. The basal body projects into the cytoplasmic matrix. Lead hydroxide, X 32,800. FIG. 18. A cilium lying within a deep luminal recess possibly about to erupt into the lumen. Lead hydroxide, X 19,380. FIG. 19. A fully erupted cilium lies in a cup-shaped niche. Note that the plasma membrane of the cell covers the surface of the cilium down to the upper limit of the basal body. Uranyl acetate, X 19,760.

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been studied, the shafts have had the same configuration as the centrioles, that is a 9 + 2 or 9 + 0 arrangement (Barnes, 1961). Only two exceptions have been noted. Shapiro et al. (1961) showed that a single central unit with a ring of nine doublet filaments is consistently found in the sperm tail of the flatworm Haematoloechus medioplexus, and Satir (1962) found amongst the enormous number of cilia of the gills of the fresh water mussel Elliptio complanatus that he examined a single cilium with twelve peripheral and one central unit of doublet filaments. In this instance the variation was considered a developmental error. The 7 + 1 doublet filament pattern has been seen so consistently in our material that we doubt that it is either a fixation artifact, or a chance variation. We are unable to conclude whether these cilia are functionally effective, since only those with a 9 + 2 arrangement of fibrils are known to be motile (Barnes, 1961) . Normal biliary epithelial cells are provided with attachment plates (terminal bars) close to the lumen of the ductules, and usually have one or, at most, two desmosomes on their lateral cell walls, as seen in any given plane of section. The presence of desmosomes is, however, not invariable. We have noted previously that during the first 14 days following ligation of the common bile duct, there is a considerable increase in the number of desmosomes (Carruthers and Steiner, 1961). This increase is maintained after the day 14 of cholestasis. It is not unusual to find as many as six along a single lateral cell membrane. We have suggested previously that this increase may be an expression of the need for closer cohesion of proliferating labile cells (Carruthers and Steiner, 1961). One may, however, also postulate that both the desmosomes and the frequently complex cell interdigitations may have a part to play in the regulation of cell multiplication, a concept suggested as an explanation for the existence of these structures in the colon where frequent desquamation of cells into the lumen of the gut has been noted (Florey, 1960). SUMMARY The ductular of the common The cells show Thin havior clearly

cell reaction in the liver of rats during the second to sixth bile duct is in part an organized proliferation of ductular an invariable tendency to surround lumina.

(% p) sections examined in the light microscope of ductular cells since the lumina of the penultimate visualized than in routine sections.

weeks following ligation (biliary epithelial) cells.

assist in the understanding bile-conduction channels

of the becan be more

Biliary epithelial cells from proliferated ductules in rats killed two to six weeks after the ligation, show by electron microscopy a persistence of some of the changes noted as occurring during the first 14 days of their proliferation, a paucity of microvilli, edema of microvilli, an increase of the endoplasmic reticulum, and an increase of free ribosomes. Additional abnormalities develop in ductular cells after day 14 of cholestasis. The Golgi zones multiply and become prominent. Smooth-surfaced vesicles increase in number. Some cells become increasingly electron-opaque, and have been designated as ‘dark’ cells, Other cells may acquire a unique one-centriole type of cilia which have an abnormal 7 + 1 doublet filament arrangement. The functional effectiveness of these cilia could not he assessed, owing to the absence of information about any other comparable cilia with a known motility. It was suggested that the prominent production by proliferating cells rather The prominence of Golgi zones and evidence of secretory activity involving form as a result of a sudden discharge

ER and RNP granules may signify an increased protein than evidence of elaboration of a protein-rich secretion. of smooth-surfaced vesicles was interpreted as possible water and electrolytes. ‘Dark’ biliary epithelial cells may of such secretion into ductular lumina.

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ACKNOWLEDGMENTS We wish to thank Professor A. C. Ritchie criticism. We also acknowledge the excellent Main, and of Mr. G. Doornewaard.

for reviewing the manuscript technical help of Miss Barbara

and for his constructive Lambert, Miss Barbara

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STEINER,