SOME ASPECTS OF EXPERIMENTAL
ARTERIOSCLEROSIS IN THE DOG

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MICHIGAN STATE UNIVERSITY

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SOME ASPECTS OF EXPERIMENTAL ARTERIOSCLEROSIS

IN THE DOG

BY

Charles C. Middleton

AN ABSTRACT
Submitted to the College of Veterinary Medicine
Michigan State University of Agriculture and

Applied Science in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE
Department of Surgery and Medicine

1961

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ABSTRACT

Some aspects of experimental arteriosclerosis of the
mature male dog were studied. Thirty-six dogs were
thyroidectomized taking care to preserve the blood supply
of the parathyroid glands. Vitamin D was administered at
the rate of 100,000 units daily starting the first post-
surgical day. Six control dogs were not thyroidectomized
and received no Vitamin D.

Symptoms of Vitamin D toxicity became evident between
the fifteenth and thirtieth day post-thyroidectomy.
Debilitation was marked by the time of death which occurred
by the seventy-third day post-thyroidectomy. Collateral
circulation studies were attempted by transacting a pelvic
limb and immediately reapposing the transected tissues. The
femur, femoral artery and vein, and the femoral and sciatic
nerve were left intact. This procedure was performed on
the forty-ninth, fiftieth or fifth-first day post-thyroidec-
tomy. 0n the eighteenth day following transection the
femoral artery and vein were ligated. Limbs of the thy-
roidectomized animals that survived the transection did not
survive the ligation as evidenced by necrosis distal to the

site of transection in all but one dog.

 

 

 

 

 

Charles C. Middleton

Electrocardiograms were recorded. Serum calcium
levels showed a significant increase in the thyroidecto-
mized dogs during the course of the experiment. Serum
cholesterol levels were progressively elevated in the
thyroidectomized dogs. Limb temperatures were recorded
at regular intervals.

Thyroidectomy coupled with vitamin D administration
proved to be a relatively quick method of producing
lesions of arteriosclerosis. Due to severe debilitation

collateral circulation studies were not conclusive.

SOME ASPECTS OF EXPERIMENTAL ARTERIOSCLEROSIS

IN THE DOG

BY

Charles C. Middleton

A THESIS

Submitted to the College of Veterinary Medicine
Michigan State University of Agriculture and
Applied Science in partial fulfillment of
the requirements for the degree of

MASTER OF SCIENCE

Department of Surgery and Medicine

1961

 

ACKNOWLEDGMENTS

The author is sincerely grateful to Dr. William V. Lumb,
Associate Professor, Department of Surgery and Medicine,
for his help and guidance throughout the course of this study.

Gratitude is also extended to Dr. Gabel Conner, Professor,
Department of Survery and Medicine, for his help with the
preparation of this thesis, and to Dr. Clayton Lewis, Con—
sultant to The School of Veterinary Medicine, for his help
and suggestions with this work.

The author is also very appreciative of the help with
the photomicrographs which Dr. Esther Smith, Associate
Professor, Department of Anatomy, so graciously offered and
to Mrs. Emerson Colby for her assistance with the typing
and proofreading of this work.

Appreciation is also extended to Dr. David K. Detweiler
Of The School of Veterinary Medicine, University of Penn-
SYlvania, for his suggestions concerning electrocardio-
graphy and to Dr. Donald F. Patterson of The School of
Veterinary Medicine, University of Pennsylvania, for his
time and effort in interpreting the electrocardiograms.

Finally the author wishes to thank Dr. William Baten,

Agricultural Experimental Station, Michigan State University,

ii

for the statistical analysis of the data; Dr. Ethard Van
Stee, Department of Surgery and Medicine, for the serum
protein electrophoresis determinations; and his wife,
Barbara, for her help in various ways with the preparation
of this manuscript.

This work was made possible by The National Institute

of Health grant H—4507 A.

iii

CHAPTER

II.

III.

IV.

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . . . .

REVIEW OF THE LITERATURE . . . . . . . . .

PPS”?

DJ

I.

Introduction
Paleopathology

Dietary Relationships

Spontaneous Arteriosclerosis and
Atherosclerosis in Animals

The Effects of Hypervitaminosis D on
Man and Animals

The Interrelation of Elastic Tissue and
Calcium in the Genesis of Arterio—
sclerosis

Vascular Degenerative Changes in
Hypothyroidism

Theories of the Genesis of
Arteriosclerosis

l. The Senescence Theory

2. The Mechanical Trauma Theory

3. The Toxin Theory

4. The Anoxemia Theory

Collateral Circulation

MATERIALS AND METHODS . . . . . . . . . .

RESULTS 0 o o o o o o o o o o o o o o o o o

A.
B.
C.

General Considerations
Thyroidectomized Dogs
Control Dogs

DISCUSSION . . . . . . . . . . . . . . . .

UOPS’

General Considerations
Thyroidectomized Dogs
Control Dogs

General Discussion

iv

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13

23

24

25
26
3O
3O
31
4O

50
57
57
57
6O
69
69
69

72
73

CHAPTER PAGE

VI. SUMMARY AND CONCLUSIONS . . . . . . . . . . 75
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . 77
APPENDICES

I. Data on the Individual Thyroidectomized
Dogs . . . . . . . . . . . . . . . . . . . 82

II. Data on the Individual Control Dogs at

Time of Euthanasia . . . . . . . . . . . . 85
III. Dogs That Died Prior to Transection . . . . 86
IV. Electrocardiographic Data and
Interpretations . . . . . . . . . . . . . . 89
V. Gross Pictures and Photomicrographs of
Thyroidectomized Dogs . . . . . . . . . . 93

TABLE

II.

III.

IV.

LIST OF TABLES

PAGE

Body weights of thyroidectomized dogs
Body weights of control dogs . . . . . . . . . 61
Serum calcium and serum cholesterol levels in

thyroidectomized dogs
Serum calcium and serum cholesterol levels in

control dogs . . . . . . . . . . . . . . . . 62
Limb temperatures of thyroidectomized dogs
Limb temperatures of control dogs . . . . . . 65
Post-transection data on thyroidectomized

dogs . . . . . . . . . . . . . . . . . . . . 67
Transection data and gross lesions for

O O O O O O 68

control dogs . . . . . . . . .

vi

CHAPTER I
INTRODUCTION

Cardiovascular disease found in ancient mummies would
suggest that this condition is as old as recorded civili-
zation.(MOodie, 1923).

It was not until early in the twentieth century that
the condition was found to be related to a dietary etiology
or experimentally produced in animals (Duff, 1935). There
has been a vast amount of theorization and research since
that time but cardiovascular disease continues to be a
perplexing problem.

Most of the early experimental work in the field of
atherosclerosis and arteriosclerosis was done with the
rabbit and chicken. Until the dog and other domestic
animals were used for the experimental production of car-
diovascular disease, very little attention was focused
upon the spontaneous occurrence of the conditions in
domestic animals. This brought to light the fact that
arteriosclerosis and atherosclerosis do occur in domestic
animals spontaneously and thus animals used for a con-

trolled experiment must be within a selected age group.

The experimental production of arteriosclerosis and
atherosclerosis is rather easily and rapidly accomplished
in the rabbit and chicken but is met with more difficulty
in the dog. It was found that depressing thyroid function
(Smith and Jones, 1957) or removal of the gland (Smith
and Jones, 1957) greatly enhanced the experimental pro—
duction of the disease in the dog.

Since cholesterol and calcium aid in the formation of
the lesions, attempts have been made to correlate an ele-
vation of serum cholesterol and serum calcium with the
spontaneous and experimental occurrence of atherosclerosis
and arteriosclerosis.

Hypertension and electrocardiographic changes are
frequently associated with cardiovascular disease and blood
pressures and electrocardiograms are an important diagnostic
and prognostic aid.

There has been an increased interest in recent years in
revascularization of partially thrombosed areas and in the
natural collateral circulation development to an ischemic
area.

The purpose of this study was to experimentally produce
arteriosclerosis in thyroidectomized dogs so that serum

calcium and cholesterol levels could be studied in relation

 

 

 

 

 

 

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to the histopathological changes produced. It was also
hoped that blood pressure and electrocardiographic changes
could be obtained. The development of collateral circu-
lation in a transected limb of an arteriosclerotic animal

was a point of considerable interest.

CHAPTER II
REVIEW OF LITERATURE

A. Introduction

The experimental production of arteriosclerosis and
atherosclerosis has been accomplished in many species and
by many methods. The main emphasis in the literature
reviewed here will be on arteriosclerosis but due to the
close relationship between the two conditions, some of

the more pertinent literature on atherosclerosis will

also be cited.

B. Paleopathology

That cardiovascular disease is not a new disease to
man is well attested to by the findings of Moodie (1923).
He reported on the histopathological and gross lesions of
Egyptian mummies. The mummy of King Merneptah, the reputed
pharoah of the Hebrew exodus, was found at Thebes, Egypt
in the tomb of Amenhoted II who reigned in Egypt from
1448-1420 B.C. The ruptured aorta of Merneptah's mummy
would dispute the theory that he was drowned in the Red

Sea during the Hebrew exodus.

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Histological study of the aorta showed the typical
calcification of the aorta, the bony parallel elastic
lamellae being perfectly preserved, and the interlamellar
material thickly strewn with calcium phosphate.

Other mummies of the eighteenth to the twenty-seventh
dynasties (1580—527 B.C.) also revealed lesions of arterio—
sclerosis which were exactly the same as are associated
with the disease today. Williams (1948) examined two
Peruvian mummies, probably between two or three centuries
of A.D. 700. Microscopic examination revealed arterio-
sclerotic changes which included calcification of the aorta

and a calcified thrombus.

C. Dietary Relationships

When Ignatovski, of the Russian Imperial Military
Medical Academy, reported in 1908 that rabbits fed milk
and egg yolks developed severe arteriosclerosis, he gave
mankind the key to control of a disease which was becom-
ing more prevalent as living standards improved (anonymous
editorial, 1958).

Duff (1935), in a general review, further stated that
Ignatovski was studying the effects of high protein diets

in rabbits, When he noticed the occurrence of fatty plaques

 

 

 

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on the intimal surface of the aorta of some animals. He

attributed the lesions to the effect of an animal protein
rich diet on a herbivorous animal. Starokadomsky the
following year, 1909, repeated Ignatovski's experiment

and drew the same conclusions. Stuckey reported in 1910
that by feeding various combinations of whole milk, egg
yolk, egg white and meat juice, the diets containing egg
yolk produced marked fatty changes in the aorta. He later
produced identical lesions by feeding brain tissue. These
findings led to the idea that the lesions were not due to
the protein fraction of the diet but rather to some lipid
common to the egg yolks and brain tissue.

Anitschkow and Chalatow in 1913 demonstrated that
feeding pure cholesterol dissolved in oil could produce
lesions in rabbits identical to those described by previous
investigators (Duff, 1935).

Wacker and Hueck reported at about the same time that
pure cholesterol added to ordinary food of rabbits could
produce the lesions and that it also resulted in a marked
increase in blood cholesterol levels (Duff, 1935).

It has been shown that while neither cholesterol feeding
nor high fat diet had appreciable effects on plasma chole—

sterol in dogs, a diet containing both fat and cholesterol

had a decided effect, and the plasma level rose even higher
if the diet was deficient in protein. Experiments by
numerous investigators have gradually made it apparent that
rabbits and chickens absorb cholesterol efficiently on diets
low in fats, but dogs, monkeys, and man do not absorb much
biliary or dietary cholesterol unless the diet contains
sufficient fat. In man the bile provides daily as much
cholesterol as would five to ten eggs and the absorption
of this cholesterol is increased when the diet is rich in
animal fat (anonymous editorial, 1958).

Recent work shows that no foods surpass eggs and butter-
fat in raising blood cholesterol of rabbits and man, and

none have a more striking effect in acceleration of thrombus

formation (anonymous editorial, 1958).

D. Spontaneous Arteriosclerosis and
Atherosclerosis in Animals

Dauber and Katz (1942) reported that chickens show
atherosclerotic lesions as early as the end of the second

year of life.
Spontaneous arteriosclerosis was reported by Dauber

(1944) to occur in the aorta in 45% of commercial roosters

and hens over one year of age. There appeared to be no sex

 

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differences in the incidence of macroscopic lesions. Only

hens showed fatty lesions of the intima of the ascending
aorta and aortic arch, while both roosters and hens com-
monly exhibited intimal lesions of the abdominal aorta.

The lesions of spontaneous arteriosclerosis in the chicken

resemble human arteriosclerosis lesions. If chickens are

to be used for experimental arteriosclerosis studies they

should be less than six months old since spontaneous lesions

start to occur at this age.

Spontaneous arteriosclerotic lesions have been reported
in cattle, dogs, swine and other species, but only rarely
is the arteriosclerotic disease clinically significant
(Smith and Jones, 1957).

While conducting an experiment on dogs unrelated to
vascular changes, Morehead and Little (1945) noted degener-

ative and reparative processes in the vascular system which

they concluded to be spontaneous in nature. This View was

strengthened further by the fact that the vascular changes

in the experimental animals were identical morphologically
with those which have been described in a large percentage

Of human aortas removed from persons dying of various

causes.

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The above authors then obtained eleven mongrel dogs
chosen at random for further study of spontaneous arterio-
sclerosis. These animals were sacrificed along with five
puppies approximately six months old and three ten day
old pups. Nine additional dogs from another terminated

experiment that was thought to have no relationship to

cardiovascular changes were also included.

Aortic lesions observed were:

1. Focal loss of elastic tissue with grouping of
smooth muscle cells was most pronounced in the
ascending aorta and arch. This was seen in vary-
ing degrees in all animals but was much less

prominent in the ten day old pups.

2. Medial necrosis with cyst formation was pronounced
in two dogs, but seen in varying degrees in eight
other dogs.

3. Intimal thickening with splitting and reduplicating
of the internal elastic lamina was present in
fourteen animals ans was confined to the abdominal
aorta. Seven dogs had nodular formations project-

ing into the lumen of the vessel. In every animal
certain sections revealed a separation of the

lamellae of the intima resulting in the formation

10

of irregular cystic spaces. This was best seen in

the ascending aorta and arch of the vessel.

Localized areas of hyalinized collagenous tissue

4.
with calcification were observed in five dogs and
was limited to the ascending aorta.

5. One dog had minimal sclerotic changes of the

coronary artery.

Kollisch examined the aortas of 110 dogs and found

arteriosclerosis in 23 of them. He included all degenera-

tive vascular changes as lesions of arteriosclerosis

(Morehead and Little, 1945).
Morehead and Little (1945) cited another group of dogs

ranging from one to five years of age used to study spon-

taneous arteriosclerosis. Only one animal in the younger

group was demonstrated to have true arteriosclerosis and

in this instance the term was restricted to intimal degen-
eration with fatty deposits and plaque formation. In the

older group of animals, arteriosclerosis was a fairly com-

mon lesion.
HUeper (1945) examined the aortas of fifty dogs between

The majority of these animals

one and eight years of age.
He concluded

were thought to be under four years of age.

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that arteriosclerosis of the aorta is exceptional in dogs
before the age of five. The arterial lesions encountered
were predominately of the sclerosing type. He considers
the highly edematous state of the inner part of the media
of the ascending portion of the aorta frequently seen in
dogs to be either a normal anatomical condition or an
agonal phenomenon resulting from circulatory disturbances.

A study on 26 dogs, 14 of which were eight to seven—
teen years of age was done by Lindsay §£_§l, (1952). This
work states that the incidence of spontaneous arterio-
sclerosis of the aorta is high in dogs. The majority of
the older dogs showed some evidence of cardiac disease,
including coronary arteriosclerosis and myocardial infarction
or fibrosis.

Medial aortic changes were observed in the majority
of the dogs and were characterized by deposition of mucoid
substance and collagen. This was often associated with
focal proliferation of smooth muscle cells and focal
alteration of elastic tissue.

The outstanding lesion of spontaneous canine arterio-
sclerosis is intimal fibrosis associated with deposition

of a mucoid ground substance and collagen. The internal

12

elastic membrane may be altered without intimal disease
and is almost invariably altered when intimal thickening

is present.

Lipids appeared to play no part in the pathogenesis of

the earliest intimal lesions, although lipids were present

in later lesions. Lipid infiltration appeared to be a
minor process even in the arteriosclerotic plaques of
older dogs.

Cholesterol was not found in older lesions even where
other lipids had infiltrated the intimal fibrous plaques
(Lindsay g3; 11,, 1952).

The work of Detweiler (1959) showed that spontaneous
arteriosclerosis was fairly frequent in dogs and increased
in incidence with advancing age. It was also associated
with chronic renal disease and was especially extensive in
dogs with renal disease in which postmortem evidence of
arterial hypertension was also observed.

The prominent lesions of canine arteriosclerosis, in
the aorta, according to Detweiler, consist of intimal
fibrosis with deposition of mucoid ground substance and
collagen. The internal elastic membrane is usually altered

by reduplication and fragmentation. Lipids do not occur in

13

the early intimal lesions and are not important in older
ones. Recent observations reveal a high incidence of
intimal changes in the posterior abdominal aorta and

iliac arteries in dogs more than eight years old. Intimal
lesions were frequently observed in coronary arteries
associated with myocardial damage. Most older dogs were
found to have medial sclerosis of the aorta, characterized
by deposition of mucoid substances and collagen. This
recent work indicates that arterial disease plays an
important role in the pathogenesis of myocardial damage

in older dogs and in some animals is associated with
arterial hypertension and renal disease.

E. The Effects of Hypervitaminosis D
on Man and Animals

Vanderveer (1931) used a group of rabbits varying in
age from three months to one year for the production of
arteriosclerosis. This was accomplished by feeding
irradiated ergosterol biweekly until death. The earliest
calcareous deposits in blood vessels were in the form of
incrustations about the elastic fibers in the media.

Smooth muscle degeneration of the media appeared later.

 

 

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Pathological calcification may be divided into two
broad groups according to Ham (1932). The first group
depends upon degenerative changes in the recipient tissues.
The mechanism may be that described by Klotz (1905) which
depends on the breaking—down of fats with the formation
of soaps and later more permanent calcium deposits, or
that described by wells (1925) which depends on the tissues
assuming characteristics more physical than chemical,
which enables them to bind calcium. The second group of
pathological calcification is etiologically different
according to Ham (1932). In this instance the blood be-
comes unable to retain all its calcium in solution. Ham
(1932) was concerned with studying the sequence of events
in tissues in an attempt to classify the calcifications.
Rats were given enormous single doses of irradiated
ergosterol which resulted in massive calcification of the
aorta, coronary vessels, and cardiac musculature as soon
as forth-eight hours following administration. Tissues
examined 24 hours after administration showed nothing that
would presage what was observed at 48 hours, so that the
calcification did not appear to depend on degenerative

changes in the recipient tissues. However, the rapidity

15

and massiveness of calcification suggested that the prime
factor in this process was the inability of the serum to
retain all of the calcium in solution. It was also sug-
gested that a change in carbon dioxide tension, addition
of other ions which would force precipitation of calcium
salts, or a continued liberation of ions from the non-
diffusable calcium after the diffusable calcium had reached
its point of saturation may bring about these changes.
Goormaghtigh and Handovsky (1938) studied the effects
of pure calciferol, administered orally, on the vascular
and renal systems of the dog. They stated that vitamin D,
a biological substance, given in adequate doses, caused
almost simultaneously hypertension and hypertrophy of the
arteriolar media. Four factors were thought to be respon—
sible for the hypertension: (a) the thyrotropic effect
of calciferol (vitamin D2), (b) the increased tone of the
hypertropic muscle cells, (c) the increased sensitivity
of the arteriolar wall to epinephrine, (d) a central ex—
citation. This hypertensive effect was elicited by daily
dosage of irradiated ergosterol ranging from 70 to 90
micrograms of vitamin D per kilogram during a period of

from 15 to 25 days. Hypervitaminosis D with marked

2

hypercalcemia led to glomerular, tubular and interstitial

16

lesions of the kidney with a distinct tendency toward uremia.

With thyroidectomy, thyroid aplasia, or marked hypo—
thyroidism, the dosage causing the renal damage will also
cause gross lesions of the aorta. A value of 13 milli-
grams per hundred cubic centimeters is the blood calcium
level which is the prelude to massive calcium excretion,
tubular distention, and subsequent glomerular regression
(Goormaghtigh and Handovsky, 1938).

Follis (1955) administered acutely toxic amounts of
vitamin D to rats and rabbits. This led to profound bio-
chemical and anatomical changes. The serum calcium, and
to a lesser extent serum phosphorus, concentrations rose
abruptly. Calcium values reached their peak on the third
or fourth day and then fell. Serum phosphorus followed a
similar pattern. Alkaline phosphatase values began to
fall and low levels were noted at the time of death. There
was an unexplained rise in the arterial blood pH. Values
as high as pH 7.8 were observed from the fourth to sixth
day of the experiment. Renal damage was increasingly
evident both microscopically and by chemical evidence of
rising calcium and phosphorus concentrations in the renal
tissue. Concentrations were also greatly elevated in the

myocardium. Rachitic changes were noted in the bones but

 

 

 

 

 

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no change in calcification of the epiphyseal cartilage was
observed. Due to inanition, growth ceased, however, wide
osteoid borders appeared which indicated that osteoblastic
activity continued. The reason for the lack of calcifi-
cation in such osteoid tissue in the presence of increased
concentrations of calcium and phosphorus was not clear.
Lesions resembling human arteriosclerosis were exper—

t al, (1960). They

imentally produced in rabbits by Hass
used irradiated ergosterol dissolved in peanut oil. This
was administered at daily, biweekly or triweekly intervals.
Initially the dosage was regulated at levels just below the
quick lethal range. Pathological changes developed rapidly
accompanied by weight loss and death within six weeks. The
dosage and interval of dosage in later groups of rabbits
was varied to facilitate studying the pathological vascular
changes at various stages.

Serum calcium, total phosphorus and inorganic phos—
phorus were determined at one and two week intervals. The
histopathological lesions produced by this method resem-
bled the M6nckeberg type of changes seen in human clinical
cases. There were no persistent or consistent elevations
in the average levels of serum calcium, however, there was

an upward trend in serum inorganic phosphate. There was

 

 

 

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no correlation between the serum calcium and inorganic
phosphate levels and the degree of calcific medial disease.
Abnormal deposition of calcium salts were found in many
extra—osseous tissues. The calcium deposition in the
vascular system was in the direction of arterial blood

flow and extended further from the heart with extended
periods of experimentation (Hass, 1960).

Overdosage with vitamin D in pups resulted in bloody
diarrhea, anorexia, listlessness, greasy hair, vomition
and loss of body weight. Necropsy revealed severe cal-
cification of the stomach, heart, lungs, kidneys and muscles.
The blood became more viscid with increased non-protein
nitrogen and serum calcium. This would indicate progres-
sive renal failure (Steiner, 1946).

Smith and Jones (1957) stated that massive doses of
vitamin D caused an abnormally high level of blood calcium
with metastic calcification especially of the arterial
media.

A total surgical thyroidectomy and massive dosage of
vitamin D orally was employed by Crisp e§_§l, (1958) to
produce arteriosclerosis in dogs. All of the animals under

experimentation developed severe gross arteriosclerotic

 

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lesions. The microscopic lesions included medial deposi-
tion of calcium and internal proliferation.

HaWk g; a1, (1954) gave the chief symptoms of vitamin D
toxicity due to overdosage, as anorexia and polyuria. In
advanced stages, calcification of soft tissues, particu—
larly of the renal arterioles and the aorta was seen,
hypertension was not encountered. Vitamin D overdosage is
indicated by a rise in serum calcium.

In rats, Sodeman (1956) listed the effects of prolonged
excessive vitamin D intake as loss of weight, anorexia,
nausea, vomition, muscular weakness, apathy, hypercalcemia
and phosphatemia and the calcification of cartilage, blood
vessels and organs including the kidneys. The same con-
ditions have developed in arthritic humans treated with
200,000 to 1,000,000 units of vitamin D. In an extensive
literature review of arteriosclerosis, Hueper (1944)
reviewed the changes associated with excessive doses of
vitamin D.

1. Vitamin D, synthetic or natural, if given in
massive doses caused toxic symptoms and arteriosclerotic
lesions. Hypertension is reported in dogs and cats main—

tained on toxic doses of vitamin D. This is attributed

 

 

 

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to the renal arteriosclerosis that develops. Severe myo—
cardial damage resulting from vitamin D poisoning may cause
hypertension.

2. There is general agreement that the toxic functional
and anatomical effects elicited by excessive doses of
vitamin D are related to the ensuing hypercalcemia and
hyperphosphatemia. An elevation in serum cholesterol has
been observed in cats, rabbits and man.

3. The arterionecrotic action of hypervitaminosis D
has been demonstrated in monkeys, dogs, cats, rabbits,
guinea pigs, rats, mice, chickens and to a limited extent
in man. The lesions are identical in their extent, ana-
tomic character, and organic distributions, as well as in
their causative mechanism to those associated with para—
thyroid hormone poisoning.

4. The majority of investigators agree that the
arterial and metastic organic calcification are of a
secondary nature, i.e., they follow the primary degenera—
tion and necrosis of the tissues brought about by the
circulatory disturbances. However, there have been some
workers reporting calcification in the absence of degen-

erative changes in the rat.

 

 

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21

5. Low toxic doses of vitamin D, moreover, show
degenerative vascular changes without calcification. These
changes consist of loosening and swelling, glassy vesicular
cells, disintegration of cells and fibrils, hyalinization
of the media and fibrous thickening of the intima. These
lesions are in contrast with extensive medial calcification
and necrosis in the aorta and the coronary arteries seen
in response to massive doses of vitamin D.

6. Hypertrophy of the renal arterioles and medial
calcinosis of the afferent arterioles were described in
dogs subjected to hypervitaminosis D by one group of
workers. Another group of investigators reported gross
aortic changes in dogs only after thyroidectomy.

7. It has also been reported that vascular injury,
due to vitamin D, was transmitted from the mother to the
young rabbits. Other reports failed to confirm this
observation in rabbits, guinea pigs and rats.

8. Exposure to ultraviolet rays, such as those con-
tained in the solar spectrum and those produced by ultra—
violet ray lamps, elicit in the skin of man and animals
photochemical reactions. These include the production

of vitamin D, the generation of melanin from a precursor

of epinephrine, tyrosine, the destruction of vitamin C,

 

.- mu an a: 2“
.~u 3. I. I. . Ce. L. .3 . .
u“ .u“ .3 .C ... v a. .s. ~.
2‘ - u u- . . n . .

 

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22

and the release of histamine. Following solarization,
there is a gradual drop in blood pressure due to peripheral
vasodilation, which lasts for several days. The action of
vasopressor proteolytic substances and a drop in blood
tyrosine, indicate a deficiency of epinephrine. With
repeated exposure to ultraviolet rays and increased pig-
mentation, the blood pressure rises and parallels the
degree of melanosis, while the blood tyrosine remains low.
There may be an increase in blood calcium.

9. Children exposed to solar radiation for several
months evidenced no signs of hypervitaminosis D as a
result of this treatment. Similar results were noted with
mice exposed to ultraviolet rays in amounts suitable for
the production of actinic cancer.

10. Rabbits fed nonirradiated ergosterol and sub-
sequently exposed to ultraviolet rays or intense solar
rays exhibited mild lesions of arteriosclerosis.

11. In a group of rats which were exposed to car-
cinogenic doses of ultraviolet rays, an excessive inci-
dence of hyaline and calcified medial lesions in the

aorta, coronary, pulmonary, pancreatic, renal, adrenal
and testicular arteries were observed. These lesions

increased in frequency, extent and severity with the

duration of treatment.

 

 

 

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23

12. Hypercalcemia resulting from excessive intake of
calcium salts or from acidosis producing chemicals mobili-
zing the calcium in the osseous structures or from exten-
sive destruction of bone tissue results in calcification
of the arterial walls and certain organs similar to those
found with hypercalcemias of parathyroid hormone and
vitamin D origin. The same vascular changes occur in
osteoporosis such as the cancers, multiple myeloma, endo-
crine disturbances of pregnancy and athyreosis and
nephrosclerosis (Hueper, 1944).

F. The Interrelation of Elastic Tissue

and Calcium in the Genesis
of Arteriosclerosis

It has been shown by Blumenthal e§_al, (1950) that the
splitting and fragmentation of the internal elastic
lamella are the initial changes in the aging vessel. It
has also been shown that the intensity and the rate of
calcium deposition are directly proportional to the inten-
sity and rate of the elastic tissue changes.

The association of elastic tissue changes and calcium
deposition is not limited to man. The condition has been
described in several species and is more pronounced in

the rabbit, cow and bird.

-v

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b.

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24

Calcification of the arterial wall becomes visible
before the formation of intimal plaques in man. This is
thought to be the case in animals as well. If this is
true, then if sufficiently old animals are used, the ease
with which athreomatous plaques are produced by cholesterol
administration is dependent upon the state of development
of the spontaneous process of elastic calcification.

The alterations that constitute arteriosclerosis depend
chiefly on the colloidal properties of elastin, and that
the changes in the elastic elements of arteries are in
striking agreement with the behavior of aging colloids in
general. The alterations with age noted are: (a) reduced
capacity to bind water, (b) decrease in elasticity, (c)
decrease in permeability, and (d) a tendency for the gel
to be transformed into a granular state with a marked
decrease in the colloidal properties. Water loss is con-
sidered the primary process, with a resulting decrease in
the capacity of the colloids to hold soluble constituents
such as cholesterol and calcium in solution.

G. Vascular Degenerative Changes
in Hypothyroidism
Kountz (1950) reported that persons with low basal

metabolic rates as well as athyroid states had a definite

 

 

 

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25

tendency toward degenerative changes in the smooth muscle
of the blood vessels. Elderly persons that had a normal
basal metabolic rate over a period of years prior to death
did not have as advanced arterial changes.

It was also noted that calcification of the media
proceeded intimal plaque formation and that the elastic
fibers were primarily involved. The mechanism involved
was thought to be a primary factor in the destructive effect
on capillaries since the media of larger vessels is well
supplied with vasa vasorum and this degeneration of the
media could be related to a decreased blood supply or stag—
nated blood in the vasa vasorum. The thyroid may also play

an important part in muscle metabolism.

H. Theories of the Genesis of Arteriosclerosis

In a general review of arteriosclerosis by Hueper (1944),
several factors were listed which must be explained if a
theory on the genesis of the disease is to be valid. These
are:

a. The absence of appreciable arteriosclerotic lesions

in people is fairly frequent.

b. The infrequent occurrence of degenerative and

sclerosing lesions in young persons.

26

The absence of arteriosclerotic lesions in certain
species of aged animals and the relatively early
incidence of the lesions in other species.

The irregular topographic involvement of the
arterial system by these changes.

The pronounced differences in anatomical types of
arterial lesions and their relationship to struc-
tural vascular locations.

The relation of certain degenerative and sclerosing
changes to sex and perhaps to race.

A correlation of these vascular changes to definite
endogenous or exogenous causal factors in many
cases.

The experimental production of various types of
arteriosclerotic lesions in various species by

markedly different chemical or physical means.

Senescence Theory

The senescence theory of arteriosclerotogenesis as-

sumes that the degenerative and sclerosing changes observed

in arteries are in general the result of the physiological

process of aging. This may be altered in individual cases

by additional vasoactive factors. This is supported in

27

part by the increase in incidence of arteriosclerosis with
increase in age. Additional support for this theory is
derived from the demonstration of physical and histological
changes in arteries, both large and small, related to
aging. Observations have established the fact that the
relative width, circumference of the vascular lumen, the
length of the vessels, and thickness increase with age.
Microscopic investigations have revealed that a degeneration
of the elastic elements accompanied by a peculiar axial
crystallization of the elastic lamellae during the fourth
and fifth decade. These lamellae later undergo granular
disintegration and develop discontinuities filled with
collagenous tissue. This results in a reduction of tensile
strength. These so—called senile vascular changes are the
basis for subsequent alterations of the lipoidal, hyaline
and calcifying types. These factors, according to the
supporters of the senescence theory of arteriosclerosis,
are an integral and important preparatory process in the
formation of arteriosclerotic lesions.

The physiochemical reactions in the tissues underlying
these morphologic senile changes and their secondary
arteriosclerotic sequela consist of the colloidal gels

composing the cellular and the intercellular matter. In

o-r‘1

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28

youth, these are finely dispersed hydrates of alkaline
reaction. They become dehydrated with increasing age,
denser, more coarsely dispersed and more acid. During
this period, processes of precipitation and disintegration
associated with the separation of complex colloidal
material favoring the formation of deposits occur.
Simultaneously, the mucin content of the vascular wall
increases, the extracellular collagenous and elastic
fibrils and hyaline membranes undergo impregnation and
disimpregnation with various chemicals. This results in
molecular transformation of the cement substances and
physical and chemical changes of the elastic substances.
Nitrogenous materials increase and an accumulation of
waste metabolites such as calcium and cholesterol, in the
aging dehydrated tissues leads to precipitation of these
products. The diffusion process in such bradytrophic
tissues, having no vessels of their own, depend, moreover,
on the presence of large surfaces and decrease therefore
With the density of the tissues. The localized nutritive
disturbances in the tissues affected by these physico—
chemical reactions are aggravated by the precipitates of
calcium and cholesterol, especially since these are usually

located near the vessel lumen.

 

 

 

 

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29

Atherosclerosis, according to these concepts, is
essentially the direct result of physiologic senescing
processes of a physicochemical nature affecting the ves~
sels and not the reaction product of alimentary hyper-
cholesteremia or disturbances in cholesterol metabolism.

Investigations have shown that these physicochemical
and morphologic vascular changes are not related to any
appreciable quantitative disturbances in the cholesterol
and calcium contents of the blood since they are not con—
sistently elevated during old age.

The appearance of arteriosclerosis has been connected
with a physiologic decrease in function of the thyroid
gland during advanced years. Cessation of functional
activity of the sexual glands is thought to be related to
arteriosclerosis. This is evidenced by the incidence of
arteriosclerosis found in women after oophorectomy and
menopause, and sometimes in men after castration and dur-
ing climacteric.

The fact that appreciable arteriosclerosis is not a
consistent finding in old age and occasional demonstration
Of local as well as generalized arteriosclerotic, arterio-

calcinotic and atherosclerotic lesions in babies, children

 

 

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30

and young adults precludes the validity of the senesence

theory of arteriosclerosis (Hueper, 1944).

The Mechanical Trauma Theory

The mechanical trauma theory considers trauma of the
muscular walls resulting from the presence of exogenous
physical forces or of physiologic or pathologic hemodyna—
mic forces as a principal or an important contributory cause
of the development of chronic degenerative arterial disease.
The evidence, however, indicates that such physiologic
or pathologic influences play a minor and secondary role,

if any, in the development of arteriosclerosis (Hueper, 1944).

The Toxin Theory

The toxin theory is favored by some investigators
according to Hueper. This is the concept that certain
substances, of bacterial origin or of endogenous or exo—
genous chemical nature, exert a specific and direct cyto-
toxic effect on the vascular wall and thereby give rise to
atherosclerotic, arteriosclerotic and arteriocalcinotic
lesions.

This theory,as originally proposed by Virchow, assumes
that bacteriotoxins present in the blood injure the intima

'and thus predispose it to subsequent atheromatous changes

 

P" 1

Afr'

5v.-

Ry:

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31

by increasing its permeability to cholesterol and fats
contained in the plasma. It further considers arterio-
sclerosis and hypertension as sequelae of the toxins'
action.

This theory is not generally applicable as there are
other mechanisms by which arteriosclerotic lesions es-
pecially those of the intima and atheromatous types are
produced. The fact that the toxins do not always produce
vascular changes would also question the validity of this

theory (Hueper, 1944).

The Anoxemia Theory

The anoxemia theory of arteriosclerotic genesis, as
reviewed by Heuper (1944), contends that the common funda—
mental action through which the various types of causal
agents and their different causal mechanisms affect the
vascular walls is represented by a severe and short, or a
moderate and prolonged, but frequently recurring or per—
Sistent interference with the oxidative metabolism and
nutrition of the vascular wall.

The validity of this concept on which the classifi-
cation of the causal agents are based is supported by

evidence obtained from an analysis of the degenerative

 

 

 

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32

organic lesions found in association with the vascular
changes. The organic degenerative manifestations are
elicited by an appreciable number of agents in the brain,
heart, stomach, blood, thyroid gland, and the testes,
where they are caused by a local anoxemia related to the
arteriosclerotogenic mechanism. These include such agents
as carbon monoxide, lead, manganese, mercury and others
which lead to softening in the basal ganglions in the
brain which frequently give rise to symptoms of the
Parkinsonion complex. Ischemic myocardial degeneration
is characteristic of poisoning with epinephrine, nicotine,
digitalis glucosides, parathyroid hormone, vitamin D,
histamine, nitrites, and reduced oxygen pressure. Peptic
ulcers on the basis of local ischemic vasospasm or stagnant
anoxemic, are found in mercury, carbon monoxide, carbon
disulfide, cyanides and nicotine. Chronic anoxemia of
various types and genesis are responsible for the degen-
eration of the spermatogenic epithelium of the testes in
exposure to reduced atmospheric oxygen pressure and in
poisoning such as carbon monoxide, carbon disulfide, ni—
trites, nicotine, etc.

The thyroid gland, an organ which has an important

function in oxygen metabolism of the body, becomes

 

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hyperactive after poisoning with carbon monoxide, mercury,
lead, manganese, cyanides, thiourea and sulfonamide.

Some factors producing anoxemia such as hypotonia of
the vessels due to vasodilating agents, followed by stag-
nation anoxemia, disturb the metabolic processes since the
media of large arteries depend entirely on diffusion pro—
cesses for their nourishment and gaseous exchange. The
outer two-thirds of the vessel receives oxygen and nutri—
tive substances from the vasa-vasorum and with severe
hypotonia, stasis develops in these small vessels.

Many toxic chemicals will bring about vascular hypo—

tonus as will traumatic shock, hypopituitarism, hypoadren

alism and hypothyroidism. Chronic pathologic hypotensive
states are not common, but it is well established that
with prolonged and intensive relaxation of the arterial
walls with a resultant fall in blood pressure and slowing
of blood flow, an intimal proliferation and medial degen-
eration ensue. Endogenous agents as histamine, acetyl-
choline, anaphylactic shock, orthostatic shock, and anoxia
are all factors in hypotonia and stagnation anoxemia.
Exogenous agents include nitrates and nitrites, carbon

monoxide, arsenic, aromatic hydrocarbons, manganese, mercury

I

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34

as well as agents producing traumatic vasoparesis, lead
to hypotonia and stagnation anoxemia with resulting arterio—
sclerotic vascular changes.

Another type of anoxemia is hypertonia followed by
vasoconstriction which results in anoxemia of the vessel
wall. Hypertonic agents elicit a contraction of the mus-
cular elements of the vascular wall and thereby a reduction
of the vascular bed. This results in a compensatory
acceleration of blood flow due to an increase in blood
pressure. Prolonged or frequently recurring excessive
vasomotion results in a disturbance of the nutritive con—
ditions and oxygenation of the tissues. This results in
degenerative vascular changes. The mechanism entails the
following events: (a) inordinate vasoconstriction, (b)
reduction of the blood flow of the vasa vasorum, (c)
contracting media, with ischemic hypoxemia, (d) accumulation
of metabolic waste products. The increased functional
activity of the muscle cells results in an increased
production of these metabolic waste products. The increased
density of the constricted vascular structures hinders the

movements of the interstitial fluid and thus reduces

diffusion.

W
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35

The validity of the hypertonia anoxemia etiological
concept is attested by the myocardial and arteriosclerosis
lesions in animals subjected to lethal poisoning by para-
thyroid hormone, vitamin D and digitalis glucosides. In
evaluating the causative factors responsible for the
hemorrhages and myocardial necrosis observed under these
conditions, it is significant that the topographic rela-
tions on the myocardial arteries and arterioles to the
surrounding muscular tissue of the heart are approximately
the same as those of the vasa vasorum to the muscle tissue
of the arterial media. The cardiac effect, of excessive
doses of the three aforementioned substances, consists of
an extraordinary prolongation and accentuation of the
systolic contraction and an incompleteness and shortening
0f the diastolic dilation of the ventricles. This creates
a circulatory stasis in the myocardial vessels which results
in myocardial anoxemia. This is most pronounced in the
myocardial tissue located near or beyond the end of the
blood vascular supply, i.e., the subendocardial and papillary
regions where the ischemic necrosis and hemorrhages are
seen. Similar myocardial changes occur in connection with

epinephrine poisoning.

 

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36

Before concluding that hypertension and arteriosclerosis
are related, one must determine if the hypertension is due
to hypertonia or if it is due to increased cardiac output,
increased blood volume, increased blood viscosity or
reduction of elasticity of the vascular walls. It must
also be remembered that not all hypertensive cases due to
hypertonia result in arteriosclerosis.

Some of the endogenous and exogenous vasoconstricting
agents, which have been employed in the experimental
production of arteriosclerosis, include the following:
vitamin D, epinephrine, physostigmine, nicotine, epinephrine
derivatives and precursors (ephedrine, amphetamine, tyramine,
pyrocatechin, tyrosine etc.). Also included are: psychic
strain which is based on life insurance studies of various
groups of people: adrenal cortical hormone which have a
vasoconstrictor action as does the synthetic desoxycorti-
costerone, pituitary hormones, as pitressin, are associated
with hypertension, and arteriosclerosis is noted in numerous
cases having basophilic adenoma encroaching upon the pos-
terior pituitary gland: parathyroid hormone in excess, but
not toxic levels, results in hypercalcemia, hyperphospha-

temia, bradycardia and hypertonia.

 

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Other vasoconstricting agents which have been observed
clinically to produce vascular sclerotic changes are hydra-
stine, ergot, barium chloride, lead, thyrosin and iodine,
cold, physical trauma, renal hypertension and essential
hypertension. These arteriosclerotic vascular changes are
due to anoxemia according to this theory (Heuper, 1944).

With the increasing interest in space medicine recent
workers have studied, among other things, the effect of
altitude on the cardiovascular system. Highman and Altland
(1949) studied a group of 14 day old rats exposed to
25,000 feet of simulated altitude for four hours daily.

An important effect of this procedure was the development
of polycythemia. Pathological observations consisted of
gross cardiac hypertrophy, varying degrees of myocardial
damage, and nodular thickening of the heart valves. There
were also renal infarcts and other lesions not involving
the cardiovascular system.

Aortic insufficiency was produced by Altland and
Highman (1959) in one group of dogs and mitral insuffi—
ciency in other. Following thirty days post surgical rest,
these animals were subjected to simulated high altitudes.
The dogs were gradually taken to thirty thousand feet and

maintained at this altitude for four hours daily. After a

a n
A:

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2.

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38

ten day interval, surviving animals were then exposed to
32,000 feet, to 34,000 feet, and to 36,000 feet respectively.

The most constant pathological findings were visceral
engorgement, increased cellularity of the bone marrow,
cardiovascular lesions, and renal hemosiderosis. The
severity of the lesions varied markedly. The cardio—
vascular lesions included thickening of the mitral and
aortic valves with a large amount of metachromatic material
and material staining like acid mucopolysaccharide. The
myocardial arteries frequently had a thickened adventitia
and occasionally cellular proliferation of the intima.
Some animals showed intimal hyaline changes, necrosis, or
hyalinization of the media. Some plaques were seen in the
aorta and pulmonary artery and occasionally the coronary
arteries. The thoracic and abdominal aorta showed arterio-
sclerotic lesions. The arteriosclerotic plaques were
attributed to hypoxia.

Rabbits were fed a high cholesterol diet and exposed
23 hours daily to 16,000 feet simulated altitude for a
maximum of 17 weeks by Altland and Highman (1960). The
lesions produced were compared with identically fed rabbits
maintained at ground level. The high altitude group

showed much more severe atherosclerotic lesions in the

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pulmonary.vessels than did the ground level control group.

However, lesions in the descending and abdominal aorta were
milder. The high altitude group also showed a much higher

incidence of calcification. It was thought that, at least

in part, the greater severity of lesions in the high alti—

tude group could be attributed to hypoxia.

Rinehart and Greenberg (1948) reported the occurrence
of arteriosclerotic lesions in Rhesus monkeys subjected to
pyridoxine deficiency. These same workers (1949) reported
in more detail the investigative findings of pyridoxine
deficiency in arteriosclerosis production. In 1951,
Rinehart and Greenberg again reported on 18 Rhesus monkeys
in which arteriosclerosis was experimentally produced in
the same manner as their earlier work. They state that
the sclerotic arterial lesions are related to pyrodoxine
deficiency without a doubt, and that lesions so produced
have not been found in other deficient states.

Calves, in which low magnesium levels were produced,
developed arteriosclerotic lesions which were not asso-
ciated with elevations of either serum calcium or phosphorus.
This work was done by Hartman (1959) to further investigate

work reported in the 1930's on calves on low magnesium

diets.

 

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40

White muscle disease, which has as its etiology a vita-
min E deficiency, is associated with metastic calcification
of the heart, skeletal muscle, kidneys, pancreas, fundal
portion of the abomasum, duodenum, small intestine and
lungs. This disease and the metastic calcification asso—
ciated with it occurred in thirty lambs out of a flock of

400 in western Australia (Gardiner, 1961).

I. Collateral Circulation

It was observed by Plummer years ago that patients with
exophthalmic goiter exhibited widespread dilation of the
peripheral blood vessels. This prompted Herrick gp_al,
(1933) to administer graduated doses of desiccated thyroid
to dogs to see if the same results could be produced. This
procedure resulted in an increase in blood flow from 200
to 300% in the femoral artery.

Ferris and Harvey (1925) using dogs, placed ligatures
around the upper part of the femoral artery. Limb tem-
peratures fell abruptly, about four degrees centigrade,
following ligation. In about one hour there was 1.5
degree centigrade rise. The second hour the temperature
fell 1.5 degrees centigrade and then rose suddenly to

slightly above normal. During the following six hours the

 

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temperature gradually decreased 12 degrees centigrade. This
was at eleven hours post—ligation. There was a gradual
rise during the next 75 minutes when suddenly within 15
minutes a 10.5 degree centigrade rise was recorded. There
was a further abrupt rise of two degrees centigrade and it
remained at this plane for the next 17 hours at which time
recordings were terminated. One animal had only a slight
temperature drop in the limb following ligation of the
femoral artery. This was considered to be due to a very
efficient collateral circulation already in existence.

These investigators concluded from rapidity of the
temperature rise, that the first step in the establishment
of a collateral circulation is a surprisingly abrupt
vasomotor reaction, the mechanism of which was not
determined.

Ligation experiments and temperature studies con—
ducted on dogs by Mulvihill and Harvey (1931) consisted
of (a) ligation of external iliac artery, (b) ligation of
both external iliac arteries, (c) ligation of both exter-
nal iliac arteries and unilateral removal of the tribu-
tary sympathetic ganglia.

Ligation of one or both external iliac arteries, close

to the aorta, was always followed by a temperature drop in

 

 

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and

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the corresponding foot of from ten to thirty degrees. In
a majority of the dogs, a rise in temperature and sub-
sequent return to the previous level was noted in about
13 hours post-ligation.

It was the opinion of Mulvihill and Harvey (1931) that
anatomical channels sufficient to care for the circulation
is a vasomotor phenomenon. This was further substantiated
in that the temperature of a limb which had dropped to
room temperature post-ligation of the external iliac, rose
to normal soon after removal of the tributary sympathetic
ganglia and that simultaneous removal of tributary sympa-
thetic ganglia with ligation of the artery prevented the
lowering of the temperature of that limb.

In dogs with external iliac ligations, Harvey and
Halpert (1931) studied the effects of: (a) transection
of the spinal cord at the lumbar level, (b) transection of
all posterior roots on one side below the level of the
first lumbar vertebra, (c) removal of the tributary
sympathetic ganglia on limb temperatures. This procedure
caused a prompt rise in the temperature of the corresponding
foot from room temperature to nearly that of the limb prior

to ligation of the external iliac. This rise also occurred

in animals with the external iliac artery ligated and the

 

 

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spinal cord transected and also in the dogs with all the
posterior roots cut below the level of the first lumbar
vertebra.

Eckstein g;_§l, (1941) designed an experiment to
study the magnitude, time of development and pressure
relations of collateral circulation following femoral,
carotid and coronary artery ligation.

The femoral artery was ligated in the upper third and
a canulla was placed in the main artery or in a side branch
peripheral to the point of ligation. Immediately after
occlusion, the flow and pressure values are small, how—
ever, within a minute retrograde pressures and flow increase
and continue to do so for hours. Within 15 seconds follow-
ing femoral artery ligation the peripheral pressure drops
from the control level of 180/130 mm. Hg to 20/20 mm. Hg
and the pulse disappears. After 90 minutes the pressure
increased to 98/93 mm. Hg. Blood flow rises from 10 cc.
per minute 15 seconds following ligation to 50 cc. per
minute in 90 minutes. Once open, collaterals tend to
remain open since after one hour when the ligature was
removed, the retrograde pressure remained at about 98/93

mm. Hg and replacement of the ligature resulted in a drop

 

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44

to 72 mm. Hg compared to 20 mm. Hg following the first
occlusion. Two hundred and fifty days following ligation,
the retrograde pulse was 160/125 mm. Hg and the flow was
200 cc. per minute.

These workers, Eckstein §;_al, (1941), concluded from
this experiment that collateral circulation development
is influenced by: (a) the openings of pre-existing but
non-functional collaterals, (b) by a combination of dif-
ferential pressures, metabolites, nerve action and the
formation of new collaterals.

Collateral circulation development in the presence of
an arteriovenous fistula, is not due to the Lewis Theory—-
that the tissue needs beyond the arteriovenous fistula
excite development of collateral circulation, because
amputation of the potentially ischemic tissues beyond the
fistula does not interfere with collateral circulation
development. The important condition for collateral
circulation development was found to be the ready access
of blood to the site of the fistula by retrograde flow
through a widely patent distal artery and its branches.
The most efficient deterrent to the development of col—

lateral circulation is to ligate the artery just distal

 

 

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to the fistula, leaving no branches intervening between
the fistula and the site of ligation (Holman, 1949).

The collateral circulation which develops following
ligation of a large artery depends upon transforming high
end pressure in the parent artery into lateral pressure
which directed into its branches, results in an increased
volume flow through them. This volume flow distends the
collaterals and opens up their prearteriolar and arterio—

lar beds (Holman, 1949).

4
(Pl- P2)r
Applying Poiseuille's Law of flow Q =

L" '
the most important value is r, for if the radius of the
capillary is doubled, the flow within it is increased 16—
fold. When 6% of the total blood volume is accommodated
in the capillaries, 94% of the blood volume is either
approaching or returning to them. This is the basis for
Learmonth's (1950) approach to collateral circulation.

The therapeutic objective is to keep the pressure as high
as possible on the arterial end of the capillary and keep
its caliber as wide as possible. To do this it is neces-
sary to maintain adequate blood volume and normal systemic
arterial pressure which is not due to vasoconstriction.

Another consideration is the architecture of collateral

Circulation Which falls into two groups: (a) after a short

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course in alternative channels the arterial blood is
returned into the main artery or arteries of the part,
(b) arterial blood never returns to the main arteries,
usually because they have been blocked by extensive
thrombosis. In the normal arterial tree 20 mm. Hg are
expended in forcing blood into arterioles, but with occlu-
sion, and only small branches available, 60 mm. Hg are
expended. These two groups make it obvious that to
prevent ischemia the main and terminal arterial branches
must be kept dilated to allow as much blood as possible
to reach the arterioles. The arterioles must also be kept
widely dilated to increase the amount of blood reaching
the capillaries. These objectives are accomplished by the
following methods: (a) use of drugs such as the dihydro—
genated alkaloids to produce peripheral vasodilation, (b)
blocking vasoconstrictor impulses at their synapses in the
sympathetic ganglia with such drugs as tetraethyl ammonium
bromide or chloride, (c) stimulation of vasodilator nerves
with such agents as butylsympatol, (d) heat application to
the part, (e) sympathectomy of vasoconstrictor nerves in
the precarious region.

Collateral circulation is divided into three types of

distribution, whether or not the main collateral branches

(F

47

or accessory collateral branches are participating in its
formation (Krahl _t_§l,, 1954).

1. Areas of hypervascularization--These areas are
mainly formed by accessory collateral branches,
very small in size, which carry blood to an area
in which the normal blood supply has been cut off.

2. Supplementary branches-—These may arise either
from main or accessory collateral branches and are
large enough to be identified by arteriogram. They
usually run parallel to the occluded main artery
but vary in size and distribution.

3. Bridging collaterals-—Most of these are formed by
main collateral branches. They delimit a segmen—
tal obstruction of a main artery and are actually
responsible for the functional diversion of blood
to the main artery distal to the obstruction. The
blood flow is reversed in those bridging collaterals

which originate from the main artery distal to the

block.

The formation and development of new vascular channels
may be modified or controlled by factors other than purely

mechanical or reflex adjustments (Rosenberg and Liebow,

on a
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48

1954). This is suggested by inhibition of vascularization
to an irritated cornea When previous or subsequent admin—
istration of corticotropin. Faulty or delayed wound heal—
ing is also reported in man and animals treated with
cortisone. Growth hormone and desoxycorticosterone acetate
have been shown to have a stimulating effect on healing.
It has also been observed that bronchial arterial colla-
teral development following pulmonary artery ligation, was
much more rapid in newborn puppies than adult dogs. Rats
with the iliac artery ligated and receiving five mg. per
day of cortisone showed less collateral circulation
development than did the controls. Rats that received
growth hormone developed more collateral circulation than
did the controls. The most surprising observation was that
old rats developed the best collateral circulation. This
would suggest that the initial size of the potential
collateral channels may be a determining factor in their
ultimate size. Perhaps the final size of the animal may
thus influence collateral circulation.

Sewell and Koth (1958) employed the method first used
by Halsted in 1922, when he transected the thigh of the
dog except for the femoral and sciatic nerves, femoral

artery, vein and femur. The transected muscles were

 

 

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49

reapposed and allowed to heal. If the femoral artery was
ligated within two weeks of surgery, the limb became
gangreneous. Division of the vessel after two weeks did
not cause gangrene. Sewell and Koth ligated the femoral
arteries between the sixteenth and twentieth day. All of
the transected dogs walked within a week after surgery but
following ligation two out of five limped. The other
three walked normally by the third week following ligation

of the femoral artery.

 

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'5.

CHAPTER III
MATERIALS AND METHODS

In this study, 42 male dogs were used. Of this number,
36 were thyroidectomized and six served as controls. Their
weights ranged from 13 to 45 pounds. They were estimated
to be between one and four years of age. The dogs were
housed in a central pen for some time after procurement
from the pound before starting them on experimentation.
Only dogs that appeared to be in good health at the end
of the holding period were used.

Fecal examinations were run on all dogs and they were
given vermifuges as indicated. None of the dogs were
vaccinated against distemper, hepatitis or leptospirosis.
Dry kibbled meal* was fed free choice until the dogs
became anorexic at which time the diet was changed to
canned dog food.**

After the initial holding period a total thyroidec—
tomy was performed in all but the control dogs. The dogs

were fasted twelve hours prior to surgery. Sodium

* Fromm Complete Dog Meal, Federal Foods Inc., Thiens-

Ville, Wisconsin.
** Hill Packing Co., Topeka, Kansas.

50

 

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pentobarbital was given intravenously for general anes-

thesia and endotracheal intubation was performed after

induction. At this time the dogs were weighed and a blood

sample was drawn from the jugular vein for serum protein

electrophoresis, serum calcium and serum cholesterol

determinations.

E1ectrocardiograms* were recorded with the dog placed

in the right lateral position with the left fore and hind

limbs slightly extended. Leads I, II, III, AVR, AVL,

AVF, CR6U, CR6L, CL6U, CL6L, CF6U, and CF6L were recorded.
The surgical procedure was performed with the dog

secured in the dorsal recumbent position with the head

extended. The surgical area was clipped and scrubbed with

liquid germicidal detergent.**

A midrline incision was made extending from the larynx

caudally for four inches. By blunt dissection the thyroid

glands were exposed and the parathyroid glands were care-

fully dissected free with iris scissors taking care to

preserve their blood supply. The thyroid vessels were

then ligated and the glands removed. The right carotid

* Edin Electronic Cardiograph Model 220, Worchester 8,

Massachusetts.
** Parke, Davis and Co., Detroit, Michigan.

52

artery was next exposed by blunt dissection and the blood

pressure was recorded by means of a direct reading mer-

cury manometer. A one-inch 20 gauge needle was used for

cannulation. After the blood pressure was recorded, bull-

dog clamps were placed on the carotid artery above and

below the puncture and left in place until a clot formed

‘at the puncture site. The muscle fascia was sutured and

the skin incision closed with simple interrupted sutures

of number 50 nylon.

On the first post-operative day, the dogs were given

100,000 units of vitamin D* orally. This was continued

daily throughout the experiment.

On the forty—ninth, fiftieth or fifty—first day fol—

lowing thyroidectomy, the dogs were weighed and a blood

sample was drawn from the jugular vein. Thiamylal sodium**

was given for general anesthesia and the dogs intubated.

The gastrocnemius muscle temperature was recorded from

both hind limbs using a direct reading thermometer*** with

a trochar electrode. This was inserted about an inch into

* Deltalin, Eli Lilly & Co., Indianapolis, Indiana.
** Surital Sodium, Parke, Davis and Co., Detroit, Michigan.

*** Electronic Thermometer Model TMB Tri-R Instruments,
Jamaica 35, New York.

 

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the muscle. An electrocardiogram was obtained from dogs

33(B), 26(B), 2(W), BSN, and 13(Y), and a packed cell

volume was determined on dogs 16(W), 37(W), 38(W), 39(W),

12(Y), and 26(B). The dogs were next placed in a lateral

recumbency with the pelvic limb to be transected suspended

by means of a nylon cord. The limb was clipped and scrubbed

with germicidal detergent. The transection was made mid—

way between the stifle and coxofemoral joints and was

complete except for the femur, sciatic nerve, femoral

artery, vein and nerve. Bleeding vessels were ligated

as they were encountered. After transection was complete

and hemostasis established, the muscles were carefully

reapproximated and sutured with interrupted Vetafi1*

sutures. Skin closure was routine.

I

The day following transection, dogs 26(B), 2(W), 33(B)
BSN, and 13(Y) were given one grain of thyroid extract per
orum and this was continued every fourth day until death.
The other dogs did not receive thyroid extract.

Eighteen days following transection, the dogs were
again anesthetized with thiamylal sodium and intubated.

limb temperatures were taken in the same manner as

_.__

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54
previously described. The dogs were then scrubbed for

surgery and a skin incision was made over the femoral

vessels. The femoral artery and vein were exposed by

blunt dissection, double ligated with vetafil, and sec-

tioned between the ligatures. Closure was routine.

Pelvic limb temperatures were recordedenmuy'seventh

day following ligation until death of the animal.

At the time of death, the aorta, cardiac muscle,

lung and kidneys were collected and fixed in ten percent

formalin. The aorta was rolled so that an entire longi—

tudinal section could be made. A piece of all tissues

collected was placed in a Decal* for calcium removal.
The tissues were sectioned at six microns and stained with

Von Kossa's, hemotoxyln and eosin, and Weigert's-Van

Gieson stains.

Control dogs were not thyroidectomized, and were not

given vitamin D. At the time they were started on exper-

iment their body weight was recorded, a jugular vein blood

sample was drawn, and the right carotid blood pressure was

obtained as previously described.

* Scientific Products Division, American Hospital Supply
Corp., Evanston, Illinois.

 

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On the forty—ninth, fiftieth or fifty—first day, the

body weight was recorded and the pelvic limb was tran—

sected in the same manner as the thyroidectomized group.

Eighteen days following transection, limb temperatures

were obtained and the femoral artery and vein were ligated

and transected as previously described. Every seventh

day following ligation, limb temperatures were taken as

with the thyroidectomized group. On the twenty—first

post—ligation day, each control dog was anesthetized, limb

temperature was taken and body weight obtained. A blood

sample was drawn from the jugular vein and the carotid

blood pressure was determined. The dogs were then

euthanized and the aortas were collected and fixed in ten

percent formalin. The tissues were prepared and sectioned

as previously described.

Blood samples collected during the experiment were

allowed to clot. The serum was collected and frozen

until serum calcium, cholesterol, and protein paper

electrophoresis determinations* were made.

Calcium determinations were made by means of the

* Model R Paper Electrophoresis System, Beckman Instru-
ments Inc., Belmont, California.

 

 

56

flame photometer.* Serum cholesterol determinations were

performed by the method of Ferro and Ham.**

* Coleman Instruments. Inc-. Maywood, Illinois.
** Ferro, p, v° and Ham, A. B. A simple direct method

for the determination of total serum cholesterol.
Publication of Dade Reagents, Inc., Miami, Florida.

 

 

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CHAPTER IV
RESULTS
A. General Considerations

Dogs that were housed together in a large pen for three
weeks or longer prior to experimentation were in better

physical and nutritional condition and survived longer than

dogs kept for shorter periods. Death occurred within

eleven days in some of the dogs that were used for the

experiment shortly after receipt from the pound. Many of

them were asymptomatic and others showed early clinical

symptoms of canine distemper prior to death. Larger dogs

remained in better condition longer than smaller ones in

most instances.

Two dogs died as a result of fighting. After these

losses the dogs were caged individually for the remainder

of the experiment.

B. Thyroidectomized Dogs

Lethargy and anorexia became evident between the
fifteenth and thirtieth day following thyroidectomy. At

this time the diet was changed to a mixture of dry kibbled

57

 

 

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meal and canned dog food mixed with water. This ration

was eaten better, but the appetite remained poor. By the

time of death, lethargy, anorexia, dehydration, mental

depression and a rough greasy hair coat were noticed in

all thyroidectomized dogs. Loss of body weight was pro—

nounced as indicated in Table I. Decubitis ulcers and

eczema occurred in most of the dogs, but to a variable

degree. All thyroidectomized dogs were quite debilitated

by the time of limb transection. Edema following tran-

section and ligation was minimal. Limbs that survived

transection did not survive femoral artery and vein liga-

tion as evidenced by necrosis distal to the site of

transection. See Appendix V, figure 3. Figures 1 and 2

in Appendix V show the limb condition of a dog that sur-

vived limb transection.

Gross arteriosclerotic lesions in thyroidectomized
dogs were quite variable as indicated in Table IV. Some

dogs had minimal cardiovascular lesions at the time of

death 73 days following thyroidectomy. Other dogs that

died as early as the thirty-fifth post-thyroidectomized
day had extensive thick plaques and intimal elevations

throughout the aorta and its large branches. Gross lesions

were more pronounced and seen earlier in the smaller dogs

In most instances.

 

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59

Microscopic lesions are described in the photomicro-

graphs of Appendix V. Gross cardiovascular lesions are

shown and described in Appendix V also.

Day of death post-transection, limb condition, and

the presence of gross lesions of the thyroidectomized and

transected dogs are shown in Table IV.
There was electrocardiographic evidence of myocardial

damage. See Appendix IV for electrocardiographic results

and interpretations.
Serum calcium and cholesterol levels increased signi-
ficantly during the course of the experiment as shown in
Table II. Statistical analysis showed this increase to be
Dogs 30(W), 813,

on the order of five standard deviations.

and 955 were not included due to a possible error in label-

ing of the blood samples.

The albumin-globulin ratios as determined by paper

electrophoresis were within normal limits.

Limb temperatures are compared in Table III. However

they were not statistically significant.

Dogs 26(b), 2(W), BSN, B(Y), and 33(B) received one

grain of desiccated thyroid orally starting with the first
Post—transection day, and given every fourth day until

death. Thyroid administration caused no visible change

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60

in the behavior or general physical condition of the dogs.

The effect that thyroid supplement had on gross, and micro~

scopic lesions is not known.

C. Control Dogs

Control dogs evidenced no appreciable loss of body

weight. They remained alert and active throughout the

experiment. Growth rate of hair following clipping was

normal and the hair coat was unaffected. The appetite

remained good throughout the experiment. Neither decu-

bitis ulcers nor eczema was a problem. All dogs survived

limb transection and vessel ligations. There was con-

siderable edema distal to the transection for about five

days following surgery. A slight reoccurrence of edema

was noted following vessel ligation.

Body weights at the time the dogs were started on the

experiment are compared with the weights at termination in

Table I. Serum calcium and cholesterol levels were not

altered appreciably during the course of the experiment

as shown in Table II. The transection data and gross

lesions are shown in Table V. Paper electrophoresis re-

vealed no appreciable changes in the albumin-globulin ratios.

At necropsy, one dog was found to have minimal gross

arteriosclerotic lesions at the aortic valves.

61

TABLE I

Body weights of the Thyroidectomized Dogs

Dog Numbers Weight in Pounds Following Thyroidectomy

 

3 (W) Day 1 Day 50* Day 68**
3 (W) 22 15 -—
16(W) 37 21 --
37(W) 3O 20 16
38(W) 35 25 20
39(W) 32 17 —-
11(Y) l7 -- ——
12(Y) 27 25 ——
13(Y) 31 2O 15
3 (Y) 17 —- --
33(B) 39 27 21
BSN 40 30 -—
2 (W) 25 13 __
26(B) 3O 2O --
24(w) 30 20 --

Body weights of the Control Dogs

Dog Numbers Body weight in Pounds

 

Day 1 Day 50 Day 89
15(8) 24 23 23
955 17 l9 17
24(3) 20 24 22
32(B) 30 35 32
22(W) 35 50 37
35(w) 31 32 27

* Day of limb transection
** Day of vessel ligation

 

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TABLE II

62

Serum Calcium and Serum Cholesterol Levels

in Thyroidectomized Dogs

 

Dog Day Post- Calcium Cholesterol Day of
Number operative Mg % Mg % Death Post-
Thyroidectomy

26(B) 1 12.1 148.6

51 20.0* 300.0

69 20.0 238.0 72
813 1 ---— 323.0

37 ---— 340.4

63 20.0* 212.8 66
37(W) 1 13.0 166.0

49 18.6 --—- 73
13(Y) 1 13.4 166.0

51 13.2 190.0 72
21(B) 1 12.2 160.0

32 17.7 -—-- 35
8 (W) 1 15.0 132.2

33 --—- 256.0 51
958 1 10.0 181.8

36 ---— 298.8 57
39(W) 1 9.9 152.0

51 —--- 200.0 51
3 (Y) 1 10.5 ---- 50
2 (w) 1 10.5 -—--

50 20.0* -—-- 55
38(W) 1 11.2 165.0

49 19.8 162.6 73

* Maximum capacity of flame photometer was 20 mg.%.

63

TABLE II (Continued)

 

 

Dog Day Post- Calcium Cholesterol Day of
Number operative Mg %» Mg % Death Post—
thyroidectomy

33(B) 1 11.0 --——

49 18.4 —-—-

67 20.0* 190.0 68
36(B) 1 14.4 122.0

32 15.2 ——-- 45
8 (B) 1 —--- 144.0

35 9.6 —--— Euthanized

63 14.8 ---- 63
16(W) 1 12.3 ----

31 12.2 ---- 59
3 (W) 1 16.0 86.2

32 —--- 258.0 55
24(W) 1 14.5 153.8

34 20.0* ---- 51
ll(Y) 1 15.7 78.0 35
12(Y) 1 ---- 230.0

50 ___— 238.0 56
30(w) 1 17.7 206-0

30 12.8 ---- 34

* Maximum capacity of flame photometer was 20.0 mg. %.

64

TABLE 11 (Continued)

Serum Calcium and Serum Cholesterol

Levels in Control Dogs

 

Dog Day Post- Calcium Cholesterol Day
Number operative Mg. % Mg. % Euthanized
955 1 12.9 ---—

30 11.0 ----

61 8.8 248.0

89 14.4 182.0 89
15(B) 1 —-—— ----

30 9.8 ----

61 11.3 160.0

89 10.3 130.0 89
35(W) 1 13.8 144.7

30 10.2 144.7

61 12.8 153.1

89 17.4 153.1 89
24(B) 1 13.6 153.1

30 10.2 170.0

61 15.1 148.4

89 12.0 182.9 89
32(B) 1 13.2 126.0

30 10.9 170.0

61 12.8 152.0 89
22(W) 61 9.6 180.0 89

65

TABLE III

Limb Temperatures of Thyroidectomized Dogs

 

Dog Limb Limb Post-operative
Number Transected Measured Temperatures in O C.
Day 50* Day 68**
13(Y) Right Right 35.75 27.00
Left 34.50 28.25
33(B) Right Right 37.25 35.90
Left 36.00 35.90
BSN Left Left 35.00
Right 34.50
2 (w) Right Right 33.00
Left 34.50
26(B) Left Left 35.50 35.50
Right 35.50 37.00
37(W) Right Right 33.50 34.00
Left 32.75 36.00
38(W) Left Left 36.00 32.00
Right 36.50 31.00
39(w) Right Right 28.00
Left 30.50
12(Y) Left Left 35.00
Right 36.00

* Day of limb transection
** Day of femoral artery and vein ligat1on

 

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66

TABLE III (Continued)

Limb Temperatures of Control Dogs

Dog Limb Limb
Number Transected Measured

Post-operative

I O
Temperatures 1n C.

 

Day 68** Day 75 Day 82
15(B) Right Right 37.00 34.80 34.00
Left 37.50 37.00 36.00
955 Left Left 35.50
Right 37.50
24(B) Right Right 34.00
Left 36.50
32(B) Left Left 34.50 35.80
Right 38.50 37.75
22(W) Right Right 36.90 38.10 35.50
Left 37.25 38.50 36.50
35(W) Left Left 36.00 36.00 35.50
Right 36.00 37.00 36.50

* Day of femoral artery and vein ligation.

Day 89

36.50
38.00

36.25
37.10

37.00
36.60

36.90
37.25

36.00
37.75

36.00
38.00

 

a.)

 

 

67

TABLE IV

Post-transection Data on Thyroidectomized Dogs

 

Dog Day of Limb Condition Gross Lesions
Number Death Post- at Death of Blood Ves-
transection sels. Degree
of Plaque
Formation
3(W) 6th Necrot1c distal Marked
to transection
16(W) 10th Early necrosis Moderate
37(W) wnd Early necrosis Moderate
38(W) 24th Early necrosis Moderate
39(W) Same day ---- Marked
12(Y) 6th Viable Minimal
13(Y) 20th Viable Marked
Functional
33 d t
(B) 19th and healed Mo era e
BSN 7th Viable None
26(B) let Necrotic Moderate
2 (W) 5th Viable Minimal
813 3rd Viable Marked
8(W) 1st Viable Moderate
24(W) Same day Viable Marked

* See Appendix I for more detailed information

(‘1

(.1

 

68

TABLE V

Transection Data and Gross Lesions for Control Dogs

 

Dog Condition of Limb Condition of Limb Gross
Number at Time of Vessel at Time of Exper— Lesions
Ligation iment Termination
955 Healed and Healed and None
functional functional
Healed and Not
24(B) functional functional None
Healed and Foot ulcerated Moderate
32(B) functional limping plaques
at aortic
valves
Healing No healing
22(W) incomplete on anterior None
surface. Not
functional
Healed and Healed and
35 W
( ) functional functional None
Healed and Healed and
15
(B) functional functional None

Additional data in Appendix II

 

'VD

 

A.

V.

CHAPTER V

DISCUSSION

A. General Considerations

Pound dogs that are used for experimental purposes
present numerous problems. The age of the animals is a
rough estimation. Shipments usually contain whatever sex,
breed and size of dogs are available when the order is
filled. The stress that these dogs have recently been
subjected to prior to receipt predispose them to outbreaks
of distemper, hepatitis, leptospirosis and other diseases.
Parasitism, both external and internal, also debilitate
the dogs. In this study these factors lead to numerous
early death losses which were not encountered in dogs that
were maintained on a good nutritional and health plan

three weeks prior to use as an experimental animal.

1

B. Thyrodectomized Dogs

t 21, (1958) was employed in this

The method of Crisp
study to produce experimental arteriosclerosis. Problems
Of anorexia, early death losses and debilitation were
encountered by the author in this study, however, Crisp

EL 31. (1958) do not mention any of these problems.

69

 

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Perhaps the reason for this would be that thyroidectomy
was not complete in their study and thus metabolism was
not as greatly disrupted. If smaller doses of vitamin D
were administered over a longer period, or if the inter—
val of time between administration were greater, it might
be possible to produce lesions of arteriosclerosis with-
out such severe vitamin D toxicity. If this is possible
it would make collateral circulation studies more feasible.
The lack of uniformity in the size of the dogs used
in this experiment made it difficult to evaluate the time
required to produce arteriosclerotic lesions since all dogs
received the same amount of vitamin D irrespective of size.
There was, as a rule, earlier development of lesions in
the smaller dogs and death occurred sooner in this group.
The lesions seen by producing hypervitaminosis D in
these dogs were in agreement with those described by
Crisp £5 31. (1958), Smith and Jones (1957), and similar
to those described in rabbits by Follis (1955) and by
Sodeman (1956) in rats.
The elevations of serum calcium levels obtained in
this experimental study agree with the reports in dogs by

Detweiler (1958), Smith and Jones (1957) and Hueper (1944).

 

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71

Similar elevations were reported in rats subjected to
massive doses of vitamin D by Follis (1955). Hass _£.§l.
(1960) reported no persistent or consistent elevations in
serum calcium levels in animals subjected to hypervitamin—
osis D. No reports were found as to changes in the serum
cholesterol levels in animals on a hypervitaminosis D
regime.

The albumin-globulin ratio of this group of dogs
remained within normal limits. No change was expected and
no literature was found in relation to this.

Variations in limb temperatures were not statistically
significant. If more sensitive recording equipment had
been used and if the temperature had been recorded shortly
after limb transection, there might have been significant
differences. This, however, is an assumption. Hueper
(1944) reported hypertension due to renal arteriosclerosis,
and vasoconstriction due to overdose with vitamin D. This
could influence the development of collateral circulation
and limb temperatures. If the vasoconstriction was mild,
the hypertension would aid in the development of collateral
Circulation. The dogs used in this experiment were too

debilitated to risk direct blood pressure recordings,

 

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72

therefore it was not known if they were hypotensive, normo-
tensive or hypertensive. More modern electronic recording

equipment such as a transducer would alleviate the problem

of blood pressure recordings.

Of the four dogs which had an electrocardiogram prior
to and following transection, two showed definite signs of
myocardial disease, one had changes suggestive of myocar-
dial disease, and one showed no significant changes. The
other dogs were started on experimentation sooner and at
that time the early death loses were not anticipated so
that death occurred before a second electrocardiogram was

recorded.

C. Control Dogs

The control group of dogs were not thyroidectomized
and received no vitamin D. They did have a limb transected
in a manner identical to that of the thyroidectomized group.

Serum calcium and cholesterol levels were not altered
from the normal range and the albumin—globulin ratios
remained within normal limits as was expected.

One control dog, 32(B), exhibited minimal gross arterio-
sclerotic lesions at the semilunar valves. Microscopic

examination revealed the area to be calcified from the

 

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73

intima to the adventitia. This dog was estimated to be
about four years old and the lesions were thought to be
spontaneous in origin. This finding is in accord with
the reports of spontaneous arteriosclerosis by Morehead
and Little (1945), Hueper (1945), Detweiler (1958) and
other investigators.

Limb survival following transection agrees with the
work Of Sewell (1958) , however, the dogs in this study did
not use the transected limb as well following vessel liga-

tion as did the dogs reported.by'Sewe11 (1958).

D. General Discussion

It would seem that much valuable information could
be gained from a more thorough study of domestic animals
in the age range that spontaneous cardiovascular disease
becomes quite common. Detweiler _t_gl, (1960) states that
in dogs over eleven years of age, 415 in every 1,000 would
be expected to have some form of cardiovascular disease.
Kountz (1950) compared the incidence of vascular
disease in a group of people with low basal metabolic
rates, a number of years prior to death, to those having
a normal basal metabolic rate, a number of years prior to

death, and found a considerably higher incidence in the

 

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74

former group. This might well be an important aid in
detecting or avoiding vascular disease in man. The ease
with which cardiovascular disease can be produced in
animals by depressing thyroid function or by thyroidectomy
would lend greater weight to a more extensive use of basal
metabolic rate studies.

A low basal metabolic rate in conjunction with cardio-
vascular lesions would help to substantiate the anoxemia

theory of arteriosclerosis.

 

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CHAPTER VI
SUMMARY AND CONCLUSIONS

Forty—two male dogs were used in this study. Thirty-
six were thyroidectomized taking care to preserve the
parathyroid glands. The thyroidectomized dogs were given
100,000 units of vitamin D orally daily throughout the
course of the experiment. Six animals served as controls.
The age of the dogs was estimated to be between one and
four years. Body weight ranged from 13 to 45 pounds.

Dogs housed a considerable period prior to starting on
experimentation survived longer and remained in better
health as a rule. All thyroidectomized dogs started
showing signs of vitamin D toxicity between the fifteenth
and thirtieth day. This was generally more pronounced in
the smaller dogs.

Of the thirty—six dogs which were thyroidectomized,
l4 survived to the time of pelvic limb transection on
the forty-ninth, fiftieth or fifty-first day following
tthoidectomy. Twenty—eight thyroidectomized dogs died
Prior to the time of limb transection. The femoral artery
and vein were ligated on the eighteenth day post-transection.

Five dogs lived to ligation date and nine died prior to

75

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this procedure. Limb temperatures were taken at regular
intervals from the gastrocnemius muscle.

Dogs receiving thyroid supplement following limb
transection did not survive long enough to determine the
effects of this procedure. Gross and microscopic lesions
were variable in this group.

Gross lesions of arteriosclerosis varied from none,
to heavy plaques throughout the aorta and its branches in
the thyroidectomized group. The histopathologic findings
consiSted of disruption of the internal elastic lamellae,
intimal plaques and medial and adventitial calcification.

Myocardial damage was evidenced by the electrocardio-
graphic changes.

Thyroidectomy and oral administration of 100,000 units
of vitamin D daily proved to be a rapid means of producing
arteriosclerotic lesions. However the toxic effect of the
vitamin D was too debilitating to permit a good collateral

circulation study.

h
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V.

BIBLIOGRAPHY

Atland, P. D., Highman, B., and Roshe, J.: Effects of
altitude on dogs with valvular heart disease. Arch.
Path. 68 (1959): 475-486.

Altland, P. D., and Highman, B.: Effects of high altitude
on cholesterol fed rabbits. A.M.A. Arch. Path. 70
(1960): 349—357.

Anonymous editorial, Research in arteriosclerosis--The
first fifty years. Ann. Int. Med. 49 (1958): 699-773.

Blumenthal, H. T., Lansing, A. I., and Gray, S. H.: The
interrelation of elastic tissue and calcium in the
genesis of arteriosclerosis. Am. J. Path. 26 (1950):
989—1009.

Canine Medicine, 2nd ed. American Veterinary Publications
Inc. Santa, Barbara, Calif. (1959).

Crisp, N. W. Jr., Root, H. D., Jenson, C. B., Imamoglu, K.
Yonehiro, E. G.: Experimental production of arterio—
sclerosis in the dog. Surgical Forum 11 (1958): 304-
307.

l

Dauber, D. V., and Katz, L. N.: Experimental cholesterol
atheromatosis in an omnivorus animal, the chick.
Arch. Path. 34 (1942): 937-950.

Dauber, D. V.: Spontaneous arteriosclerosis in chickens.
Arch. Path. 38 (1944): 46-51.

Detweiler, D. K.; Heart in "Canine Medicine." 2nd ed.
American veterinary Publications Inc. Santa Barbara,
Calif. (1959).

Detweiler, D. K., Hubben, K., Patterson, D. F.: Survey
of cardiovascular disease in dogs--preliminary report
on the first 1,000 dogs screened. Am. J. Vet. Res.
21 (1960): 329—359.

77

 

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‘0‘:

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01'
(I)

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‘0‘

78

Duff, Gil.: Experimental cholesterol arteriosclerosis
and its relationship to human arteriosclerosis. Arch.
Path. 20 (1935): 81-91.

Eckstein, R. W., Gregg, P. E., and Pritchard, W. H.: The
magnitude and time of development of the collateral
circulation in occluded femoral carotid and coronary
arteries. Am. J. Physio., 132 (1941): 351—361.

Ferris, H. W., and Harvey, S. C.: Development of col-
lateral circulation of dog. A physiological study of
the development of the collateral circulation in the
leg of the dog. Proc. Soc. Exp. Biol. & Med. 22
(1924-1925): 383-386.

Follis, R. H. Jr.: Studies on hypervitaminosis D. Am.
J. Path. 31 (1955): 568-569.

Gardiner, M. R., Metastic calcification associated with
white muscle disease in lambs. J.A.V.M.A. 138 (1961):
553—556.

Goormaghtigh, N., and Handovsky, H.: Effect of vitamin D2
(calciferol) on the dog. Arch. Path. 25 (1938): 1144-
1182.

Ham, A. W.: Mechanism of calcification in the heart and
aorta in hypervitaminosis D. Arch. Path. 14 (1932):
613-626.

Hartman, H. A. Jr.: Histopathologic changes in experi-
mental magnesium deficiency of calves. Thesis for the
degree of M.S., Michigan State University, 1959.

Harvey, S. C., and Halpert, B.: Studies on collateral
circulation II. Thermic changes after arterial
ligation, section of spinal cord or posterior roots
and ganglionectomy. Jour. Clin. InVest. 10 (1931):
431-434.

Hass, G. M., Trueheart, R. D., Hemmens, A.: Experimental
arteriosclerosis due to hypervitaminosis D. Am. J.
Path. 37 (1960): 521-549.

 

 

 

u».

 

u

‘\

 

 

79

Hawk, P. B., Oser, B. L., and Sommerson, W. H.: Practical
Physiological Chemistry. 13th ed. The Blakiston Co.
Incl, New YOrk, Toronto (Printed by Maple Press Co.,
York, Pa.).

HerriCk, J. F., Essex, H. E., Mann, F. C. and Baldes, E. J.:
The effect of feeding desiccated thyroid gland on the
flow of blood in the femerol artery of the dog. Am. J.
Physio. 105 (1933): 434—435.

Highman, B., Altland, P. D.: Acclimatization response
and pathologic changes in rats at an altitude of 25,000
feet. Arch. Path. 48 (1949): 503—575.

Holman, B.: Conditions controlling collateral circulation
in the presence of an arteriovenous fistula, following
the ligation of an artery. Surg. 26 (1949): 889-917.

Hueper, W. C.: Aortic abnormalities in dogs used for
experimental purposes. Arch. Path. 39 (1945): 375—
380.

Hueper, W. C.: General reviews: Arteriosclerosis. Arch.
Path. 38 (1944): 162—181, 245-285, 350—364.

Klotz, O. J. of Exper. Med. 7 (1905): 633-675.

Kountz, W. B.: Vascular degeneration in hypothyroidism.
Arch. Path. 50 (1950): 765-777.

Krahl, E., Pratt, G. H., Rousellot, L. M., and Ruzicka,
F. F.: The collateral circulation in the arterial
occlusive disease of the lower extremity. Surg. Gyn.
Obst. 98 (1954): 324-330.

Iearmonth, J.: Collateral circulation, natural and
artificial. Surg., Gyn. Obst. 90 (1950): 385-392.

Idndsay, S., Chaikoff, I. G., and Gilmore, J. W.: Arterio—
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aorta and coronary arteries. Arch. Path. 53 (1952):
281-300.

Nbodie, R. L.: The Antiquity of Disease. The University
of Chicago Science Series (1923).

 

 

 

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nov-

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1""
kfic

(I)
I

 

80

Morehead, R. P., and Little, J. M.: Changes in the blood
vessels of apparently healthy dogs. Am. J. Path. 21
(1945): 339-355.

Mulvihill, D. A., and Harvey, S. C.: Studies on collateral
circulation. I. Thermic changes after arterial liga-
tion and ganglionectomy. J. Clin. Invest. 10 (1931):

Patterson, D. F.: University of Penn. School of Vet. Med.
Cardiovascular Research Center. Personal communication.

Rinehart, J. F., and Greenberg, L. D.: Pathogenesis of
experimental arteriosclerosis in pyridoxine deficiency.
Arch. Path. 51 (1951): 12-18.

Rinehart, J. F., and Greenberg, L. D.: Arteriosclerotic
lesions in pyridoxine deficient monkeys. Federation

Proc. 7 (1948): 278.

Rinehart, J. F., and Greenberg, L. D.: Arteriosclerotic
lesions in pyridoxine deficient monkeys. Am. J. Path.

25 (1949): 481-491.

Rosenberg, M. Z., and Liebow, A. A.: Effects of age.
growth hormone, cortisone and other factors on colla-
teral circulation. A.M.A. Arch. Path. 57 (1954):

89-105.

Sewell, W. H., and Koth, D. R.: A basic observation on
the ability of newly formed capillaries to develop into
collateral arteries. Surgical Forum 11 (1958): 227-229.

Smith, H. A., and Jones, T. C.: Veterinary Pathology.
Lea & Febiger, 1957. Philadelphia.

Sodeman, W. A.: Pathologic Physiology Mechanisms of
Disease. 2nd ed. W. B. Saunders Co. Philadelphia

& London (1956).

Steiner, A., and Kendall, F. E.: Atherosclerosis and
arteriosclerosis in dogs following incestation of
cholesterol and thiouracil. Arch. Path. 42 (1946):
433-444.

81
Vanderveer, H. L.: Hypervitaminosis D and arteriosclero-
sis. Arch. Path. 12 (1931): 941-955.

wells, H. C.: Chemical Pathology. 5th ed. W. B.
Saunders Co. Philadelphia (1925).

Williams, H. A. Gross and microscopic anatomy of two
Peruvian mummies. Arch. Path. 4 (1927): 26—33.

 

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APPENDIX I
DATA ON THE INDIVIDUAL THYROIDECTOMIZED DOGS

Dog 3(W) was anemic at the time of transection. Two
hundred cc. of whole blood were administered intravenously
following surgery. Death occurred the sixth post-
operative day. The transected limb was necrotic distal to
the transection.

Dog 16(W) died ten days after the transection. A
massive pocket of purulent exudate was found at the site
of transection. There was no evidence of healing. The
limb distal to the transection was becoming necrotic. Lung
abcesses comparable to those of chronic distemper were seen
at necropsy.

Dog 37(W) died the second post-transection day. The
transected limb was becoming gangrenous distal to the
transection.

Dog 38(W) died the sixth post—ligation day. The limb
was becoming necrotic distal to the transection.

Dog 39(W) was anemic at the time of transection. Two
hundred cc. of whole blood were administered following

surgery, however, death ensued shortly.

82

 

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Dog 12(Y) was fairly active and alert at the time of
transection, however, he died six days later. The site
of transection was purulent and there was no evidence of
healing. Lung abscesses were evidenced as seen in chronic
distemper.

Dog 13(Y) was too weak to stand by the time of vessel
ligation. A purulent exudate was coming from the tran-
section site. Death occurred two days following ligation.

Dogs 33(B) died on the first post—ligation day. The
limb was functional and well healed prior to death.

Dog (BSN) developed generalized sarcoptic mange. Death
occurred the seventh post-transection day. There was no
evidence of healing at the site of transection.

Dog 26(B) died the third day following ligation. The
line of transection was open on the anterior surface of
the limb. The limb was sloughing distal to the site of
transection.

D09 2(W) developed a generalized eczema by the time
Of limb transection. Death was on the fifth post-
transection day. There was very little evidence of healing
at the transection. Vascular lesions were confined to the

region of the aortic valves. These were minimal.

 

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Dog 813 was transected on day 63; sodium pentobarbital
was the anesthetic used. The dog died the third post-
transection day. Anesthetic recovery was not complete at
the time of death. Necropsy revealed well defined vascular
lesions throughout the aorta and pulmonary artery.

Dog 8(W) died on the first post-transection day.
Vascular lesions were well defined in the pulmonary artery
and aorta.

Dog 24(W) died at the time of limb transection, proba-
bly due to anesthesia. well defined vascular lesions were
found in the aorta, pulmonary and carotid arteries. Mini-

mal lesions were noted in the femoral arteries.

 

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APPENDIX II

DATA ON THE INDIVIDUAL CONTROL DOGS
AT TIME OF EUTHANASIA

Dog 955. The transected limb was healed and func-
tional. No gross lesions were seen at necropsy.

Dog 24(B). The dog did not use the transected limb.
The site of transection was healed.

Dog 32(B). The dorsal surface of the second and third
digits started to necrose seven days post-ligation. At
euthanasia the lesion was to the bone. The dog used the
limb but limped.

Dog 22(W). This dog licked out the sutures on the
anterior surface of the transection. These were replaced
tWice but were gone at the time of euthanasia. There was
an open pocket exuding serosanguineous fluid where lick-
ing had occurred. Limb function due to contraction of
the muscles on the posterior aspect of the limb was poor.
This was pronounced so that the limb was somewhat flexed.
Necropsy revealed a hypertrophic periosteal reaction of
the femur at the site of transection.

Dog 35(W). Function of the transected limb approached

normal.

85

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APPENDIX III

DOGS THAT DIED PRIOR TO TRANSECTION

Dog 3(Y). Emaciation and icterus were pronounced at
the time of death on the fiftieth day. Moderate lesions
were evident from the aortic valves to the renal arteries.

Dog ll(Y). By the time of death on the thirty-fifth
day, moderate lesions were evident in the pulmonary,
femoral, anterior mesenteric, splenic, internal and
external thoracic arteries, and from the aortic valves to
the renal arteries.

Dog 958. At necropsy on the fifty-seventh day,
vascular lesions were observed in the region of the aortic
valves and were minimal.

Dog 8(B). Due to the severe generalized moist eczema
and debilitation this dog was euthanized on the sixty-third
day. Marked gross lesions were observed in the pulmonary,
carotid, external iliac and femoral arteries, and the
aorta.

Dog 7(8). At death, on the forty—third day, necropsy

revealed well defined lesions of the aorta.

86

 

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Dog 6(8). Death was on the sixty-first day. Minimal
lesions were found at the aortic valves, and from the renal
to the external iliac arteries.

Dog 21(B). At death on the thirty-fifth day, minimal
lesions were evident at the aortic valves.

Dog 36(B). Death occurred on the forty-fifth day.
Moderate lesions were observed from the aortic valves to
the femoral arteries.

Dog 18(B). This dog died following a fight on the
twenty-fifth day. No vascular lesions were present.

Dog 21(W). Death resulted from a fight on the
thirtieth day. There were moderate lesions throughout the
aorta at the origin of the intercostal ascending branches.

Dog 30(W). Death was on the thirty-fourth day at which
time vascular lesions were minimal and confined to the
pulmonary artery.

Dog 22(8). At death on day 33, no vascular lesions
were observed.

Dogs 3(B) and 7(W). These dogs died on the twenty-
second and twenty—third days respectively. They were not
necropsied.

Dog 5(R). This dog went into tetany the third day

:flfllowing thyroidectomy. Twenty cc. of a calcium

88

solution* was administered intravenously and tetany abated.
Death ensured two days following treatment; however, no
further signs of tetany were observed prior to death. At
the time of thyroidectomy two parathyroid glands were

located. The blood supply of one was interrupted during

the course of surgery.

Dog 7(B). This dog exhibited tonic-clonic convulsions
the first day post-thyroidectomy. The seizures occurred
at about 15 minute intervals. Three such seizures were
observed. No treatment was administered and no further

convulsions were evidenced during the course of the

experiment.

Dog 25. This dog was found comatose five days after
it was started on the experiment. A radiograph revealed a
prostatic calculus. This was removed surgically: however,
death occurred the second post—operative day. Bilateral

hydronephrosis was seen at necropsy.
Dogs 36(W), 21(W), 25W). 7(3). 22(W). 16(W), 5(R).
18(W), and 25(B) died in eleven days or less following

thyroidectomy. None of these dogs were necropsied.

* Calsem, S. E. Massengill Co., Fifth St.,Bristol,
Tenn.

APPENDIX IV
ELECTROCARDIOGRAPHIC DATA AND INTERPRETATIONS

Dog 33(B)

5-5-60
This tracing appears within normal limits, except for
lead AVR which has apparently been mislabelled.

6-23-60
There is a bigeminal rhythm produced by coupled
ventricular premature beats which occur every other
beat. There is depression of the S-T segment in leads
facing the left ventricle (II,III,AVF) and reciprocal
elevation in lead AVR. Precordial leads over the free
wall of the left ventricle also show S-T segment
depression.

7-11-60
The bigeminal rhythm is still present, the extra-
systolic QRS complexes being of similar configuration
to those seen on 6—23-60. S-T segment depressions seen
on that date (6—23-60) are more prominent. CF6U and
AVL may have been switched in the labelling in this
dog. The tracings labelled CF6U appear more character-
istic of AVL in the dog, and AVL looks more like CF6U

should look.

89

9O

Interpretations:

The changes in the latter two tracings are definite

evidence of myocardial disease.

29.8%

5—5-60
This tracing is within normal limits. The QRS axis
is approximately 800.

6-24-60
The QRS has shifted to 900, a slightly more vertical
axis. The P waves are higher in amplitude. These
changes might well be due to positional changes of the
dog during taking of the tracings and probably are not
significant.

Interpretations:

The EKG changes in this dog are not significant.
Doq720(B)

5—21-60
This tracing is within normal limits. The QRS axis is

approximately 600.

91

7-11—60
There is a marked sinus tachycardia. The QRS axis is
slightly more vertical, but no other significant
changes have occurred.

7-29-60
The T waves have broadened and deepened, and the S—T
segments sag in I, II, III, AVF, and the left
precardial leads.

Interpretations:
The T wave changes, although suggestive of myocardial

disease, are not conclusive evidence.
Doq 13(Y)

4-25-60

Within normal limits.
7-5-60

There has been a slight shift in the T Vector,
resulting in inversion of T in II, III and AVF. T was
previously upright in these leads on 4-25-60.

7-24-60

There are numerous ventricular premature beats, at

times giving a bigeminal rhythm. Runs of paroxysmal

Ventricular tachycardia occur in several places.

92

Interpretations:

The last tracing is indicative of definite myocardial
disease.

The T wave changes observed in the second tracing may
be indicative of myocardial abnormality, but are not

clear-cut enough to be reliable evidence.

APPENDIX V

Gross Pictures and Photomicrographs
of Thyroidectomized Dogs

93

Figure 1. Condition of transected limb of
thyroidectomized dog prior to
vessel ligation.

Figure 2. Close—up of the same limb.

 

Figure 3. Illustration of transection technique showing
femoral artery, vein and nerve in left fore-
ground and sciatic nerve in right background.

Figure 4. Transected limb of thyroidectomized dog on the

fifth post surgical day. Necrosis well
ev1denced distal to site of transection.

 

 

Figure 5.

Figure 6.

Gross arteriosclerotic plaques from
experimental dog.

Close-up view of the above picture.

 

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Figure 8.

Gross arteriosclerotic plaques of the external
iliac arteries extending through the femoral

arteries.

Gross arteriosclerotic plaques of the common
carotid artery and a portion of the aorta.

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Figure 9. Coronary artery showing calcification and h“
fragmentation of the internal elastic membrane. ‘1

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Figure 10.

Intimal proliferation and calcification of
the aorta. (H&E x150) 39(W)

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Figure 11.

Medial degeneration and calcification of
renal artery.
(H&E X154) 8 (B)

 

 

Figure 12.

Calcification and fragmentation of the
internal elastic membrane of a coronary
artery. Moderate intimal proliferation.
(H&E x152) 2(W)

 

 

 

Figure 13.

Coronary artery with medial calcification,
calcification of the internal elastic
membrane, and intimal proliferation.

(H&E x151.25) ll(Y)

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Figure 14. Coronary artery showing intimal proliferation

and sub-intimal calcification. f
(H&E x150) 37(W)

 

 

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Figure 15. Myocardial degeneration and hemorrhage.
(H&E x157) 35(W)

 

 

Figure 16. Calcification of the myocardium. Intimal
calcification and degeneration of the internal ‘5
elastic membrane of the coronary artery. f
(H&E x157) 38(W)

 

 

Figure 17.

Calcification of the semilunar valve.
(H&E x154) 32(8)

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Figure 18. Sub-intimal calcification and intimal
proliferation of a coronary artery.
(H&E x154) 813 V)

 

 

 

 

Figure 19.

Endocardial calcification.
(H&E x170) 8 (B)

 

Figure 20. Calcification and fragmentation of the
internal elastic membrane of a coronary
artery.

(H&E x152) 2(W)

 

Figure 21.

Decalcified aorta showing voids from which
calcium was removed from the media.
(H&E x54) ll(Y)

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Figure 22 Diff
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(H&E X50) 24(W) c1f1cation of the aorta.

 

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Figure 23. Aortic medial calcification with disruption
of the intima.

(Von Kossa's x53) ll(Y)

 

 

Figure 24.

Intimal proliferation, sub-intimal
calcification and deep medial
calcification of an aorta.

(H&E x50) 39(W)

l

 

Figure 25.

Calcification of the endocardium.
(H&E x154) 813

 

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n ’

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Figure 26. Sub-intimal and medial calcification of the
myocardium.
(H&E x174)

 

Figure 27. Calcification of the intima and internal
elastic membrane of the renal artery.
(H&E x58) ll(Y)

 

Figure 28.

Calcification of Bowman's membrane and the
basement membrane of the tubules.
(H&E x146) 813

 

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Figure 29.

Renal tubular calcification of the medulla.
(H&E x157) 8 (B)

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A

m

 

 

Calcific deposits in the renal medulla.

Figure 30.

 

(H&E x154) 8(B)

 

 

 

 

Figure 31. Calcific deposits within the renal tubules.
(H&E x149) 2(w)

 

 

 

Figure 32.

Hyaline casts in the renal tubules.
(H&E x149) 8 (B)

 

 

 

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lri 1‘ Ely”..l\si.¥"|h hurt “Ill... t‘. q. 1 Oh“ fibfi‘t.fl.\!~l All it, i

 

Calcium deposits in the renal tubules.
2(W)

(H&E x152)

Figure 33.

 

 

Figure 34. Calcification of the pulmonary veins.
(H&E x53) 13(Y)

 

Figure 35. Pulmonary alveolar calcification and
fragmentation.
(H&E x154) 2 (w)

 

Figure 36.

Calcification of the elastic fibers of a
pulmonary vein and intimal proliferation.
(H&E x154) 13(y)

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h

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Figure 37. Calcification of the elastic fibers of a
pulmonary vein.
(H&E x150) 37(W)

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_ 15.11. 84.9%... .

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,q .

H-Ms
.\ O . o
. I
c . I

 

Figure 38. Trabecular artery calcification and

fragmentation of the spleen.
(H&E x150) 8 (B)

 

 

 

Figure 39. Medial necrosis and calcification of a
trabecular artery in the spleen.
(H&E x152) 8(B)

 

 

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