Gastroenterologia Japonica Copyright (~ 1977 by The Japanese Society of Gastroenterology
Vol. 12, No. 6 Printed in Japan
--Original Article
S C A N N I N G ELECTRON M I C R O S C O P Y OF THE BILE D U C T U L E T a t s u y a I T O S H I M A , M.D., Ph.D., K e n Y O S H I N O , M.D., K a z u h i d e Y A M A M O T O , M.D., W a t a r u O H T A , M.D., Masahiro K U B O T A , M.D., M i n o r u U K I D A , M.D., Toshio I T O , M . D . , Hiroyasu H I R A K A W A , M.D., F u m i o M U N E T O M O , M.D. a n d Yoshihiro S H I M A D A , M.D., Ph.D.
Firsf. Department of Internal Medicine, Okayama University Medical School, Okayama 700, Japan
Sunxmary Scanning electron microscopy of a liver biopsy specimen from a patient 6 months after the onset of acute hepatitis revealed a normal appearing bile ductule. The bile ductule was 13 ~tm in outer diameter and 1.5-2.7 txm in inner diameter. The duetular lumen was surrounded by two ductular cells and ampullary dilated at the canalicular side. I n the lumen, intracytoplasmic diverticles were observed with an orifice d ameter of 0.9 ~tm. About 15 microvilli 0.4-0.6 ~xm in length and 0.1 ~tm in thickness were observed on 1 0tm2 of luminal surface. The number of microvilli was calculated to be approximately 1,500 per ductular cell. A single cilium 0.15 ~tm in diameter at the base and 0.10 ~m at the trunk, and 7-15 ~tm in length was found on each ductular cell. The cilium arose from a recess at the canalicular side of the ductular cell, and the free-end was on the bile-duct side. The cilia seemed to play an important role in bile flow to the duct. Sometimes between the ductular cells at the edge of the basal portion a cell was intercalated with thin processes. Such thin processes were not observed on ductular cells.
Key Words: bile ductule, scanning electron microscopy. N o r m a l 1~) a n d proliferated bile ductules 2,4-7) have b e e n studied by transmission
study the bile d u c t u l e of a liver biopsy speci-
electron microscopy ( T E M ) in e x p e r i m e n t a l
acute hepatitis.
animals a n d m a n .
m e n of a p a t i e n t in convalescent stage of
However, limitations are
i n h e r e n t in T E M observations on such subjects, as the bile ductules are small a n d few, a n d occupy only a small part of the specimen.
Materials and Methods A cylinder-shaped liver biopsy tissue (2 m m in diameter a n d 10 m m i n length) was ob-
I n contrast, s c a n n i n g electron microscopy (SEM) is suited for observations of small areas of larger specimens.
tained by a V i m - S i l v e r m a n needle u n d e r peritoneoscopy from a 29-year-old male in
Recently, Brooks et al. 8) observed proliferated bile ductules after bile duct ligation
onset of hepatitis was 6 m o n t h s earlier. U s i n g a syringe needle, the tissue was perfused with
u n d e r SEM. W e also reported on a bile d u c t u l e in cirrhotic n o d u l e 9).
R i n g e r solution a n d
I n the present paper, S E M was used to
convalescent stage of acute
hepatitis.
The
then re-perfused with
0.1 M phosphate buffer solution c o n t a i n i n g 2 % g l u t a r a l d e h y d e ( p H 7.4) 1~ T h e tissue
December 1977
Bile Ductule
was cut into blocks and fixed in the re-perfused solution overnight. The blocks were osmicated for electric conduction by the revised tannin-osmium me thod ~1), dehydrated through a graded series of ethanol and dried in liquid carbon dioxide by the critical point method. The dried specimens were cracked with a scalpelg, 12~. A bile ductule was observed in the portal triad on the cracked surface and on the matched surface by SEM (JSM-U3, S-310, HFS-2) with accelerating voltages of 4 to 15kV. For stereoscopic observation, pairs of scanning electron micrograpbs were taken at two angles differing by 7 degrees. For light microscopy, the biopsy tissue was embedded in paraffin and sectioned for staining by hematoxylin and eosin, azan, orcein, digest PAS and silver impregnation.
Results Peritoneoscopy revealed a normal size liver with reddish-brown tint, slightly rounded edges, somewhat increased consistency and smooth surface with several small depressions. T h e surface had regular markings formed by the terminal portal triads. By light microscopy, lymphocytes, plasma cells and macrophages were moderately infiltrated in the portal triad, but there was no fibrosis and no enlargement of the portal triad. T h e interlobular bile ducts and bile ductules showed no remarkable changes. T h e hepatocytes showed no remarkable chalages except for slight hydropic degeneration and fatty metamorphosis in the central zone. Some orcein positive hepatocytes were observed singulary and sporadically in the lobules. M a n y Kupffer cells were mobilized and phagocytized in the central zone. SEM clearly the Disse spaces, tissue spaces and all cleaned by
demonstrated the sinusoids, and the portal triad vessels, the bile ductule which were puncture perfusion 9~ (Figs.
477
l a , b). The air crackings tended to occur between the cells and disclosed the intercellular surfaces 9) (Figs. 1-5). A ductule 13 ~m in outer diameter was found at the border of a portal triad 120 ~m in diameter with a portal vein branch 50 ~m in diameter (Figs. l a , b). The bile dnctule had sinusoids at the lobule side, and had tissue spaces and a capillary 3) 7 ~m in diameter at the portal area side (Figs. 2a, b). The antiluminal border of the ductule was rather smooth and devoid of microvilli (Figs. 3a~ b). The intercellular surfaces of the ductular cells were disclosed and observations of matched surfaces revealed that the bile ductular lumen was surrounded by two ductular cells (Figs. 1-5). The inner diameter of the ductule ranged from 1.6 to 2.7 Exm. The lumen was ampullary dilated (2.7 txm in diameter) at the canalicular side (Figs. 2-4, 6), but the canalicular-ductular .junction was not evident. The ductular cell was measured to be about 18 ~zm in length. In the ductular lumen there were intracytoplasmic diverticles with a 0.9 ~m diameter orifice (Fig. 6). Microvilli and a single cilium were found on the luminal surface of the ductular cell (Figs. 3-7). Bleb formation was not encountered. An average of 15 (range 8-20) microvilli 0.4 to 0.6 ~m in length and 0.1 vtm in thickness were observed on 1 ~xm2 of luminal surface (Figs. 3-7). The distribution of microvilli was not uniform but dense at the marginal flap and in the diverticle of the ductular cell 5) (Figs. 4--7). T h e number of microvilli was calculated to be approximately 1,500 per ductular cell when the microvilli number per 1 ~m 2 of luminal surface, the luminal diameter and the length of the cell were 15, 1.8 ~m and 18 ~m. A single cilium was present on a ductular cell (Figs. 2-7). The cilium was 0.15 ~m in diameter at the base, 0.I0 &m at the trunk
478
Vol. 12, No. 6
7. 1 T O S H I M A E T AL.
and 7-15 ~m in length. The cilium arose from a recess at the canalicular side of the ductular cell and the free-end was on the bileduct side (Figs. 4-7). The distance from the luminal to antiluminal side of the intercellular surface of the ductular cell was 5 to 6 ~xm. T h e intercellular surface 1.0 to 1.5 btm from the luminal side was smooth, but it was often attached by the cracked cytoplasm of the opposite cell (Figs. 4, 5, 7). The outer area of the intercellular surface was undulated (Figs. 3-5, 7), but without interdigitation which is a feature of the large interlobular bile duct 13). Sometimes between the ductular cells at the edge of the basal portion, a cell was intercalated with thin processesl,6, 7) (Figs. 2, 3). The ductular cell surfaces corresponding to the intercalated cell were smoothly depressed and formed a canal 3 ~tm in diameter parallel to the long axis of the ductule (Figs. 2, 3).
Discussion The bile ductule is located on the border of the portal triad and lobule and connects the bile canaliculi with the interlobular bile duct. T h e bile ductule is composed of 2 to 4 cellsT, 14). The outer diameter of the bile ductule measures about 10 ~tm2). The luminal diameter is 1 to 3 ~tm 7). The bile ductule of the present study was consistent with these reported measures. By light microscopy, the bile ductules and interlobular bile ducts of the present case showed no remarkable changes. Moreover by SEM, only one bile ductule was found on the cracked surface of a portal triad, and the ductule had no bleb formation2,4"7),,~ These findings indicate that this bile ductule was not a proliferative but almost normal ductule. The presence of ampulla where two or more bile canaliculi drain 15) is still undecided. By T E M , some 16) observed the ampulla but
othersl, tT~ did not. In our study, the bile ductule was ampullary dilated at the canalicular side (Figs. 3, 4, 6) where the intracytoplasmic diverticulum 5, 77 (Fig. 6) and recess (Fig. 6) were located. The cilium originated from the recess. Drainage from the bile canaliculi was not clear. The number of bile ductule luminal microvilli ranges from 5 to 30 per sectioned cell surface 2~. In our study the number of microvilli was calculated to be about 1,500 per ductular cell. The number is almost the same as that of the interlobular bile duct cell (500 to 2,000) in normal guinea pig 18~. The cilium of the ductular cell has been observed by T E M s-7) and SEMa, 9). Steiner and Carruthers 4"6) considered the cilium as metaplasia. Sasaki et al. 7~ observed ductular cilia occasionally in both normal and abnormal liver. T h e present study confirmed that every ductular cell has a single cilium 4~ as in cirrhotic nodules and in interlobular bile duct epithelium 18~. Thus, a cilium is molst likely a normal and constant condition in the bile ductule epithelium. The cilium was relatively long (7-15 btm) compared with the ductular lumen (1.6-2.7/zm). The cilium had a base at the canalicular side and the free-end at the duct-side of the cell (Fig. 3-7). The cilium has been reported as having a motile structureS, 4). These findings suggest that the cilia play an important role in bile flow to the duct. The present study showed an intercalated cell with thin processes parallel to the long axis of the ductule. This cell did not reach the lumenS, 6~ (Figs. 2, 3). The morphology of the intercalated cell was different from the ductular cell and mesenchymal cell. Other studies on regions around the bile ductule are presently under way.
Acknowledgment The authors are grateful to Prof. Hideo
Bile Ductule
December 1977 Nagashima
for
helpful
criticism
and
Dr.
H i r o s h i Shigei, P r e s i d e n t o f Shigei H o s p i t a l , for a l l o w i n g us to use the S-310 s c a n n i n g e l e c t r o n microscope. Messrs. N o b u o H a y a shi a n d N o b o r u K i s h i m o t o , E l e c t r o n M i c r o scope D i v i s i o n of the
Research
Laboratory
o f o u r school, a n d M r . I c h i r o N a i t o , D i v i s i o n o f C l i n i c a l R e s e a r c h L a b o r a t o r y , Shigei H o s p i tal, K u r a s h i k i , p r o v i d e d t e c h n i c a l help. References
l) Steiner JW, Carruthers JS: Studies on the fine structure of the terminal branches of the biliary tree. I. The morphology of normal bile canaliculi, bile pre-ductule (duct of Hering) and bile ductules. Am J Path 38: 639-661, 1961 2) Schaffner F, Popper H: Electron microscopic studies of normal and proliferated bile ductules. Am J Path 38: 393-410, 1961 3) Sternlieb I: Functional implications of human portal and bile ductular ultrastructure. Gastroenterology 63: 321-327, 1972 4) Steiner JW, Carruthers JS, Kalifat SR: The ductular cell reaction of rat liver in extrahepatic eholestasis. I. Proliferated biliary epithelial cells. Exp Mol Path 1: 162-185, 1962 5) Steiner JW, Carruthers JS: Experimental extrahepatic biliary obstruction: Some aspects of the fine structural changes of bile ductules and preductules (duct of Hering). Am J Path 40:253 270, 1962 6) Steiner JW, Carruthers JS: Electron microscopy of hyperplastic ductular cells in ct-naphthyl isothiocyanate-induced cirrhosis, Lab Invest 16: 471498, 1963 7) Sasaki I~, Schaffner F, Popper H: Bile ductules in cholestasis: morphologic evidence for secretion
479
and absorption in man. Lab Invest 16: 84-95, 1967 8) Brooks SEH, Reynolds P, Audretsch JJ, Haggis G: Scanning electron microscopy of proliferating bile duetules. Lab Invest 33:311 315, 1975 9) ltoshima T, Yoshino K, Yamamoto K, Shimada Y: Scanning electron microscopy of cirrhotic nodules. Submitted for publication. 10) Murakami T: Puncture perfusion of small tissue pieces for scanning electron microscopy. Arch Histol Jpn 39: 99-105, 1976 11) Murakami T: A revised tannin-osmium method for non-coated scanning electron microscope specimens. Arch Histol Jpn 36:189 193, 1974 12) Itoshima T, Shimada Y, Hayashi N, Murakami T: A scanning electron microscopy of subcellular structures of the human hepatic cell. Arch Histol Jpn 39:15 21, 1976 13) Itoshima T, Shimada Y, 34urakami T: Scanning electron microscopy of the intrahepatic bile duct. Igaku No Ayumi 93: A469-473, 527-528, 1975
(in Jpn). 14) Sasaki I~: Ultrastructure of intrahepatic biliary epithelial cell. Nihon Rinsho 23:1439 1455, 1965 (in Jpn). 15) Clara M: Untersuchungen an der menschlichen Leber. I. Ueber den Uebergang der Gallenkapillaren in die Gallengaenge. Ztschr Mikr Anat Forsch 20: 58z1~607, 1930 16) Daems WT: The micro-anatomy of the smallest biliary pathways in mouse liver tissue. Acta Anat 46: 1-24, 1961 17) Cossel L: Electronenmikroskopische Befunde am Uebergang der intralobulaeren Gallenkanaelchen in die Gallengaenge. Virchow Arch Path Anat 335: 647-653, 1942 18) Itoshima T, Yoshino K, Yamamoto K, et ah A scanning electron microscopic observation of peribiliary blood vessels of guinea pig. Jpn J Gastroenterol 74: 34-39, 1977 (Jpn text with Engl abstr)
Received September 8, 1977. Accepted November 14, 1977. Address requestsfor reprints to : Dr. Tatsuya Boshima, M.D., First Department of Internal Medicine, Okayama University Medical School, Okayama, 700 Japan.
480
T. I T O S H I M A E T AL.
Vol. 12, No. 6
Explanations of Figures Scanning electron micrographs of a bile ductule. Fig. 1, 2 and 3 each show matched pairs of cracked specimens. The arrow indicates the presumed direction of bile flow. Fig. 1.
Fig. 9.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
A portal triad (120 ~m in diameter) and its surroundings. A bile ductule (in the rectangle) is is on the margin of the triad. P, Portal vein (50 ~m in diameter) ; A, arteriole; H, hepatic cell. x380. High power views of the bile ductule (BD) 13 t~m in outer diameter in the rectangle of Fig. l a and b, respectively. Intercellular surfaces of the ductular cells and the lumen of the bile ductule are horizontally exposed. An intercalated cell (IC) is located parallel to the long axis of the ductule between the basal parts of the ductular cells. C, Capillary; K, Kupffer cell; S, sinusoid; L, lymphocyte;*, space for intercalated cell. x 1,500. Higher power views of the bile duetule. The ductule (1.6-2.7 t~m in inner diameter) is composed of two cells and dilated at the left area of Fig. 3a and the right area of Fig. 3b. The ductular cell has numerous microvilli and a cilium. IC, Intercalated cell; *, space for intercalated cell; IS, intercellular surface of the ductular cell; IA, intercellular space, x 3,000. Stereoscopic pair of a high power view of Fig. 3a. The left area of the ductule is ampullary dilated. A cilium (curved arrow) 15 t~m in length starts from a recess at the left area. The intercellular surface (IS) is wavy. The protruded part (*) corresponds to the intercellular space of Fig. 5. x5,300. Stereoscopic pair of a high power view of Fig. 3b. The right area of the ductule is ampullary dilated. A cilium (curved arrow) 7 ~m in length has its origin on the right side and free-end on the left. IA, Intercellular space; N, probable nerve fiber, x5,300. A detailed view of the ampullary dilated area of Fig. 4a. The cilium (curved arrow) starts from a recess. There is a diverticulum (D) in the cell. Mierovilli appear like fingers (0.1 ~m in thickness, 0.4-0.6 ~m in length). • 19,000. A detailed view of the bile ductule in Fig. $b. The cilium (curved arrow) is 0.10 ~m in thickness at the trunk and 0.15 txm at the base. Microvilli are denser at the marginal flap. The flat zone (Z) of the intercellular surface probably corresponds to the zonula occludens. X 11,000.
December 1977
Bile Ductule
481
482
T. I T O S H I M A E T AL.
Vol. 12, No. 6
Vol. 12, No. 6 Printed in Japan
--Original Article
S C A N N I N G ELECTRON M I C R O S C O P Y OF THE BILE D U C T U L E T a t s u y a I T O S H I M A , M.D., Ph.D., K e n Y O S H I N O , M.D., K a z u h i d e Y A M A M O T O , M.D., W a t a r u O H T A , M.D., Masahiro K U B O T A , M.D., M i n o r u U K I D A , M.D., Toshio I T O , M . D . , Hiroyasu H I R A K A W A , M.D., F u m i o M U N E T O M O , M.D. a n d Yoshihiro S H I M A D A , M.D., Ph.D.
Firsf. Department of Internal Medicine, Okayama University Medical School, Okayama 700, Japan
Sunxmary Scanning electron microscopy of a liver biopsy specimen from a patient 6 months after the onset of acute hepatitis revealed a normal appearing bile ductule. The bile ductule was 13 ~tm in outer diameter and 1.5-2.7 txm in inner diameter. The duetular lumen was surrounded by two ductular cells and ampullary dilated at the canalicular side. I n the lumen, intracytoplasmic diverticles were observed with an orifice d ameter of 0.9 ~tm. About 15 microvilli 0.4-0.6 ~xm in length and 0.1 ~tm in thickness were observed on 1 0tm2 of luminal surface. The number of microvilli was calculated to be approximately 1,500 per ductular cell. A single cilium 0.15 ~tm in diameter at the base and 0.10 ~m at the trunk, and 7-15 ~tm in length was found on each ductular cell. The cilium arose from a recess at the canalicular side of the ductular cell, and the free-end was on the bile-duct side. The cilia seemed to play an important role in bile flow to the duct. Sometimes between the ductular cells at the edge of the basal portion a cell was intercalated with thin processes. Such thin processes were not observed on ductular cells.
Key Words: bile ductule, scanning electron microscopy. N o r m a l 1~) a n d proliferated bile ductules 2,4-7) have b e e n studied by transmission
study the bile d u c t u l e of a liver biopsy speci-
electron microscopy ( T E M ) in e x p e r i m e n t a l
acute hepatitis.
animals a n d m a n .
m e n of a p a t i e n t in convalescent stage of
However, limitations are
i n h e r e n t in T E M observations on such subjects, as the bile ductules are small a n d few, a n d occupy only a small part of the specimen.
Materials and Methods A cylinder-shaped liver biopsy tissue (2 m m in diameter a n d 10 m m i n length) was ob-
I n contrast, s c a n n i n g electron microscopy (SEM) is suited for observations of small areas of larger specimens.
tained by a V i m - S i l v e r m a n needle u n d e r peritoneoscopy from a 29-year-old male in
Recently, Brooks et al. 8) observed proliferated bile ductules after bile duct ligation
onset of hepatitis was 6 m o n t h s earlier. U s i n g a syringe needle, the tissue was perfused with
u n d e r SEM. W e also reported on a bile d u c t u l e in cirrhotic n o d u l e 9).
R i n g e r solution a n d
I n the present paper, S E M was used to
convalescent stage of acute
hepatitis.
The
then re-perfused with
0.1 M phosphate buffer solution c o n t a i n i n g 2 % g l u t a r a l d e h y d e ( p H 7.4) 1~ T h e tissue
December 1977
Bile Ductule
was cut into blocks and fixed in the re-perfused solution overnight. The blocks were osmicated for electric conduction by the revised tannin-osmium me thod ~1), dehydrated through a graded series of ethanol and dried in liquid carbon dioxide by the critical point method. The dried specimens were cracked with a scalpelg, 12~. A bile ductule was observed in the portal triad on the cracked surface and on the matched surface by SEM (JSM-U3, S-310, HFS-2) with accelerating voltages of 4 to 15kV. For stereoscopic observation, pairs of scanning electron micrograpbs were taken at two angles differing by 7 degrees. For light microscopy, the biopsy tissue was embedded in paraffin and sectioned for staining by hematoxylin and eosin, azan, orcein, digest PAS and silver impregnation.
Results Peritoneoscopy revealed a normal size liver with reddish-brown tint, slightly rounded edges, somewhat increased consistency and smooth surface with several small depressions. T h e surface had regular markings formed by the terminal portal triads. By light microscopy, lymphocytes, plasma cells and macrophages were moderately infiltrated in the portal triad, but there was no fibrosis and no enlargement of the portal triad. T h e interlobular bile ducts and bile ductules showed no remarkable changes. T h e hepatocytes showed no remarkable chalages except for slight hydropic degeneration and fatty metamorphosis in the central zone. Some orcein positive hepatocytes were observed singulary and sporadically in the lobules. M a n y Kupffer cells were mobilized and phagocytized in the central zone. SEM clearly the Disse spaces, tissue spaces and all cleaned by
demonstrated the sinusoids, and the portal triad vessels, the bile ductule which were puncture perfusion 9~ (Figs.
477
l a , b). The air crackings tended to occur between the cells and disclosed the intercellular surfaces 9) (Figs. 1-5). A ductule 13 ~m in outer diameter was found at the border of a portal triad 120 ~m in diameter with a portal vein branch 50 ~m in diameter (Figs. l a , b). The bile dnctule had sinusoids at the lobule side, and had tissue spaces and a capillary 3) 7 ~m in diameter at the portal area side (Figs. 2a, b). The antiluminal border of the ductule was rather smooth and devoid of microvilli (Figs. 3a~ b). The intercellular surfaces of the ductular cells were disclosed and observations of matched surfaces revealed that the bile ductular lumen was surrounded by two ductular cells (Figs. 1-5). The inner diameter of the ductule ranged from 1.6 to 2.7 Exm. The lumen was ampullary dilated (2.7 txm in diameter) at the canalicular side (Figs. 2-4, 6), but the canalicular-ductular .junction was not evident. The ductular cell was measured to be about 18 ~zm in length. In the ductular lumen there were intracytoplasmic diverticles with a 0.9 ~m diameter orifice (Fig. 6). Microvilli and a single cilium were found on the luminal surface of the ductular cell (Figs. 3-7). Bleb formation was not encountered. An average of 15 (range 8-20) microvilli 0.4 to 0.6 ~m in length and 0.1 vtm in thickness were observed on 1 ~xm2 of luminal surface (Figs. 3-7). The distribution of microvilli was not uniform but dense at the marginal flap and in the diverticle of the ductular cell 5) (Figs. 4--7). T h e number of microvilli was calculated to be approximately 1,500 per ductular cell when the microvilli number per 1 ~m 2 of luminal surface, the luminal diameter and the length of the cell were 15, 1.8 ~m and 18 ~m. A single cilium was present on a ductular cell (Figs. 2-7). The cilium was 0.15 ~m in diameter at the base, 0.I0 &m at the trunk
478
Vol. 12, No. 6
7. 1 T O S H I M A E T AL.
and 7-15 ~m in length. The cilium arose from a recess at the canalicular side of the ductular cell and the free-end was on the bileduct side (Figs. 4-7). The distance from the luminal to antiluminal side of the intercellular surface of the ductular cell was 5 to 6 ~xm. T h e intercellular surface 1.0 to 1.5 btm from the luminal side was smooth, but it was often attached by the cracked cytoplasm of the opposite cell (Figs. 4, 5, 7). The outer area of the intercellular surface was undulated (Figs. 3-5, 7), but without interdigitation which is a feature of the large interlobular bile duct 13). Sometimes between the ductular cells at the edge of the basal portion, a cell was intercalated with thin processesl,6, 7) (Figs. 2, 3). The ductular cell surfaces corresponding to the intercalated cell were smoothly depressed and formed a canal 3 ~tm in diameter parallel to the long axis of the ductule (Figs. 2, 3).
Discussion The bile ductule is located on the border of the portal triad and lobule and connects the bile canaliculi with the interlobular bile duct. T h e bile ductule is composed of 2 to 4 cellsT, 14). The outer diameter of the bile ductule measures about 10 ~tm2). The luminal diameter is 1 to 3 ~tm 7). The bile ductule of the present study was consistent with these reported measures. By light microscopy, the bile ductules and interlobular bile ducts of the present case showed no remarkable changes. Moreover by SEM, only one bile ductule was found on the cracked surface of a portal triad, and the ductule had no bleb formation2,4"7),,~ These findings indicate that this bile ductule was not a proliferative but almost normal ductule. The presence of ampulla where two or more bile canaliculi drain 15) is still undecided. By T E M , some 16) observed the ampulla but
othersl, tT~ did not. In our study, the bile ductule was ampullary dilated at the canalicular side (Figs. 3, 4, 6) where the intracytoplasmic diverticulum 5, 77 (Fig. 6) and recess (Fig. 6) were located. The cilium originated from the recess. Drainage from the bile canaliculi was not clear. The number of bile ductule luminal microvilli ranges from 5 to 30 per sectioned cell surface 2~. In our study the number of microvilli was calculated to be about 1,500 per ductular cell. The number is almost the same as that of the interlobular bile duct cell (500 to 2,000) in normal guinea pig 18~. The cilium of the ductular cell has been observed by T E M s-7) and SEMa, 9). Steiner and Carruthers 4"6) considered the cilium as metaplasia. Sasaki et al. 7~ observed ductular cilia occasionally in both normal and abnormal liver. T h e present study confirmed that every ductular cell has a single cilium 4~ as in cirrhotic nodules and in interlobular bile duct epithelium 18~. Thus, a cilium is molst likely a normal and constant condition in the bile ductule epithelium. The cilium was relatively long (7-15 btm) compared with the ductular lumen (1.6-2.7/zm). The cilium had a base at the canalicular side and the free-end at the duct-side of the cell (Fig. 3-7). The cilium has been reported as having a motile structureS, 4). These findings suggest that the cilia play an important role in bile flow to the duct. The present study showed an intercalated cell with thin processes parallel to the long axis of the ductule. This cell did not reach the lumenS, 6~ (Figs. 2, 3). The morphology of the intercalated cell was different from the ductular cell and mesenchymal cell. Other studies on regions around the bile ductule are presently under way.
Acknowledgment The authors are grateful to Prof. Hideo
Bile Ductule
December 1977 Nagashima
for
helpful
criticism
and
Dr.
H i r o s h i Shigei, P r e s i d e n t o f Shigei H o s p i t a l , for a l l o w i n g us to use the S-310 s c a n n i n g e l e c t r o n microscope. Messrs. N o b u o H a y a shi a n d N o b o r u K i s h i m o t o , E l e c t r o n M i c r o scope D i v i s i o n of the
Research
Laboratory
o f o u r school, a n d M r . I c h i r o N a i t o , D i v i s i o n o f C l i n i c a l R e s e a r c h L a b o r a t o r y , Shigei H o s p i tal, K u r a s h i k i , p r o v i d e d t e c h n i c a l help. References
l) Steiner JW, Carruthers JS: Studies on the fine structure of the terminal branches of the biliary tree. I. The morphology of normal bile canaliculi, bile pre-ductule (duct of Hering) and bile ductules. Am J Path 38: 639-661, 1961 2) Schaffner F, Popper H: Electron microscopic studies of normal and proliferated bile ductules. Am J Path 38: 393-410, 1961 3) Sternlieb I: Functional implications of human portal and bile ductular ultrastructure. Gastroenterology 63: 321-327, 1972 4) Steiner JW, Carruthers JS, Kalifat SR: The ductular cell reaction of rat liver in extrahepatic eholestasis. I. Proliferated biliary epithelial cells. Exp Mol Path 1: 162-185, 1962 5) Steiner JW, Carruthers JS: Experimental extrahepatic biliary obstruction: Some aspects of the fine structural changes of bile ductules and preductules (duct of Hering). Am J Path 40:253 270, 1962 6) Steiner JW, Carruthers JS: Electron microscopy of hyperplastic ductular cells in ct-naphthyl isothiocyanate-induced cirrhosis, Lab Invest 16: 471498, 1963 7) Sasaki I~, Schaffner F, Popper H: Bile ductules in cholestasis: morphologic evidence for secretion
479
and absorption in man. Lab Invest 16: 84-95, 1967 8) Brooks SEH, Reynolds P, Audretsch JJ, Haggis G: Scanning electron microscopy of proliferating bile duetules. Lab Invest 33:311 315, 1975 9) ltoshima T, Yoshino K, Yamamoto K, Shimada Y: Scanning electron microscopy of cirrhotic nodules. Submitted for publication. 10) Murakami T: Puncture perfusion of small tissue pieces for scanning electron microscopy. Arch Histol Jpn 39: 99-105, 1976 11) Murakami T: A revised tannin-osmium method for non-coated scanning electron microscope specimens. Arch Histol Jpn 36:189 193, 1974 12) Itoshima T, Shimada Y, Hayashi N, Murakami T: A scanning electron microscopy of subcellular structures of the human hepatic cell. Arch Histol Jpn 39:15 21, 1976 13) Itoshima T, Shimada Y, 34urakami T: Scanning electron microscopy of the intrahepatic bile duct. Igaku No Ayumi 93: A469-473, 527-528, 1975
(in Jpn). 14) Sasaki I~: Ultrastructure of intrahepatic biliary epithelial cell. Nihon Rinsho 23:1439 1455, 1965 (in Jpn). 15) Clara M: Untersuchungen an der menschlichen Leber. I. Ueber den Uebergang der Gallenkapillaren in die Gallengaenge. Ztschr Mikr Anat Forsch 20: 58z1~607, 1930 16) Daems WT: The micro-anatomy of the smallest biliary pathways in mouse liver tissue. Acta Anat 46: 1-24, 1961 17) Cossel L: Electronenmikroskopische Befunde am Uebergang der intralobulaeren Gallenkanaelchen in die Gallengaenge. Virchow Arch Path Anat 335: 647-653, 1942 18) Itoshima T, Yoshino K, Yamamoto K, et ah A scanning electron microscopic observation of peribiliary blood vessels of guinea pig. Jpn J Gastroenterol 74: 34-39, 1977 (Jpn text with Engl abstr)
Received September 8, 1977. Accepted November 14, 1977. Address requestsfor reprints to : Dr. Tatsuya Boshima, M.D., First Department of Internal Medicine, Okayama University Medical School, Okayama, 700 Japan.
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Explanations of Figures Scanning electron micrographs of a bile ductule. Fig. 1, 2 and 3 each show matched pairs of cracked specimens. The arrow indicates the presumed direction of bile flow. Fig. 1.
Fig. 9.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
A portal triad (120 ~m in diameter) and its surroundings. A bile ductule (in the rectangle) is is on the margin of the triad. P, Portal vein (50 ~m in diameter) ; A, arteriole; H, hepatic cell. x380. High power views of the bile ductule (BD) 13 t~m in outer diameter in the rectangle of Fig. l a and b, respectively. Intercellular surfaces of the ductular cells and the lumen of the bile ductule are horizontally exposed. An intercalated cell (IC) is located parallel to the long axis of the ductule between the basal parts of the ductular cells. C, Capillary; K, Kupffer cell; S, sinusoid; L, lymphocyte;*, space for intercalated cell. x 1,500. Higher power views of the bile duetule. The ductule (1.6-2.7 t~m in inner diameter) is composed of two cells and dilated at the left area of Fig. 3a and the right area of Fig. 3b. The ductular cell has numerous microvilli and a cilium. IC, Intercalated cell; *, space for intercalated cell; IS, intercellular surface of the ductular cell; IA, intercellular space, x 3,000. Stereoscopic pair of a high power view of Fig. 3a. The left area of the ductule is ampullary dilated. A cilium (curved arrow) 15 t~m in length starts from a recess at the left area. The intercellular surface (IS) is wavy. The protruded part (*) corresponds to the intercellular space of Fig. 5. x5,300. Stereoscopic pair of a high power view of Fig. 3b. The right area of the ductule is ampullary dilated. A cilium (curved arrow) 7 ~m in length has its origin on the right side and free-end on the left. IA, Intercellular space; N, probable nerve fiber, x5,300. A detailed view of the ampullary dilated area of Fig. 4a. The cilium (curved arrow) starts from a recess. There is a diverticulum (D) in the cell. Mierovilli appear like fingers (0.1 ~m in thickness, 0.4-0.6 ~m in length). • 19,000. A detailed view of the bile ductule in Fig. $b. The cilium (curved arrow) is 0.10 ~m in thickness at the trunk and 0.15 txm at the base. Microvilli are denser at the marginal flap. The flat zone (Z) of the intercellular surface probably corresponds to the zonula occludens. X 11,000.
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