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Electron and light microscopy of choline deficiency in the mouse; with special reference to six-hour hepatic liposis.

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Electron and Light Microscopy of Choline Deficiency
in the Mouse; with Special Reference to
Six-hour Hepatic Liposis'
ROLAND D. MEADER
Department of A n a t o m y , Lozrisiana State University School of Medicine,
N e w Orleans, Louisiana
In a previous investigation (Meader
and Williams '57) the authors reported
changes induced in the livers of mice maintained on a choline-deficient diet from 24
hours through 10 months. In this report
it was clearly demonstrated that fat accumulation proceeded rapidly as evidenced
by the appearance of stainable fat in the
form of very small cytoplasmic droplets
within the centro-lobular parenchymal
cells as early as 24 hours after restriction
to the choline-deficient diet. The lipid was
clearly demonstrated by staining frozen
sections with Sudan dyes, including Sudan
black and IV, and osmic acid. At this
time (24 hours of restriction) portal zones
of parenchyma were devoid of fat except
for a few cytoplasmic droplets in a minority of the cells. At the end of three days
of choline deficiency, fat was present as
cytoplasmic droplets in the parenchymal
cells of all of the lobular zones. A significant increase of fat was observed after 7
days of choline deficiency. The separate
small cytopalsmic droplets of fat then
fused to form large, single droplets which
in most instances completely N e d the parenchymal cells and displaced the nuclei
to one side. This process continued progressively during the next three weeks.
The present study has been made in an
attempt to make a more detailed cytological
observation on the initial deposition of fat
during the first 24 hours of the cholinedeficient diet, using methods additional to
those employed in the earlier study. Electron microscopy was used since it seemed
probable that submicroscopic lipohepatosis
is present in the liver some time before any
change can be detected and followed with
the light microscope.
MATERIALS AND METHODS
A. Diets, animals and
experimental groups
A diet (modified Gyorgy) that had previously produced hepatic lesions characteristic of choline deficiency in mice
(Meader and Williams, '57) was selected
for use in this study and has been designated as the basal diet in this report.
The composition of this diet is as follows:
gm
Vitamin free casein
Sucrose
Lard
Salt Mixture (no. 2, U.S.P. XIII)
I-cystine
Cod liver oil
Vitamin powder
80.0
480.5
380.5
40.0
5.0
4.5
10.0
The Vitamin powder contains:
Thiamine hydrochloride
Riboflavin
Pyridoxine hydrocholoride
Calcium pantothenate
Nicotinic acid
Powdered sugar
0.500
0.250
0.200
1.000
1.000
997.050
Mice ( 6 weeks of age with a weight
range of 10-12 gm) of the Canvorth Farm
strain were used. These mice were subjected to the following experimental procedures.
1. Basal (choline-deficient) diet. One
hundred and twenty-six female mice were
fed this diet. Three mice were killed every
15 minutes for the first 6 hours, and thereafter at hourly intervals through 24 hours.
2. Basal diet plus choline chloride. A
survey of 6 mice showed the 1.0% supplementation with choline to be adequate and
This study was supported by a research grant
from the National Institutes of Health ( A - 3 3 6 4 ) ,
and from the Medical Research Fund of the
Louisiana State University School of Medicine.
Department of Anatomy, Louisiana State University School of Medicine, New Orleans, Louisiana.
1
2
ROLAND D. MEADER
84 additional mice were fed the thus supplemented diet for 24 hours. Two mice
were killed every 15 minutes for 6 hours,
and thereafter at hourly intervals through
24 hours.
3. Choline therapy. Ten mice were fed
the basal (choline-deficient) diet for 7 days
and then received the same diet plus 1.O%
choline for three days and were killed. The
purpose of this group was to demonstrate
the lipotropic action of this level of choline
supplementation in mice that had received
the deficient diet for one week.
4. Controls. Twenty-four mice of the
Canvorth Farm strain were fed the standard diet of “Fox Chow.” One animal was
killed every hour through 24 hours.
B . Histologic Procedures
All animals were killed by compression
of spinal cord at the cervical level.
Tissues were prepared for electron microscopy by fixation in buffered osmium
tetroxide, dehydrated in graded ethyl alcohol solutions and embedded in N-butyl
methacrylate. Sections of approximately
0.02-0.05 IL were selected for examination
by using their interference colors to judge
their thickness. Electron micrographs
were made at original magnifications of
5000-9000 and enlarged photographically
to the desired size.
To determine the initial appearance of
cytoplasmic lipid demonstrable by staining methods in the hepatic parenchyma of
choline-deficient mice, as well as to facilitate interpretation of the electron micrographs, blocks of the same livers were fixed
for light microscopy in formalin and frozen sections were stained with Sudan dyes,
including Sudan black and IV, and osmic
acid. Routine hematoxylin-eosin preparations were also made.
OBSERVATIONS
Lipohepatosis uiith the light microscope.
It is generally agreed that stainable lipid
does not occur in hepatic parenchymal cytoplasm of rats (Glynn, ’47; Best, ’50) or
mice (Williams, ’51;Meader and Williams,
’57) fed adequate diets. This is true for
the stock of mice used here.
Livers of mice fed either the “Fox Chow”
diet or the choline supplemented diet contained no stainable lipid detectable by light
microscopy (fig. 1). Neither was there any
evidence of stainable lipid in the cytoplasm
of parenchymal cells during the first hour
of choline deficiency (fig. 2).
The initial centrolobular deposition of
stainable fat in the form of very small individual cytoplasmic droplets within the
parenchymal cells was first obvious during
the 4th hour of restriction to the cholinedeficient diet (fig. 3 ) . During the subsequent two hours there not only was a progressive increase of intracytoplasmic lipid
which was accomplished by formation of
larger globules or droplets as a result of
fusion of smaller globules but also the
centrolobular liposis spread in a peripheral
direction and usually involved most of the
lobular zones during the 6th hour of restriction to the choline-deficient diet (fig.
4). The least amount of fat was in the
parenchyma of the peripheral zones.
There appeared to be no specific centralperipheral intralobular gradient in the
quantity of lipid in the zonal cells, since
mid-zonal cells often contained more stainable lipid than central cells. The lipid was
clearly demonstrable by staining frozen
sections with Sudan dyes, including Sudan
black and IV, and by osmic acid.
Lipohepntosis as revealed by the electron microscope. The initial appearance
of intracytoplasmic lipid as revealed by the
light microscope during the 4th hour of
restriction to the choline-deficient diet, was
the determining point for the initiation of
the electron microscopic study, which was
undertaken, in order to see if submicroscopic lipid was present in the hepatic
parenchyma prior to its initial appearance
as revealed by light microscopy.
In the livers of mice fed either the standard laboratory ration of Purina Fox Chow
or the choline supplemented diet, occasional small cytoplasmic droplets of fat
were observed in a few of the parenchymal
cells with no confinement to any particular
zonal area of the lobule (fig. 5 ) .
In the livers of mice maintained on the
choline-deficient diet, electron microscopy
revealed the presence of submicroscopic
lipid in the cytoplasm of the centrolobular
parenchymal cells beginning with the first
hour after restriction to the choline-deficient diet (fig. 6 ) and increasing in quantity through the 4th hour (fig. 7 ) at .which
CHOLlNE DEFICIENCY I N THE MOUSE
3
Williams, '51) and cortisone (Williams
and Davis, '59).
Much attention has been directed to the
patterns of liposis in mice (Buckley and
Hartroft, '55; Meader and Williams, '57)
and rats (Hartroft and Ridout, '51; Hoffbauer and Wittenburg, '54) fed hypolipotropic diets.
It was originally concluded that initial
liposis begins centrally and ultimately
spreads in a peripheral direction eventually
involving the entire lobule when the choline-deficient diet (containing lard as fat)
was fed to rats (Hoffbauer and Wittenburg,
'54) and to mice (Meader and Williams,
'57).
In a previous study (Williams, Cardle
and Meader, '59) an initial and continuing
peripheral liposis was observed in mice
maintained on a choline-deficient diet in
DISCUSSION
which the dietary fat was a Cls-saturated
triglyceride or predominantly (50% of
The purpose of this study was to see if total dietary fat) a C6-saturatedtriglyceride.
electron microscopy could detect the pres- A similar pattern has been described in
ence of submicroscopic lipid in hepatic rats (Shils and Stewart, '54) fed cholineparenchyma before it could be revealed by deficient diets containing corn meal as the
light microscopy with no differentiation be- major source of protein. This type of
tween deposition of fat synthesized in vivo liposis does not substantiate the original
in mice fed an adequate diet from that sup- explanation that the fairly broad susceptiplied by the basal (choline-deficient) diet. bility of centrolobular cells to a variety of
The electron micrographs of the present injurious agents and conditions is due to
investigation indicate that submicroscopic a regional deficiency of essential bloodlipid was present in the hepatic paren- borne elements in central zones because of
chyma of normal, well-nourished animals prior utilization of such materials in the
and in the experimental animals three more peripheral portions of the lobules
hours prior to its initial appearance as re- (Deane, '44; Himsworth, '47; Williams,
vealed by the light microscope.
'51). It seems clearly demonstrated that
Submicroscopic pattern of liposis. The the intralobular pattern of hepatic liposis
initial centrolobular appearance and subse- is controlled to a considerable degree by
quent general distribution of hepatic liposis the chemical composition of the lipid comobserved here is identical with that de- ponents of the choline-deficient diets (Wilscribed in rats (Best and Huntsman, '32; liams, Cardle and Meader, '59).
MacLean and Best, '34; Gyorgy and Goldblatt, '39, '42, '49; Lillie, Ashburn, Sebrell,
SUMMARY
Daft and Lowry, '42; Hartroft, '50), in
mice (Meader and Williams, '57) and in
1. Electron microscopy revealed the
guinea pigs (Casselman and Williams, presence of submicroscopic lipid in the
'54 ) .
centrolobular hepatic parenchyma of mice
An initial and immediate centrolobular three hours before its initial appearance
liposis is a frequent response of hepatic was revealed by light microscopy.
parenchyma of several species including
2 . The initial centrolobular liposis
mice to a variety of insults including the which involved most of the lobular zones
action of choline-deficient diets (Meader at the end of 6 hours of choline deficiency,
and Williams, '57), starvation (Williams, was observed in mice fed a choline-deficient
'5 1 ) , polyhalogens (Stowell and Lee, '50 j diet containing lard as the lipid component,
time it was first detectable by light microscopy. During the subsequent two hours ( 6
hours of restriction) there was a progressive increase of intracytoplasmic lipid in
the cells of the hepatic lobule (fig. S ) , and
the submicroscopic pattern resembled that
observed by the light microscope. A characteristic feature observed only in many of
the smaller globules of lipid was the presence of a dense component or inclusion
body (fig. 6 ) which appeared as a small
rod somewhat similar in appearance to the
dense components or "crystalloid structures" described in the eosinophilic granules of normal human blood cells (Low
and Freeman, '58). Whether or not these
structures have any functional significance in the initial formation of fat globules awaits further investigation.
4
ROLAND D, MEADER
LITERATURE CITED
Best, C. H. 1950 Protection of livers and kidneys by dietary factors. Choline and its precursors as lipotropic agents. Fed. Proc., 9:
506-511.
Best, C. H., and M. E. Huntsman 1932 The
effects of the components of lecithin upon the
distribution of f a t in the liver. Am. J. Physiol.,
75: 405-412.
Buckley, G . F., and W. S . Hartroft 1955 Pathology of choline deficiency in the mouse.
Arch. Path., 59: 185-197.
Casselman, W. G. B., and G. R. Williams 1954
Choline-deficiency in the guinea pig. Nature,
173: 210-211.
Deane, H. W. 1944 A cytological study of storage and secretion in the developing liver of
the mouse. Anat. Rec., 88: 161-173.
Glynn, L. E. 1947 Production and modification
of liver disease by diet. Nutr. Abstr. Rev., 16:
751-762.
Gyorgy, P., and H. Goldblatt 1939 Hepatic injury on a nutritional basis in rats. J. Exp. Med.,
70: 185-192.
1942 Observations on the conditions of
dietary hepatic injury (necrosis, cirrhosis) i n
rats. Ibid., 75: 355-368.
1949 Further observations on the production and prevention of dietary hepatic injury in rats. Ibid., 89: 245-268.
Hartroft, W. S. 1950 Accumulation of fat i n
liver cells and in lipodiastaemata preceding experimental dietary cirrhosis. Anat. Rec., 106:
61-87.
Hartroft, W. S., and J. H. Ridout 1951 Pathogenesis of the cirrhosis produced by cholinedeficiency. Escape of lipid from fatty hepatic
cysts into the biliary and vascular systems. Am.
J. Path., 27: 951-989.
Himsworth, H. P. 1947 Derangements of the
hepatic circulation in disease. Pub. of Josiah
Macy, Jr. Foundation, Trans. Sixth Liver Injury Conference, pp. 73-74.
Hoffbauer, F. W., and B. Wittenburg 1954 Dietary hepatic necrosis in the rat. Absence of
cirrhosis following recurrent episodes. Ann.
N. Y. Acad. Sci., 57: 843-860.
Lillie, R. D., L. L. Ashburn, W. H. Sebrell, F. S .
Daft and J. V. Lowry 1942 Histogenesis and
repair of the hepatic cirhosis in rats produced
on low protein diets and preventable with choline. Pub. Health Rep., 57: 502-508.
Low, F. N., and J. A. Freeman 1958 Electron
Microscopic Atlas of Normal and Leukemic
Human Blood. McGraw Hill Book Co., New
York.
MacLean, D. L., and C. H. Best 1934 Choline
and liver fat. Brit. J. Exp. Path., 15: 193-199.
Meader, R. D., and W. L. Williams 1957 Choline deficiency in the mouse. Am. J. Anat.,
100: 167-204.
Shils, M. E., and W. B. Stewart 1954 Develoument of uortal fatty liver i n rats on corn
diets: response to lipotropic agents. Proc. SOC.
Exp. Biol. Med., 85: 298-303.
Stowell, R. E., and C. S. Lee 1930 Histochemical studies of mouse liver after a single feeding of carbon tetrachloride. Arch. Path., SO:
519-537.
Williams, W. L. 1951 Cytoplasmic changes i n
hepatic parenchyma of mice during starvation
and carbon tetrachloride induced injury. Yale
J. Biol. Med., 23: 629-652.
Williams, W-.L., J. B. Cardle and R. D. Meader
1959 The nature of dietary f a t and the pattern of hepatic liposis in choline-deficient mice.
Ibid., 31: 263-270.
Williams, W. L., and R. L. Davis 1959 Effects
of cortisone and of vitamin B12 on starved
mice. Anat. Rec., 134: 7-24.
CHOLINE DEFICIENCY IN THE MOUSE
Roland D. Meader
PLATE 1
A11 figures show frozen sections of liver stained with osmic acid. X 295. The central vein is shown
in each.
1 Photomicrograph of liver from a normal well-nourished mouse. Note the complete absence of
stainable lipid which if present would appear black.
2 One hour of choline deficiency. Note the total lack of stainable lipid.
3 Four hours of choline deficiency. Note the initial appearance of stainable lipid (L), by light
microscopy, in the form of small, individual blackened droplets within the centrolobular parenchymal
cells.
4 Six hours of choline deficiency. Most of the lobular zones now contain lipid. Observe the increase in the number and size of lipoid droplets ( L ) over those shown i n figure 3.
CHOLINE DEFICIENCY IN THE MOUSE
Roland D. Meader
PLATE 2
5 Electron micrograph of a portion of the same liver as illustrated i n figure 1. Note here the presence of submicroscopic lipid ( L ) within the parenchymal cells, the presence of which was not detectable by light microscopy in figure 1. A t this magnification, small, dense particles (179 A) which
are clearly visible in the cytoplasmic matrix ( C M ) at the upper left (see arrows) are thought to be
the precursors of the larger droplets of submicroscopic lipid ( L ) . This micrograph includes a cross
section of a typical hepatic sinusoid ( H S ) and numerous mitochondria (M). Portions of two nuclei
( N ) are visible. X 13,000.
6
CHOLINE DEFICIENCY IN THE MOUSE
Roland D. Meader
PLATE 3
6 Electron micrograph of a portion of the same liver as shown i n figure 2. Again, observe here the
presence of submicroscopic lipid ( L ) which was not visible by light microscopy in the hepatic
parenchyma of figure 2. Note the presence of the peculiar dense component or “inclusion body” contained i n the droplets of submicroscopic lipid (L) at A and B. The cytoplasmic matrix (CM) contains numerous mitochondria ( M ) and an aggregation of rough-surfaced endoplasmic reticulum
(ER). X 19,000.
7
CHOLINE DEFICIENCY IN THE MOUSE
Roland D. Meader
PLATE 4
7 Electron micrograph of a portion of a liver cell from the same liver as that illustrated i n figure 3. The nucleus ( N ) is at the lower left and shows a pattern of densities corresponding to the
chromatin of light microscopy. Note the presence of the large lipid droplets ( L ) and observe the
characteristic alternating light and dark shadowed areas appearing as broad parallel bands on the
surface of the lipid droplets ( L ) , a physical feature apparently due to the cutting quality of the
lipid itself. The pale faintly granular background is the cytoplasmic matrix (CM) which contains
several mitochondria ( M ) and both parallel aggregations and isolated strands of rough-surfaced
endoplasmic reticulum (ER). X 35,000.
CHOLINE DEFICIENCY IN THE MOUSE
PLATE 5
Roland D. Meader
8 Electron micrograph of a portion of a liver cell from the same liver as that illustrated in figure 4. The nucleus ( N ) is at the lower right and exhibits a pattern of densities corresponding to
the chromatin of light microscopy. Note the increased number of lipid droplets ( L ) present over
that shown i n figure 7, and observe the characteristic alternating light and dark shadowed areas
appearing as broad parallel bands on the surface of the lipid droplets ( L ) , a physical feature apparently due to the cutting quality of the lipid itself. The pale granular cytoplasmic matrix (CM)
contains a number of mitochondria ( M ) and parallel aggregations and isolated strands of roughsurfaced endoplasmic reticulum ( E R ) . X 27,000.
9
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