- Email: [email protected]
V. Serial sections
V. S E R I A L S E C T I O N S By means of serial sectioning we wanted to make sure that no direct communication exists between the canaliculi and the space of Disse. Such a negative proof is difficult to obtain, but different methods lead us to the same (negative) conclusion. Phase contrast
In the phase contrast microscope, the bile canaliculi could sometimes be confused with the space of Disse. Therefore we t o o k livers in which the peroxidase had already been secreted into the bile canaliculi. It appears as compact, r o u n d or tubular profiles between the adjacent hepatocytes, surrounded most often by intracellularly situated black granules. By means of these two characteristics, bile canaliculi can easily be distinguished f r o m the space of Disse, which has a greyish, fuzzy appearance. In the thirty series examined, we followed each bile canaliculus (about 10-20 per series) to determine whether any canaliculus joined with the space of Disse. We found no joinings except artifactual ones, where two hepatocytes had been disrupted by mechanical procedure at the border of the tissue block. The same artifact can cause difficulties in the interpretation of the retrograde injection experiments and will be discussed below. On the other hand, we will show one series (Fig. 20a-p) that demonstrates an important feature, namely the extremely close relationship between the bile canaliculi and the space of Disse. In this series one can follow the serial sections of a bile canaliculus (arrow), from Fig. 20a to 20f. Already, in the following section, the spot that corresponds to the bile canaliculus has been taken by an extension of the space of Disse. This gives us a maximal distance of 0.1 /~, assuming that section thickness is 0.1 #, as indicated by the interference colors. We have no statistical data on the frequency of such regions of minimal distance between the bile canaliculi and the space of Disse, but we estimate that about every 20th to 30th bile canaliculus (in our sections) shows this close proximity to the space of Disse. FIG. 20a-p. Serial sections of a portion of rat liver, injected intravenously with horseradish peroxidase 30 rain before fixation. A bile canaliculus is filled with peroxidase; reaction product can also be seen in the pericanalicular dense bodies, in the space of Disse, and in sinusoids. Red blood cells appear very dark. In section g the bile canaliculus has disappeared and is replaced by a portion of the space of Disse, easy to follow in the next sections. This series shows an intimate relationship between the bile canaliculi and the space of Disse, but no communication. The minimal distance between the two compartments is about 0.1 # (--about one section thickness). × 1800.
i
2 0 ¢ ~:
Fins. 21-24. Serial sectioning of portions of two hepatocytes, whose microvilli are covered by the endothelium of the sinusoid. The space of Disse forms a little lacuna (Figs. 21 and 22D). In section No. 1 (Fig. 21 a, top left) a bile canaliculus is forming intracellularly (arrow), as in section No. 24 (Fig. 23 b). These little cavities change after 2 or 3 sections from their intracellular to an intercellular position, tightly closed at each side by the junctional complex. The bile canaliculi are very near the space of Disse, in one section (No. 32, Fig. 24a) separated from it by a cytoplasmic lamella of only 0.1/~, but never opening into the space of Disse. The big arrow in section No~ 32 indicates the direction of the view of Fig. 26; the little arrows, the plane of the section. The arrow in section No. 44 (Fig. 24c) indicates the direction of the view of Fig. 25. × 20,000.
i
~i~ii,~
¸¸i
~
•
,
i~!!
i
i ¸
!i~ ~
i,
¸
.....
~
3 - - 69[[833 Matter
bc
bc
35
MORPHOLOGICAL ASPECTS OF BILE FORMATION
2~
F~G. 25. The two bile canaliculi of the foregoing series drawn in parallel perspective with the Perspektomat P-40, in the direction indicated by the arrow in section No. 44 (Fig. 24 e). The dotted lines give the back. Each curve represents one section; the 45 sections give a thickness of about 2.5 #.
Electron microscope We have examined 20 series of 60-80 sections of livers which have not been treated with peroxidase. The perfusion fixed livers mostly show wide open bite canaliculi, which are therefore easy to follow. But, for the interpretation, all characteristics of a bile canaliculus (see Section III) have to be carefully considered: microvilli with terminal web, junctional complex, pericanalicular organelles (Golgi, lysosomes), thereby excluding the canaliculus-like protrusions and lacunae of the space of Disse (Fig. 21a-a, 22e). We have never found a direct communication between the space of Disse and the bile canaliculus. It is interesting that we have found the same regions of minimal distance of 0.1 # between these two structures in about one in every 30 bile canaliculi examined. The same artifact of ruptured junctional complexes with a widely open intercellular space is also sometimes seen. In the series shown in this paper (Figs. 21-.27) we want to demonstrate the regions of minimal distance between the space of Disse and two bile canaliculi, and a typical lacuna of the space of Disse, which could easily be mistaken for a bile canaliculus if only one picture were available (Fig. 22a). The bile canaliculi begin most often intracellularly and, after a short distance go between two, seldom three adjacent hepatocytes. Occasionally, the bile canaliculi run intracellularly for long distances of 1-3 #. In such a series, the continuity of the different parts of the junctional complex may also be seen. Although varying in length (50-100 m#), the region of the closecontact-points (zonula occludens) appears whereas the zonula adhaerens often fails to appear and the randomly distributed macula adhaerens is seen more or less as a
36
M A T T E R ET AL.
27
1:;i6.26. The bile canaliculus appearing at the left of Fig. 25 is shown as indicated by the large arrow in section No. 32 (Fig. 24a), sectioned in the plane of the little arrows. The figure shows the intimate relationship between the bile canaliculus and the space of Disse. FIG. 27. A simplified reconstruction of the Fig. 26. The region of minimal distance is shown by a circle. dot-like structure (Figs. 25-27). Because of the wide opening of the bile canaliculi, their microvilli are neither n u m e r o u s n o r very long. The g r a p h i c a l r e c o n s t r u c t i o n in parallel perspective (Figs. 25-27) gives the threed i m e n s i o n a l a p p e a r a n c e of the two bile canaliculi with their j u n c t i o n a l complexes. A section p e r p e n d i c u l a r to the p l a n e of the p h o t o g r a p h s shows the view on a region of m i n i m a l distance between one of the bile canaliculi a n d the space of Disse. A possible function of these regions is c o n s i d e r e d in the general discussion.
In the phase contrast microscope, the bile canaliculi could sometimes be confused with the space of Disse. Therefore we t o o k livers in which the peroxidase had already been secreted into the bile canaliculi. It appears as compact, r o u n d or tubular profiles between the adjacent hepatocytes, surrounded most often by intracellularly situated black granules. By means of these two characteristics, bile canaliculi can easily be distinguished f r o m the space of Disse, which has a greyish, fuzzy appearance. In the thirty series examined, we followed each bile canaliculus (about 10-20 per series) to determine whether any canaliculus joined with the space of Disse. We found no joinings except artifactual ones, where two hepatocytes had been disrupted by mechanical procedure at the border of the tissue block. The same artifact can cause difficulties in the interpretation of the retrograde injection experiments and will be discussed below. On the other hand, we will show one series (Fig. 20a-p) that demonstrates an important feature, namely the extremely close relationship between the bile canaliculi and the space of Disse. In this series one can follow the serial sections of a bile canaliculus (arrow), from Fig. 20a to 20f. Already, in the following section, the spot that corresponds to the bile canaliculus has been taken by an extension of the space of Disse. This gives us a maximal distance of 0.1 /~, assuming that section thickness is 0.1 #, as indicated by the interference colors. We have no statistical data on the frequency of such regions of minimal distance between the bile canaliculi and the space of Disse, but we estimate that about every 20th to 30th bile canaliculus (in our sections) shows this close proximity to the space of Disse. FIG. 20a-p. Serial sections of a portion of rat liver, injected intravenously with horseradish peroxidase 30 rain before fixation. A bile canaliculus is filled with peroxidase; reaction product can also be seen in the pericanalicular dense bodies, in the space of Disse, and in sinusoids. Red blood cells appear very dark. In section g the bile canaliculus has disappeared and is replaced by a portion of the space of Disse, easy to follow in the next sections. This series shows an intimate relationship between the bile canaliculi and the space of Disse, but no communication. The minimal distance between the two compartments is about 0.1 # (--about one section thickness). × 1800.
i
2 0 ¢ ~:
Fins. 21-24. Serial sectioning of portions of two hepatocytes, whose microvilli are covered by the endothelium of the sinusoid. The space of Disse forms a little lacuna (Figs. 21 and 22D). In section No. 1 (Fig. 21 a, top left) a bile canaliculus is forming intracellularly (arrow), as in section No. 24 (Fig. 23 b). These little cavities change after 2 or 3 sections from their intracellular to an intercellular position, tightly closed at each side by the junctional complex. The bile canaliculi are very near the space of Disse, in one section (No. 32, Fig. 24a) separated from it by a cytoplasmic lamella of only 0.1/~, but never opening into the space of Disse. The big arrow in section No~ 32 indicates the direction of the view of Fig. 26; the little arrows, the plane of the section. The arrow in section No. 44 (Fig. 24c) indicates the direction of the view of Fig. 25. × 20,000.
i
~i~ii,~
¸¸i
~
•
,
i~!!
i
i ¸
!i~ ~
i,
¸
.....
~
3 - - 69[[833 Matter
bc
bc
35
MORPHOLOGICAL ASPECTS OF BILE FORMATION
2~
F~G. 25. The two bile canaliculi of the foregoing series drawn in parallel perspective with the Perspektomat P-40, in the direction indicated by the arrow in section No. 44 (Fig. 24 e). The dotted lines give the back. Each curve represents one section; the 45 sections give a thickness of about 2.5 #.
Electron microscope We have examined 20 series of 60-80 sections of livers which have not been treated with peroxidase. The perfusion fixed livers mostly show wide open bite canaliculi, which are therefore easy to follow. But, for the interpretation, all characteristics of a bile canaliculus (see Section III) have to be carefully considered: microvilli with terminal web, junctional complex, pericanalicular organelles (Golgi, lysosomes), thereby excluding the canaliculus-like protrusions and lacunae of the space of Disse (Fig. 21a-a, 22e). We have never found a direct communication between the space of Disse and the bile canaliculus. It is interesting that we have found the same regions of minimal distance of 0.1 # between these two structures in about one in every 30 bile canaliculi examined. The same artifact of ruptured junctional complexes with a widely open intercellular space is also sometimes seen. In the series shown in this paper (Figs. 21-.27) we want to demonstrate the regions of minimal distance between the space of Disse and two bile canaliculi, and a typical lacuna of the space of Disse, which could easily be mistaken for a bile canaliculus if only one picture were available (Fig. 22a). The bile canaliculi begin most often intracellularly and, after a short distance go between two, seldom three adjacent hepatocytes. Occasionally, the bile canaliculi run intracellularly for long distances of 1-3 #. In such a series, the continuity of the different parts of the junctional complex may also be seen. Although varying in length (50-100 m#), the region of the closecontact-points (zonula occludens) appears whereas the zonula adhaerens often fails to appear and the randomly distributed macula adhaerens is seen more or less as a
36
M A T T E R ET AL.
27
1:;i6.26. The bile canaliculus appearing at the left of Fig. 25 is shown as indicated by the large arrow in section No. 32 (Fig. 24a), sectioned in the plane of the little arrows. The figure shows the intimate relationship between the bile canaliculus and the space of Disse. FIG. 27. A simplified reconstruction of the Fig. 26. The region of minimal distance is shown by a circle. dot-like structure (Figs. 25-27). Because of the wide opening of the bile canaliculi, their microvilli are neither n u m e r o u s n o r very long. The g r a p h i c a l r e c o n s t r u c t i o n in parallel perspective (Figs. 25-27) gives the threed i m e n s i o n a l a p p e a r a n c e of the two bile canaliculi with their j u n c t i o n a l complexes. A section p e r p e n d i c u l a r to the p l a n e of the p h o t o g r a p h s shows the view on a region of m i n i m a l distance between one of the bile canaliculi a n d the space of Disse. A possible function of these regions is c o n s i d e r e d in the general discussion.