morphology of the tight junctional network around the canalicular lumen of the hepatocytes inrat liver afterexperimental bile-duct ligation. The more or less ...
Br. J. exp. Path. (1978) 59, 220
MORPHOLOGIC CHANGES OF THE JUNCTIONAL COMPLEX OF THE HEPATOCYTES IN RAT LIVER AFTER BILE DUCT LIGATION R. DE VOS AND V. J. DESMET From the Laboratory of Hi8tochemi8try and Cytochemi8try, Academi8ch Ziekenhuis Sint Rafael,
Univermity of Leuven, Belgium Received for publication November 30, 1977
Summary.-Using freeze-fracture techniques, we have examined the changes in the morphology of the tight junctional network around the canalicular lumen of the hepatocytes in rat liver after experimental bile-duct ligation. The more or less regular belt of parallel strands formed by the tight junctions around the canalicular lumen of normal hepatocytes is changed after extrahepatic obstruction. A more irregular network is formed with a reduced number of strands which also extend more in an abluminal direction with formation of irregular loops. Striking changes are seen at the gap junctions: the small patches normally situated within the tight junctional network become less numerous; at some canaliculi they are even absent; also the larger gap junctional areas normally present in deeper abluminal extensions of the lateral cell membrane become hard to find or are even absent. This altered tight junctional pattern suggests an increased permeability so that the pathway of intercellular escape of biliary constituents towards the blood stream in cholestasis becomes as plausible as transhepatocytic regurgitation. The disappearance of the gap junctions would result in a lack of intercellular communication and uncoupling of liver cells, which may lead to a more individual behaviour of adjacent hepatocytes, explaining the heterogeneity in canalicular changes in cholestasis.
BILE canaliculi between liver cells are separated from the intercellular space by tight junctions which form a barrier to large molecule exchange between the biliary and the blood compartment. Tight junctions are specialized membrane areas on the intratrabecular domain of the livercell membrane. In thin sections a tight junction appears as a fusion of the outer leaflets of the plasma membranes of two adjacent cells (Farquhar and Palade, 1963). Its structure is better revealed by the freeze-etch replica technique. In this technique, the frozen tissue is cleaved; the cleaving occurs in the hydrophobic region of the cell membrane, thus revealing intramembranous particles and exposing the inner side of the outer leaflet of the cell membrane
(E face) and the outer side of the cytoplasmic leaflet (P face). In such replicas the tight junctions form bandlike, parallel and sometimes branching ridges and particles on the P face (Branton et al., 1975) and complementary furrows or grooves and pits on the E face (Staehelin, 1974). The structural design of these tight junctional strands or network -which seal spaces between cells-plays an important role in the physiological properties and especially the permeability characteristics (Claude and Goodenough, 1973) of the tissue, although recent findings indicate that other features of the junctions or the fibrils themselves, not yet revealed by electronmicroscopy, play a central role in regulating epithelial permeability (Martinez-Palomo and Erlij, 1975;
Correspondence: R. de Vos, Laboratorium voor Histochemie en Cytochemie, Department Medische Navorsing, A.Z. Sint Rafael, B-3000 Leuven, Belgium.
JUNCTIONAL COMPLEX IN CHOLESTASIS
Mollgard, Malinowska and Saunders, 1976). At variable distances from the canalicular gap junctions occur between liver cells. The gap junction (nexus or macula communicans) is a highly ordered plasma membrane specialization which establishes very thin cell-to-cell communication channels. It can be recognized by its characteristic morphology: a hexagonal lattice after staining with lanthanum is visible (Goodenough and Revel, 1970). In freeze-etched replica's gap junctions appear as patches of associated globular particles of very regular size in the P face, and closely packed pits in the E face whose spacing is identical to that of the particles in the P face. Gap junctions are the sites for intercellular passage of ions and larger molecules involved in intercellular communication, giving signals from cell to cell; they correspond to low-resistance sites and sites of electrotonic coupling (Goodenough and Revel, 1970; Goodenough and Stoeckenius, 1972). The anatomical pathway of bile regurgitation in cholestasis remains undefined; increased permeability of tight junctions remains a possible pathway (Desmet, 1975). In cholestasis a great variability in canalicular morphology is observed (Desmet, 1972), suggesting a possible disturbance in intercellular communication and coordination along the canalicular channel. Several experiments suggest that tight and gap junctions undergo rapid configurational changes depending on various experimental conditions (Elias and Friend, 1976; Montesano et al., 1975; Montesano et al., 1976; Wade and Karnovsky, 1974). Therefore the present study is intended to follow the changes and rearrangement, if any, of tight and gap junctions in rat liver as revealed by the freeze fracture technique after experimental bile duct ligation. MATERIALS AND METHODS
Male albino rats from the Wistar R-A strain weighing approximately 150-200 g were used.
The animals were fed on a laboratorv stock diet and tapwater. The rats were divided into 2 series. Group 1: the control series, contained 3 normal rats and 2 normal rats which had undergone sham operation. Group 2: comprised 8 rats with bile-duet ligation. During constant ether flow anaesthesia, the ductus choledochus was exposed by blunt dissection, doubly ligated and sectioned between the 2 ligatures. All animals were killed by exsanguination, liver biopsy specimens were taken from the left liver lobule. The time between the bile-duct operation and the death of the animals varied from 3 to 21 days. The effectiveness of the bile-duct ligature was controlled by morphological and histochemical staining on paraffin sections. Thin slices 1 1 mm were fixed by immersion for 1 h in cold (40) 2.5% glutaraldehyde solution in phosphate buffer, pH 7-2, followed by buffer rinse overnight. The slices were then infiltrated for 20 min each in 10-2o5% solutions of glycerol in phosphate buffer. From these slices small pieces of liver tissue were prepared: from Group 1, 41 blocks and from Group 2, 85 blocks were prepared for for freeze-etched replicas. These blocks were rapidly frozen in Freon 12 cooled in liquid nitrogen, fractured at -1000 in a Balzers BAF 300 Freeze-Etch apparatus (Balzers AG. Balzers Lichtenstein) and shadowed with platiniumcarbon according to the technique of Moor and Miihlethaler (1963). After thawing of the tissue, the replicas were cleaned in a sodium hypochlorite solution overnight, rinsed in distilled water and mouinted on copper grids. Replicas were examined in a Zeiss EM 10 electronmicroscope. RESULTS
In freeze-etch replicas of normal and sham-operated rat livers the bile canalicular lumen is sealed from the intercellular space by a bundle of 4 to 5 more or less parallel tight junctional strands, below the level of the microvilli. The junctions are composed of ridges and particles in the P face, grooves and pits in the E face, and occupy a rather shallow zone on the lateral cell membrane. At several points there are vertical connections between the parallel strands. From these parallel bundles, some free-ending strands and loops more or less perpendicular to the canalicular lumen are
R. DE VOS AND V. J. DESMET
2~~~~~~~~~~~~~~~~~~ FIG. 1.
Freeze-fractured bile canaliculus from a control rat. The tight junctions are recognizable as more or less parallel strands of ridges in the P face and grooves in the E face. Small gap junctions (-+) can be seen in the tight junctional compartment while the larger ones are seen more abluminally at the lateral cell membrane (P face). Cross-sections of microvilli (V). x 19,150. FIG. 2. Detail of tight junction of a control rat: parallel running stran(is (P face). Bile canalicular microvilli (V). x 37,550.
JUNCTIONAL COMPLEX IN CHOLESTASIS
4 FIG. 3-6. Freeze-fractured preparations of rats with bile-duct ligation. FIG. 3. Tight junctional network (E face) showing an irregular pattern with a reduced number of junctional strands after 21 days' bile-duct obstruction. Cross-sections of microvilli (V). x 19,150. FIG. 4. Detail of the irregular tight junctional network (P face) after 21 days' bile-duct obstruction. Compare with Fig. 2. x 37,550.
R. DE VOS AND V. J. DESMET
6 FIG. 5. Detail of the irregular tight junctional pattern after 11 days' bile-duct obstruction (E face). Numerous free ending strands and loops extending more abluminally are see (-4). x 37,550. FIG. 6. Disarranged tight junctional pattern of a ptitative Type I canaliculus (E face). Note irregu-lar lumen and small number of microvilli. x 37,550.
seen, running across a larger area of the lateral cell membrane. Gap junctions occur as patches in small "compartments" of the network of tight junctions, and are also found in deeper abluminal extensions of the lateral cell membrane, where they occupy large independent areas of ± 1 f2 (Fig. 1, 2).
In the livers of rats of Group 2 the junctional pattern is changed. Instead of the more or less regular belt of parallel strands formed by the tight junctions around the canalicular lumen, a more irregular network is formed with a reduced number of strands, which also extend more in an abluminal direction. An in-
JUNCTIONAL COMPLEX IN CHOLESTASIS
creased number of loops and free ending strands of tight junctions is formed (Fig. 3-5). In all obstructed rats, similar changes can be seen. The alteration seems to be more pronounced as the obstruction time is longer: the loops become more frequent and the junctional network more irregular. These changes are seen around canaliculi with dilated lumina and with a reduced number of cross-sections of microvilli as compared to normal ones. Striking changes are seen at the gap junctions: the small patches normally situated within the tight junctional network become less numerous, at some canaliculi they are even absent; also the larger gap junctional areas become hard to find or are even absent. At some acinar arrangements of hepatocytes-as far as can be estimated from freeze-etched replicas-the composing hepatocytes show a different behaviour with regard to their tight junctional configuration: some hepatocytes have an irregular shallow network, others have a broad zone of parallel belts. Some canaliculi are observed with small irregular lumina-different from the normal one-with only a few cross-sections of microvilli, under which a shallow but very irregular pattern of tight junctional network can be found; no gap junctions could be detected here (Fig. 6). DISCUSSION
Numerous studies have been devoted to the physiological properties of tight junctions in different tissues (Bockman, 1974; Hull and Staehelin, 1976; Machen, Erlij and Wooding, 1972; Metz et al., 1977; Montesano et al., 1975; Schatzki, 1971). The general conclusion is that the structural pattern of tight junctions may influence the permeability properties of a tissue. On this basis various epithelia have been classified according to their transepithelial permeability into "leaky" and "tight" epithelia (Claude and Goodenough, 1973). The junctional complex of the liver cell
has been generally considered, with regard to its morphological and physiological tightness, as a relatively tight type of junction, constituting an efficient barrier for transport between the canaliculus and the intercellular space. Several findings indicate, however, that the liver cell tight junction might not be as tight as generally assumed and that a transjunctional permeability under physiological control may exist (Diamond, 1974; Papadimitriou and Walters, 1968; Pavel, Petrovici and Bonaparte, 1972; Wheeler, 1975). In livers of bile duct ligated rats the tight junctional pattern becomes strikingly different from normal: an irregular network replaces the belts of more or less parallel strands surrounding the canaliculi. Furthermore, the junctional strands extend further abluminally on the lateral cell membrane with formation of irregular loops. This pattern shows a striking resemblance to the altered junctional patterns described by Montesano et al. (1976), in rat liver after chronic phalloidin administration, a condition which also induces intrahepatic cholestasis. In extrahepatic cholestasis, as in the present study, similar alterations were recently described by Metz et al. (1977). The structural pattern of the altered tight junctions after bile duct ligation suggests an increased permeability, due to an increased number of discontinuities in the strands and the formation of numerous free-ending loops. Metz et al. (1977) confirmed an increased permeability by demonstrating passage of the tracer horseradish peroxidase from the blood to the bile canaliculus. It is not yet established whether this increased permeability is selective for certain compounds, depending on molecular size on the one hand and the size of the openings in the tight junctional network on the other hand. Conceivably, also the changes of the glycocalix (Phillips et al., 1976) with associated changes in electric charge may influence the tight junction. In any event, it seems that in extrahepatic obstruction with increased canali-
R. DE VOS AND V. J. DESMET
cular pressure the pathway of intercellular escape of biliary constituents towards the bloodstream becomes as plausible as transhepatocytic regurgitation (Desmet, 1972, 1975). According to Hull and Staehelin (1976), the establishment of a loose tight junctional network indicates the ability of a tissue to adapt itself to changes in mechanical stress. The formation of long abluminally running strands and loops may favour an increased mechanical coherence of liver cells, counteracting the increased intracanalicular pressure. This, together with the preservation of marginal microvilli and the overlapping of the marginal ridge (Vial, Simon and Mackinnon, 1976) may enforce intercellular adherence and prevent disruption of canaliculi. A second important observation in the present study, also recently confirmed by others (Metz et al., 1977), is the remarkable decrease, and even disappearance of the gap junctions. Since the main function of the gap junctions consists in transmitting signals from cell to cell and regulating intercellular transports (Goodenough and Revel, 1970; Goodenough and Stoeckenius, 1972; Peracchia, 1973) the present finding suggests that in extrahepatic cholestasis the intercellular communication is impeded with resultant uncoupling of liver cells. This may lead to a more individual behaviour of adjacent hepatocytes, explaining the heterogeneity in canalicular changes in cholestasis (Desmet, 1972), and the variation observed in tight junctional patterns even in adjacent hepatocytes, particularly so in so-called "secondary canaliculi" or acinar arrangements of liver cells. Whether the proliferation of tight junctions occurs at the expense of preexisting gap junctions or occurs de novo by the assembly of particles as described in foetal rat liver (Montesano et al., 1975) remains uncertain. Working with an in vitro system for the
modulation of tight and gap junctional differentiation, Elias and Friend (1976) came to the conclusion that there exists for both types of junctions a common pool of membrane particles, and that the type of stimulus determines which type of junction is formed or broken down. The special canaliculi, observed in the present study, and characterized by an irregular lumen and an irregular tight junctional network, may well correspond to canaliculi Type I described previously in cholestatic rat liver (De Vos et al., 1975). In analogy with similar structures observed in foetal rat liver (De Wolf-Peeters et al., 1974), they were interpreted as newly formed canaliculi, implying also newly established tight junctions (De Vos et al., 1975). It is well known that the formation of new tight junctions is a very rapid and dynamic process (Hudspeth, 1975). It is tempting to speculate on the possible role of the cytoskeletal system (microtubules, microfilaments) in the modulation of tight junctional structures. The present study does not establish such a relationship. Nevertheless, impressive changes of microfilaments have been observed in different forms of cholestasis (De Vos et al., 1975; French, 1976; Phillips et al., 1975), and several studies have shown that the cytoskeletal system plays an important role in the trasmembrane control and topography of membrane particles (Berlin et al., 1975; Nicolson and Poste, 1976). This study was supported by a grant of the "Fonds voor Wetenschappelijk Geneeskundig Onderzoek". The authors with to thank Mrs Vuelemans-Weckx for typing the manuscript and Mr Rooseleers for the preparation of the photographs. REFERENCES BERLIN, R. D., OLIVER, J. M., UKENA, T. E. & YIN, H. H. (1975) The Cell Surface. New Engl. J. Med., 292, 515. BOCKMAN, D. E. (1974) Route of Flow and Micropathology Resulting from Retrograde Intrabiliary
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