It has long since been recognized that the development of atherosclerosis is
considered to be closely related to the degree and duration of
hypercholesterolemia, and lipid metabolism is important in the pathogenesis of
Among the factors related to disturbance of lipid metabolism, there is abundant
evidence in the literature that one of the main factors which influences the
metabolism of lipids and cholesterol is the secretion of the endocrine glands
(Duell, 1955; Pincus, 1959). The role of the thyroid in controlling the level of
plasma lipids and cholesterol, is best established, and other endocrine glands,
such as pituitary, adrenals, gonads, and pancreas also effect the utilization of
lipids and cholesterol (Rosenman et al., 1951 & 1952b; Friedman et at.,1952a & b;
Stamler, 1953). The mechanism by which the thyroid hormone affects cholesterol
metabolism is not yet known, but there is evidence to suggest that cholesterol
destruction is elevated in hyperthyroid rats (Byers et al., 1952c). Hence, it is
assumed that the thyroid hormone acts on cholesterol metabolism cia the liver
(Rosenman et al., 1952b).
The liver is the organ where cholesterol synthesis predominantly occurs
(Cornforth, 1959), and is the main source for the plasma cholesterol (Friedman et.
al., 1951a & b). Although biliary concentration of cholesterol is a reliable index
of the rate of hepatic synthesis of this substance (Byers & Friedman, 1952a & b),
cholesterol synthesis may also occur elsewhere (friedman et at., 1955; Beher et al.
1956). This is strongly supported by the fact that the concentration of desmosterol
in several tissues may be higher than that observed in the liver and serum.
Therefore, the levels of circulating cholesterol seems to 1)e regulated by a
dynamic interchange of cholesterol between blood and tissue stores (reversible
pool), which would depend upon the amount of cholesterol available to the different
areas of the body.
Induced hypercholesterolemia, when sufficiently Prolonged, results in
atherosclerosis in many animals. Dogs and especially rats are resistant; resistance
in dogs, however, can be overcome by feeding large amounts of cholesterol during
induced hypothyroidism(Steiner St Kendall, 1946). The rat is generally considered
to be very resistant to development of atherosclerosis. Although numerous attempts
to produce atherosclerosis in this species have been recorded, most have been
completely unsuccessful (Horlick & Havel, 1948; Kendall, 1949; Marx et at., 1949;
Altschul, 1950; Katz & Stamper, 1953), and the few reported instances of deposition
of lipid in rat arteries have apparently had little resemblance to human
atherosclerosis (Page & Brown, 1952; Bragden & Boyle, 1952; Wissler, et at., 1954;
Fillios, et al., 1956).
The nature of this resistance to the development of experimental atherosclerosis
is unknown, and its elucidation on mechanisms is vital to further knowledge of the
pathogenesis of the disease. One of the factors involved in the process of
resistance may be hormonal(Steiner and Kendall, 1946).
Therefore, in rats a few causes for natural resistance may be considered: 1)
endogenous destruction of the additional cholesterol; 2) excretion of the
additional amount ingested via the bile and the gastrointestinal tract; 3)
diminished endogenous synthesis of cholesterol in balance the additional amount
ingested; 4) storage of the additional cholesterol outside the blood stream.
The majority of the studies, particularly in rats, have dealt mainly with the
fluctuation of plasma lipid levels and the production of atherosclerosis, butt very
little attent-ion huts been paid to lipid distribution within various organs. Also
it is recognized that the level of plasma lipid alone cannot adequately explain the
varied features of the diseases resulting from disturbances in lipid metabolism.
Therefore, study of the Pattern of tissue lipid distribution in hypothyroid and
partial hypatectomized, alone or combined, rats seems appropriate and important.
Moreover in order to consider the resistance of the production of atheromatous
arterial lesions in rats, it is important to evaluate the factors which regulate
The purpose of this study is to characterize the patterns of tissue lipid
distribution and define the nature of the morphological changes in various organs
in rats, after prolonged administration of high concentrations of cholesterol. At
the same time, the effect of Tapazole (antithyroid) and of partial hepatectomy was
evaluated as to the mechanism(5) involved in their action on tissue lipid
MATERIALS AHD METHODS
Healthy male albino rats weighing around 200 Grams were used. A total of 59 rats
were subjected to the experiment. The animals were divided into 4 major groups:
Group Ⅰ consisted of 7 rats as normal controls; Group Ⅱ consisted of 6 rats
receiving cholesterol only; Group Ⅲ consisted of 8 rats receiving cholesterol and
Tapazole; Group Ⅳ consisted of 38 rats having a partial hepatectomy. Group Ⅳ was
subdivided into 5 minor groups: Group "a" consisted of 8 rats receiving partial
hepatectomy only; Group "b" consisted of 9 rats receiving cholesterol only after
partial hepatectomy; Group "c" consisted of 8 rats receiving Tapazole only after
partial hepatectomy; Group "d" consisted of 8 rats receiving cholesterol and
Tapazole after partial hepatectomy; Group "e" consisted of 5 rats receiving
cholesterol and thyroxine after partial hepatectomy.
Partial hepatectomy was performed by the method of Higgins and Anderson (1931),
removing the entire median lobes of the liver, around 40% of the entire liver
weight, under ether anesthesia and with aseptic precautions.
Cholesterol was given in 5 gm. doses daily per kg. until the end of the
experiment, and Tapazole in 0.17mg. doses daily per kg. parenterally, and
L-thyroxine sodium, in 0.2mg. doses per kg., was given intramuscularly every other
day until the end of the experiment.
During the 3 months' experimental period, body weight was measured weekly. The
surviving animals were sacrificed at the end of the experimental period. The total
serum cholesterol levels were determined by the method of Kingsley and
Schaffert(1949). The organs, such as heart, kidney, adrenal, and liver, were
weighed. For the study of tissue lipid distribution, Oil Red O stain after frozen
sections was performed on the adrenal, small intestine, liver, kidney, heart and
the aorta. For the histopathologic examination of the organs, paraffin sections of
all specimens, were prepared for hematoxylin-eosin stain and in addition, periodic
acid Schiff's reaction, aldehyde fuchsin stain of Gomori, and the colloidal-iron
method of Rinehart-Abul-Haj, were used for special tissue alterations.
RESULTS AND DISCUSSION
The level of total serum cholesterol was slightly elevated in the
cholesterol-only treated group compared with that of normal controls, and was more
elevated in the Tapazole treated groups such as Group Ⅲ, Group c and d, compared
with that of the cholesterol-only treated group. In contrast, the cholesterol level
appeared in the thyroxine treated group lower than that of the normal controls. In
all groups the fluctuation of the cholesterol levels was minimal, and partial
hepatectomy had no particular effect.
Histopathologic examination of the thyroid showed slight hyperplastic changes in
the cholesterol-only treated group with decreased colloid content and increased
P.A.S. positive granules in the lining epithelium. The thyroid gland in the
Tapazole treated group disclosed marked hyperplasia composed of irregular,
small-sized follicles with decreased colloid content and alto reduced P.A.S.
positive granules in the lining epithelium. However, the thyroxine treated group
showed intermingled minimal hyperplastic or hypoplastic changes with slight
increased P.A.S. positive granules in the lining epithelium, which appeared to be
an effect of cholesterol rather than thyroxine. Bernick et al.(1962) stated that
a1% cholesterol diet in several kinds of animals caused the thyroid glands of
hamster and rats to become hyperplastic, but the glands of rabbits and guinea pigs
to become hypo-plastic in appearance, and he concluded that the latter animals have
a relatively low thyroid activity and their liver is unable to efficiently
metabolize exogenous lipids or cholesterol. Hence, they show both hyperlipemia and
hypercholesterolemia, conditions which are factors in their susceptibility to
The adrenal gland showed increased sudanophilic material particularly in the zona
fasciculata in the cholesterol-only treated group,―a finding which was, more
marked in the Tapazole treated groups and thyroxine treated group, but partial
hepatectomy had no particular effect. The adrenal gland of the thyroxine treated
group showed increased mitotic activity in the zona fasciculata, bona reticularis
and in the medulla.
The small intestine revealed more increased sudanophilic material in the lining
epithelium and the stroma of the mucosa in the cholesterol-only treated group, the
Tapazole treated groups and the thyroxine treated group, as compared with normal
controls, without significant differences among the groups, and there was also no
particular effect following partial hepatectomy. Abell et at. (1956) studied
cholesterol metabolism in the dog, and obtained a result of 75 to 83% fecal
excretion of administered high cholesterol regardless of the thyroid state, they
found no demonstrable effect upon the intestinal absorption of cholesterol. They
concluded that the increase in bile acid output was equivalent to the increased
cholesterol absorption from the diet. Also it was noted that dogs receiving a high
cholesterol diet plus thiouracil have an impaired capacity to convert excess
dietary cholesterol into bile acids. In this study increased sudanophilic material
in the intestinal mucosa is considered microscopically to be evidence of increased
absorption of cholesterol on the high cholesterol diet. Moreover, it appeared that
there were no particular differences in the absorption of cholesterol among the
cholesterol-only treated group, the Tapazole treated groups, and the thyroxine
In the cholesterol-only treated group, the liver, showed a minimal degree of
sudanophilic material in the Kupffer cells and immediately beneath the hepatic cell
membrane, particularly around the central veins. In the Tapazole treated groups,
sudanophilic material was markedly and diffusely increased throughout the entire
lobes, but in the thyroxine treated group, sudanophilic material had almost
completely disappeared. There were also no significant differences following
partial hepatectomy though it appeared to be slightly increased in the hepatectomy
groups compared with those of non-hepatectomy groups. Franz et al. (1954) studied
the relationship between the level of serum cholesterol and the concentration of
hepatic cholesterol. They found a relative constancy of the concentration of serum
cholesterol, despite large differences in the cholesterol content of the liver may
postulated the existance of a homeostatic mechanism, and stated that, the liver
appeared to act as a buffer for serum cholesterol. So, if large amounts of
cholesterol are absorbed from the intestinal tract, the liver quickly removed most
of the cholesterol from the blood. As the cholesterol content of the liver rises,
hepatic synthesis of cholesterol is depressed. Many investigators have noted the
depressed rate of hepatic cholesterol synthesis (endogenous) following the
administration of high cholestelol (exogenous) diet in dogs (Gould et at. 1953) and
in rats (Tomkins et al., 1953; Frantz et al., 1954). Thess is a negative feedback
mechanism which inhibition of endogenous cholesterol synthesis in the liver is
followed by administration of large amounts of exogenous cholesterol.
The kidney showed a minimal degree of sudanophilic material in the renal tubules
and glomeruli in the cholesterol-only treated group, and a moderate degree of
sudanophilic material in the Tapazole treated groups. In contrast, in the thyroxine
treated group, sudanephilic material was rarely seen. Partial hepatectomy was
followed by no significant change nor effect in and of these groups.
The coronary artery and aortas showed neither gross atheromatous changes nor
microscopic lipid deposits nor alteration of the acid mucopolysaccharides. However,
several instances, particularly of the Tapazole treated groups, showed minimal
interruption of the internal elastic membrane in the coronary artery. This appeared
to be similar to the coronary artery lesion described by Bernick et al. (1972) who
concluded it only minimal atherosclerotic lesions despite no atheromatous nor
lipomatous lesions of the coronary artery. However, in the face of these equivocal
findings further studies are needed to prove whether or not actual early
atheromatous changes are present.
1. Exogenous high cholesterol feeding did not elevate the total serum cholesterol
in normal and partial hepatectomized rats, but did cause significant alterations in
the tissue lipid distribution-particularly in the adrenal gland, small intestine,
liver, and kidney The thyroid gland became hyperplastic.
2. The combined administration of Tapazole and cholesterol caused only a slight
elevation of the serum cholesterol level as compared with that of controls, but
very significant alterations in the tissue lipid distribution in the adrenal gland
3. The combined administration of thyroxine and cholesterol caused only a
tendency to minimal decrement of serum cholesterol level as compared with that of
controls, but produced a significant inhibition of tissue lipid accumulation in the
liver and kidney.
4. Partial hepatectomy caused neither the changes in serum cholesterol level, nor
effect of the tissue lipid distribution.
5. Lipid accumulation in the coronary artery and aorta could not be demonstrated,
although there was some alteration in the serum cholesterol level and in the tissue
It appears that, in the rats, there was no paricular alteration of the intestinal
absorption of cholesterol regardless of the thyroid status, and in the pathways of
cholesterol metabolism after absorption. Partial hepatectomy did not induce
particular effect, but changes in the thyroid function did show measurable effects.
However, the fact that there were no significant elevations in the serum
cholesterol and tissue lipid in the liver suggests that homeostatic mechanisms may
halve a greater role in the high resistance to the development of atherosclerosis
than does an actively functioning thyroid gland.