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Electron microscopy and morphometry of canalicular differentiation in fetal and neonatal rat liver
EXPERIMENTAL
AND
MOLECULAR
21,
PATHOLOGY
339-350
(1974)
Electron Microscopy and Morphometry of Canalicular Differentiation in Fetal and Neonatal Rat Liver1 C. DE WOLF-PEETERS, Laboratorium Vniuersiteit
2 R. DE Vos, 2 V. DESMET,
UOOT Histochemie en Cytochemie, Leuven, 3000 Leuuen, Belgium, Electron Microscopy, Uniuersity Received
2 L. BIANCHI, 8 AND H. P. ROHR3
Academisch Ziekenhuis St. Rafad, Katholieke 2 and Institute of Pathology, Department of of Basel, Basel, Switzerland 3
February
X,1974
In order to complement previous electron microscopic studies on the differentiation of the biliary pole of the rat hepatocyte during perinatal maturation, a morphometric analysis was undertaken of the bile canaliculi in fetal and adult rat liver. Four morphologic canalicular types were found and morphometrically characterized using a surface/volume relation (s/v index) as key parameter. The surface/volume relation of the canaliculus in fetal and postnatal maturation as well as in the adult rat liver supports the previously formulated hypothesis that these different types correspond to successive maturation stages of the biliary pole of the hepaocyte. Furthermore, the s/v index of type 3 canaliculus supports the concept that this canalicular type represents a canalicular structure inefficient for secretion.
INTRODUCTION In a previous study (De Wolf-Peeters et al., 1972), the differentiation of the biliary pole of the rat hepatocyte was studied during late fetal and early neonatal development in order to identify a morphologic substrate for the well-known immaturity (De Wolf-Peeters et al., 1971; Schenker, 1963) of the bile secretory function in this period. Four different prototypes of canaliculi have been distinguished; it was suggested that they represent successive stages during the maturation of the biliary pole of the liver cell. This paper deals with the results of a morphometric analysis of the biliary pole of fetal and neonatal rat liver cells, which was undertaken to provide an objective and quantitative basis for the previously formulated hypothesis. MATERIAL
AND METHODS
Rats of Albino Wistar strain were obtained commercially. During the study all rats were fed a laboratory stock diet and tap water ad E&turn. Female rats were matched with males overnight and examined the next morning for the presence of sperm in the vaginal smear. The day of observation of sperm was considered day 1 of pregnancy. The fetuses were obtained by successive Cesarean section performed on mother rats under a constant flow ether anesthesia. 1 This Onderzoek
work was supported of Belgium.
by a grant
from
the
339 Copyri ht 0 1974 by Academic Press, Inc. AU rig i: ts of reproduction in any form reserved.
Fends
voor
Wetenschappelijk
Geneeskundig
DE WOLF-PEETERS
340
ET AL.
Twenty-three fetuses and newborn rats as well as 3 adult male rats (weight 150 g) were studied. These animals were divided into 7 different groups: rats of 16 days of fetal age; rats of 20 days of fetal age; rats at birth; rats 24 hr after birth; rats 4 days after birth; rats 10 days after birth, and adult rats. The liver was removed quickly after decapitation of the animal. A tissue specimen of the right half of the median liver lobe was taken, cut into thin slices and fixed by immersion for 2 hr in cold (4°C) 6% glutaraldehyde and 3.04% formaldehyde cacodylate buffer pH 7.2 (Toro et al., 1966) followed by buffer rinse overnight. A post fixation was carried out in 1% Os04 in phosphate buffer for 1 hr at 4°C (Millonig, 1961), folIowed by dehydrating in graded alcohols and embedding in Epon. Morphometric methods
One liver specimen was taken from at least 3 animals per group. From each of these biopsies, 5 blocks were used for preparation of ultrathin sections, which were examined after staining with uranyl acetate (Watson, 1958) followed by lead citrate (Reynolds, 1963). From each experimental group 3040 micrographs were taken at random at a primary magnification of 9.500 with an electron microscope Zeiss EM 9, and enlarged together with a double lattice test screen ( Weibel, 1969; Fig. 5). In addition, in order to characterize the different canahcular types by morphometry, 3040 micrographs were taken of each prototype (from the different groups) and processed as described for the groups. To determine the volume density of the bile canalicular lumen the fine test points lying inside the ‘canalicular lumen (1089 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. 6). Calculations and statistical analysis (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
The criterion
applied in this as well as in previous studies (De Wolf-Peeters is its limitation by two or three junctional complexes which separate it from the rest of the intercellular space. According to their morphologic appearance, the canaliculi observed in all liver specimens studied can be divided into 4 principal types (De Wolf-Peeters et al., 1972) : Type 1 canaliculus can be defined as an intracytoplasmic invagination of two adjacent cell membranes into one of the two neighboring hepatocytes (Fig. 1). et al., 1972) to accept a structure as “canalicular”
Type 2 canaliculus is provided with a lumen, which is irregular in form and
often partly filled with a clump of electron-dense material, interpreted as a fragment of sequestrated cytoplasm (Fig. 2). Type 3 canaliculus is characterized by a wide but regular lumen, around
FETAL
AND NEONATAL
BAT LIVER
341
FIG. 1. Bile canaliculus type 1. X 36,800
which microvilli are scanty or even absent. An amorphous electron-dense material may be found in such a lumen (Fig. 3). Type 4 canaliculus has a smaller lumen, virtually filled with numerous microvilli. Its morphologic characteristics correspond to those of normal adult canaliculi ( Fig. 5 ) .
DE WOLF-PEETERS
342
FIG. 2. Bile
canaliculus
type
ET AL.
2. X 36,800
Trarwiticd
forms between types 3 and 4 are also found. These canaliculi show a widened regular lumen similar to type 3, ,but a variable length of the surrounding cell membrane is provided with regular microvilli (Fig. 4). Depending on the age of the animal, one type of canaliculus is found in strikingly higher number than the other ones, although some other canalicular types may be found in one and the same specimen. From 16 until 19 days of fetal age most canaliculi are of type 1. During the following days canaliculi type 2 become more numerous. From the 21st day on, canaliculi type 3 appear. Just before and after birth most canaliculi are of type 3. Twelve hours after birth many transitional forms between types 3 and 4 can be seen, Their number decreases from the 3rd day on, while canaliculi type 4 are found in increasing numbers.
For results see Tables I-III
and Text Fig. l-3. DISCUSSION
The different canalicular types previously described by simple morphology and histochemistry during maturation of the fetal and neonatal rat liver (De Wolfparameters. Peeters et al., 1972) have been investigated by morphometric As shown in Table II(a) each canalicular type reveals a proper mean volume
FETAL AND NEONATAL
FIG.
3.
RAT LIVER
Bile canaliculus type 3.
X
343
36,800
whereby the very high figure for type 3 canaliculus is outstanding. In addition, the different types of canaliculi are fairly well distinguishable by their surface values. Primary parameters, their standard deviation and significances are given in Tables I-III, The fact, that the mean Ic value of type 3/4 (Table I a) shows no significant difference from that of types 3 and 4, respectively, supports the
DE WOLF-PEETERS
344
FIG.
ET AL.
4. Bile canaliculus type 3-4. X 36,800
previously advanced hypothesis that this ~canalicular type represents a transitional form between types 3 and 4. By characterizing the canalicular types by means of their surface/volume ratio (Table IIa) a different value valid for types 2, 3, and 3/4 is obtained. The fact that type 1 and type 4 give similar values with respect to surface/volume ratio
FETAL
AND NEONATAL
FIG. 5. Bile canaliculus
type
RAT LIVER
4.
345
x 36,800
may be explained by their different irregular three-dimensional configuration (compare Fig. 1 with Fig. 4), as seen from the individual secondary parameters for surface and volume. The low surface/volume value typical for type 3 canaliculus by itself represent a very unfavorable morphologic configuration for secretory function. This morphometric finding in type 3 canaliculus might be part of an explanation for an excretory inefficiency of this canalicular type. In fact, canaliculus type 3 is considered to be typical for cholestatic conditions in man and animals ( Desmet, 1972). By morphometric analysis one can observe distinct periods during the maturation of fetal rat liver according to a changing surface/volume ratio of randomly chosen samples of canaliculi. In this respect, the fetal liver at 16 days of gestational age is quite different from the rat liver at birth and the adult liver. At the 16th gestational day the surface/volume ratio is very close to that of canaliculus type 1, predominantly found in this period. It corresponds also to type 4 for the reasons explained above. The close relationship of the surface/volume ratio on the 16th gestational day with that of canaliculus type 1 supports the concept that most of these canaliculi represent newly forming biliary poles. At birth and 24 hr after birth a very low surface/volume ratio is found. This may be easily explained by the predominance of type 3 canaliculi during this period.
346
FIG. 6. Bile canalicuhs
DE WOLF-PEETERS
type 3-4 with superimposed
ET AL.
double lattice test screen. X 15,000.
Adult rat liver also shows a quite typical surface/volume ratio in its canalicular pattern. The finding of a lower surface/volume value in the adult liver than that of type 4 canaliculus correlates with the observation that also in adult liver different canalicular types may be present, although canaliculus type 4 predominates. The periods corresponding to the 20th fetal day, the 4th day and 10th day after birth do not show a distinguishable canalicular surface/volume ratio. These morphometric results also fit in with the previously reported morphologic observation that in these periods one canalicular type is progressively being replaced by another one. At 20 days of fetal age not all canaliculi are of type 1, but type 2
FETAL
AND
NEONATAL TABLE
PRIMARY
DATA
BAT
347
I
OF MORPHOMETRIC EVALUATION POLE OF THE HEPATOCYTE A Types
LIVER
OF THE BILIARY
of Canaliculi IC
Type Type Type Type Type
1
2 3 2 4
PPL
?a
SE
e&
16.85 26.03 36.53 42.07 39.85
1.97 2.75 3.5 4.21 5.88
0.007 0.037 0.167 0.071 0.018
B Groups
According
Ic
PPL
54.18 22.65 25.6 70.36 41.94 22.05 21.82
TABLE SECONDARY
SE
‘rii
5.77 2.4 2.7 6.73 2.55 2.32 2.1
0.039 0.024 0.025 0.262 0.127 0.039 0.009
Type Type Type
0.007 0.037 0.167 0.071 0.018
3 2 4
B Groups
Adult rat 10 days postnat. 4 days postnat. 24 hr after birth Birth 20 days fetal age 16 days fetal age
0.005 0.002 0.006 0.056 0.020 0.007 0.002
OF THE
of Canaliculi svc (m*/cm”)
Type 1 Type 2
SE
II
PARAMETERS OF MORPHOMETRIC EVALUATION BILIARY POLE OF THE HEPATOCYTE A Types
0.001 0.004 0.025 0.014 0.003
to Age
?a
Adult rats 10 days postnat. 4 days postnat. 24 hr after birth Birth 20 days fetal age 16 days fetal age
SE
According
0.323 0.499 0.701 0.807 0.764
svclTvvL
(mz/cm3) 43.303 13.282 4.183 11.354 41.721
to Age SVC/vVVL
VVCL
svc
(cm’)
(mz/cm3)
(ma/cm*)
0.039 0.024 0.025 0.262 0.127 0.039 0.009
1.039 0.434 0.491 1.35 0.804 0.423 0.418
26.417 17.526 19.417 5.137 6.321 10.733 42.387
Adult rat 10 days postnatal 4 days postnatal 24 hr after birth Birth 20 days fetal age 16 days fetal age
Type 1 Type2 Type 3 Type2 Type4
S S S S S S
IC
-
S S
ll.S.
KS.
lLS.
S
S
-
S S S S S
S
PPL
10 d p.n.
IO
KS.
S S S S
PPL
S S S S
PPL
adult rat
-
Type 1
S S D.S. S
IC
S S S
S
Ic
S S S
S
PPL
LS.
n.s.
S S n.s. S
ll.S.
S
PPL
4 d p.n.
S S n.s. ns.
S ns.
Ic
T-test)
S S
S S S
S S S -
S S
Jl.S.
S S S
24 h p.n. IC PPL
-
S S
PPL
T-test)
Type 3 S S
Ic
B Significance (Student’s
-
Type2
TABLE III A Significance (Student’s
S S
S S S S
Ic
-
birth
R.S.
S S
S S S n.6.
‘PPL
-
TYPf n.8. S n.s.
Ic
S
S S S
PPL
n.s.
-
S
S S
I1.8.
S
LS.
PPL
20 d f.a.
n.s. S S
n.s.
S
IC
-
Type4 S S n.s. n.s.
IC
S n.s. n.s. S S n.s.
-
S S S S S S
PPL
16 d f.a.
S S S S
IC
PPL
F
2
E!
3 G 2 2
iii
FETAL
AND NEONATAL
RAT LIVER
349
Svc (m2/cm3)
I 0.600 0.400 0.200
type 1
type 2
type 3
type+&
S,,
type
L
(m2/cm3
1
1.500 c
L/L J
0.500
It 16d
ItI 20d Oh 2l.h
> 4d
TEXT FIG. 1. SVCpro canalicular
X)d "
adult
type; SVCpro group.
canaliculi are found as well as type 3, as exemplified by the comparison of group values with type values. Four as well as 10 days after birth canaliculi of types 3, 314, and 4 are found by conventional ultrastructural examination. This finding is confirmed by morphometric values given in Text Fig. 3. In conclusion, the results of this morphometric analysis of the biliary pole in fetal, neonatal and adult rat liver support the previously reported findings and advanced hypothesis that the different types represent successive maturation stages. The present findings furthermore suggest a structural inadequacy of type
0.150 -
0.100 O.lOOt 0.050 0.050-
I-
t
tYP* t
0.300
type 2
(Ypc 3
c
VP+*
type I
V,,
km”)
10d
T&r-
0.200
0.100
ll!L
16d
20d
Oh 2Lh
Id
TEXT FIG. 2. VvL pro canalicular
type; VVL pro group.
DE WOLF-PEETERS
350
ET AL.
Svc I VVL lm2 /cm3 1 I
40 I\;:: S,,/V,,
(m*/cm3)
30
/
20
10
16d
TEXT
20d
Oh 21h
Id
10d
.dult
FIG. 3. Sv~/Vvr, pro canalicular type; SVC/VVL pro group.
3 canaliculus as well as of the random sample of canaliculi at birth, both characterized by a very low surface/volume ratio. This morphologic substrate may be partly responsible for a secretory inefficiency of this type of lcanaliculus and of this period in the maturation. ACKNOWLEDGMENTS The authors are most grateful to Mrs. L. Seys-Tanghe, Mr. D. Gozin and Mr. M. Rooseleers for technical help, and to Mrs. S. Smets-Honsia for preparation of the manuscript.
REFERENCES V. (1972). Morphologic and histochemical aspects of cholestasis. In “Progress in Liver Diseases” (H. Popper et ~2. Ed.), Vol. IV, p, 97. Grune & Stratton, New York. DE WOLF-PEETERS, C., DE Vos, R., and DESMET, V. ( 1971). Histochemical evidence of a cholestatic period in neonatal rats. Pediut. Bes. 5, 704-709. DE WOLF-PEETERS, C., DE Vos, R., and DESMET, V. (1972). Electron microscopy and histochemistry of canalicular differentiation in fetal and neonatal rat liver. Tissue & CeZZ 4, 379-388. MILLONIG, G. ( 1961). Advantages of phosphate buffer for OsOn solutions in fixation. J. Appl. DESMET,
Physics 32,1637. REYNOLDS, E. S.
(1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. BaoZ. 17, 208-212. Rorm, H. P., and RIEDE, U. N. (1973). Experimental metabolic disorders and the subcellular reaction pattern. Current Topics in Pathol. 58, 1. SCXENI(ER, S. (1963). Deposition of bilirubin in the fetus and the newborn. Ann. N.Y. Acad. sci. III, 303-306. TORO, I., -and Joo, G. (1966). An aldehyde mixture as a fixative for the preservation of both fine structure and acid phosphatase activity. Acta BioZ. Acad. Sci. Hungary 17, 265-279. WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy meals. J. Biophys. Biochem. CytoZ. 4, 475-478. WEIBEL, E. ( 1969). Stereological principles Znt. Rev. CytoZ. 26, 235-302.
for morphometry
in electron microscopic cytology.
AND
MOLECULAR
21,
PATHOLOGY
339-350
(1974)
Electron Microscopy and Morphometry of Canalicular Differentiation in Fetal and Neonatal Rat Liver1 C. DE WOLF-PEETERS, Laboratorium Vniuersiteit
2 R. DE Vos, 2 V. DESMET,
UOOT Histochemie en Cytochemie, Leuven, 3000 Leuuen, Belgium, Electron Microscopy, Uniuersity Received
2 L. BIANCHI, 8 AND H. P. ROHR3
Academisch Ziekenhuis St. Rafad, Katholieke 2 and Institute of Pathology, Department of of Basel, Basel, Switzerland 3
February
X,1974
In order to complement previous electron microscopic studies on the differentiation of the biliary pole of the rat hepatocyte during perinatal maturation, a morphometric analysis was undertaken of the bile canaliculi in fetal and adult rat liver. Four morphologic canalicular types were found and morphometrically characterized using a surface/volume relation (s/v index) as key parameter. The surface/volume relation of the canaliculus in fetal and postnatal maturation as well as in the adult rat liver supports the previously formulated hypothesis that these different types correspond to successive maturation stages of the biliary pole of the hepaocyte. Furthermore, the s/v index of type 3 canaliculus supports the concept that this canalicular type represents a canalicular structure inefficient for secretion.
INTRODUCTION In a previous study (De Wolf-Peeters et al., 1972), the differentiation of the biliary pole of the rat hepatocyte was studied during late fetal and early neonatal development in order to identify a morphologic substrate for the well-known immaturity (De Wolf-Peeters et al., 1971; Schenker, 1963) of the bile secretory function in this period. Four different prototypes of canaliculi have been distinguished; it was suggested that they represent successive stages during the maturation of the biliary pole of the liver cell. This paper deals with the results of a morphometric analysis of the biliary pole of fetal and neonatal rat liver cells, which was undertaken to provide an objective and quantitative basis for the previously formulated hypothesis. MATERIAL
AND METHODS
Rats of Albino Wistar strain were obtained commercially. During the study all rats were fed a laboratory stock diet and tap water ad E&turn. Female rats were matched with males overnight and examined the next morning for the presence of sperm in the vaginal smear. The day of observation of sperm was considered day 1 of pregnancy. The fetuses were obtained by successive Cesarean section performed on mother rats under a constant flow ether anesthesia. 1 This Onderzoek
work was supported of Belgium.
by a grant
from
the
339 Copyri ht 0 1974 by Academic Press, Inc. AU rig i: ts of reproduction in any form reserved.
Fends
voor
Wetenschappelijk
Geneeskundig
DE WOLF-PEETERS
340
ET AL.
Twenty-three fetuses and newborn rats as well as 3 adult male rats (weight 150 g) were studied. These animals were divided into 7 different groups: rats of 16 days of fetal age; rats of 20 days of fetal age; rats at birth; rats 24 hr after birth; rats 4 days after birth; rats 10 days after birth, and adult rats. The liver was removed quickly after decapitation of the animal. A tissue specimen of the right half of the median liver lobe was taken, cut into thin slices and fixed by immersion for 2 hr in cold (4°C) 6% glutaraldehyde and 3.04% formaldehyde cacodylate buffer pH 7.2 (Toro et al., 1966) followed by buffer rinse overnight. A post fixation was carried out in 1% Os04 in phosphate buffer for 1 hr at 4°C (Millonig, 1961), folIowed by dehydrating in graded alcohols and embedding in Epon. Morphometric methods
One liver specimen was taken from at least 3 animals per group. From each of these biopsies, 5 blocks were used for preparation of ultrathin sections, which were examined after staining with uranyl acetate (Watson, 1958) followed by lead citrate (Reynolds, 1963). From each experimental group 3040 micrographs were taken at random at a primary magnification of 9.500 with an electron microscope Zeiss EM 9, and enlarged together with a double lattice test screen ( Weibel, 1969; Fig. 5). In addition, in order to characterize the different canahcular types by morphometry, 3040 micrographs were taken of each prototype (from the different groups) and processed as described for the groups. To determine the volume density of the bile canalicular lumen the fine test points lying inside the ‘canalicular lumen (1089 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. 6). Calculations and statistical analysis (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
The criterion
applied in this as well as in previous studies (De Wolf-Peeters is its limitation by two or three junctional complexes which separate it from the rest of the intercellular space. According to their morphologic appearance, the canaliculi observed in all liver specimens studied can be divided into 4 principal types (De Wolf-Peeters et al., 1972) : Type 1 canaliculus can be defined as an intracytoplasmic invagination of two adjacent cell membranes into one of the two neighboring hepatocytes (Fig. 1). et al., 1972) to accept a structure as “canalicular”
Type 2 canaliculus is provided with a lumen, which is irregular in form and
often partly filled with a clump of electron-dense material, interpreted as a fragment of sequestrated cytoplasm (Fig. 2). Type 3 canaliculus is characterized by a wide but regular lumen, around
FETAL
AND NEONATAL
BAT LIVER
341
FIG. 1. Bile canaliculus type 1. X 36,800
which microvilli are scanty or even absent. An amorphous electron-dense material may be found in such a lumen (Fig. 3). Type 4 canaliculus has a smaller lumen, virtually filled with numerous microvilli. Its morphologic characteristics correspond to those of normal adult canaliculi ( Fig. 5 ) .
DE WOLF-PEETERS
342
FIG. 2. Bile
canaliculus
type
ET AL.
2. X 36,800
Trarwiticd
forms between types 3 and 4 are also found. These canaliculi show a widened regular lumen similar to type 3, ,but a variable length of the surrounding cell membrane is provided with regular microvilli (Fig. 4). Depending on the age of the animal, one type of canaliculus is found in strikingly higher number than the other ones, although some other canalicular types may be found in one and the same specimen. From 16 until 19 days of fetal age most canaliculi are of type 1. During the following days canaliculi type 2 become more numerous. From the 21st day on, canaliculi type 3 appear. Just before and after birth most canaliculi are of type 3. Twelve hours after birth many transitional forms between types 3 and 4 can be seen, Their number decreases from the 3rd day on, while canaliculi type 4 are found in increasing numbers.
For results see Tables I-III
and Text Fig. l-3. DISCUSSION
The different canalicular types previously described by simple morphology and histochemistry during maturation of the fetal and neonatal rat liver (De Wolfparameters. Peeters et al., 1972) have been investigated by morphometric As shown in Table II(a) each canalicular type reveals a proper mean volume
FETAL AND NEONATAL
FIG.
3.
RAT LIVER
Bile canaliculus type 3.
X
343
36,800
whereby the very high figure for type 3 canaliculus is outstanding. In addition, the different types of canaliculi are fairly well distinguishable by their surface values. Primary parameters, their standard deviation and significances are given in Tables I-III, The fact, that the mean Ic value of type 3/4 (Table I a) shows no significant difference from that of types 3 and 4, respectively, supports the
DE WOLF-PEETERS
344
FIG.
ET AL.
4. Bile canaliculus type 3-4. X 36,800
previously advanced hypothesis that this ~canalicular type represents a transitional form between types 3 and 4. By characterizing the canalicular types by means of their surface/volume ratio (Table IIa) a different value valid for types 2, 3, and 3/4 is obtained. The fact that type 1 and type 4 give similar values with respect to surface/volume ratio
FETAL
AND NEONATAL
FIG. 5. Bile canaliculus
type
RAT LIVER
4.
345
x 36,800
may be explained by their different irregular three-dimensional configuration (compare Fig. 1 with Fig. 4), as seen from the individual secondary parameters for surface and volume. The low surface/volume value typical for type 3 canaliculus by itself represent a very unfavorable morphologic configuration for secretory function. This morphometric finding in type 3 canaliculus might be part of an explanation for an excretory inefficiency of this canalicular type. In fact, canaliculus type 3 is considered to be typical for cholestatic conditions in man and animals ( Desmet, 1972). By morphometric analysis one can observe distinct periods during the maturation of fetal rat liver according to a changing surface/volume ratio of randomly chosen samples of canaliculi. In this respect, the fetal liver at 16 days of gestational age is quite different from the rat liver at birth and the adult liver. At the 16th gestational day the surface/volume ratio is very close to that of canaliculus type 1, predominantly found in this period. It corresponds also to type 4 for the reasons explained above. The close relationship of the surface/volume ratio on the 16th gestational day with that of canaliculus type 1 supports the concept that most of these canaliculi represent newly forming biliary poles. At birth and 24 hr after birth a very low surface/volume ratio is found. This may be easily explained by the predominance of type 3 canaliculi during this period.
346
FIG. 6. Bile canalicuhs
DE WOLF-PEETERS
type 3-4 with superimposed
ET AL.
double lattice test screen. X 15,000.
Adult rat liver also shows a quite typical surface/volume ratio in its canalicular pattern. The finding of a lower surface/volume value in the adult liver than that of type 4 canaliculus correlates with the observation that also in adult liver different canalicular types may be present, although canaliculus type 4 predominates. The periods corresponding to the 20th fetal day, the 4th day and 10th day after birth do not show a distinguishable canalicular surface/volume ratio. These morphometric results also fit in with the previously reported morphologic observation that in these periods one canalicular type is progressively being replaced by another one. At 20 days of fetal age not all canaliculi are of type 1, but type 2
FETAL
AND
NEONATAL TABLE
PRIMARY
DATA
BAT
347
I
OF MORPHOMETRIC EVALUATION POLE OF THE HEPATOCYTE A Types
LIVER
OF THE BILIARY
of Canaliculi IC
Type Type Type Type Type
1
2 3 2 4
PPL
?a
SE
e&
16.85 26.03 36.53 42.07 39.85
1.97 2.75 3.5 4.21 5.88
0.007 0.037 0.167 0.071 0.018
B Groups
According
Ic
PPL
54.18 22.65 25.6 70.36 41.94 22.05 21.82
TABLE SECONDARY
SE
‘rii
5.77 2.4 2.7 6.73 2.55 2.32 2.1
0.039 0.024 0.025 0.262 0.127 0.039 0.009
Type Type Type
0.007 0.037 0.167 0.071 0.018
3 2 4
B Groups
Adult rat 10 days postnat. 4 days postnat. 24 hr after birth Birth 20 days fetal age 16 days fetal age
0.005 0.002 0.006 0.056 0.020 0.007 0.002
OF THE
of Canaliculi svc (m*/cm”)
Type 1 Type 2
SE
II
PARAMETERS OF MORPHOMETRIC EVALUATION BILIARY POLE OF THE HEPATOCYTE A Types
0.001 0.004 0.025 0.014 0.003
to Age
?a
Adult rats 10 days postnat. 4 days postnat. 24 hr after birth Birth 20 days fetal age 16 days fetal age
SE
According
0.323 0.499 0.701 0.807 0.764
svclTvvL
(mz/cm3) 43.303 13.282 4.183 11.354 41.721
to Age SVC/vVVL
VVCL
svc
(cm’)
(mz/cm3)
(ma/cm*)
0.039 0.024 0.025 0.262 0.127 0.039 0.009
1.039 0.434 0.491 1.35 0.804 0.423 0.418
26.417 17.526 19.417 5.137 6.321 10.733 42.387
Adult rat 10 days postnatal 4 days postnatal 24 hr after birth Birth 20 days fetal age 16 days fetal age
Type 1 Type2 Type 3 Type2 Type4
S S S S S S
IC
-
S S
ll.S.
KS.
lLS.
S
S
-
S S S S S
S
PPL
10 d p.n.
IO
KS.
S S S S
PPL
S S S S
PPL
adult rat
-
Type 1
S S D.S. S
IC
S S S
S
Ic
S S S
S
PPL
LS.
n.s.
S S n.s. S
ll.S.
S
PPL
4 d p.n.
S S n.s. ns.
S ns.
Ic
T-test)
S S
S S S
S S S -
S S
Jl.S.
S S S
24 h p.n. IC PPL
-
S S
PPL
T-test)
Type 3 S S
Ic
B Significance (Student’s
-
Type2
TABLE III A Significance (Student’s
S S
S S S S
Ic
-
birth
R.S.
S S
S S S n.6.
‘PPL
-
TYPf n.8. S n.s.
Ic
S
S S S
PPL
n.s.
-
S
S S
I1.8.
S
LS.
PPL
20 d f.a.
n.s. S S
n.s.
S
IC
-
Type4 S S n.s. n.s.
IC
S n.s. n.s. S S n.s.
-
S S S S S S
PPL
16 d f.a.
S S S S
IC
PPL
F
2
E!
3 G 2 2
iii
FETAL
AND NEONATAL
RAT LIVER
349
Svc (m2/cm3)
I 0.600 0.400 0.200
type 1
type 2
type 3
type+&
S,,
type
L
(m2/cm3
1
1.500 c
L/L J
0.500
It 16d
ItI 20d Oh 2l.h
> 4d
TEXT FIG. 1. SVCpro canalicular
X)d "
adult
type; SVCpro group.
canaliculi are found as well as type 3, as exemplified by the comparison of group values with type values. Four as well as 10 days after birth canaliculi of types 3, 314, and 4 are found by conventional ultrastructural examination. This finding is confirmed by morphometric values given in Text Fig. 3. In conclusion, the results of this morphometric analysis of the biliary pole in fetal, neonatal and adult rat liver support the previously reported findings and advanced hypothesis that the different types represent successive maturation stages. The present findings furthermore suggest a structural inadequacy of type
0.150 -
0.100 O.lOOt 0.050 0.050-
I-
t
tYP* t
0.300
type 2
(Ypc 3
c
VP+*
type I
V,,
km”)
10d
T&r-
0.200
0.100
ll!L
16d
20d
Oh 2Lh
Id
TEXT FIG. 2. VvL pro canalicular
type; VVL pro group.
DE WOLF-PEETERS
350
ET AL.
Svc I VVL lm2 /cm3 1 I
40 I\;:: S,,/V,,
(m*/cm3)
30
/
20
10
16d
TEXT
20d
Oh 21h
Id
10d
.dult
FIG. 3. Sv~/Vvr, pro canalicular type; SVC/VVL pro group.
3 canaliculus as well as of the random sample of canaliculi at birth, both characterized by a very low surface/volume ratio. This morphologic substrate may be partly responsible for a secretory inefficiency of this type of lcanaliculus and of this period in the maturation. ACKNOWLEDGMENTS The authors are most grateful to Mrs. L. Seys-Tanghe, Mr. D. Gozin and Mr. M. Rooseleers for technical help, and to Mrs. S. Smets-Honsia for preparation of the manuscript.
REFERENCES V. (1972). Morphologic and histochemical aspects of cholestasis. In “Progress in Liver Diseases” (H. Popper et ~2. Ed.), Vol. IV, p, 97. Grune & Stratton, New York. DE WOLF-PEETERS, C., DE Vos, R., and DESMET, V. ( 1971). Histochemical evidence of a cholestatic period in neonatal rats. Pediut. Bes. 5, 704-709. DE WOLF-PEETERS, C., DE Vos, R., and DESMET, V. (1972). Electron microscopy and histochemistry of canalicular differentiation in fetal and neonatal rat liver. Tissue & CeZZ 4, 379-388. MILLONIG, G. ( 1961). Advantages of phosphate buffer for OsOn solutions in fixation. J. Appl. DESMET,
Physics 32,1637. REYNOLDS, E. S.
(1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell. BaoZ. 17, 208-212. Rorm, H. P., and RIEDE, U. N. (1973). Experimental metabolic disorders and the subcellular reaction pattern. Current Topics in Pathol. 58, 1. SCXENI(ER, S. (1963). Deposition of bilirubin in the fetus and the newborn. Ann. N.Y. Acad. sci. III, 303-306. TORO, I., -and Joo, G. (1966). An aldehyde mixture as a fixative for the preservation of both fine structure and acid phosphatase activity. Acta BioZ. Acad. Sci. Hungary 17, 265-279. WATSON, M. L. (1958). Staining of tissue sections for electron microscopy with heavy meals. J. Biophys. Biochem. CytoZ. 4, 475-478. WEIBEL, E. ( 1969). Stereological principles Znt. Rev. CytoZ. 26, 235-302.
for morphometry
in electron microscopic cytology.