EXPERIMENTAL
AND
MOLECULAR
Significance
of Liver
PATHOLOGY
12-34
( 19%)
Canalicular Changes Bile Duct Ligation I92
DE Vos R., DE WOLF-PEETERS, Departement Academisch Belgium
23,
C., DESMET
after
V., BIANCHI,
L., AND ROHR, H. P.
Medische Navorsing, Laboratorium voor Histochemie Ziekenhuis St. Rafad, Katholieke Universiteit Leuven, and Institute of Pathology, Department of Electron University of Bad, Basel, Switzerland Received
September
Experimental
en Cytochemie, B-300 Leuven, Microscopy,
30, 1974
An ultrastructural morphologic, histochemical, and morphometric study was made on the canalicular structures in rat liver after experimental bile duct ligation (BDL). Four different canalicular structures are distinguished, which are proved by morphometry to correspond to the different types observed during perinatal maturation. Canaliculus type 4, corresponding to the excretory pole of a normal adult hepatocyte, is characterized by a lumen filled with regular, slender microvilli. Canaliculus type 3, corresponding to the canalicular structure described in cholestasis, has a wide lumen devoid of microvilli and is found 2-3 days after BDL. A transitional canalicular structure between type 3 and 4, partly provided with microvilli, is found from the first day after BDL. After 3 days of BDL an increasing number of other canalicular types is observed. Canaliculi type 2, showing an irregular lumen with irregular microvillus-like projections, and canaliculi type 1, representing intracytoplasmic invaginations of two adjacent cell membranes, are found together with canaliculi type 3 and type 3-4. the enzyme activity is detected in After incubation for alkaline phosphatase, canaliculi type 1, 3-4, and type 4. The striking analogy of the sequential changes in canalicular structures after BDL and during perinatal maturation leads to the following interpretation: ( 1) Canaliculi type 1 and 2, more frequently observed in longstanding cholestasis correspond to newly formed excretory poles of the hepatocytes. (2) Canaliculus type 3, or the so-called cholestatic canalicu!ar type, reflects an inactive secretory function. Morphometrically this canalicular type shows a very low S/V relation which may be partly responsible for this secretory inactivity. (3) Canaliculus type 34 reflects a disturbed secretory function after BDL and a beginning secretory activity during perinatal maturation.
INTRODUCTION microscopic and histochemical studies have been devoted and canalicular changes in human rat liver in different forms of intrahepatic and extrahepatic cholestasis (Desmet, 1972). From these it appears that dilatation of the canaliculi and disappearance of their microvilli may Numerous
to
electron
hepatocellular
1 This work was supported by a grant from the “Fonds voor Onderzoek” of Belgium. * Part of this work has been presented at the 7th Meeting the study of the liver. Arnhem, The Netherlands, September 12 Copyright Ail rights
0 1975 by of reproduction
Academic in any
Press, Inc. form reserved
Wetenschappelijk of the 1972.
European
Geneeskundig Association
for
CANALICULAR
CHANGES
IN CHOLESTASIS
I.3
be regarded as the ultrastructural hallmark of cholestasis. However, the significance of other aberrant morphological appearances of canaliculi remains unclear. Recently, morphologic and histochemical evidence was produced, both at the microscopical (De Wolf-Petters et al., 1971) and ultrastructural (De WolfPeeters et al., 1972) level and completed by morphometric analysis (‘De WolfPeeters et al., 1974), that cholestatic types of canaliculi occur in the liver of normal newborn rats shortly after birth. Tllis corrc1utt.s with ;ul tlscretory disturbance as has been proved by biochemical studies (Bakken, 1970; Dawkins, 1966; Gamter et al., 1969; Whelan et al., 1970). Such cholestatic features were found to represent only a transient phase in a complex chronologic sequence of the canalicular changes, suggestive of successive stages in canalicular development and maturation. These findings indicated that a careful comparative study of neonat,al and cholestatic livers might shed new light on the significance of canalicular changes in cholestasis. The present study was undertaken in order to make a detailed analysis of canalicular morphologic and histochemical changes as they occur in the liver of the rat after ligation of the common bile duct, and to compare them to the various morphologic and histochemical characteristics of canaliculi as found in the perinatal period. Since zonal differences are know to exist in the normal liver lobule (Ma et al., 1968; Loud, 1968), and in different types of the liver pathology, including cholestasis ( Wachstein, 1963), this study was restricted to the changes in canalicular morphology and histochemistry in the periportal region. Quantitative data were obtained by morphometry in order to complete the qualitative observations. MORPHOLOGY
Materials
AND HISTOCHEMISTRY
and Methods
Male rats of albino Wistar R strain, weighing 150-200 g were obtained commercially. The animals were fed a laboratory stock diet and tap water. Twenty-one Wistar rats were subjected to bile duct ligation (BDL). During constant ether flow anesthesia the ductus choledochus was exposed by blunt dissection, doubly ligated and sectioned between two ligatures. All animals were killed by exsanguination; the time interval between the surgical intervention and the sacrifice of the animals varied from 1 to 2, 3, 4, 9, 20, and 40 days. It must be mentioned that in this group no animals were included which had a choleperitoneum (rupture of the proximal choledochal stump) or which showed fibrous connections between the blind-ending choledochus and the gut (masking an eventual fistula). The animals were divided into eight groups: normal adult rats, rats 1 day after BDL, rats 2 days after BDL, rats 3 days after BDL, rats 4 days after BDL, rats 9 days after BDL, rats 20 days after BDL, and rats 40 days after BDL. Specimens were taken from the left anterior liver lobe. Each specimen was divided in two parts. One part was immediately quenched in liquid nitrogen-cooled isopentane, g-pm-thick cryostat sections were cut, fixed for 15 min in cold formol-calcium chIorid+dextran (Planteydt, 1961)) and stained for alkaline phosphatase, using
1.4
DE VOS ET AL.
FIG . 1. Normal rat liver. Fresh-frozen section prepared for cytochemical study. Peripol rtally locali: zed canaliculi are weakly positive for alkaline phosphatase. X160, inset x400.
the (:alcium cobalt method of Gomori (Gomori, 1939; Takamatsu, 1939). The incul 3ation time was 20 min at 37°C. Frl om the second part, thin Aices (l/1/10 mm) were fixed by immersion for 2 hr in cold (4°C) 6.00 g/100 ml glutaraldehyde and 3.04 g/100 ml forma Jdehyde cacodylate buffer (Tijro et al., 1966), pH 7.2, followed by buffer I inse over1light.
CANALICULAR
CHANGES
IN CHOLESTASIS
15
For morphological study, portions of each specimen were postfixed in 1% OsOa in phosphate buffer, pH 7.2 (Millonig, 1961)) for 1 hr at 4”C, dehydrated in graded alcohols, ‘and embedded in Epon (Luft, 1961). For cytochemical study, 40-pm-thick sections, prepared with the TC2 Smith and Farquhar tissue sectioner, were incubated for alkaline phosphatase activity buffer, with beta-glycerophosphate (2 mg/ml) as substrate in 0.05 M Tris-HCL
FIG. 2. Rat liver 2 days after extrahepatic bile duct obstruction. Alkaline phosphatase positive canaliculi with irregular pattern and intense staining reaction. X160, inset x400.
16
FIG. 3. Normal rat liver. Thin acetate and lead citrate. Normal villi. Type 4. X24,000.
DE
VOS
ET AL.
section prepared for morphologic study, stained with canaliculus; the lumen is filled with numerous regular
uranyl micro-
pH 8.2, for 30 min at 37°C. Lead citrate was used as capture reagent (Saito et al., 1968). Control sections were incubated in substrate-free incubation medium and in incubation medium with a specific inhibitor: EDTA 0.05 M. After incubation the sections were rinsed in cacodylate buffer, postfixed for 1 hr in 1% Os04 in phosphate buffer, and embedded in Epon. From each specimen, semithin sections were cut, stained for 1 min, with toluidine blue (Lynn, 1965 ) and examined under the optical microscope. These
CANALICULAR
CHANGES
IN
CHOLESTASIS
17
FIG. 4. Rat liver 1 day after extrahepatic bile duct obstruction. Thin section prepared for morphologic study, stained with uranyl acetate and lead citrate. Canaliculus with dilated lumen and a few short microvilli. Transitional form between type 4 and type 3. X24,000.
sections served to identify specific zones of the hepatic Iobule; only the specimens containing a portal field were selected for further cutting of thin sections. Periportal areas were trimmed so that they included a recognizable portion of a portal tract and at most seven hepatocytic cell diameters surrounding it (Loud, 1968). Thin sections were cut with an OM U 3 Reichert ultramicrotome, stained with uranyl acetate (Watson, 1958) followed by lead citrate (Reynolds, 1963) and studied with a Zeiss EM 9 electron microscope.
1s
DE
VOS
ET
AL.
FIG. 5. Rat liver 2 days after extrahepatic bile duct obstruction. Thin section morphologic study, stained with uranyl acetate and lead citrate. Dilated canaliculi lumen without microvilli. Type 3 canaliculi. X18,000.
prepared for with empty
Results Optical microscopy. In normal rat liver the lead precipitate after incubation for alkaline phosphatase is localized on the canaliculi of the periportal zone; in the centrilobular zone the reaction is virtually negative (Fig. 1). One day after bile duct obstruction an intensely positive reaction is observed in all canalicular structures (Fig. 2). This enzyme activity remains high during the
CANALICULAR
CHANGES
IN
19
CHOLESTASIS
ensuing days of obstruction while the morphology of the abnormal pattern. Electron microscopic morphology. In normal rat liver, the canaliculi, located between two adjacent hepatocytes and tional complexes, is filled with numerous long and regular zone of pericanalicular ectoplasm can be observed (Fig.
canaliculi
shows an
lumen of periportal sealed off by juncmicrovilli; a narrow 3). One day after
FIG. 6. Rat liver 4 days after extrahepatic bile duct obstruction. Thin section prepared for morphologic study, stained with uranyl acetate and lead citrate. Canaliculus with irregular lumen; presence of secondary extensions separated from the main cavity by cytoplasmic bridges. Type 2. X18,oQQ,
20
DE
VOS
ET AL.
FIG. 7. Rat liver 10 days after extrahepatic bile duct obstruction. Thin section prepared for morphologic study, stained with uranyl acetate and lead citrate. Canaliculus-like structure without real lumen formed by an intracytoplasmic invagination of the outer cell membranes located between junctional complexes. Type 1 canaliculus. X38,000.
obstruction numerous canaliculi show a slightly dilated lumen, the microvilli are irregular (Fig. 4); the pericanalicular ectoplasm is often widened. Two to three days after bile clurt obstruction a higher number of canaliculi shows a dilated lumen while most of their microvilli have disappeared (Fig. 5); their lumen may be free, or occluded by an edematous bleb, or filled with amorphous electron-
CANALICULAR
FIG. 8, Normal acetate and lead on the microvillar
CHANGES
IN
CHOLESTASIS
rat liver. Thin section prepared for cytochemical citrate. Canaliculus type 4 with positive reaction membranes. X 18,000.
study, stained for alkaline
with u~i any1 phospha tase
dense material. Tlhc morphologic appearance of the latter material is ql rite similar to the pericanalicular ect.oplasm. In this period, but even more frequently 3 to 4 days and later on after obstr ‘UCtion, segments of hepatocellular plasma membranes, located between junctic ma1 are complexes, show invaginations and interdigitations. These invaginations usually found more extensively into the cytoplasm of only one of two adjac :ent hepatocytes (Fig. 7). Some of these membrane invaginations, appearing as
22
DE
VOS
ET
FIG. 9. Rat liver 4 days after extrahepatic bile duct cytochemical study, stained with uranyl acetate and showing alkaline phosphatase-positive type 4 canaliculi and practically negative type 3 canaliculi ( b ) with remnants of microvilli. X7600.
AL.
obstruction. Thin section prepared for lead citrate. A low-power photograph ( + ) , positive type 1 canaliculi ( D ), a thick
ectoplasmic
zone
and
a few
canaliculus-like structures because of the presence of two limiting tight junctions, may isolate a cytoplasmic sequester from the rest of the cytoplasm of the hepatocyte in which they are extending.
CANALICULAR
CHANGES
FIG. 10. Rat liver 10 days after extrahepatic for cytochemical study, stained with many1 positive type 1 canaliculus. X38,000.
bile acetate
IN
23
CHOLESTASIS
duct and
obstruction. lead citrate.
Thin section prepared Alkaline phosphatase-
Also during this period canaliculus~~like structures are found with a very irregular lumen, provided with a few cytoplasmic finger-like extensions; in their vicinity smaller cavities can be found in the hepatocytic cytoplasm which are separated from the main lumen by cytoplasmic bridges (Fig. 6). From 9 to 20 days after obstruction one still fiuds dilated canaliculi, but there is a higher number of the above-mentioned canaliculus-like membrane invaginations without real lumen and of canaliculi with irregular lumen.
24
-
DE
VOS
FIG. 11. Rat liver 4 days after extrahepatic for cytochemical study, stained with uranyl canaliculus; the lumen is filled with amorphous X38,000.
ET AL.
bile duct obstruction. Thin acetate and lead citrate. material showing a positive
section prepared Dilated type 3 lead precipitate.
The pericanalicular ectoplasm surrounding most of these canaliculus-like strutures ‘and irregular canaliculi is sometimes strongly widened and appears swollen and edematous. After 40 days of obstruction 011est.ill finds an increasing number of canaliculuslike structures. In this period, however, such membrane invaginations are usually located between three or four neighboring hepatocytes. It should also be
CANALICULAR
FIG. 12. Rat liver 1 day cytochemical study, stained superimposed double-lattice
CHANGES
IN
25
CHOLESTASIS
after extrahepatic bile duct obstruction. with uranyl acetate and lead citrate. test screen. X 12,000.
Thin section prepared Canaliculus type 43
for with
stressed that during the whole obstructive period one can always find a few canaliculi with normal morphologic appearance. Alkaline Phosphatase Hislochemisty. In normal liver tissue a positive reaction is found on the microvillous membrane of the canaliculi after incubation for alkaline phosphatase (Fig. 8). After obstruction a strongly positive reaction is observed in all canaliculi and canaliculus-like structures, with the exception of broadly widened canaliculi devoid of microvilli (Figs. 9 and 10). [When, however, their lumen contains electron-dense amorphous material a positive lead precipitate is found on the latter (Fig. ll)]. After incubation in substrate-free media and in normal media containing a specific inhibitor no lead precipitate could be detected in any of the sections at the canalicular cell membrane. MORPHOMETRY Methods For morphometric study five blocks, contaiuing a portal field-identified as described above-from each rat liver tissue sample, were used for preparation of ultrathin sections. Pro group 40-50 micrographs were taken ,at random at a primary magnification of 9500 and enlarged together with a double-lattice test screen (Weibel, 1969) (Fig. 12). I n order to characterize the different canalicular types by morphometry, 3040 micrographs were taken of each prototype, and processed as described for the groups.
26
DE
VOS
ET
AL.
To determine the volume density of the bile canalicular lumen the fine test points lying inside the canalicular lumen (IO89 points per test area) were counted. To evaluate the surface density of the canaliculus, the number of intersections of the test lines with the canalicular membrane was counted (Fig. 12). C,alculations and statistical analyses (mean value, standard deviation, standard error, Student’s t test) were performed with the so-called multipurpose morphometric computer program on an Olivetti P. 602 microcomputer system (Rohr et al., 1973). Results Morphometric
results
can be seen in Tables I to VI. DISCUSSION
The criteria applied in this and previous studies (De Wolf-Peeters et al., 1972, 1974) to consider a structure as a canaliculus or its equivalent is its limitation by two or three junctional complexes which separate it from the rest of the intercellular space. All morphologic variants of canalicular structures observed in this experiment may be grouped into four prototypes, as described previously in perinatal
TABLE PRIMARY
DATA
I
OF MORPHOMETRIC EVALUATION .4~ THE HEPATOCYTE~
OF BILIARY
POLE
PPL
SE Types Type Type Type Type Type Group
SE
of canaliculi 14.46 22.64 30.54 .50.81 39.85
1
2 3 3-4 4 according
Adult normal 1 day BDL 2 days BDL 3 days BDL 4 days BDL 9 days BDL 20 days BDL 40 days BDL
to duration rats
1.74 2.14 2.42 4.83 *5.88
0.007 0.022 0.122 0.072 0.018
0.001 0.002 0.013 0.011 0.003
5.77 3.50 2.68 2.43 1.79 1.64 2.72 2.14
0.039 0.051 0.04*5 0.054 0.058 0.046 0.020 0.023
0.005 0.006 0.008 0.008 0.007 0.006 0.007 0.004
obstruction Fi4.18 35.42 32.12 29.49 30.54 22.76 20.44 21.34
a hhbreviat,ions. L = Lumen of the bile canalicuhw. C = Contour of the bile canaliculus. Pp = Poinl. den&y (cm”). Vv = Volllme densit,y ((km”). IT, = Intersections (cm-l). Sv = Surface density (m”/cm”). VVL = Volume of the bile canaliculus per unit volume of surrounding liver tissue. SVL = Surface of bile canalicuhls per unit volume of surrounding liver t,iessue. I = Intersections. M = Mean value. SE = Standard error. S = Significant. NS = Nonsignificant.
CANALICULAR
CHANGES
IN CHOLESTASIS
TABLE SECONDARY
PARAMETERS
II
OF MORPHOMETRIC EVALUATION POLE OF THE HEPATOCYTE~ SVC W/cm”)
Type
27
VVCL
OF THE BILIARY
S~C/VVCL
(cm”)
(m2/cm”)
of canaliculi
Type Type Type Type Type Group
1
0.277 0.434 0.586 0.975 0.764
2 3 34 4 according
Adult normal l-day BDL a-days BDL a-days BDL 4-days BDL O-days BDL 20-days BDL 40-days BDL a See footnote
Table
to duration
0.007 0.022 0.122 0.072 0.018
35.007 19.563 4.788 13.366 41.721
0.039
26.417 13.191 13.509 10.351 9.948 9.412 19.090 17.620
obstruction
rat
1.039 0.679 0.616 0.565 0.568 0.436 0.392 0.409
0.051 0.045 0.054 0.058 0.046 0.020 0.023
I.
rat liver (De Wolf-Peeters et al., 1972) whereby the frequency of each canalicular type depends upon the duration of the BDL. Type 4 canaliculus is characterized by a lumen which is virtually filled with numerous long and regular microvilli. Such are the canaliculi in normal adult rat liver. Transitional forms between type 4 and 3 show a widened lumen with a few short microvilli. This type is mainly found during the first days after bile duct obstruction, Type 3 canaliculi, found from the second day on, have a dilated lumen with only a few microvilli. The lumen is empty or filled with amorphous electrondense material or with an edematous swollen bleb. They correspond to the dilated canaliculi devoid of microvilli described in the literature as the characteristic hallmark of cholestasis. From the present investigation it appears that these canaliculi do not show alkaline phosphatase activity on their limiting membrane. Only their luminal TABLE SIGNIFICANCE
ACCORDING
Type I, Type Type Type Type a For
abbreviations
1
2 3 4 see Table
TO TYPE
1 PPL
I.
III
Type
2
(STUDENT’S Type
t TEST)~ 3
Type
4
1.
PPL
1,
PPL
1,
PPL
S
NS
s s
s s
s s s
s s s
rat
abbreviations
day BDL days BDL days BDL days BDL days BDL days BDL days BDL
1 2 3 4 9 20 40
a For
adlllt
Normal
I.
-
-
see Table
PPL
adult
IO
Normal
rat,
PI’L
h
-
-< h
-
BDL
L
1 day
~IG~YIFICANCE
NS -
-
b‘(
P’PL
BL)L
NS
s
L
2 days
.~XORI~ING
TABLE
NS NP
8
1,
3 days
TO &RATION
IV
NS NS
s
PPI, .~__
BDI,
NS NS NS
h~I
1,
4 days
OF OIJSTRI~CTIOS
NS NS NS NV8
S h
NS
P
PPL
BI>L
b’
s
NS
L
9 days
I TEST)
NS NS
h’
PPL
BDL
(~TI.UXNTS’
BDL
PI’L
20 days
L
Ns NS
40 days
b-’ NS
BIJL
CANALICULAR
CHANGES
IN
TABLE
29
CHOLESTASIS
V
C~MPMZISON OF THE SURFACE/VOLUME RELATION OF THE C~N~LI~UL~R TYPES OF RAT LIVER DURING PERINATAL MATURATION (De Wolf-Peeters et al., 1974) AND THE CANALICULAR TYPES OF ADULT RA,~ LIVER DURING CHOLESTASISQ Values of morphometric parameters for perinatal canaliculi
(cm?
VVCL Type Type Type Type Type a For
1 3 3-4 4
abbreviations
see Table
carraliculi
SVO (m2/cm3)
0.007 0.037 0.167 0.071 0.018
2
Values of morphometric parameters for cholest,at,ir
VVCL
0.323 0.499 0.701 0.801 0.764
(cm?
SVC (m”/cm3)
0.007 0.022 0.122
0.277 0.434 0.586
0.072
0.975
I.
content, if present in the form of amorphous material (bile constituents) shows a positive reaction. Type 2 canaliculi have an irregular lumen which shows secondary extensions, separated from the main cavity by cytoplasmic bridges; their lumen is partly occupied by finger-like cytoplasmic extensions from the adjacent hepatocytes, or with a cytoplasmic sequester. Type 1 canaliculus is an irregular invagination of two adjacent cell membrane segments, located between junctional complexes, into the cytoplasm of one of two neighboring hepatocytes. The latter two types are found together with dilated canaliculi from 3 days on after BDL. Their number increases with longer duration of the cholestasis. In analogy with our previous study (De Wolf-Peeters et al., 1974) the different canalicular types were morphometrically characterized by a S/V ratio (Figs. 13, 14, 15). Furthermore, a comparison of the primary data of these types was made with the data of the canalicular types during neonatal maturation (Table IV). These results prove that different canalicular types can be recognized and that these types are very similar to those described in perinatal rat liver. TABLE SIGNIFICANCE
(STUDENT’S
Cholestatic canaliculi
Perinatal Type
Type 1 Type 2 Type Type Q For
VI
3 4
abbreviations
1
Type
2
t TEST)” canaliadi ‘be
10
PPL
1,
PPL
1,
NS S S S
NS S S S
NS S S S
S S NS S
S NS NS S
see Table
I.
3 PPL
S NS S S
S S NS NS
6 S S NS
DE
VOS
ET AL.
I__*
01234
FIG.
13. Surface
9
density
20 DAYS
of the
canaliculi
pro
group.
Therefore, the present morphologic and histochemical investigation, supported by morphometric analysis, points towards an evolution in the morphologic appearance of the biliary pole of the hepatocytes after BDL as well as to the appearance of newly formed canalicular structures. In our previous study (De Wolf-Peeters et al., 1974) it has been suggested that the different canalicular types represent successive stages in the maturation of the biliary pole, whereby type 1 canaliculus corresponds to the most primitive stage of the developing canalicular structure, changing progressively into type 4 canaliculus. It may, therefore, be assumed that canaliculi type 1, found after long-standing cholestasis, correspond to newly formed excretory poles of the hepatocytes. This interpretation would fit in with data from the literature, that after common bile duct obstruction liver cell regeneration is known to occur early (Kram, 1965; MacDonald et al., 1961; Stroun, 1966). We would exclude the possibility that canaliculi type 1 represent collapsed canalicular structures type 3 after a decompression of the bile duct obstruction as this epiphenomenon was excluded by the experimenta conditions. The possibility cannot be excluded that new canalicular structures also develop on preexisting hepatocytes. The fact that type 3 canaliculi are generally reported as the hallmark of cholestasis means that only the most striking changes have been described. The present findings, although they confirm the general statement that canaliculi type 3 are a characteristic hallmark of cholestasis, also show that cannlicular changes in cholestasis are of a more complex nature.
0,234
FIG.
14. Volume
9
density
20
of the canaliculi
’ DAYS
pro
4o
group.
CANALICULAR
CHANGES
IIIII
15. Ratio
of surface
CHOLESTASIS
9
01234
FIG.
IN
density
20
to volume
density
” DAYS
31
40
of the
canaliculi
pro
group.
As in the previous study a mean value of the canalicular S/V ratio was measured pro period after BDL (Figs. 16, 17, 18). A comparison between Fig. 15 and Fig. 18 together with the findings of conventional electron microscopy support the conclusion that the different canalicular types found in cholestasis reflect the dynamic evolution of the canalicular alterations. The sequence of these changes is summarized in Fig. 19. Immediately after BDL a number of normal canaliculi undergo morphologic and enzyme histochemical changes. Their lumen becomes slightly dilated and some microvilli disappear. The transitional type 34 canaliculi stain intensely positive
AND
MOLECULAR
Significance
of Liver
PATHOLOGY
12-34
( 19%)
Canalicular Changes Bile Duct Ligation I92
DE Vos R., DE WOLF-PEETERS, Departement Academisch Belgium
23,
C., DESMET
after
V., BIANCHI,
L., AND ROHR, H. P.
Medische Navorsing, Laboratorium voor Histochemie Ziekenhuis St. Rafad, Katholieke Universiteit Leuven, and Institute of Pathology, Department of Electron University of Bad, Basel, Switzerland Received
September
Experimental
en Cytochemie, B-300 Leuven, Microscopy,
30, 1974
An ultrastructural morphologic, histochemical, and morphometric study was made on the canalicular structures in rat liver after experimental bile duct ligation (BDL). Four different canalicular structures are distinguished, which are proved by morphometry to correspond to the different types observed during perinatal maturation. Canaliculus type 4, corresponding to the excretory pole of a normal adult hepatocyte, is characterized by a lumen filled with regular, slender microvilli. Canaliculus type 3, corresponding to the canalicular structure described in cholestasis, has a wide lumen devoid of microvilli and is found 2-3 days after BDL. A transitional canalicular structure between type 3 and 4, partly provided with microvilli, is found from the first day after BDL. After 3 days of BDL an increasing number of other canalicular types is observed. Canaliculi type 2, showing an irregular lumen with irregular microvillus-like projections, and canaliculi type 1, representing intracytoplasmic invaginations of two adjacent cell membranes, are found together with canaliculi type 3 and type 3-4. the enzyme activity is detected in After incubation for alkaline phosphatase, canaliculi type 1, 3-4, and type 4. The striking analogy of the sequential changes in canalicular structures after BDL and during perinatal maturation leads to the following interpretation: ( 1) Canaliculi type 1 and 2, more frequently observed in longstanding cholestasis correspond to newly formed excretory poles of the hepatocytes. (2) Canaliculus type 3, or the so-called cholestatic canalicu!ar type, reflects an inactive secretory function. Morphometrically this canalicular type shows a very low S/V relation which may be partly responsible for this secretory inactivity. (3) Canaliculus type 34 reflects a disturbed secretory function after BDL and a beginning secretory activity during perinatal maturation.
INTRODUCTION microscopic and histochemical studies have been devoted and canalicular changes in human rat liver in different forms of intrahepatic and extrahepatic cholestasis (Desmet, 1972). From these it appears that dilatation of the canaliculi and disappearance of their microvilli may Numerous
to
electron
hepatocellular
1 This work was supported by a grant from the “Fonds voor Onderzoek” of Belgium. * Part of this work has been presented at the 7th Meeting the study of the liver. Arnhem, The Netherlands, September 12 Copyright Ail rights
0 1975 by of reproduction
Academic in any
Press, Inc. form reserved
Wetenschappelijk of the 1972.
European
Geneeskundig Association
for
CANALICULAR
CHANGES
IN CHOLESTASIS
I.3
be regarded as the ultrastructural hallmark of cholestasis. However, the significance of other aberrant morphological appearances of canaliculi remains unclear. Recently, morphologic and histochemical evidence was produced, both at the microscopical (De Wolf-Petters et al., 1971) and ultrastructural (De WolfPeeters et al., 1972) level and completed by morphometric analysis (‘De WolfPeeters et al., 1974), that cholestatic types of canaliculi occur in the liver of normal newborn rats shortly after birth. Tllis corrc1utt.s with ;ul tlscretory disturbance as has been proved by biochemical studies (Bakken, 1970; Dawkins, 1966; Gamter et al., 1969; Whelan et al., 1970). Such cholestatic features were found to represent only a transient phase in a complex chronologic sequence of the canalicular changes, suggestive of successive stages in canalicular development and maturation. These findings indicated that a careful comparative study of neonat,al and cholestatic livers might shed new light on the significance of canalicular changes in cholestasis. The present study was undertaken in order to make a detailed analysis of canalicular morphologic and histochemical changes as they occur in the liver of the rat after ligation of the common bile duct, and to compare them to the various morphologic and histochemical characteristics of canaliculi as found in the perinatal period. Since zonal differences are know to exist in the normal liver lobule (Ma et al., 1968; Loud, 1968), and in different types of the liver pathology, including cholestasis ( Wachstein, 1963), this study was restricted to the changes in canalicular morphology and histochemistry in the periportal region. Quantitative data were obtained by morphometry in order to complete the qualitative observations. MORPHOLOGY
Materials
AND HISTOCHEMISTRY
and Methods
Male rats of albino Wistar R strain, weighing 150-200 g were obtained commercially. The animals were fed a laboratory stock diet and tap water. Twenty-one Wistar rats were subjected to bile duct ligation (BDL). During constant ether flow anesthesia the ductus choledochus was exposed by blunt dissection, doubly ligated and sectioned between two ligatures. All animals were killed by exsanguination; the time interval between the surgical intervention and the sacrifice of the animals varied from 1 to 2, 3, 4, 9, 20, and 40 days. It must be mentioned that in this group no animals were included which had a choleperitoneum (rupture of the proximal choledochal stump) or which showed fibrous connections between the blind-ending choledochus and the gut (masking an eventual fistula). The animals were divided into eight groups: normal adult rats, rats 1 day after BDL, rats 2 days after BDL, rats 3 days after BDL, rats 4 days after BDL, rats 9 days after BDL, rats 20 days after BDL, and rats 40 days after BDL. Specimens were taken from the left anterior liver lobe. Each specimen was divided in two parts. One part was immediately quenched in liquid nitrogen-cooled isopentane, g-pm-thick cryostat sections were cut, fixed for 15 min in cold formol-calcium chIorid+dextran (Planteydt, 1961)) and stained for alkaline phosphatase, using
1.4
DE VOS ET AL.
FIG . 1. Normal rat liver. Fresh-frozen section prepared for cytochemical study. Peripol rtally locali: zed canaliculi are weakly positive for alkaline phosphatase. X160, inset x400.
the (:alcium cobalt method of Gomori (Gomori, 1939; Takamatsu, 1939). The incul 3ation time was 20 min at 37°C. Frl om the second part, thin Aices (l/1/10 mm) were fixed by immersion for 2 hr in cold (4°C) 6.00 g/100 ml glutaraldehyde and 3.04 g/100 ml forma Jdehyde cacodylate buffer (Tijro et al., 1966), pH 7.2, followed by buffer I inse over1light.
CANALICULAR
CHANGES
IN CHOLESTASIS
15
For morphological study, portions of each specimen were postfixed in 1% OsOa in phosphate buffer, pH 7.2 (Millonig, 1961)) for 1 hr at 4”C, dehydrated in graded alcohols, ‘and embedded in Epon (Luft, 1961). For cytochemical study, 40-pm-thick sections, prepared with the TC2 Smith and Farquhar tissue sectioner, were incubated for alkaline phosphatase activity buffer, with beta-glycerophosphate (2 mg/ml) as substrate in 0.05 M Tris-HCL
FIG. 2. Rat liver 2 days after extrahepatic bile duct obstruction. Alkaline phosphatase positive canaliculi with irregular pattern and intense staining reaction. X160, inset x400.
16
FIG. 3. Normal rat liver. Thin acetate and lead citrate. Normal villi. Type 4. X24,000.
DE
VOS
ET AL.
section prepared for morphologic study, stained with canaliculus; the lumen is filled with numerous regular
uranyl micro-
pH 8.2, for 30 min at 37°C. Lead citrate was used as capture reagent (Saito et al., 1968). Control sections were incubated in substrate-free incubation medium and in incubation medium with a specific inhibitor: EDTA 0.05 M. After incubation the sections were rinsed in cacodylate buffer, postfixed for 1 hr in 1% Os04 in phosphate buffer, and embedded in Epon. From each specimen, semithin sections were cut, stained for 1 min, with toluidine blue (Lynn, 1965 ) and examined under the optical microscope. These
CANALICULAR
CHANGES
IN
CHOLESTASIS
17
FIG. 4. Rat liver 1 day after extrahepatic bile duct obstruction. Thin section prepared for morphologic study, stained with uranyl acetate and lead citrate. Canaliculus with dilated lumen and a few short microvilli. Transitional form between type 4 and type 3. X24,000.
sections served to identify specific zones of the hepatic Iobule; only the specimens containing a portal field were selected for further cutting of thin sections. Periportal areas were trimmed so that they included a recognizable portion of a portal tract and at most seven hepatocytic cell diameters surrounding it (Loud, 1968). Thin sections were cut with an OM U 3 Reichert ultramicrotome, stained with uranyl acetate (Watson, 1958) followed by lead citrate (Reynolds, 1963) and studied with a Zeiss EM 9 electron microscope.
1s
DE
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ET
AL.
FIG. 5. Rat liver 2 days after extrahepatic bile duct obstruction. Thin section morphologic study, stained with uranyl acetate and lead citrate. Dilated canaliculi lumen without microvilli. Type 3 canaliculi. X18,000.
prepared for with empty
Results Optical microscopy. In normal rat liver the lead precipitate after incubation for alkaline phosphatase is localized on the canaliculi of the periportal zone; in the centrilobular zone the reaction is virtually negative (Fig. 1). One day after bile duct obstruction an intensely positive reaction is observed in all canalicular structures (Fig. 2). This enzyme activity remains high during the
CANALICULAR
CHANGES
IN
19
CHOLESTASIS
ensuing days of obstruction while the morphology of the abnormal pattern. Electron microscopic morphology. In normal rat liver, the canaliculi, located between two adjacent hepatocytes and tional complexes, is filled with numerous long and regular zone of pericanalicular ectoplasm can be observed (Fig.
canaliculi
shows an
lumen of periportal sealed off by juncmicrovilli; a narrow 3). One day after
FIG. 6. Rat liver 4 days after extrahepatic bile duct obstruction. Thin section prepared for morphologic study, stained with uranyl acetate and lead citrate. Canaliculus with irregular lumen; presence of secondary extensions separated from the main cavity by cytoplasmic bridges. Type 2. X18,oQQ,
20
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ET AL.
FIG. 7. Rat liver 10 days after extrahepatic bile duct obstruction. Thin section prepared for morphologic study, stained with uranyl acetate and lead citrate. Canaliculus-like structure without real lumen formed by an intracytoplasmic invagination of the outer cell membranes located between junctional complexes. Type 1 canaliculus. X38,000.
obstruction numerous canaliculi show a slightly dilated lumen, the microvilli are irregular (Fig. 4); the pericanalicular ectoplasm is often widened. Two to three days after bile clurt obstruction a higher number of canaliculi shows a dilated lumen while most of their microvilli have disappeared (Fig. 5); their lumen may be free, or occluded by an edematous bleb, or filled with amorphous electron-
CANALICULAR
FIG. 8, Normal acetate and lead on the microvillar
CHANGES
IN
CHOLESTASIS
rat liver. Thin section prepared for cytochemical citrate. Canaliculus type 4 with positive reaction membranes. X 18,000.
study, stained for alkaline
with u~i any1 phospha tase
dense material. Tlhc morphologic appearance of the latter material is ql rite similar to the pericanalicular ect.oplasm. In this period, but even more frequently 3 to 4 days and later on after obstr ‘UCtion, segments of hepatocellular plasma membranes, located between junctic ma1 are complexes, show invaginations and interdigitations. These invaginations usually found more extensively into the cytoplasm of only one of two adjac :ent hepatocytes (Fig. 7). Some of these membrane invaginations, appearing as
22
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ET
FIG. 9. Rat liver 4 days after extrahepatic bile duct cytochemical study, stained with uranyl acetate and showing alkaline phosphatase-positive type 4 canaliculi and practically negative type 3 canaliculi ( b ) with remnants of microvilli. X7600.
AL.
obstruction. Thin section prepared for lead citrate. A low-power photograph ( + ) , positive type 1 canaliculi ( D ), a thick
ectoplasmic
zone
and
a few
canaliculus-like structures because of the presence of two limiting tight junctions, may isolate a cytoplasmic sequester from the rest of the cytoplasm of the hepatocyte in which they are extending.
CANALICULAR
CHANGES
FIG. 10. Rat liver 10 days after extrahepatic for cytochemical study, stained with many1 positive type 1 canaliculus. X38,000.
bile acetate
IN
23
CHOLESTASIS
duct and
obstruction. lead citrate.
Thin section prepared Alkaline phosphatase-
Also during this period canaliculus~~like structures are found with a very irregular lumen, provided with a few cytoplasmic finger-like extensions; in their vicinity smaller cavities can be found in the hepatocytic cytoplasm which are separated from the main lumen by cytoplasmic bridges (Fig. 6). From 9 to 20 days after obstruction one still fiuds dilated canaliculi, but there is a higher number of the above-mentioned canaliculus-like membrane invaginations without real lumen and of canaliculi with irregular lumen.
24
-
DE
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FIG. 11. Rat liver 4 days after extrahepatic for cytochemical study, stained with uranyl canaliculus; the lumen is filled with amorphous X38,000.
ET AL.
bile duct obstruction. Thin acetate and lead citrate. material showing a positive
section prepared Dilated type 3 lead precipitate.
The pericanalicular ectoplasm surrounding most of these canaliculus-like strutures ‘and irregular canaliculi is sometimes strongly widened and appears swollen and edematous. After 40 days of obstruction 011est.ill finds an increasing number of canaliculuslike structures. In this period, however, such membrane invaginations are usually located between three or four neighboring hepatocytes. It should also be
CANALICULAR
FIG. 12. Rat liver 1 day cytochemical study, stained superimposed double-lattice
CHANGES
IN
25
CHOLESTASIS
after extrahepatic bile duct obstruction. with uranyl acetate and lead citrate. test screen. X 12,000.
Thin section prepared Canaliculus type 43
for with
stressed that during the whole obstructive period one can always find a few canaliculi with normal morphologic appearance. Alkaline Phosphatase Hislochemisty. In normal liver tissue a positive reaction is found on the microvillous membrane of the canaliculi after incubation for alkaline phosphatase (Fig. 8). After obstruction a strongly positive reaction is observed in all canaliculi and canaliculus-like structures, with the exception of broadly widened canaliculi devoid of microvilli (Figs. 9 and 10). [When, however, their lumen contains electron-dense amorphous material a positive lead precipitate is found on the latter (Fig. ll)]. After incubation in substrate-free media and in normal media containing a specific inhibitor no lead precipitate could be detected in any of the sections at the canalicular cell membrane. MORPHOMETRY Methods For morphometric study five blocks, contaiuing a portal field-identified as described above-from each rat liver tissue sample, were used for preparation of ultrathin sections. Pro group 40-50 micrographs were taken ,at random at a primary magnification of 9500 and enlarged together with a double-lattice test screen (Weibel, 1969) (Fig. 12). I n order to characterize the different canalicular types by morphometry, 3040 micrographs were taken of each prototype, and processed as described for the groups.
26
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AL.
To determine the volume density of the bile canalicular lumen the fine test points lying inside the canalicular lumen (IO89 points per test area) were counted. To evaluate the surface density of the canaliculus, the number of intersections of the test lines with the canalicular membrane was counted (Fig. 12). C,alculations and statistical analyses (mean value, standard deviation, standard error, Student’s t test) were performed with the so-called multipurpose morphometric computer program on an Olivetti P. 602 microcomputer system (Rohr et al., 1973). Results Morphometric
results
can be seen in Tables I to VI. DISCUSSION
The criteria applied in this and previous studies (De Wolf-Peeters et al., 1972, 1974) to consider a structure as a canaliculus or its equivalent is its limitation by two or three junctional complexes which separate it from the rest of the intercellular space. All morphologic variants of canalicular structures observed in this experiment may be grouped into four prototypes, as described previously in perinatal
TABLE PRIMARY
DATA
I
OF MORPHOMETRIC EVALUATION .4~ THE HEPATOCYTE~
OF BILIARY
POLE
PPL
SE Types Type Type Type Type Type Group
SE
of canaliculi 14.46 22.64 30.54 .50.81 39.85
1
2 3 3-4 4 according
Adult normal 1 day BDL 2 days BDL 3 days BDL 4 days BDL 9 days BDL 20 days BDL 40 days BDL
to duration rats
1.74 2.14 2.42 4.83 *5.88
0.007 0.022 0.122 0.072 0.018
0.001 0.002 0.013 0.011 0.003
5.77 3.50 2.68 2.43 1.79 1.64 2.72 2.14
0.039 0.051 0.04*5 0.054 0.058 0.046 0.020 0.023
0.005 0.006 0.008 0.008 0.007 0.006 0.007 0.004
obstruction Fi4.18 35.42 32.12 29.49 30.54 22.76 20.44 21.34
a hhbreviat,ions. L = Lumen of the bile canalicuhw. C = Contour of the bile canaliculus. Pp = Poinl. den&y (cm”). Vv = Volllme densit,y ((km”). IT, = Intersections (cm-l). Sv = Surface density (m”/cm”). VVL = Volume of the bile canaliculus per unit volume of surrounding liver tissue. SVL = Surface of bile canalicuhls per unit volume of surrounding liver t,iessue. I = Intersections. M = Mean value. SE = Standard error. S = Significant. NS = Nonsignificant.
CANALICULAR
CHANGES
IN CHOLESTASIS
TABLE SECONDARY
PARAMETERS
II
OF MORPHOMETRIC EVALUATION POLE OF THE HEPATOCYTE~ SVC W/cm”)
Type
27
VVCL
OF THE BILIARY
S~C/VVCL
(cm”)
(m2/cm”)
of canaliculi
Type Type Type Type Type Group
1
0.277 0.434 0.586 0.975 0.764
2 3 34 4 according
Adult normal l-day BDL a-days BDL a-days BDL 4-days BDL O-days BDL 20-days BDL 40-days BDL a See footnote
Table
to duration
0.007 0.022 0.122 0.072 0.018
35.007 19.563 4.788 13.366 41.721
0.039
26.417 13.191 13.509 10.351 9.948 9.412 19.090 17.620
obstruction
rat
1.039 0.679 0.616 0.565 0.568 0.436 0.392 0.409
0.051 0.045 0.054 0.058 0.046 0.020 0.023
I.
rat liver (De Wolf-Peeters et al., 1972) whereby the frequency of each canalicular type depends upon the duration of the BDL. Type 4 canaliculus is characterized by a lumen which is virtually filled with numerous long and regular microvilli. Such are the canaliculi in normal adult rat liver. Transitional forms between type 4 and 3 show a widened lumen with a few short microvilli. This type is mainly found during the first days after bile duct obstruction, Type 3 canaliculi, found from the second day on, have a dilated lumen with only a few microvilli. The lumen is empty or filled with amorphous electrondense material or with an edematous swollen bleb. They correspond to the dilated canaliculi devoid of microvilli described in the literature as the characteristic hallmark of cholestasis. From the present investigation it appears that these canaliculi do not show alkaline phosphatase activity on their limiting membrane. Only their luminal TABLE SIGNIFICANCE
ACCORDING
Type I, Type Type Type Type a For
abbreviations
1
2 3 4 see Table
TO TYPE
1 PPL
I.
III
Type
2
(STUDENT’S Type
t TEST)~ 3
Type
4
1.
PPL
1,
PPL
1,
PPL
S
NS
s s
s s
s s s
s s s
rat
abbreviations
day BDL days BDL days BDL days BDL days BDL days BDL days BDL
1 2 3 4 9 20 40
a For
adlllt
Normal
I.
-
-
see Table
PPL
adult
IO
Normal
rat,
PI’L
h
-
-< h
-
BDL
L
1 day
~IG~YIFICANCE
NS -
-
b‘(
P’PL
BL)L
NS
s
L
2 days
.~XORI~ING
TABLE
NS NP
8
1,
3 days
TO &RATION
IV
NS NS
s
PPI, .~__
BDI,
NS NS NS
h~I
1,
4 days
OF OIJSTRI~CTIOS
NS NS NS NV8
S h
NS
P
PPL
BI>L
b’
s
NS
L
9 days
I TEST)
NS NS
h’
PPL
BDL
(~TI.UXNTS’
BDL
PI’L
20 days
L
Ns NS
40 days
b-’ NS
BIJL
CANALICULAR
CHANGES
IN
TABLE
29
CHOLESTASIS
V
C~MPMZISON OF THE SURFACE/VOLUME RELATION OF THE C~N~LI~UL~R TYPES OF RAT LIVER DURING PERINATAL MATURATION (De Wolf-Peeters et al., 1974) AND THE CANALICULAR TYPES OF ADULT RA,~ LIVER DURING CHOLESTASISQ Values of morphometric parameters for perinatal canaliculi
(cm?
VVCL Type Type Type Type Type a For
1 3 3-4 4
abbreviations
see Table
carraliculi
SVO (m2/cm3)
0.007 0.037 0.167 0.071 0.018
2
Values of morphometric parameters for cholest,at,ir
VVCL
0.323 0.499 0.701 0.801 0.764
(cm?
SVC (m”/cm3)
0.007 0.022 0.122
0.277 0.434 0.586
0.072
0.975
I.
content, if present in the form of amorphous material (bile constituents) shows a positive reaction. Type 2 canaliculi have an irregular lumen which shows secondary extensions, separated from the main cavity by cytoplasmic bridges; their lumen is partly occupied by finger-like cytoplasmic extensions from the adjacent hepatocytes, or with a cytoplasmic sequester. Type 1 canaliculus is an irregular invagination of two adjacent cell membrane segments, located between junctional complexes, into the cytoplasm of one of two neighboring hepatocytes. The latter two types are found together with dilated canaliculi from 3 days on after BDL. Their number increases with longer duration of the cholestasis. In analogy with our previous study (De Wolf-Peeters et al., 1974) the different canalicular types were morphometrically characterized by a S/V ratio (Figs. 13, 14, 15). Furthermore, a comparison of the primary data of these types was made with the data of the canalicular types during neonatal maturation (Table IV). These results prove that different canalicular types can be recognized and that these types are very similar to those described in perinatal rat liver. TABLE SIGNIFICANCE
(STUDENT’S
Cholestatic canaliculi
Perinatal Type
Type 1 Type 2 Type Type Q For
VI
3 4
abbreviations
1
Type
2
t TEST)” canaliadi ‘be
10
PPL
1,
PPL
1,
NS S S S
NS S S S
NS S S S
S S NS S
S NS NS S
see Table
I.
3 PPL
S NS S S
S S NS NS
6 S S NS
DE
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ET AL.
I__*
01234
FIG.
13. Surface
9
density
20 DAYS
of the
canaliculi
pro
group.
Therefore, the present morphologic and histochemical investigation, supported by morphometric analysis, points towards an evolution in the morphologic appearance of the biliary pole of the hepatocytes after BDL as well as to the appearance of newly formed canalicular structures. In our previous study (De Wolf-Peeters et al., 1974) it has been suggested that the different canalicular types represent successive stages in the maturation of the biliary pole, whereby type 1 canaliculus corresponds to the most primitive stage of the developing canalicular structure, changing progressively into type 4 canaliculus. It may, therefore, be assumed that canaliculi type 1, found after long-standing cholestasis, correspond to newly formed excretory poles of the hepatocytes. This interpretation would fit in with data from the literature, that after common bile duct obstruction liver cell regeneration is known to occur early (Kram, 1965; MacDonald et al., 1961; Stroun, 1966). We would exclude the possibility that canaliculi type 1 represent collapsed canalicular structures type 3 after a decompression of the bile duct obstruction as this epiphenomenon was excluded by the experimenta conditions. The possibility cannot be excluded that new canalicular structures also develop on preexisting hepatocytes. The fact that type 3 canaliculi are generally reported as the hallmark of cholestasis means that only the most striking changes have been described. The present findings, although they confirm the general statement that canaliculi type 3 are a characteristic hallmark of cholestasis, also show that cannlicular changes in cholestasis are of a more complex nature.
0,234
FIG.
14. Volume
9
density
20
of the canaliculi
’ DAYS
pro
4o
group.
CANALICULAR
CHANGES
IIIII
15. Ratio
of surface
CHOLESTASIS
9
01234
FIG.
IN
density
20
to volume
density
” DAYS
31
40
of the
canaliculi
pro
group.
As in the previous study a mean value of the canalicular S/V ratio was measured pro period after BDL (Figs. 16, 17, 18). A comparison between Fig. 15 and Fig. 18 together with the findings of conventional electron microscopy support the conclusion that the different canalicular types found in cholestasis reflect the dynamic evolution of the canalicular alterations. The sequence of these changes is summarized in Fig. 19. Immediately after BDL a number of normal canaliculi undergo morphologic and enzyme histochemical changes. Their lumen becomes slightly dilated and some microvilli disappear. The transitional type 34 canaliculi stain intensely positive
