幼若 家兎 食餌性 動脈硬化症의 可逆性에 關한 硏究

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.