EXPERIMENTAL AND MOLECULAR PATHOLOGY 16, 170-190 (1971) Production of Advanced Coronary Atherosclerosis, Myocardial Infarction and “Sudden Death” in Swine’ K. T. LEE, J. JARMOLYCH, D. N. KIM, C. GRANT, J. A. KRASNEY, IV. A. THOMAS, AND A. M. BRUNO Departments of Pathology, Medicine and Physiology, Albany Medical College, and V.A. Hospital, Albany, N. Y., and National Heart and Lung Institute, Bethesda, Maryland Received April 1,197l The purpose of this study was to develop a model in swine for advanced coronary atherosclerosis and myocardial infarction. The approach was to combine a number of techniques that were thought to induce the development of athero- sclerosis, including high-fat, high-cholesterol diets plus propylthiouracil and X-ir- radiation to the precordial region. The dose of X-irradiation that was used produced no significant changes by itself, but seemed only to enhance the effect of atherogenic diets. Among 28 swine fed the severe atherogenic diet and X-irradiated twice, 24 developed myocardial infarcts. All had advanced coronary atherosclerosis. Many died “suddenly” in the sense that no clinical signs of illness were observed prior to death. Coronary angiography was done on some swine and direct measure- ments of coronary blood flow on a few others. Coronary atherosclerosis and myocardial infarction were also produced in a few swine without propylthiouracil using a combination of a high-fat, high-cholesterol diet and X-irradiation. Numerous experimental animal models are available for the study of athero- sclerosis. Most studies of such models are designed to help elucidate the patho- genesis of atherosclerosis and may ultimately lead to the development of practi- cal methods for prevention. Meanwhile, thousands of people are developing ad- vanced atherosclerosis and dying of complications such as myocardial infarction and “sudden death” (The National Health Education Committee). Another type of model, utilizing experimental animals with advanced atherosclerosis and its complications, might provide information that would be useful in preventing death in people who already have severe atherosclerosis. The availability of such animals might also allow the scientific trial of therapeutic devices and surgical procedures prior to their use in man. The purpose of the current study was to develop such a model in swine. Since the object was to provide a model for study of effects of advanced atherosclero- sis, it was not considered essential that the etiology and pathogenesis of the lesion be precisely the same as in man. It was only necessary that the final product have features reasonably similar to those in man. For the model to be of maximum usefulness it should be possible to produce advanced disease in a reasonably short time. The approach chosen in the current study was to combine a number of techniques thought to enhance the development of atherosclerosis, 1 Supported by NIH Contract 69-2050 and HE-7155. 170
Transcript
EXPERIMENTAL AND MOLECULAR PATHOLOGY 16, 170-190 (1971)
Production of Advanced Coronary Atherosclerosis, Myocardial
Infarction and “Sudden Death” in Swine’
K. T. LEE, J. JARMOLYCH, D. N. KIM, C. GRANT, J. A. KRASNEY, IV. A. THOMAS, AND A. M. BRUNO
Departments of Pathology, Medicine and Physiology, Albany Medical College, and V.A.
Hospital, Albany, N. Y., and National Heart and Lung Institute, Bethesda, Maryland
Received April 1,197l
The purpose of this study was to develop a model in swine for advanced
coronary atherosclerosis and myocardial infarction. The approach was to combine a number of techniques that were thought to induce the development of athero- sclerosis, including high-fat, high-cholesterol diets plus propylthiouracil and X-ir-
radiation to the precordial region. The dose of X-irradiation that was used
produced no significant changes by itself, but seemed only to enhance the effect of atherogenic diets.
Among 28 swine fed the severe atherogenic diet and X-irradiated twice, 24 developed myocardial infarcts. All had advanced coronary atherosclerosis. Many died “suddenly” in the sense that no clinical signs of illness were observed prior
to death. Coronary angiography was done on some swine and direct measure- ments of coronary blood flow on a few others. Coronary atherosclerosis and
myocardial infarction were also produced in a few swine without propylthiouracil using a combination of a high-fat, high-cholesterol diet and X-irradiation.
Numerous experimental animal models are available for the study of athero- sclerosis. Most studies of such models are designed to help elucidate the patho- genesis of atherosclerosis and may ultimately lead to the development of practi- cal methods for prevention. Meanwhile, thousands of people are developing ad- vanced atherosclerosis and dying of complications such as myocardial infarction and “sudden death” (The National Health Education Committee). Another type of model, utilizing experimental animals with advanced atherosclerosis and its complications, might provide information that would be useful in preventing death in people who already have severe atherosclerosis. The availability of such animals might also allow the scientific trial of therapeutic devices and surgical procedures prior to their use in man.
The purpose of the current study was to develop such a model in swine. Since the object was to provide a model for study of effects of advanced atherosclero- sis, it was not considered essential that the etiology and pathogenesis of the lesion be precisely the same as in man. It was only necessary that the final product have features reasonably similar to those in man. For the model to be of maximum usefulness it should be possible to produce advanced disease in a reasonably short time. The approach chosen in the current study was to combine a number of techniques thought to enhance the development of atherosclerosis,
1 Supported by NIH Contract 69-2050 and HE-7155.
170
MYOCARDIAL INFARCTION AND SUDDEN DEATH IN SWINE 171
including high-fat, high-cholesterol, propylthiouracil and X-irradiation. None of the techniques chosen was expected to produce significant permanent changes in the swine except in relation to the arteries. Thus it was expected that when advanced atherosclerosis had developed propylthiouracil and X-irradiation could be discontinued and the complications of advanced atherosclerosis studied in a euthyroid animal maintained on a high-fat, high-cholesterol diet.
MATERIAL AND METHODS
A total of 100 male miniature swine of the Pitman-Moore strain were used in developing the methods presented in this report. All were weanlings at the outset of the experiment. Three types of atherogenic diets (“mild,” ‘(severe,” and “milk-cholesterol”j and a control stock diet were used; and these are presented in Table I.
All swine received 2500 Cal/day. Eighty-two swine were pre-fed the milk-cho- lesterol diet for 2 months and then given the severe diet; 14 swine were given the mild diet throughout the experiment; and 4 swine were given the stock diet. When the severe diet was used, it was mixed with commercial mash for the first 3 weeks of a conditioning period in the following proportions: first week i/4 severe, 3/4 mash ; second week i/s severe, i/s mash ; third week s/4 severe, i/ mash ; fourth week and thereafter, full severe diet. During the course of the experiment when any swine appeared weak and less active than usual, a portion of the severe diet was replaced with either whole-milk powder or mash for a few days until the swine recovered and regained his strength.
Sixty-four swine were given X-irradiation of 1500 r to the precordial region twice at 4- to 7-week intervals, after anesthetizing with pentobarbital sodium, by using a Y?o-teletherapy unit at a distance of 60 cm with an 8.7~cm square
TABLE I
COMPOSITION OF DIETS CONSUMED PER D.~Y
Ingredient
Casein Sucrose
Butter
Peanut oil Salt mix, Wesson Vitamin, mix Choline chloride
Cellulose
Cholesterol Sodium cholate Propylthiouracil
Total
Stock
143.0 227.0
57.0 57.0 30.0 12.5
2.5 121.0
0
0 0
650
Diet, amount consumed (pm)
Mild Severe Milk-cholesterol
143.0 95.0 Dry whole-milk 227.0 140.0 powder 506
57.0 87.0 57.0 87.0 30.0 30.0 30 12.5 12.5 12.5
2.5 2.5 - 113.0 24.5 -
8.0 14.0 8 0 7.0 0 0 0.5 0
650 500 550.5
Calories 2506 2506 2506 2510
172 LEE ET AL.
portal. Some swine received 2500 r instead of 1500 r as their second dose of X-irradiation. A few swine received a third dose of 1500 r. Seven swine were given X-irradiation only once and 29 swine received no X-irradiation. Autopsies were performed on all swine. Animals found dead in the pens or cages were autopsied as soon as possible. When the swine were sacrificed for experimental purposes, autopsies were performed immediately. The heart and aorta were re- moved intact; all major organs were examined routinely and appropriate sections taken for light microscopy.
The hearts were examined systematically, both grossly and microscopically, for infarcts or other abnormalities. After fixation in 10% formalin, the amount and severity of coronary atherosclerosis were measured by ocular micrometer (Daoud et al., 1964). In brief, the procedure for the examination of the coronary arteries consists of a series of cross-sectional cuts along the course of the three main vessels. The cuts are made at 5-mm intervals until an external diameter of less than 1 mm is reached. Alternate 5-mm segments of vessel are then removed (Figs. 1 and 2)) processed, and microscopic sections are prepared. Diameter of the artery, diameter of the lumen, and wall thickness in its thickest and thinnest areas were measured. Average diameter of the lumen and average wall thickness were then calculated for all three main arteries. All other microscopic observa- tions, such as hemorrhage into a plaque, calcific deposits, and virtual or complete occlusion were also recorded.
Electron microscopy observation was made on the coronary arteries of two swine that were fed the severe diet and X-irradiated, and one stock-fed control swine.
Three swine were studied during life by coronary angiography. The procedure is that of routine clinical coronary arteriography, carried out on a fully anesthe- tized swine. Thiopental sodium was used for anesthesia and the Sones catheter was introduced via carotid artery, and maneuvered into each coronary ostium in turn. Eight selective coronary arteriograms, including tine and larger film of each vessel in both oblique views, were taken.
Electrocardiograms were taken on 54 swine before and after completion of X-irradiation. Standard six limb leads and six precordial leads were taken in supine position without anesthesia.
Studies of coronary hemodynamics were carried out on anesthetized swine (pentobarbital sodium; six control and two X-irradiated severe diet-fed swine) via left lateral thoracotomy by means of the electromagnetic flow meter (Bio- tronex BL 610) using noncannulating flow transducers.
Serum cholesterol levels were determined by the method of Leffler (1959) in all swine before they were put on experimental diets. They were also determined periodically to assess the effect of diets and before the animals were sacrificed.
Serum thyroxine iodine (T4) levels were determined in 20 swine by the method of Pileggi and Kessler (1968).
RESULTS
During the early phase of the experiment (during the first 2 months when the swine were still on the milk-cholesterol diet) 16 swine died. In seven swine the
MYOCARDIAL INFARCTION AND SUDDEN DEATH IN SWINE 173
-. .L- -_
FIG. 1. Gross photograph of anterior view of heart of a swine fed the severe atherogenic
diet for 58 weeks and X-irradiated twice. The anterior wall of the left ventricle has been partially cut away to demonstrate the large recent, well-demarcated infarct (arrow). A fibrinous deposit can be seen in patches on the pericardial surface. Segments of the coronary
arteries have been previously removed.
cause of death was attributed to respiratory infection; in eight swine no apparent cause of death was found, and one died soon after bleeding for cholesterol measurement. In no case were atherosclerotic lesions found. Sixty-six swine sur- vived for 2-14 months before they either died in cages or were killed for experi- mental purposes and 18 swine are still living.
The results are summarized in Tables II-‘VIII.
174 LEE ET AL.
1 the fro1
the the
m this
right
Seque e diet
of viri corona
tntial, alternate segments of the three main coronary arteries of a
for 40 weeks and X-irradiated twice (TSC 3 in Table II). The I taken prior to sacrifice, are shown in Fig. 10 and can be directly cc
tual occlusion seen grossly in these segments. The dark areas of the
ry artery were grossly hemorrhagic.
swine fed mgiogr nams Impare d to ! lesion .s of
MYOCARDIAL INFARCTION AND SUDDEN DEATH IN SWINE 175
TABLE II
Dana CONCERNING SWINE FED SEVERE ATHEROGENIC DIET AND X-IRR.~DIATED TWICE, SURVIVING UNTIL TERMINATION OF EXPERIMENT
Pig
G&J
Ef%’ first X-ray (weeks)
IS
/
nterval between X-rays Iweeks)
urvived after first
X-ray lweeks)
1 4 4 12
2 8 7 28
3 29 6 11.5 4 39 4.4 9
5 40.5 6 15.5 6 40.5 6 15.5
7 40.5 6 15.5 8 40.5 6 15.5
9 40.5 6 14 10 40.5 6 17
-
I Final
Weight (kg)
nitial 1
__ -
15.5 23.4
15.5 9.1
15.9 16.5
- 33.9 26.4 24.5
26.7
- -
21.3 38.4
15.7 57 18 65
- Serum
cholesterol h$%)
--
1
.-
T-
nitial Final Avg
lmina diam (mm)
- - 247 .80 55 269 .58
159 1176 .40 114 532 .63
59 1300 .73 120 739 .58
150 543 .73 89 1119 .33
98 668 .70 109 678 .58
Coronary arteries
Avg wall
thick- ness (mm)
.23
.48 .73 .45
.43
.53 .30 .68
.50 .35
t 1 c
Vir- ual 0 total ICClU- sion
- I
Infarct
r R
-
.ec.” Old
l-
L
* Recent infarct: the same applies in all subsequent Tables.
Clinical observations during life. The swine usually t#olerated the diets well and ate the entire amount of food that was offered. The initial body weight and weight gain for individual swine were not uniform. Some gained considerably but others only slightly and a few swine that died early even lost some weight. Although they were in a hypothyroid state in general most swine grew fairly well and appeared healthy (Fig. 3).
Starting at approximately four weeks after the last X-irradiation, 22 swine fed the severe diet died rather suddenly without apparent signs of sickness. They were found dead in cages or pens. One swine died in the cage 2 hours after being transported to another location for special studies.
Among the 14 swine fed the mild diet exclusively, one died after the first X-irradiation. Another died immediately after the second X-irradiation and nine died 1 to 3 weeks after the second X-irradiation. There was an epidemic of respiratory infection at that time in our animal facilities. Only three survived for 9 weeks or longer.
Autopsy observations. No gross visible lesions were found in coronary arteries or aortas of the four stock-fed swine even though they were X-irradiated twice. In one swine slight fibrous pericardial thickening was noted.
In the mild diet-fed swine, no atherosclerotic lesions were found in animals that died within 4 weeks after the second X-irradiation. In most of the swine that died early the cause of death was bronchopneumonia. In the three swine that survived 9 and 10 weeks after the last irradiation, either healed and/or recent myocardial infarcts were found. The main coronary arterial walls were markedly thickened in only one swine but secondary coronary branches were markedly narrowed in all three. These three animals showed fibrous pericarditis.
176 LEE ET AL.
TABLE III
DATA CONCERNING SWINE FED SEVERE ATHEROGENIC DIET AND X-IRRADIATED
TWICE, DYING SUDDENLY PRIOR TO TERMINATION OF EXPERIMENT
Pig no.
TSC
-
c
Serum Cholesterol
(w%)
Weight (kg)
Coronary arteries Infarct
nterval between X-rays :weeks)
i iurvived after first
X-ray (weeks)
6 11.0
7 17.0
7 16.0
6 10.0
6 12.0
7 14.0
6 8.0 6 9.5
5 10.0
4 4.5 4 13.0
6 15.0
6.3 16.0 6 16.0
6 18.0
6 18.0
6 18.5
6 18.5
On diet before
first X-ray
[weeks)
t
I
-
Aw umina diam (mm)
1
Aw wall t
thick- ness ( (mm) --
- -
.43 .55 .50 .75
.63 .38
.40 .68
.25 .90
.23 .65
.65 .60
.53 .60
.93 .30
.65 .65
.93 .28
.73 .23
.58 .65
.98 .35
.80 .30
.70 .63
.53 .48 -
Final I
E f
-
nitial nitial I Final
16.8 15.9 98 1136
18.2 23.6 81 1620
20.0 36.8 95 3020
23.5 30.0 88 1740
16.1 31.4 68 1400
15.2 30.0 95 1840
13.2 22.5 106 1265
12.3 18.8 122 1382 10.2 34.1 81 1195
29.3 37.0 94 1380 29.5 52.5 65 618
24.5 40.5 95 654
8.0 13.6 105 951
16.0 72.0 133 584 25.0 63.5 90 586
25.0 72.8 103 1149 22.0 35.5 99 1597
14.5 21.6 95 977
Vir- ual 0 total xclu sion
Old Rec.
- I
-
I
-
r 1
. -
-
11 12
13 14
15 16
17 18 19
20 21
22
23 24
25
26 27 28
+ + + + + + + + +
+
+
+ +
+
+
+ + + + +
+ + +
+ +
10.0 4.0
8.0 10.0
10.0 8.0
29.0 29.0 30.0
46.5 40.5
40.5
39.0 40.5 40.5
49.5
40.5 40.5
-
Among the severe diet-fed swine seven were X-irradiated only once because they died or were killed for evaluation of lesions before the second X-irradiation was scheduled. Four of them showed severe atherosclerotic lesions with virtual occlusions of coronary arteries but the remaining four showed either no, or only a slight degree of, atherosclerosis. None had a myocardial infarct and only one showed a pericardial reaction. Twenty-five severe diet-fed swine received two doses of X-irradiation and three received three doses. Of these 28, 24 had my- ocardial infarcts, either healed, recent, or both. Both healed and recent infarcts were found in 14 swine, recent infarct alone in four and healed infarct alone in six. The size of infarcts varied greatly and some involved large portions of left ventricle and interventricular septum. In two swine there was a slight degree of aneurysmal dilatation of the left ventricular wall due to extensive infarcts in- volving the entire thickness of the left ventricular wall. In most of the swine with infarcts the main coronary arteries were virtually occluded by atherosclerotic plaques; in the few in which the main coronaries were not occluded the secondary branches were severely narrowed or occluded. The aortas of swine fed the severe diet for over 30 weeks displayed yellow-gray plaques of varying size, several millimeters to 5 cm in length, with their long axes parallel to the blood flow. The
MYOCARDIAL INFARCTION AND SUDDEN DEATH IN SWINE 177
TABLE IV DATA CONCERNING SWINE FED SEVERE ATHEROGENIC DIET AND
X-IRRADIATED ONLY ONCE
Weight 04
Serum cholesterol
hw%)
Coronary arteries Infarct
l- r
Rec. Old
On diet before X-ray
(weeks)
Pig no.
TSC
I Vumbel of
times I-rayed
Survives after
X-ray (weeks)
Initial
29 4 Once 6.0 20 30 8 Once 8.0 17 31 28 Once 4.5 19.1 32 28 Once 4.5 10.7 33 28 Once 4.5 14.5 34 28 Once 4.5 11.8 35 29 Once 6.0 13.6
-
1
--
Initial Final Aw
lmina diam (mm)
89 228 .95 171 .73
90 2480 .83 104 2080 .40 124 1643 .73 65 1103 .80
144 1585 .55
Vir- .ual 0 total
occlu- sion
Avg wall
thick- ness (mm)
.18
.23
.88
.90
.28
.30
.40
Final
17.5 -
18.0 15.9 23.2 25.0 23.0
+ + +
+
TABLE V DATA CONCERNING SWINE FED SEVERE ATHEROGENIC DIET BUT NOT X-IRRADIATED
plw ies i were more striking in the abdominal aorta than elsewhere and often tended to be located around the orifices of vessels leaving the aorta. An aneu- rysm of the abdominal aorta was found in two swine that did not receive S-irradiation but received the severe diet for 55 weeks. In twenty-three, fibrin- ous or fibrous pericarditis was noted.
Thirteen swine were given no X-irradiation; they are the ones that died before
178 LEE ET AL.
TABLE VI DATA CONCERNING SWINE FED MILD ATHEROGENIC DIET AND X-IRRADIATED TWICE -
I I
Serum cholesterol
bg%) Weight (kg) Coronary arteries Infarct
iurvived after
1st X-ray
(weeks)
4.0 4.0
4.0 4.0
4.5 4.5
4.5 4.5
4.5
4.5 5.0 9.0
10.0 10.0
On diet , before 1
first X-ray ,
(weeks)
--
7
7 7
7 7
7 7
7.5 7.5
7.5
7.5 7.5
7.5 7.5
nterva jetweer X-rays [weeks)
nitial Final
132 100 165 179 181 158 153 246 149 213 135 227
315 142
Vir- tual
to% ,cclu- sion
Pig
p”s”;:
1 2 3
4 5
6 7
8 9
10
11 12
13 14
Avg Avis Lumi- wall
nal :hick- diam ness (mm) (mm)
- -
.85
.78
.93
.80
.78
.93
.85
-
.13
.13
.18
.13
.15
.13
.18 - -
.78 .15
.50 .25 - -
.40 .65
Rec. Old
+
+
.-
1 nitial Final I 1
_- 4 4
4 4
4 4
4 4
4
4 4
4 4.4
4.4
5.0 5.4
13.2 4.8
15.0 8.1
10.0 9.1
13.6 5.9
11.8 13.0
14.1 17.3
10.6 10.8 16.6 9.5
20.0 14.1
25.5 16.3 -
10.6
14.7 19.7
21.1 30.0
85 90
105 100 95 98
110 108 84
114
80 95
110 85
TABLE VII DATA CONCERNING SWINE FED STOCK DIET AND X-IRRADIATED TWICE
Pig no. PS
- 1
- Serum
cholesterol (w%)
Weight (kg) Coronary arteries Infarct
Rec. Old
--
Interval Survived between after
first X-raYs X-ray
(weeks) (weeks)
On diet before
first X-ray (weeks)
Avg Vir-
wall tual
thick- ness to:1
(mm) occlu-
sion
Final
Aw lumi-
nal diam (mm)
152 1.23 0.18
154 1.03 0.23 162 1.18 0.23
98 1.05 0.25
Initial Final Initial
20.4 17.0 24.7 11.6
35.9 95 34.5 56 31.6 84 30.0 94
12 6 9
12 6 10
12 6 10 12 6 18
X-irradiation could be scheduled. Those dead within 2 months after they were given the severe diet showed no gross lesions in coronary arteries. Among those fed the severe diet for a much longer period, four had virtual or total occlusion of main coronary arteries. One that survived for 55 weeks and received no X-irra- diation had a healed myocardial infarct. In three swine fibrous pericardial thick- ening was found.
MYOCARDIAL INFARCTION AND SUDDEN DEATH IN SWINE 179
TABLE VIII
THICKNESS OF INTIMA AND MINIMAL LUMEN OF CORONARY ARTERIES OF FOUR SWINE FED STOCK DIET AND 28 SWINE FED SEVERE ATHEROGENIC DIET AND X-IRRADIATED TWICP
Left ant desc Left circumflex Right
. . Pig no. Intima Media Intima Media Intima Media yr!F,“’ Infarct
Max Mean Mean Max Mean Mean Max Mean Mean
PS 1 .13 .04 .14 .03 .03 .19 .13 .06 .23 1.03
2 .05 .03 .16 .03 .03 .14 . 10 .04 .23 .75
3 .13 .06 .19 .13 .04 .15 .13 .07 .27 .78
4 .13 .05 .21 .03 .03 .lS .03 .03 .26 .60
TSC 1 2 3
4
5 6 7
8 9
10
11 12 13
14
15 16 17
18 19
20 21
22 23
24 25
26 27 28
-
- Stock diet group
.15 .08 .19
.95 .38 .19
.50 .21 .31
.55 .16 .13
.15 .03 .22
.73 . 26 .27
.20 .07 .17
1.03 .40 .20
.93 .26 .22
.38 .07 .I6 -
1.13
1.35
.48
.78
.70
.80
.28
.45
.35
1.65 .13
.16
.20
.60
.20
.50
.90
.38
.47
.lO
.23 .23
.27
.15
.09
.09
.61
.08
.03
.08
.14
.08
.18
.13
- -
.20 .63
.30 1.18
.26 .55
.38 .58
.23 .85
.25 .50
.22 1.00
.24 .85
.22 .43
.14 .98
.22 .13
.15 .08
.24 .93
.23 .88
.23 .15
.16 1.00
.24 .40
Severe diet group
.08 .03 .14 .08 .03 .16
.50 .25 .15 .43 .12 .35
.45 .27 .25 1.58 .69 .21
.88 .33 .16 .70 .27 .22
.35 .19 .20 .63 .43 .27
.45 .19 .20 .95 .42 .27
.88 .44 .ll .18 .06 .21
.80 .40 .16 1.00 .38 .23
.55 .21 .14 1.23 .42 .18
.63 .23 .28 .13 .05 .15
.36
.55
.24
.29
.55
.30
.46
.47
.08
.36
.05
.03
.55
.26
.08
.46
.14
-
.25
.33
.21
.16
.35
.24
.30
.22
.18
.16
.23
.lO
.23
.30
.19
.29
.20
.43 1.00
.20 1.25
2.00 1.10
1.25 1.33
.50 1.75
.25
.lO 1.18
.23
.23
1.60 1.40
-
.23
.43 .08
.51 1.01
.55
.47
.58
.09
.62
.07
.04
.66
.08
.09
.53
.88
.17
.25
.25
.28
.21
.22
.33
.23
.29
.18
.26
.22
.16
.20
.29
.38
.28
.58
<.05 <.05 <.05
.45
<.05 .23
<.05 <.05
.21
<.05 <.05
<.05 .18
<.05 <.05
<.05 <.05
< .05 .45
<.05 .55
.45 <.05
.23
.40 <.05
<.05
+ + +
0 Sections were taken at l-cm intervals from origin of each coronary artery until external diameter was less than 1 mm. Measurements of intima, media, and minimal lumen were made
on microscopic sections as explained in text. Flow measurements were made for the anterior descending artery on swine TSC-5 and TSC-7 during life and the angiograms in Fig. 10 are from swine TSC-3.
180 LEE ET AL.
FIG. 3. Picture of one of the swine fed the severe diet for 4 months and given two doses of X-irradiation. Although they are in hypothyroid state the swine grew well and appeared healthy. This swine is 7 months old and weighs 38 kg.
In 17 out of 25 swine that had myocardial infarcts there was evidence of congestive heart failure, characterized by marked congestion of the liver, peri- cardial and/or pleural effusion. However, in only three swine were there massive pleural (over 2000 ml) and peritoneal (over 1500 ml) effusions. The thyroid glands of the swine fed the severe diet for over 20 weeks were greatly enlarged.
Light microscopic observations. Histologic appearance of coronary lesions var- ied, depending upon the length of time the swine were on the diet and the length of the period after X-irradiation. The earliest. change noted was smooth muscle cell (SMC) proliferation, one to two cells thick, over normal appearing internal elastic membrane. With the progression of the disease the lesions became thicker and cytoplasm of many cells was filled with lipids (Fig. 4). In most instances the narrowed lumen was eccentric and irregularly shaped (star, diamond, slit-like, etc.). Hemorrhage was a prominent and frequent feature of these advanced le- sions (Fig. 5). The hemorrhage appeared almost always to be fresh in the intima, media or both. Necrosis and calcification also varied from case to case. In some cases they were quite prominent features (Fig. 6).
Complete or virtual occlusion of main trunks or their branches by extensive lesions with or without recent hemorrhage into the plaque were frequently observed in almost all cases (Tables II-VIII). Thrombotic occlusion was not a common feature and was seen in large arteries only in two cases. Occasionally thrombosis of small intramural branches was seen in areas of myocardial infarcts. It is of interest to note that this diet and X-ray induced coronary artery disease affects arteries of all calibers (main trunks as well as small intramural branches). In some instances it is even more severe in smaller branches than in the main trunks.
The histologic appearance of the myocardial infarcts was similar to that of human hearts (Figs. 7 and 8).
MYOCARDIAL INFARCTION AND SUDDEN DEATH IN SWINE 181
FIG. 4. Low power light micrograph of one of the points of near-occlusion of a coronary artery by a proliferative lesion. Foamy, fat-filled cells can be seen, mainly in the media, but most of the lesion is made up of smooth muscle cells. Severe diet-fed, X-irradiated swine.
The four swine that were X-irradiated and fed the stock diet and that survived nine weeks or longer after the first X-irradiation showed spindle cell intimal SMC proliferation of one to two cells thick. This degree of cellular proliferation is a common finding even in normal control swine. Acute radiation vasculitis affecting capillaries in rabbit heart as reported by Fajardo and Steward (1970) was not seen in our X-irradiated swine.
Electron microscopy observations. Electron micrographs of coronary lesions showed extensive proliferation of SMC and a large amount of collagen (Fig. 9). Many of the SMC were filled with lipid. These features are also seen in human atherosclerosis but the amount of lipid in SMC is greater in the rapidly produced coronary lesions in the swine.
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FIG. 5. Very low-power light micrograph of a coronary artery in the epicardial fat. The lesion is similar to that in Fig. 3 but shows, in addition, an area of hemorrhage in the intima and media (dark area at lower right). Severe diet-fed, X-irradiated swine.
Coronary angiograms. The coronary angiograms showed extensive luminal narrowing of all coronary arteries (Fig. 10-A, B, C, D) .
ECG observations. In one swine, electrocardiographic evidence suggesting my- ocardial infarction was obtained and this was later confirmed anatomically. Isoelectric or inverted T waves were common in our swine even before they were put on experimental diets. Occasionally a moderate degree of sinus arrhythmia was noted in swine fed the severe diet but no other significant arrhythmia was noted.
Coronary hemodynamics. Coronary flow responses to a number of vasoactive agents and to brief mechanical occlusions of the coronary artery were markedly altered in the swine fed the severe diet and X-irradiated as compared to re- sponses to these procedures in a group of six healthy control swine.
MYOCARDIAL INFARCTION .AND SUDDEN DEATH IN SWINE 183
Fro. 6. Very low power light micrograph of a coronary artery with a lesion similar to those in Fig. 4 and 5, but in addition showing extensive calcium deposits in the outer intima and inner media. Severe diet-fed, X-irradiated swine.
In the control swine, blood flow in the anterior descending branch of the left common coronary artery was initially found to be 63 to 88% higher than flow in the corresponding circumflex branch. Therefore, anterior descending flow was studied and the term coronary flow will refer to left anterior descending flow. Injections of adenosine (2.0 mg) (Afonso, 1969) into the jugular vein increased coronary flow in these control animals from 68 ml/min/lOO gm left ventricular weight (SE t20.2) to 93.8 ml/min/lOO gm (k27.4) (p < 0.025 by Student t test). This represents a 96.5% (+27) increase in flow with a 35% (--+8.6) decrease in calculated coronary vascular resistance. In addition production of histotoxic hypoxia by intravenous injection of NaCN (0.3 mg/kg), or induction of arterial hypoxemia (arterial POz, 2530 mm Hg) by ventilation with a 6% 0294% Na gas mixture (Afonso, 1969)) led to typical large increases in coronary flow (300400%) and marked decreases in coronary resistance. By comparison the effects of intravenous nitroglycerin (0.3 mg) on coronary flow were more variable than those observed with adenosine or hypoxia. Nitroglycerin increased coro- nary flow in three swine (+4.2 to +24%) and decreased coronary resistance in 4 swine (-16.8 to -38.9%). On the other hand nitroglycerin decreased coronary
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FIG. 7. Very low power light micrograph of a recent myocardial infarct found in one of the severe diet-fed, X-irradiated swine. Areas contain pale, swollen, partially necrotic muscle cells alternating with areas of marked inflammatory cell infiltrate. A small area of relatively intact myocardium is seen at the far upper right.
flow in the other 3 animals (-21 to -50.6%) while coronary resistance increased in two animals (+62%, i-123%). In this respect nitroglycerin appears to be a less potent coronary vasodilator in the swine than in the dog (Malindzak et al., 1970). However, consistent reactive hyperemia responses were observed immedi- ately following 5-set mechanical occlusions of the coronary artery by a ligature. Coronary flow increased from 40 ml/min/lOO gm (* 11.6) to 74.2 ml/min/lOO gm (k11.0) (p < 0.0025) after release of the ligature. This represents a 131% (k52.0) reactive hyperemia in response to temporary vascular occlusion.
By contrast we were unable to elicit coronary vasodilator responses to similar doses of adenosine or nitroglycerin in the swine fed the severe diet and X-irra- diated. In fact, these agents were without any appreciable effect on coronary
MYOCARDIAL INFARCTION AND SUDDEN DEATH IN SWINE 185
FIG. 8. An older, healing myocardial infarct with large, granular dark areas of calcification. Alternating patches of light and dark tissue correspond to the various stages of necrosis
proceeding to fibrosis.
flow. In addition, no reactive hyperemia was observed in these animals. Tempo- rary vascular occlusion actually transiently decreased coronary flow on the order of -6.5 to -19.4”/, immediately after release of the ligature. Observations in one severe diet-fed swine (TSC 7 in Table II) with an arterial pressure of 90 mm Hg suggest that coronary flow was reduced (18 ml/min/lOO gmj compared to the average flow values observed in the control swine. In the other swine (TSC 5 in Table 11, coronary flow appeared high (112 ml/min/lOO gm) . Since this animal had a transient episode of pulmonary edema during induction of anesthesia and was hypotensive (arterial pressure 30 mm Hgj, this flow level was perhaps related to relative hypoxemia.
At autopsy the swine with reduced coronary flow of the left anterior descend-
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FIG. 9. Low power electron micrograph of the endothelial surface and adjacent intima from a large atherosclerotic plaque of a coronary artery in a severe diet-fed, X-irradiated swine. The intimal cells, recognizable as smooth muscle cells, are surrounded by collagen and contain a moderate number of lipid vacuoles.
FIG. 10. A. Control swine, normal left coronary artery angiogram (L. D. left anterior descending; L. C. left circumflex). B. Same swine as Fig. 2 (TSC 3 in Table II). Left coronary artery angiogram shows marked narrowing of both branches (arrows). Measurement of luminal diameters at autopsy at l-cm intervals of L. D. gave the following results in millimeters: 0.85, 0.28, 0.95, 0.83, 0.73, 0.70, <0.05, <0.05, cO.05, cO.05; for L. C. values were: <0.65, 0.65, <0.05, cO.05. C. Control swine, normal right coronary artery angiogram (the proximal filling defect is considered to be catheter artefactl. D. Same swine as Fig. 2 and Fig. IOB. Right coronary artery angiogram shows extensive marked narrowing (arrow points to right sinus of valsalva which is filled with contrast medium). Measurements of luminal diameters at autopsy (as with L.D. and L.C.) were: 0.50, <0.05, cO.05, 0.23, 0.25. <0.05, <0.05, co.05.
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ing branch had a myocardial infarct in the portion of left ventricle that appeared to be supplied in part by the left anterior descending branch, while the one without reduced flow had no infarct. Comparisons at autopsy of luminal diame- ters of the extramyocardial portions of the left anterior descending branch of the two swine revealed no significant differences. The luminal diameters of the left anterior descending branch in swine TSC 7 at l-cm intervals from the origin are, in millimeters 1.10, 1.10, 1.10, 1.00, 0.90, 0.80, 0.90, and 0.80; and for the swine TSC 5 are 1.00, 1.00, 0.80, 0.80, 0.80, 0.60, and 0.60. However, microscopic examination of the left anterior descending branch from the swine TSC 7 re- vealed focal areas of intimal thickening as thick as the entire media in several areas. Similar study in the swine TSC #5 revealed no prominent foci of intimal thickening (ratio of intima to media no more than 1 to 5).
Examination of intramyocardial arteries revealed occlusion of some in TSC 7 and much less change in TSC 5. Marked differences in the rate of coronary flow that had been observed were possibly a consequence of hypoxia, intramyocardial arterial occlusion or physiological changes of arterial wall associated with inti- ma1 thickening in the main arteries.
Chemical determinations. The intitial and final serum cholesterol levels for individual swine are shown in Tables II-VII.
The mean T4 level for the control swine was 1.5 pg% and that for the swine fed the severe diet was 0.26 pg%, indicating that they were in a hypothyroid state.
DISCUSSION
Advanced occlusive coronary artery disease can be induced rapidly in swine by using a combination of injurious and atherogenic agents. The most effective combination, to date, is given in the following sequence: (1) young miniature swine are given a high-fat, high-cholesterol diet containing propylthiouracil; (2) X-irradiation of the precordium (1500 r) is given after 8 weeks or more on the diet; (3) a second X-irradiation is given 4 to 7 weeks later; (4) severe coronary artery disease and its complications begin to appear approximately 4 weeks after the last X-irradiation.
The occlusive arterial disease produced in these swine has many similarities to and some differences from that observed in man. The most important feature, from the standpoint of the objectives of the study, is that the lumen is narrowed and blood flow to parts of the myocardium is reduced. The swine frequently develop ischemic heart disease with myocardial infarction and/or sudden death, and occasionally congestive heart failure. Thus the model would appear to be useful for studies aimed at elucidating and preventing physiological derange- ments resulting from ischemic heart disease.
The lesions involve the major branches of the coronary arteries, with severity varying from branch-to-branch as in man. However, the marked luminal nar- rowing extends over longer segments than are usually seen in man. Thrombosis, which is common in man, was not seen grossly even in those with myocardial infarcts. Instead, hemorrhage into plaques was much more frequent than in man.
Histologically all features of the arterial lesions in the swine can be found in
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man. As in man the predominant cell was the smooth muscle cell, much lipid was present both intra- and extracellularly, and regions of necrosis and calcification could be found. However, the cellular component was more prominent and the necrotic debris and calcification were much less prominent than in man with advanced coronary atherosclerosis. This is to be expected in rapidly developing lesions since the early phase in both man and experimental animals appears to be largely cellular proliferation, with extensive fibrosis and large regions of necrotic debris developing later.
There were no significant features other than pericardial fibrosis that could definitely be attributed to X-irradiation per se. The swine treated with X-irra- diation alone did not develop demonstrable arterial or myocardial lesions. The effect of the X-ray was apparently to enhance the atherogenic properties of the diet and drug regimen.
The role played by the propylthiouracil is not clear. It was used because it is known to produce much higher serum levels of cholesterol than if dietary cholesterol is given without it. However, atherosclerosis with myocardial infarc- tion was produced in several animals without propylthiouracil, so it is not an absolutely essential feature of the regimen. It has the disadvantage of inducing metabolic changes that can complicate interpretation of observations related to the ischemic heart disease. For example, we do not know precisely the role played by hypothyroidism in the swine with coronary occlusive disease that died suddenly. We did observe that deaths were not frequent among hypothyroid swine with only minimal narrowing of coronary arteries. Also some euthyroid swine with occlusive coronary arterial disease appeared to have died suddenly. Thus the hypothyroid state is not a required feature for sudden death, but it may increase the likelihood of dying.
If the propylthiouracil is removed from the diet after advanced atherosclerosis has already developed, the swine return to an euthyroid state but still have occlusive coronary disease. Only a few swine were treated in this manner and these were observed for only a short time in the euthyroid state. Therefore, we do not yet know whether regression of arterial lesions and decrease in mortality would eventually occur in these swine.
The immediate cause of death was not determined in most swine with ad- vanced occlusive coronary artery disease. Some of the swine died with intercur- rent infections and a few with rather severe congestive failure. In men with coronary disease dying suddenly, it is usually speculated that they died of ventricular fibrillation but proof of this is difficult to obtain. Further study of these swine with continuous electrocardiograms may provide some insight into the exact mechanism of death.
We have used the phrase “sudden death” to refer to any death in which preceding severe illness was not recognized in the swine. Since most of those that died suddenly had one or more myocardial infarcts, it is obvious that they did actually have severe preceding disease. This has led us to review our recent experience in Albany with medico-legal autopsies on people who (by the same definition of sudden death) die suddenly and unexpectedly. We found that ap- proximately one half of these also had recent myocardial infarcts (manuscript in
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preparation). This whole aspect of sudden death needs further investigation both in man and in experimental animals. The swine model developed in the current study should be of considerable value in such studies.
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