(The) study on the reversibility of experimental cholesterol atherosclerosis in young rabbits.V
Authors
윤기호
Issue Date
1967
Description
의학과/박사
Abstract
[한글]
The Study on the Reversibility of Experimental Cholesterol Atherosclerosis in Young
Rabbits
Ki Ho Yoon, M.D.
Department of Pathology
Yonsei University College of Medicine Seoul, Korea
(Directed by professor Dong Sik Kim, M.D.)
Arteriosclerosis in elderly persons is a well known condition through a lot of
investigations using human materials and animal experiments on the pathogenesis and
treatment. But the study on arteriosclerosis in young persons was rare, and
recently has been increasingly reported. At first Zeek (1930) collected 98
necropsied cases under 20 years of age in the world literature and reviewed the
most comprehensive study of juvenile arteriosclerosis. Following her study,
investigators have been enormously interested; Willius and Smith(1933) reported on
the incidence and the degree of coronary and aortic sclerosis in 5060 post-mortem
examinations under 9 years of age; Stryker(1946), Gruenwals(1949) and Cochrane and
Browden(1954) on the incidence and morphology of coronary arteries and aortas in
necropsied cases of still birth. More recently in Korea, Kim and Shin(1963)studied
histochemical changes in the fetal arteries of Koreans with 76 necropsied cases of
still birth, under 40 weeks gestation and Pak(1965) collected 207 necropsied cases
under 31 years of age and studied the histochemical changes of arteries of Koreans.
The reversibility of human atherosclerosis is, of course, a vital question.
Despite the difficult problem of reversibility in humans with clinically evident
atherosclerosis, the question whether or rot it is possible for experimentally
induced atherosclerotic plaques to be resorbed has been investigated several times.
Leary(1934) supported the observation of Anitschkow(1933) that withdrawal of
cholesterol from cholesterol-fed rabbits was followed by a gradual disappearance of
lipid from the plaques. Bragdon(1952) noted the spontaneous atherosclerosis of
suckling rabbits disappears after weaning, and Pollak(1953) successfully prevented
hypercholesterolemia and atherosclerosis in rabbits by the simultaneous
administration of cholesterol and sitosterol. Investigators(Dury, 1956;
Chakravarti,1956; Willis, 1957) induced regression or complete absence of aortic
atherosclerosis by cortisone therapy, thyroid and estrogen therapy to rabbits, and
vitamin C thereapy in scorbutic guinea pigs. McMillan and his colleagues(1955) also
noted that histological reorganization of plaques but no decrease in arterial lipid
content was detected chemically in animals killed at intervals up to six months.
They attributed this to the persistence of hyperlipemia, the etiological mechanism
in their experimental procedure.
Recently Taylor et al. (1961) have brought strong support to the reversibility of
human atherosclerosis, and then contended that cholesterol plaques should be
thought of as resorbable "foreign material. " Rustein et al. (1958) have provided
probably the only acceptable human data on the reversibility of human
atherosclerosis using tissue culture technique.
It is interesting that Rustein found that the deposition of lipid was completely
inhibited by the simultaneous addition of linolenic acid and potentiated by the
addition of stearic acid.
The majority of the studies have dealt mainly with atherosclerosis of elderly
persons and adult animals, and moreover, very little attention has been paid to the
probable mechanism of the development of arteriosclerosis in young persons and also
young animals. Therefore, a study of the pattern of atheroma in young rabbits and
of the serum cholesterol content seems to be appropriate and important.
Furthermore, there are still a few different theories as to the mode of action of
the various factors in the pathogenesis of reversibility, particularly in young
rabbits.
The purpose of this study is to characterize the patterns and nature of atheroma
in induced cholesterol atherosclerosis and after withdrawal of cholesterol feeding
particularly in young rabbits. It seeks to clarify the degree of
hypercholesterolemia in relation to the degree of production of atheroma and also
whether or not it is possible for induced cholesterol atherosclerotic plaques to be
resorbed in young rabbits.
Materials and Methods
Healthy young rabbits of both sexes weighing around 250 Grams were used. A total
of 75 young rabbits were subjected to the experiment. The animals were divided Into
3 major groups: Group A consisted of 5 rabbits as normal controls; Group B
consisted of 35 rabbits receiving cholesterol to the end of the experiment, and was
subdivided into 5 minor groups according to the cholesterol feeding period; Group
Ⅰ, 30 days period of cholesterol feeding, Group Ⅱ 45 days, Group Ⅲ 60 days,
Group Ⅳ 75 days, and Group Ⅴ 90 days; Group C consisted of 35 rabbits, 30 days
withdrawal after cholesterol feeding, and was also subdivided into 5 minor groups
according to the duration of the cholesferal feeding; Group Ⅵ, 30 days cholesterol
feeding, Group Ⅶ 45 days, Group Ⅷ 60 days, Group Ⅸ 75 days, and Group Ⅹ 90
days.
The cholesterol was given in 500 mg. doses daily per animal for periods of 30
days, 45 days, 60 days, 75 days, and 90 days. In Group C, withdrawal of cholesterol
feeding was done for 30 days after cholesterol feeding in each different feeding
period.
During the experiment, the body weight was measured every 15 days. The surviving
animals were sacrificed at the end of each different experimental period, and the
total serum cholesterol and free cholesterol levels were determined by Zak's
method(1954) The aortas were dissected out and grossly stained by Sudan Ⅳ after
fixation with 10% formalin, and examined grossly and microscopically. The
tophographic estimation of atherosclerotic lesions was made by the method of
Holman(1958). For the histopathologic examination of the aortas, paraffin section
were prepared for hematoxylin-eosin stale in all cases, and in addition, for van
Gieson stain, aldehyde fuchsin stain of Gomori, colloidaliron method of
Rinehart-Abul-Haj, and phosphotungustic acid hematoxylin stain (PTAH) .And Oil Red
0 stain after frozen sections was made.
Results and Discussion
The level of total serum cholesterol and of free cholesterol was markedly
elevated in Group B compared with that of normal controls. In the subdivided minor
groups of Group B, those levels showed a tendency to gradual increment toward group
V, and also similar change in the subdivided minor groups of Group C.
But there were very interesting findings; the total serum cholesterol level of
group Ⅵ was markedly reduced and the level of free cholesterol in group Ⅵ and Ⅶ
also showed relative decrement to the total cholesterol level compared with those
of group Ⅰ and Ⅱ. These findings suggest that the degree of hypercholesterolemia
in young rabbits is parallel to the amount of cholesterol administered and also to
the duration of administration. However, marked reduction of cholesterol level in
group Ⅵ and relative decrement of free cholesterol level suggest that induced
hypercholesterolemia is reversible and hepatic parenchymal tissue is not severely
damaged, particularly in a short period of cholesterol feeding with 30 days
withdrawal, but there is no significant effect on the serum cholesterol level
particularly in long-term feeding with only 30 days withdrawal.
Gross examination of the aortas in the cholesterol fed control group(Group Ⅰ,
Ⅱ, Ⅲ, Ⅳ, & Ⅴ), showed variable degrees of atheromatous change which is
particularly marked in the ascending aorta and the arch of the aorta, but also
there was a minimum or slight degree of atheromatous change in the thoracic and
abdominal aortas. These atheromatous lesions gradually progressed as the time of
cholesterol feeding lapsed. In the cholesterol feeding withdrawal group(Group Ⅵ,
Ⅶ, Ⅷ, Ⅸ, & Ⅹ), similar degrees of atheromatous change to the cholesterol
feeding group; that is not significantly different in, but significant reduction In
the thoracic and abdominal aorta, and showed a tendency to decrement in amount in
the withdrawal group grossly. These findings suggest that the degree of
atheromatous change produced by cholesterol feeding is parallel to the level of
hypercholesterolemia in young rabbits.
Microscopic examination of the aortas revealed various degrees of atheromatous
change both in Group B and C, progressively increasing as the experimental periods
were prolonged, as seen in gross examination. The lesions of shorter experimental
period were characterized by a deposit of stainable lipid along the internal
elastic membrane and in the immediately adjacent intima without significant
penetration into the media, but as the experimental period was prolonged, lipid
penetrated into the media, rather deeply in Group Ⅴ. Macrophages wert scarcely
notes. Each individual plaque appeared as a confluent mass of stainable lipid with
no associated fat-free areas. Acid mucopolysaccharides were rather increased in
amount beneath the atheromatous lesions. These findings occurred only in the
ascending aorta and in the arch but in the thoracic and abdominal aortas the
atheromatous changes were minimal, and the lipid was deposited in the intimal side,
particularly localized in the beginning of the branches of the aorta, without
significant penetration of the media.
In the cholesterol feeding withdrawal group(Group C), the atheromatous lesions of
the shorter experimental period appeared to be less intense and minimal in their
lipid deposit. Relatively many macrophages were seen on the intimal side of the
internal elastic membrane and some on the medial side. These macrophages are more
significant in number especially in group Ⅶ than Ⅵ, but are scarcely found in the
greater atheroma formation. More severe lesions in this group appeared to be
somewhat of a different pattern, being more resistant to resorption. The lipid was
aggregated into separate islands with lipid-free areas intervening. Sometimes such
Islands had a few macrophages around them but occasionally none were noted.
Sometimes there appeared to be diffusion of lipid into the inner layer of the
arterial media.
Microscopic examination of the heart showed atheroma formation of coronary
arteries both in Group B and C, progressively increasing in degree as the
experimental periods were prolonged. The atheroma formation of coronary arteries in
Group B occured mainly in the small sifted and then in the medium sifted arteries
resulting in almost complete obstruction of their lumens, but subintimal lipid
deposition was very slight particularly in the larger sized arteries. There were
also lipid diffusion into the myocardium, increasing as the experimental duration
lapsed. But in the Group C, the atheroma formation of coronary arteries was found
to be reduced mainly in the medium sized and then in the small sized arteries
compared with Group 0, but no particular subintimal lipid deposition was found in
the larger sifted arteries. Lipid diffusion into the myocardium was also reduced.
Conclusions
1. Atheromatous lesions in young rabbits can be produced experimentally with a
large amount of cholesterol feeding.
2. The degree of hypercholesterolemia was not reduced after 30 days withdrawal of
cholesterol feeding in the groups of relatively longer experimental period, but was
markedly reduced after withdrawal of cholesterol feeding particularly in the group
of shorter period, Group Ⅵ.
3. The degree of hypercholesterolemia is parallel to the extent of atheroma, but
is somewhat different from the extent of histologic lesions of aorta which appear
rather reduced
4. Atheromatous lesions may be reversible in the aorta and the heart particularly
in the groups of shorter period in which the lesions were rather superficial and
the media of the aorta was not severely damaged.
It appears that the persistence of hypercholesterolemia may be essential for the
pro-duction of the lesions. This hypercholesterolemia actually causes production of
atheroma also in young rabbits. The results of this investigation suggest that the
smaller lesions of atheroma seem to be resorbed, but more severe lesions appear
more resistant to reversal. With a more prolonged period of withdrawal of
cholesterol feeding, more reversal and interesting change might be noted in such
lesions of the aorta and the heart.
[영문]
Arteriosclerosis in elderly persons is a well known condition through a lot of investigations using human materials and animal experiments on the pathogenesis and treatment. But the study on arteriosclerosis in young persons was rare, and recently has been increasingly reported. At first Zeek (1930) collected 98 necropsied cases under 20 years of age in the world literature and reviewed the most comprehensive study of juvenile arteriosclerosis. Following her study, investigators have been enormously interested; Willius and Smith(1933) reported on the incidence and the degree of coronary and aortic sclerosis in 5060 post-mortem examinations under 9 years of age; Stryker(1946), Gruenwals(1949) and Cochrane and Browden(1954) on the incidence and morphology of coronary arteries and aortas in necropsied cases of still birth. More recently in Korea, Kim and Shin(1963)studied histochemical changes in the fetal arteries of Koreans with 76 necropsied cases of still birth, under 40 weeks gestation and Pak(1965) collected 207 necropsied cases under 31 years of age and studied the histochemical changes of arteries of Koreans.
The reversibility of human atherosclerosis is, of course, a vital question. Despite the difficult problem of reversibility in humans with clinically evident atherosclerosis, the question whether or rot it is possible for experimentally induced atherosclerotic plaques to be resorbed has been investigated several times.
Leary(1934) supported the observation of Anitschkow(1933) that withdrawal of cholesterol from cholesterol-fed rabbits was followed by a gradual disappearance of lipid from the plaques. Bragdon(1952) noted the spontaneous atherosclerosis of suckling rabbits disappears after weaning, and Pollak(1953) successfully prevented hypercholesterolemia and atherosclerosis in rabbits by the simultaneous administration of cholesterol and sitosterol. Investigators(Dury, 1956; Chakravarti,1956; Willis, 1957) induced regression or complete absence of aortic atherosclerosis by cortisone therapy, thyroid and estrogen therapy to rabbits, and
vitamin C thereapy in scorbutic guinea pigs. McMillan and his colleagues(1955) also noted that histological reorganization of plaques but no decrease in arterial lipid content was detected chemically in animals killed at intervals up to six months. They attributed this to the persistence of hyperlipemia, the etiological mechanism in their experimental procedure.
Recently Taylor et al. (1961) have brought strong support to the reversibility of human atherosclerosis, and then contended that cholesterol plaques should be thought of as resorbable "foreign material. " Rustein et al. (1958) have provided probably the only acceptable human data on the reversibility of human
atherosclerosis using tissue culture technique.
It is interesting that Rustein found that the deposition of lipid was completely inhibited by the simultaneous addition of linolenic acid and potentiated by the addition of stearic acid.
The majority of the studies have dealt mainly with atherosclerosis of elderly persons and adult animals, and moreover, very little attention has been paid to the probable mechanism of the development of arteriosclerosis in young persons and also young animals. Therefore, a study of the pattern of atheroma in young rabbits and of the serum cholesterol content seems to be appropriate and important.
Furthermore, there are still a few different theories as to the mode of action of the various factors in the pathogenesis of reversibility, particularly in young rabbits.
The purpose of this study is to characterize the patterns and nature of atheroma in induced cholesterol atherosclerosis and after withdrawal of cholesterol feeding particularly in young rabbits. It seeks to clarify the degree of hypercholesterolemia in relation to the degree of production of atheroma and also whether or not it is possible for induced cholesterol atherosclerotic plaques to be resorbed in young rabbits.
Materials and Methods
Healthy young rabbits of both sexes weighing around 250 Grams were used. A total of 75 young rabbits were subjected to the experiment. The animals were divided Into 3 major groups: Group A consisted of 5 rabbits as normal controls; Group B consisted of 35 rabbits receiving cholesterol to the end of the experiment, and was subdivided into 5 minor groups according to the cholesterol feeding period; Group Ⅰ, 30 days period of cholesterol feeding, Group Ⅱ 45 days, Group Ⅲ 60 days, Group Ⅳ 75 days, and Group Ⅴ 90 days; Group C consisted of 35 rabbits, 30 days withdrawal after cholesterol feeding, and was also subdivided into 5 minor groups according to the duration of the cholesferal feeding; Group Ⅵ, 30 days cholesterol feeding, Group Ⅶ 45 days, Group Ⅷ 60 days, Group Ⅸ 75 days, and Group Ⅹ 90
days.
The cholesterol was given in 500 mg. doses daily per animal for periods of 30 days, 45 days, 60 days, 75 days, and 90 days. In Group C, withdrawal of cholesterol feeding was done for 30 days after cholesterol feeding in each different feeding period.
During the experiment, the body weight was measured every 15 days. The surviving animals were sacrificed at the end of each different experimental period, and the total serum cholesterol and free cholesterol levels were determined by Zak's method(1954) The aortas were dissected out and grossly stained by Sudan Ⅳ after fixation with 10% formalin, and examined grossly and microscopically. The tophographic estimation of atherosclerotic lesions was made by the method of Holman(1958). For the histopathologic examination of the aortas, paraffin section were prepared for hematoxylin-eosin stale in all cases, and in addition, for van Gieson stain, aldehyde fuchsin stain of Gomori, colloidaliron method of Rinehart-Abul-Haj, and phosphotungustic acid hematoxylin stain (PTAH) .And Oil Red 0 stain after frozen sections was made.
Results and Discussion
The level of total serum cholesterol and of free cholesterol was markedly elevated in Group B compared with that of normal controls. In the subdivided minor groups of Group B, those levels showed a tendency to gradual increment toward group V, and also similar change in the subdivided minor groups of Group C.
But there were very interesting findings; the total serum cholesterol level of group Ⅵ was markedly reduced and the level of free cholesterol in group Ⅵ and Ⅶ also showed relative decrement to the total cholesterol level compared with those
of group Ⅰ and Ⅱ. These findings suggest that the degree of hypercholesterolemia in young rabbits is parallel to the amount of cholesterol administered and also to the duration of administration. However, marked reduction of cholesterol level in
group Ⅵ and relative decrement of free cholesterol level suggest that induced hypercholesterolemia is reversible and hepatic parenchymal tissue is not severely damaged, particularly in a short period of cholesterol feeding with 30 days withdrawal, but there is no significant effect on the serum cholesterol level
particularly in long-term feeding with only 30 days withdrawal.
Gross examination of the aortas in the cholesterol fed control group(Group Ⅰ, Ⅱ, Ⅲ, Ⅳ, & Ⅴ), showed variable degrees of atheromatous change which is particularly marked in the ascending aorta and the arch of the aorta, but also there was a minimum or slight degree of atheromatous change in the thoracic and abdominal aortas. These atheromatous lesions gradually progressed as the time of cholesterol feeding lapsed. In the cholesterol feeding withdrawal group(Group Ⅵ, Ⅶ, Ⅷ, Ⅸ, & Ⅹ), similar degrees of atheromatous change to the cholesterol feeding group; that is not significantly different in, but significant reduction In the thoracic and abdominal aorta, and showed a tendency to decrement in amount in the withdrawal group grossly. These findings suggest that the degree of atheromatous change produced by cholesterol feeding is parallel to the level of hypercholesterolemia in young rabbits.
Microscopic examination of the aortas revealed various degrees of atheromatous change both in Group B and C, progressively increasing as the experimental periods were prolonged, as seen in gross examination. The lesions of shorter experimental period were characterized by a deposit of stainable lipid along the internal elastic membrane and in the immediately adjacent intima without significant penetration into the media, but as the experimental period was prolonged, lipid penetrated into the media, rather deeply in Group Ⅴ. Macrophages wert scarcely notes. Each individual plaque appeared as a confluent mass of stainable lipid with no associated fat-free areas. Acid mucopolysaccharides were rather increased in amount beneath the atheromatous lesions. These findings occurred only in the ascending aorta and in the arch but in the thoracic and abdominal aortas the atheromatous changes were minimal, and the lipid was deposited in the intimal side, particularly localized in the beginning of the branches of the aorta, without significant penetration of the media.
In the cholesterol feeding withdrawal group(Group C), the atheromatous lesions of the shorter experimental period appeared to be less intense and minimal in their lipid deposit. Relatively many macrophages were seen on the intimal side of the internal elastic membrane and some on the medial side. These macrophages are more significant in number especially in group Ⅶ than Ⅵ, but are scarcely found in the greater atheroma formation. More severe lesions in this group appeared to be somewhat of a different pattern, being more resistant to resorption. The lipid was aggregated into separate islands with lipid-free areas intervening. Sometimes such Islands had a few macrophages around them but occasionally none were noted.
Sometimes there appeared to be diffusion of lipid into the inner layer of the arterial media.
Microscopic examination of the heart showed atheroma formation of coronary arteries both in Group B and C, progressively increasing in degree as the experimental periods were prolonged. The atheroma formation of coronary arteries in Group B occured mainly in the small sifted and then in the medium sifted arteries
resulting in almost complete obstruction of their lumens, but subintimal lipid deposition was very slight particularly in the larger sized arteries. There were also lipid diffusion into the myocardium, increasing as the experimental duration lapsed. But in the Group C, the atheroma formation of coronary arteries was found to be reduced mainly in the medium sized and then in the small sized arteries compared with Group 0, but no particular subintimal lipid deposition was found in the larger sifted arteries. Lipid diffusion into the myocardium was also reduced.
Conclusions
1. Atheromatous lesions in young rabbits can be produced experimentally with a large amount of cholesterol feeding.
2. The degree of hypercholesterolemia was not reduced after 30 days withdrawal of cholesterol feeding in the groups of relatively longer experimental period, but was markedly reduced after withdrawal of cholesterol feeding particularly in the group
of shorter period, Group Ⅵ.
3. The degree of hypercholesterolemia is parallel to the extent of atheroma, but is somewhat different from the extent of histologic lesions of aorta which appear rather reduced
4. Atheromatous lesions may be reversible in the aorta and the heart particularly in the groups of shorter period in which the lesions were rather superficial and the media of the aorta was not severely damaged.
It appears that the persistence of hypercholesterolemia may be essential for the pro-duction of the lesions. This hypercholesterolemia actually causes production of atheroma also in young rabbits. The results of this investigation suggest that the smaller lesions of atheroma seem to be resorbed, but more severe lesions appear more resistant to reversal. With a more prolonged period of withdrawal of cholesterol feeding, more reversal and interesting change might be noted in such lesions of the aorta and the heart.