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Ultrastructure of gallstones produced in mice fed a high cholesterol diet.

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Ultrastructure of Gallstones Produced in Mice Fed a
High Cholesterol Diet
L. c. SALAND AND L. M . NAPOLITANO
Department of A n a t o m y , University of N e w Mexico School of Medicine,
Albuquerque, New Mexico 87131
ABSTRACT
Mice fed a high cholesterol-cholic acid diet for two to six months
develop gallstones; these were studied by transmission electron microscopy after
glutaraldehyde-digitonin fixation. Examination of the contents of mouse gallbladders presents views of layered structures and surrounding amorphous material. We interpret these images of gallstones to suggest that they may arise
by cohesion of material rich in cholesterol to form more ordered structures. Gallbladder contents of mice fed the diet for five to six months were found to contain
occasional crystals and rectangular areas similar to those observed in thin sections of human gallstones (unpublished observations). Recent findings that human
gallstones can be dissolved with chenodeoxycholic acid are discussed, with reference to their applicability to studies of gallstones in mice.
The formation of gallstones of cholesterol
has been widely investigated i n humans
(Small, '70; Redinger and Small, '72;
Small and Rapo, '70). Some studies indicate that abnormal hepatic bile rich in
cholesterol and low in bile acids is a
primary factor in providing an environment
for precipitation and ultimate cholesterol
stone formation (Small and Rapo, '70;
Lahana and Schoenfield, '73). Other investigations, using animal models such as
the mouse (Tepperman et al., '64; Caldwell et al., '65; Caldwell and Levitsky,
'67), hamster (Wheeler et al., '73; Robins
and Fasulo, '73), prairie dog (Brenneman
et al., '72) and monkey (Osuga and Portman, '72), have found that feeding high
cholesterol or lipid diets for extended periods
will induce formation of gallstones and/or
cause changes in biliary ratios of cholesterol to bile acids, thereby producing a
bile supersaturated with cholesterol. Although physiological studies of stone formation are abundant, ultrastructural studies
of stones are relatively rare. Ogata and
Murata ('71) have observed human gallstones with the scanning electron microscope, and have suggested that stones develop by laying down of cholesterol crystals
from the center to the periphery. Additional scanning electron microscopic studies in humans (Wolpers and Blaschke,
'71; Osuga et al., '75) and in monkeys
(Osuga et al., '74) reveal similarities between gallstones of both species.
ANAT. REC., 187: 207-218.
The present investigation utilizes transmission electron microscopy for visualization of gallstones produced in mice fed a
high cholesterol diet. Thin sections reveal
details of forming stones which are not
readily apparent with scanning methods. A
portion of the findings has been previously
reported in abstract (Saland and Napolitano, '75).
MATERIALS A N D METHODS
Adult female mice (25 gm) were fed a
diet containing 1 % cholesterol and 0.5 %
cholic acid (Nutritional Biochemicals,
Cleveland, Ohio) as described by Tepperman
and co-workers ('64). Animals were anaesthetized with ether after being fed the diet
for two to six months. Gallbladder contents
were withdrawn with a syringe. Some gallbladders were first injected with fixative
(see below) before being removed. A number of animals was perfused through the
heart with saline followed by buffered fixative before gallbladders were injected and
removed. Contents of gallbladders from
three to six mice were pooled, immersed
in fresh fixative, and spun to a pellet with
a laboratory centrifuge. Pelleted contents
were prepared for electron microscopy
as follows: Pellets were fixed in 2 . 5 %
glutaraldehyde in 0.15 M cacodylate/HCl
buffer for 24 hours (at room temperature),
then f-med for a n additional 24 hours in
Received July 9, '76. Accepted Aug. 25, '76
207
208
L. C. SALAND AND L. M. NAPOLITANO
the same fixative plus 0.2% digitonin. The
digitonin was purified before use by reaction with cholesterol, followed by a recovery procedure according to methods modified from Sperry (’63). Preparation of the
glutaraldehyde-digitonin fixative has been
previously described (Napolitano and Scallen, ’69). Spun pellets were rinsed in buffer and postfixed for one to two hours
in 1% osmium tetroxide in 0.15 M cacodylate/HCl buffer. Some pellets were not
postfixed or stained with heavy metals
prior to examination. All specimens were
rapidly dehydrated in graded ethanols ( 3
minutes each change), and embedded in
Epon 812. Use of digitonin in fixation followed by rapid dehydration has been shown
to preserve up to 85% cholesterol in rat
sciatic nerve (Sterzing and Napolitano,
’72). Thin sections were obtained with a
Cambridge Huxley ultramicrotome using
a Dupont diamond knife. Sections were examined and photographed in a Hitachi
HS-7S or Philips 200 electron microscope.
In addition to preparation for electron
microscopy, pelleted material of four animals on the diet for two months was suspended in cold cacodylateIHC1 buffer.
Free cholesterol and cholesterol esters were
determined by the method of Bowman and
Wolf (‘62). A sample of the suspended pellets was extracted of 99% of total cholesterol using absolute ethanol. After extraction, the same pelleted material was
processed for electron microscopy as described above.
OBSERVATIONS
Mice kept on a high cholesterol-cholic
acid diet for two to six months develop
small white calculi (up to 0.5 mm) within
the greatly distended and enlarged gdlbladder. They also exhibit extensive fatty
infiltration of the liver, a condition noted
in earlier work (Tepperman et al., ’64).
Electron micrographs of fixed gallbladder
contents from animals fed the diet for two
months reveal layered structures (figs. 13), and considerable “amorphous” material (figs. 2 , 4). Some areas exhibit small
rounded substructures (“S’ in fig. 3 )
which appear to be isolated from larger
layered areas. Small regions of layered material are most often seen located within
moderately dense “amorphous” or fuzzy
areas (fig. 4). Such “amorphous” material
may also be present alone.
In nearly all of the figures, it is apparent that structured areas are composed of
layers which are varied in electron density.
Unstained sections, or those taken from
blocks which were not post-osmicated,
were similar in appearance of layered structures to sections prepared conventionally.
It is possible that the non-homogenous appearance reflects differences in chemical
composition of various portions of the
structures.
Chemical analysis of pelleted gallbladder
contents after two months of high cholesterol-cholic acid diet reveal that there is
approximately four times as much free
cholesterol as compared to esterified sterol
(in absolute terms, 36 2 0.14 p g of free
cholesterol per mg dry weight versus
10 f 0.07 p g of esterified cholesterol).
Electron microscopy of pelleted material
after extraction of 99% of total cholesterol indicates that layered structures observed in unextracted samples are present,
but may appear disrupted, with empty
spaces in the centers (fig. 6 and inset).
The amorphous material does not appear
altered.
Close examination of larger layered structures within fixed pellets also reveals areas
of needle-like “spicules” or whorls in their
centers (fig. 3 ) . The “spicules” are believed
to be artifacts caused by the presence of
digitonin i n fixatives (Williamson, ’69;
Fruhling et al., ’71; Napolitano et al., ’72)
and will be discussed below.
Gallbladder contents from mice kept
on the high cholesterol diet for five to six
months contain ultrastructural features
similar to those observed in animals sacrificed at earlier intervals. In addition, occasionalrectangular, hexagonal or parallelogram-shaped objects (fig. 5 and inset) are
observed in thin sections after the longer
feeding interval. Their appearance is suggestive of crystals, presumably rich in
cholesterol.
Mice replaced on a regular diet after
several months of high cholesterol diet
appear to revert to a normal condition. The
livers and gallbladders of such animals
appear much the same as those of routinely fed animals.
DISCUSSION
Recent clinical studies of cholelithiasis
in humans, particularly i n gallstone-prone
American Indians, have led to the hy-
ULTRASTRUCTURE OF MOUSE GALLSTONES
pothesis of abnormal biliary ratios of cholesterol to bile acids as a prime cause of
stone formation (Small, ’70; Redinger and
Small, ’72; Grundy et al., ’72). The physiological state of the gallbladder (i,e., its
contractibility and rapidness of emptying)
must also be considered as a factor in providing the proper environment for initial
cholesterol precipitation. Vagotomy in
animals and man may be followed by
gallstone formation (Cowie and Clark, ’72;
Hopton, ’73) via decreasing tone of the
gallbladder muscular wall. In addition, it
has been suggested by Cussler and coworkers (‘70) that incomplete emptying
of a sluggish gallbladder may allow microcrystals of cholesterol to remain near the
epithelium, ultimately to precipitate into
stones.
A high cholesterol-cholic acid diet will
produce gallstones in nearly 100% of mice
fed about two months, with female mice
more prone to cholelithiasis than male
mice (Tepperman et al., ’64). Such animals
have been shown in the Tepperman study
to have greatly increased bile cholesterol
concentrations as compared to controls (approximately 6 to l), which may account for
ultimate cholesterol precipitation. Thin sections of gallbladder contents from such
mice reveal layered structures which are
reminiscent of geodes (nodules of stone
having cavities lined with crystals). They
are usually surrounded by non-structured
material. In addition, we have observed
small, rounded structures which appear
attached to the larger ones. Such observations may suggest a “building up” process
of layered structures from less ordered material. Amorphous material may also contain substances which aid in stone formation. Chemical analysis of mouse gallbladder pellets shows the bulk of cholesterol to be in the free form.
The variations observed in electron
density of layers of gallbladder contents are
striking, and are considered to reflect differences i n composition within them. Extraction of up to 99% of total cholesterol
from pelleted material followed by electron
microscopy often leaves empty or partly
empty spaces in the centers of layered
structures. It may be assumed that much
cholesterol resides in such areas. In support of this view is the fact that during
growth of human gallstones, changes in
cholesterol composition have been demon-
209
strated (Sutor and Wooley, ’74). However,
how chemical or physical differences may
arise in stone formation is unknown. Multiple factors, such as variation in bile flow,
the physiological state of the gallbladder,
and composition of bile, must surely play
roles in the appearance of layered structures
in thin sections.
Sections of gallbladder contents of mice
fed the diet for up to six months reveal
occasional “crystals” in a variety of shapes.
Such structures are thought to be rich in
cholesterol. We have also observed empty
areas where such crystals may have pulled
out of the plastic sections. Sections of
“crystals” from such animals appear similar to thin sections of human gallstones
(unpublished observations). Scanning electron microscopic studies of human gallstones (Osuga et al., ’75) reveal hexagonal
as well as rectangular shaped crystals,
similar to these observed here in the
mouse. Our findings suggest that most
of the material found in gallbladders of
experimental mice represents an early
stage in gallstone formation. Layered structures formed after shorter periods on the
high cholesterol diet do not have the
“lacy” appearance of stones observed with
scanning electron microscopy (Ogata and
Murata, ’71; Wolpers and Blaschke, ’71;
Osuga et al., ’75), and may therefore differ from gallstones observed in those investigations. Olszewski and co-workers
(‘73) have noted the presence of liquid
crystals with a multilayered appearance
in human bile, and suggest that the latter
may be an additional phase of cholesterol
in bile besides crystals and micelles. It
may be that the early “stones” observed
in the mouse are a type of liquid crystal.
Buildup of larger structures from tiny
aggregates, suggested here, does appear
consistent with hypotheses of stone formation described in scanning studies (Ogata
and Murata, ’71; Wolpers and Blaschke,
’71; Osuga et al., ’75). Our preliminary
observations of human stones suggest a
semi-ordered, often crystalline appearance,
which may be the result of very long-term
buildup processes.
In the mouse material, the centers of
some layered structures also contain “spicules” and other artifacts which are associated with the use of digitonin in fixatives (Williamson, ’69; Friihling et al.,
’71; Napolitano et al., ’72). These artifacts
210
L. C . SALAND AND L. M. NAPOLITANO
are suggested to be aggregates of cholesterol-digitonin plus phospholipids and protein (Fruhling et al., '71), and are not considered by us to be exclusive sites of cholesterol-digitonide formation, as they have
been by some authors (Williamson, '69;
Scharnbeck and Schaffner, '70; Albert
and Rucker, '75). It is interesting to note
that the artifacts are removed after extraction of cholesterol, which may indicate
that digitonin in fixatives will not stabilize
material after long-term solvent treatment.
Accurate localization of cholesterol is desirable in gallstone preparations, and autoradiographic investigations using ["] digitonin (Napolitano et al., '72) are in
progress.
It was previously observed (Caldwell et
al., '65) that stones formed in mice fed
high cholesterol-cholic acid diets will dissolve if the animals are replaced on normal
diets, a finding confirmed in our laboratory. Very recently, human gallstones have
been dissolved by feeding patients chenodeoxycholic acid (Thistle and Schoenfield,
'71; Bell et al., '72; Gerulami and Muntet,
'73; Thistle and Hoffinan, '73). In addition, Yellin and co-workers, ('73) have
found that mice do not develop gallstones
when fed diets containing cholesterol and
chenodeoxycholic acid rather than cholic
acid despite reduced bile acid to cholesterol
ratios of hepatic bile in animals fed either
substance. It would be of interest for future
studies to observe effects of chenodeoxycholic acid on stones already present in
the mouse model used here.
ACKNOWLEDGMENTS
This work was supported by NIH Grant
AM09432-12. We are grateful to Doctor
J . V. Scaletti for advice and assistance with
the chemical analyses. We thank also Miss
Judi DeLongo and William Howell for
expert technical support.
LITERATURE CITED
Albert, E. N., and R. D. Rucker 1975 Electron
microscopic demonstration of cholesterol i n
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527.
Bell, G. D., B. Whitney and R. H. Dowling 1972
Gallstone dissolution in man using chenodeoxycholic acid. Lancet 11, 7789: 1213-1216.
Bowman. R. E.. and R. C. Wolf 1962 A raDid
and specific ultramicromethod for total seAm
cholesterol. Clin. Chem., 8 .302-309.
Brenneman, D. L., W. E. Connor, E. L. Forker
and L. DenBesten 1972 The formation of abnormal bile and cholesterol gallstones from
dietary cholesterol in the prairie dog. J. Clin.
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Caldwell, F. T., Jr., and K. Levitsky 1967 Gallbladder and gallstone formation. Ann. Surg.,
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Caldwell, F. T., Jr., K. Levitsky and B. Rosenberg
1965 Dietary production and dissolution of
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Cowie, A. G. A., and C. G. Clark 1972 The
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Cussler, E. L., D. F. Evans and R. G. DePalma
1970 A model for gallbladder function and cholesterol gallstone formation. Proc. Nat. Acad.
Sci., 67: 400407.
Friihling, J., W. Penasse, G. Sand and A. Claude
1971 Reactions de la digitonine avec le cholesterol et autres lipides de la corticosurrknale du
rat: ktude par microscopie klectronique. J . de
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1972 Mechanisms of lithogenic bile formation
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cholesterol in peripheral nerve: Use of 3H-digitonin for electron microscopic autoradiography.
Anat. Rec., 175: 157-164
Napolitano, L. M.,and T. J. Scallen 1969 Observations on the fine structure of peripheral
nerve myelin. Anat. Rec., 163: 1-6.
Ogata, T., and F. Murata 1971 Scanning electron microscopic study of cholesterol gallstones.
Tohoku. J. Exp. Med., 104:2544.
Olszewski, M. F., R. T. Holzbach and A. Saupe
1973 Liquid crystals in human bile. Nature,
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S. Kintaka and 0. W. Portman 1975 A scanning electron microscopic study of gallstone development in man. Lab. Invest., 31 : 696-704.
Osuga. T., and 0. W. Portman 1972 Relationship between bile composition and gallstone
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A. Alexander 1974 A morphologic study of
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composition, bile salt metabolism and gallstones.
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ULTRASTRUCTURE OF MOUSE GALLSTONES
Saland, L. C.,and L. M. Napolitano 1975 Transmission electron microscopy of diet-induced
mouse gallstones. Anat. Rec., 181: 470.
Scharnbeck, H., and F. Schaffner 1970 Electron
microscopic effects of digitonin in normal and
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Sperry, W. M. 1963 Quantitative isolation of
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21 1
man 1964 Induction of gallstones in mice by
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PLATE 1
EXPLANATION O F F I G U R E S
212
1
This electron micrograph illustrates layered structures of varied electron density. One layered structure exhibits additional smaller subunits i n its center. Small amounts of amorphous material surround the
structured areas (arrows). Mouse gallbladder contents. X 7,500.
2
An elongate, layered structure resembling a geode is illustrated. "Amorphous'' material (A) is present near the larger area. X 7,500.
ULTRASTRUCTURE O F MOUSE GALLSTONES
L. C. Saland and L. M. Napolitano
PLATE 1
213
PLATE 2
EXPLANATION OF FIGURES
214
3
This micrograph exhibits a large layered structure. Nearby are smaller
subunits ( S ) , some of which appear attached to the larger area. Other
subunits (S) lie within or near “amorphous” material (A). “Spicules”
(arrows) i n the center of the layered structure are digitonin-induced
artifacts (see text). Mouse gallbladder contents. X 12,000.
4
The larger figure illustrates an area of “amorphous” material containing structures similar to those in other illustrations. A n enlarged portion of a layered structure (inset) shows small dense subunits (S)
which appear attached to larger units. Mouse gallbladder contents.
Largefigure X 7,500. Inset X 18,500.
ULTRASTRUCTURE OF MOUSE GALLSTONES
L. C. Saland and L. M. Napolitano
PLATE 2
215
PLATE 3
EXPLANATION
5
OF FIGURES
A group of crystal-like objects is present i n this electron micrograph.
Note the variation in sizes of objects and amorphous material closely
applied to them. The inset shows a crystalline structure i n hexagonal
form. Mouse gallbladder contents, 5.5 months on diet. Figure 5 X
10,000. Inset X 5,400.
6
This electron micrograph illustrates portions of a pellet extracted for
99% of total cholesterol. Note empty areas within what were layered
structures. T h e inset (arrow) shows another area from the same specimen which h a s some material remaining within the center. Figure 6
X 20,000. Inset X 5,600.
216
ULTRASTRUCTURE O F MOUSE GALLSTONES
L . C . Saland and L. M. Napolitano
PLATE 3
217
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