Carbon monoxide-aggravated atherosclerosis in the squirrel monkey

EXPERIMENTAL

AND

Carbon

MOLECULAR

PATHOLOGY

13,36-50

(1968)

Monoxide-Aggravated in the

Atherosclerosis

Squirrel

Monkey’

W. S. WEBSTER, T. B. CLARKSON,AND H. B LOFLAND Departments of Laboratory Animal Medicine and Pathology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, North Carolina 2710.3 Received

February

25, 1970

Twenty-two squirrel monkeys, fed a cholesterol-containing diet, were placed in two groups: 12 were exposed to CO and 10were not. In this 7-month study the atherosclerosis induced in the coronary arteries was aggravated by carbon monoxide; aortic atherosclerosis was not. The greater severity in the carbon monoxide group was shown by suggestive electrocardiographic changes, relative increases in heart weights, and a greater degree of coronary arterial stenosis as a result of intimal lipid. Marked deviations from normal values were not observed in several clinical characteristics often associated with the formation of atherosclerotic lesions of the arteries. We propose several mechanisms by which carbon monoxide could have aggravated the cholesterol-induced coronary artery lesions. In the CO-treated monkeys there was more intimal lipid in each atherosclerotic coronary artery rather than more coronary arteries with intimal lipid, as compared with the control group. Few such animal studies have been reported, so it is difficult to compare these results for the squirrel monkey with those for other species.

Carboxyhemoglobin formation in man in several working environments has been documented (Ramsey, 1967; deBruin, 1967; Ayres et al., 1965), and the resulting decrease in mixed venous oxygen tension is thought to be more severe than that ascribed to several other types of hypoxic stresses(Barlett, 1968). Experimentally induced tissue hypoxia is thought to accelerate atherosclerosis in cholesterol-fed animals (Kjeldsen et al., 1968; Fillios et al., 1961; Myasnikov, 1958) and to induce (Lorenzen and Helin, 1967) and influence (Astrup, 1967) arteriosclerosis. Cholesterol-fed, CO-exposed rabbits have higher serum and aortic cholesterol levels, more visible aortic atherosclerosis, heavier hearts, more atherosclerosis of the coronary arteries, and more myocardial infarct-like areas than control cholesterol-fed animals (Astrup et al., 1967). Relatively continuous exposure to CO has produced significant increasesin the concentration of serum cholesterol in man (Kjeldsen and Damgaard, 1968). Our aim in this study was to determine if chronic exposure to carbon monoxide would enhance atherosclerosis among squirrel monkeys fed an atherogenic diet. The squirrel monkey is a small New World primate that has been used extensively in atherosclerosis research, since the animal naturally develops mild aortic and coronary artery atherosclerosis, which can be aggravated by dietary manipulation (Middleton et al., 1967; Malinow et al., 1966). i Supported in part by Grants National Institutes of Health.

FR-00180, FR-05000, HE-04722, 36

and HE-04371 from The

SQUIRREL

MONKEY

MATERIALS

ATHEROSCLEROSIS

AND

37

METHODS

Twenty-two adult female squirrel monkeys (Saimiri sciureus) of the Peruviantype were purchased from Safari Imports, Miami, Florida, tested for tuberculosis on arrival, and housed as pairs in cages 12 X 20% X 23 inches. During the first month they were fed a commercial monkey chow and given water freely; the diet was then changed to an atherogenic one (MacNintch et al., 1967) containing 0.5 % cholesterol and 25 % fat. After 2 weeks on this diet, the exposure to CO was begun and after 2 months 65 gm of the diet was fed morning and evening to two monkeys per cage for the remainder of the experiment. For exposure to the air or carbon monoxide-air mixtures, each monkey was individually confined in a 12 X 12 X 12 inch wire mesh cage, which was placed in an exposure chamber. This chamber was a wooden box, measuring 32 X 32 X 24 inches. The gas inlet port was located 2% inches from the top of the chamber. The outlet port was diagonally across the chamber and 6 inches from the bottom. Each port was 1 inch in diameter. The monkeys were divided without conscious bias into 12 test and 10 control animals. The test monkeys were exposed to an atmosphere of CO2 diluted with air from a compressor; the controls breathed undiluted air while in the chamber. The test and control monkeys were gassed for approximately 4 hours each day, for 5 days a week for 7 months. Neither group was exposed to the chamber environment for a further 3 weeks. Compressed air going to the chamber passed first through an air filte? to remove oils, an air regulator’ to maintain constant pressure, and finally through a needle valve5 which served as a flowmeter. Vinyl plastic tubing,6 with appropriate plastic fittings, conducted the gas to an air-CO mixing chamber. The mixing chamber consisted of a loo-ml glass flask with a hand-blown, rounded bottom and two l-inch side arms; one side arm received the tubing from the air source and the other received tubing from the CO source. The neck of this flask fit over a No. 1, one-hole rubber stopper which in turn was inserted into the inlet of the gas chamber. CO input was metered through a two-stage regulator’ with a relief valve and a metering valve,* via plastic tubing to one of the side arms of the mixing flask. Air was delivered to each of the two groups at a rate of 20 l./min, while the CO flow was regulated so as to attain a chamber atmospheric concentration of 200-300 ppm during the test group’s exposure. Both air and CO were delivered at 20 lb/sq in pressure. Chamber CO concentration was measured9 twice weekly, at the end of the 4-hour exposure period. CO concentrations for the first month’s exposures averaged 100 (f30) ppm; the CO flow was increased so the CO levels reached 209 (f35) 2 Commercial grade-98%

CO, A Cylinder, Air Products, Allentown, Pennsylvania. a Wilkerson air filter, Model 1137-3F, Wilkerson Corp., Englewood, Colorado. 4 Wilkerson air pressure regulator, Model 200&3H, Series D, Wilkeraon Corp., Englewood, Colorado. 6 Miniature forged needle valve, Model 3212M4B, Hoke, Cresskill, New Jersey. 6 Nalgon vinyl plastic tubing, Catalog No. 8000, Size No. 2, Preiser Scientific, Inc., Charleston, West Virginia. 7 Carbon monoxide a-stage regulator, Model 02-1000, Air Products, Allentown, Pennsylvania. 8 Fine metering valve, Model 07-5442, Air Products, Allentown, Pennsylvania. 0 Universal Gas Sampler, Model 19-0241 and CO Indicator Tubes, Model 19-5015, Bacharach Instrument Co., Pittsburgh, Pennsylvania.

38

WEBSTER,

CLARKSON,

AND

LOFLAND

ppm during the second month and 300 (~35) ppm during the third month of exposure. The latter concentration of CO induced extreme ataxia and emesis, the flow rate was gradually reduced during each exposure so that approximate concentrations of 250 ppm CO were reached at the end of each exposure period, and this level was used for the remaining 4 months of the experiment. Temperature and relative humidity were recorded before and after each exposure period with a hygrometer,‘O which remained inside the chamber. During the course of each group’s exposure in the chamber, the mean temperature increase was 4”F, while the temperature range was 73-84°F. The mean increase in relative humidity was 6 %, with a range of 37-54 %. Oxygen and carbon dioxide concentrations in the chamber atmosphere were periodically analyzed at the end of an exposure period for both groups of animals using an oxygen analyzer” and a pCOz electrode.12 The concentration of oxygen by volume had been reduced to 19 % by the end of a 4-hour exposure period, while the chamber CO2 concentration was 0.50.75 %. Two-milliliter samples of femoral blood were obtained weekly from all 22 monkeys for 3 weeks of each month during the experimental period. Carboxyhemoglobin and hemoglobin determinations, erythrocyte counts, and hematocrit analyses were made every other week on the heparinized13 blood. Blood was obtained the second week of each month from fasted animals; the serum was separated and frozen for subsequent total serum cholesterol and total serum protein determinations and serum protein electrophoresis. Body weights were recorded on all animals at this time also. All of these base line data were obtained prior to feeding the atherogenic diet and also prior to the initiation of gassing. The carboxyhemoglobin determinations were made on test monkeys, by a modification of a spectrophotometric method (Whitehead and Worthington, 1961). This method is reportedly accurate and results have a SD of ~1.0 % over a wide range of carboxyhemoglobin values (O-70 %) ; at a level of 10 % carboxyhemoglobin the SD was ho.7 %. We modified this method by using a buffer of pH 5.42 and final dilutions of test solutions of 1:8. The erythrocytes were not washed because of their fragility. Spectrophotometric readings were made on a suitable photometer.‘l Hemoglobin and hematocrit determinations and erythrocyte counts were done by conventional methods. Total serum protein was determined refractometrically with a hand refractometer.lj Serum proteins were electrophoresed on cellulose acetate strips16 (barbital buffer, pH 8.6, 1.5 mA per strip, 90 minutes at 200 V). Relative serum protein fractions were quantified with a scanner,” with pooled sera as a secondary standard. Monthly serum cholesterol determinations were made using an AutoAnalyzer.18 lo Ashton Humidiguide Hygrometer, No. 5547, Taylor Instrument Co., Asheville, North Carolina. *I Beckman Analyzer, Model 777, Beckman Instruments Co., Fullerton, California. I2 Type E5036, Radiometer A,&, Copenhagen, Denmark. I3 Lipo-Hepin, Riker Laboratories, Northridge, California. I4 Klett-Summerson Photoelectric Colorimeter, Model 8003, Philadelphia, Pennsylvania. I5 TS Meter, Model 10401, American Optical Co., Instrument Division, Buffalo, New York. i6 Elect,rophoresis Chamber for Cellulose’ Polyacetate Media, Model g-528-100, Gelman Instrument Co., Ann Arbor, Michigan. I7 Beckman Analytrol, Scan-A-Tron, Model E4940, T. M. Beckman Instruments, Inc., Fullerton, California. I8 The automated method of Block et al. was used (Clin. Chem. 12, 681-689, 1966).

SQUIRREL

MONKEY

ATHEROSCLEROSIS

39

Base line and terminal a- and p-lipoprotein cholesterol analyses were made, using a method whereby P-lipoproteins were precipitated with dextran sulfate (Kritchevsky et al., 1963). Lee and White clotting times (37°C and 30-second inspection intervals) and clot retraction times were measured on blood samplesobtained at the end of the study. Prothrombin times and indices were done terminally on most animals (Quick, 1945) with a preparation of pooled human thromboplastin being used to determine control prothrombin times. Platelets were counted in the titrated blood used for the prothrombin analyses. Electrocardiograms were recordedlg on all monkeys just before the initiation of gassing and at the end of the experiment, 7 months later. Terminal tracings were made 1 week after last exposure to the respective gases.Animals were anesthetized with thiopental sodium20(15 mg/kg body weight) and placed in a supine position. Standard unipolar limb and precordial leads were obtained according to an earlier technique (Wolf et al., 1969). At the end of the experiment the anesthetized monkeys were exsanguinated, necropsied, and weights recorded on all body organs; hearts were weighed after clots had been removed. Tissues were fixed in 10% formalin buffered with phosphate. Intact aorta and carotid arteries were removed, including the bifurcation of each carotid and the iliac bifurcation, and adventitial fat was dissected away. Each artery was split longitudinally; one-half, including the left carotid artery, was formalin-fixed for later lipid staining and histologic examination, and the other half was frozen for tissue cholesterol analysis. Each split, previously frozen aorta was later divided into thoracic (including the right carotid artery) and abdominal (including a renal artery and 3 to 5 mm of the iliac artery) portions. Aortic cholesterol determinations were made on each such section and the amount of cholesterol expressedin terms of aortic DNA (Chargaff and Davidson, 1955). Lipids were extracted from the sections into chloroform-methanol 2: 1. After centrifugation, the supernatant organic phase was evaporated, the residue resuspendedin isopropanol, and cholesterol determined. The prevalence and extent of grossly visible atherosclerosis was estimated by visual grading of the aorta after staining with Sudan IV-oil red 0.21 The thoracic and abdominal aortas were considered separately in estimating the percentage of the intimal surface occupied by fatty streaks or plaques. A consensusscore of two observers was recorded. Three blocks from each fixed heart were sampled by a method previously described (Clarkson et al., 1962) and 10-r sections cut with a cryostat. Three sections per block were chosen at random and stained with Sudan IV and hematoxylin. All coronary arteries that were transversely sectioned and clearly showedtheir structure were counted on each of the nine cryostat sections per monkey heart. The criterion for an arterial lesion was the presenceof intimal lipid; when it was present, the percentage of the lumen obliterated was estimated. The percentage of arteries with I9 Sanborn Visocardiette, Hewlett-Packard Company, Palo Alto, California. 2o Pentothal Sodium, Abbott Laboratories, North Chicago, Illinois. 21 Preparation made by combining 6 gm oil red 0, 2.4 gm Sudan IV, and 1200 ml of 700/, isopropanol.

40

WEBSTER,

CLARKSON,

AND

TABLE

LOFLAND

I

MEAN VALUES OF SERUM CHOLESTEROL, HEMATOCRIT, AND CARBOXYHEMOGLOBIN IN CHOLESTEROL-FED SQUIRREL MONKEYS CHRONICALLY EXPOSED TO AIR OR CARBON MONOXIDE Hematocrit (% f SD)

Total serum cholesterol (mg/lOO ml i SD) Control Before study After diet initiation & prior to gas exposure Exposure period (weeks)

177 f 491 f

38 116

180 f 455 f

440 f -

218

380 f -

114

400 i -

163

455 f -

125

504 i -

174

470 i -

193

423 f -

185

404 f

221

38 dz 3 42 i 4

38 f 2 42 zk 3

42 f -

15 f -

3

136

42 z!z 4 -

3

474 i -

3 2

9f3 20 f -

8

144

43 f 42 f -

5 2

395 f -

43 f 43 f -

2 2

18 f 19 f

5 5

158

42 f 42 f -

3 3

430 i -

40 f 40 f -

2 2

25 f 26 f

6 7

125

39 f 40 f -

4 4

411 f -

38 f 39 f -

2 3

21 i 16 f -

8 5

191

38 f 42 f -

4 4

489 f -

35 f 37 f -

3 2

16 f 24*7 -

3

171

39 i 40 i -

3 2

430 * -

39 f 37 f -

2 2

18 f 25 i

8 6

176

38 f 42 i -

4 3

428 f -

38 f 39 f -

3

23 f

5

175

42 f -

2

352 f

40 f -

-

TABLE HEMATOLOGIC

VALUES

FOR OR

HematocriP (%)

Group

Control

(N = 10)

Test (A’ = 12) = Mean ’ Values ’ Values

39 f 41 l

Hemoglobinb k&100 ml) 2

2

12.9 13.4

zk 0.46 zt 0.84

values & 1 SD. obtained over the experimental obtained at end of experiment.

Test

38 86

-

2 4 5 7 8 9 11 12 13 15 16 17 19 20 21 23 24 25 27 28 29 32

Control

Test

Carboxyhemoglobin (% Sat. f SD)

period.

-

-

-

II

SQUIRREL

CARBON

-

MONKEYS

EXPOSED

TO

AIR

MONOXIDE”

Platelew (No./mm’ blood)

51 x 1w * 57.2 X 10’ f

17 x 104 25.3 X 10’

Pro&d;zbinc

0.62 0.69

f f

0.114 0.088

Clottk&time

3.5 f 3.2 f

“ot.re; tractKIn (min) 0.80 0.66

69 f 55 f

25 17

SQUIRREL

MONKEY

41

ATHEROSCLEROSIS

3634-

g < 5 e g 2 4 5

32

-

28

-

24

-

22

-

20

-

18

-

16

-

14

-

12

-

10

-

8

-

6

-

4

-

2-

% of coromry Arteries with Lesions

Severity of Coronary Artery L&OtlS

Mean Valuer Carbon-monoxide

of

Mean values Air group

of

group

“Comnary Artery Index”

FIQ. 1. Coronary artery disease in hypercholesterolemic or to carbon monoxide.

squirrel

monkeys exposed to air

lesions and the percentage of lumen occluded were averaged for each monkey. The remainder of the heart blocks were parafhn-embedded, sectioned, and stained with hematoxylin and eosin. The various data were compared by the use of Student’s t test, the correlation coefficient r, or by the chi-square method. RESULTS There were no significant differences between the two groups in their final body weights or in weight gained over the experimental period. The mean body weights of the control group at the start and end of the study were 554 and 597 gm, respectively. Corresponding values for the test monkeys were 541 and 561 gm, respectively. In general, the appetites of the test monkeys were not as good as those of the control group. Three control and two test monkeys lost weight; test animal No. 190 lost more than 100 gm over the experimental period. This monkey also developed contracted tendons in both rear limbs over the course of gassing. Mean carboxyhemoglobin values are presented in Table I. The test group had

42

WEBSTER,

CLARKSON,

AND

LOFLAND

FIG. 2. A photomicrograph of transverse sections of two coronary arteries in the left ventricular myocardium of CO-treated monkey No. 185. Marked lumen obliteration is produced by atherosclerosis in both arteries. The frozen section is stained with Verhoeff’s elastic van Gieson. X 100. FIG. 3. A photomicrograph of a transverse section through the anterior descending coronary artery of CO-treated monkey No. 226. This is an example of one of the largest lipid-containing intimal lesions in a large coronary artery. The internal elastic lamina is thin and split beneath the larger plaque, while it appears completely disrupted in one area. Medial fibers, with lipid apparent among them, are disoriented below the larger intimal lesion. The section is stained with Verhoeff’s elastic van Gieson. X100.

SQUIRREL

MONKEY

ATHEROSCLEROSIS

TABLE THE EXTENT

OF CORONARY SQUIRREL MONKEYS

43

III

ARTERY ATHEROSCLEROSIS IN HYPERCHOLESTEROLEMIC EXPOSED TO CARBON MONOXIDE OR TO AIR

Carbon monoxide group

Air group Coronary Monkey Percent of arNo. teries with lesions” Severityb ;te;y c

190 233 185 241 226 65 235 195 79 176 276 194

79 51 50 29 28 23 23 16 14 13 4 0 Mean = 27.5

46 48 54 36 41 51 28 28 23 26 11 0 32.7d

62.5 49.5 52.0 32.5 34.5 37.0 25.5 22.0 18.5 19.5 7.5 0 30.v

108 74 183 234 75 83 77 193 87 236

32 30 23 20 14 10 9 9 5 1 Mean = 15.3

30 24 23 24 15 10 16 7 7 4

31.0 27.0 23.0 22.0 14.5 10.0 12.5 8.0 6.0 2.5

16.W

15.7d

a Expressed sections of the b Expressed atherosclerotic c Expressed artery lesions, d Difference

as the mean percentage of arteries seen with atherosclerotic lesions in nine-step heart. as the mean percentage of apparent lumen obliteration in arteries having lesions. as percentage of coronary arteries with lesions plus mean severity of coronary divided by 2. between mean values of the two groups is significant at .Ol > p > .OOl.

insignificantly gassing when

higher mean hemoglobin compared to the control

and hematocrit values over the period of animals (Table II). Significant differences in

hemoglobin, hematocrit, and erythrocyte counts between the two groups were observed when mean values were compared at various times during the course of the study. Increases in mean hematocrit and hemoglobin were seenin each group in the period between arrival at the vivarium (before study) and just prior to gassing, a period of about, 6 weeks. The relatively low hematologic values seen prior to the study

were reflective

of the generally

poor physical

condition

of the animals

on ar-

rival at the vivarium. Just prior to sacrifice, one test monkey (No. 190) had a 10 % hematocrit, 4.4 gm of hemoglobin/100 ml of blood, and 2.5 X 106 per mm3 erythrocytes; the anemia was normocytic and normochromic. At, the end of the study the mean prothrombin index of the CO-treated monkeys tended to be greater than that of the control monkeys, although not significantly (.2 > p > .l)

(Table

II).

The mean platelet

count

also tended

to be higher

in the

test than in the control group: the CO-exposed monkeys averaged 60 X 103 more platelets per mm3 than the control monkeys. Anemic test monkey No. 190, with the most abnormal coronary artery index, also had the highest platelet count, 24 times the values of other test animals.

44

WEBSTER,

CLARKSON,

AND LOFLAND

“6 FIG. 4. Precordial leads of base line (left column) and terminal (right column) electrocardiograms of test monkey No. 235. Notched or double-peaked R wave indicative of ventricular conduction delay or incomplete right bundle branch block is present in lead VI of the base line tracing. Leads V, and Vg in the terminal tracings demonstrate widened QRS complexes with decreased voltages, suggestive of complete right bundle branch block. Diminished P and T waves are present, particularly in leads V, and Va .

Clotting times for the two groups of monkeys were very similar at the conclusion of the study, with the control group averaging about 0.5 minutes slower than the test group. Mean clot retraction time tended to be slower in the control group. (Table II). Control monkeys No. 108 and 74, which ranked first and second in coronary artery index, had longer clot retraction times than other monkeys of their group. No such relationship was evident among test monkeys, which also had high coronary artery indices. There was a significant and equal increase in mean total serum cholesterol level within each group when base line data were compared with the data for the first month on the diet (p < .OOl for each group) (Table I). Changeswere not seenin the (Y/P lipoprotein cholesterol ratio for either group when beginning and final data on these fractions were compared. The @-lipoprotein fraction comprised about 80 % of the total lipoprotein in both test and control groups, at the start and at the end of the study. The data obtained from grading coronary arteries for atherosclerosis are summarized in Fig. 1 and Table III. The test group had both a greater mean percentage of coronary arteries with atherosclerotic lesionsand more lumen occlusion among the affected arteries. There tended to be more CO-exposed animals than control animals with over 50 % of their coronary arteries affected with lesions. On chi-square analysis

SQUIRREL

MONKEY

ATHEROSCLEROSIS

TABLE IV SERUM AND AORTIC CHOLESTEROL VALUES AND RESULTS OF VISUAL GRADINQ OF STAINED ARTERIES FOR FATTY STREAKS AND PLAQUES~ Group Air (N = 10) Carbon monoxide (N = 12)

Total* serum cholesterol 446 f 435 i

121.4 126.9

Carotid Thoracic aorta artery scorec Score0 Cholesterold 13 14

34 30

3.6 i 3.1 i

1.39 1.55

Abdominal aorta Scorec 26 19

Cholesterold 3.3 i 2.6 i

1.55 1.18

D All values expressed as mean values f 1 SD, calculated over the experimental period. * Expressed in mg cholesterol/l99 ml serum. c Mean value of two observer’s scores of percentage of intimal surface stained with the Sudan IV-oil red 0 stain. d Expressed in mg cholesterol/mg aortic DNA.

there were significantly (.Ol > p > .OOl) more CO-treated monkeys than control monkeys having 35 % or more apparent atherosclerotic stenosis among the affected arteries. Examples of coronary artery atherosclerosis are presented in Figs. 2-3. There were no differences in distribution or morphologic characteristics of coronary artery lesions between the two groups. The coronary artery atherosclerosis was most often seen in the small intramyocardial arteries. Many coronary artery lesions in both groups contained considerable medial lipid. Of all the monkeys, only one test monkey (No. 226) had mineral deposition in the coronary arteries; in this one the mineralization was in the intima and media of several arteries of the right ventricular myocardium. The results of gross scoring of the lipid-stained arterial segments are presented in Table IV. No significant differences were observed when the mean scores for the various arterial segments were compared for the two groups of monkeys. Microscopically, the aortic lesions in both groups of monkeys had sudanophiiic material in the intima and the inner third of the media. There was wide variation in aortic cholesterol content among monkeys of both groups (Table IV). There were no significant differences in the mean total serum protein values between control and test animals when compared at monthly intervals or when group means were compared over the experimental period. There were no meaningful fluctuations in the total serum protein values in individual animals throughout the exposure period, in either group. No significant changes were observed in percentages of serum albumin, globulins, or in A/G ratios, when means of two groups were compared over the period of gassing. Changes were not seen in the proportions of serum albumin, beta and gamma globulin, or A/G ratio when values of individual animals of either group were compared over the experimental period. Significant changes in heart rate, P-R interval, or axis deviation were not seen in the electrocardiograms of any of the monkeys on comparison of terminal with base line tracings. T wave inversions were not observed in any of the monkeys. However, changes were noted in the RS-T segment, the T wave amplitude, and in QRS complex duration in some animals. Altered S-T takeoffs were seen in 6 of 12

46

WEBSTER,

CLARKSON, TABLE

THE

PREVALENCE OF MONKEYS

AND

LOFLAND

V

RS-T ABNORMALITY EXPOSED

IN THE TERMINAL TO CARBON MONOXIDE AND

EKG’s TO

OF SQUIRREL

AIR

Group

No. animals

Prevalence in all leads’

Prevalence in std. leads*

Prevalence in V leadsc

co

12 10

7/12 4/10

11/36 3/30d

lo,‘36 2/30d

Air

a Expressed as number of monkeys with depressed/elevated RS-T takeoffs in any of the nine EKG leads over the number of monkeys examined electrocardiographically. * Expressed as number of depressed/elevated RS-T takeoffs in any of the standard leads over the total number of standard leads examined. c Expressed as number of depressed/elevated RS-T t,akeoffs in any of the precordial leads over the total number of precordial leads examined. d Difference in distribution within the two groups is significant. at .05 > p > .02 (x2 analysis)

test animals and 2 of 10 control animals in either/or both leads II and III, when terminal were compared to base line tracings. RS-T segment alterations are tabulated in Table V. Slurred R waves or delayed intrinsicoid deflections and prolonged QRS complexes in the precordial leads appeared to be more common in the COexposed than in the control monkeys. Slurring of the R wave with a resultant widened complex in all three V leads was found on terminal electrocardiograms of three test animals (No. 65, 185, and 235). In particular, test monkeys No. 185 and 235 had greatly widened complexes, measuring 0.05-0.06 and 0.06-0.07 secondsin the V leads, respectively. No S wave deflections were noted in any of the precordial leads of either monkey (Fig. 4). Widened QRS complexes were seenin two other test animals, in lead Vs only. Only one control monkey (No. 74) had R wave slurring and that occurred in lead Vc alone. Four CO-exposed monkeys had obviously flattened, sometimes nondiscernible T waves, in all terminal precordial leads. P waves were usually diminished in leads where the T waves were of low voltage. Depressed P and T waves are also seenin Fig. 4. The terminal electrocardiographic findings in three test monkeys (No. 65, 185, and 235) were suggestive of complete right bundle branch block. These animals with widened QRS complexes, slurred R waves, and flattened T waves had electrical patterns consistent with a diagnosis of ventricular conduction delay. Bundle branch block as produced by delays in activation at the terminal ramifications of the Purkinje network would also be consistent with the anatomic location of the coronary arterial lesions seen in these monkeys. Definitive electrocardiographic evidence of left ventricular hypertrophy was not found in any monkey. The mean heart weight: body weight ratio was significantly greater for the COexposed group than for the control group at the 2 % (0.55 vs 0.47 %) confidence level. On an absolute basis, there was no significant difference between mean heart weights of the two groups. No significant’differences in means were seen in either thyroid or adrenal weight: body weight ratios between the two groups of animals. Four of 10 control animals and 9 of 12 test animals had grossly visible thymus glands at autopsy. All nine organs from the test animals as compared to two of four of the controls, comprised over 0.06% of the total body weight, 011 an individual

SQUIRREL

MONKEY

ATHEROSCLEROSIS

47

basis. Based on presence or absence of visible thymus and the weight of individual thymus glands the control group of monkeys seemed to include a greater number of older animals. DISCUSSION Evidence for several types of cardiac abnormalities among CO-treated monkeys has been found in this study: electrocardiographic evidence of delayed depolarization of the ventricular musculature and right bundle branch block, anatomic evidence of increased heart weight: body weight ratios, and the pathologic finding of more extensive coronary artery atherosclerosis. An exacerbating effect df CO on diet-aggravated atherosclerosis of these monkeys could be documented only in the coronary arteries. This finding is similar to that in cholesterol-fed, CO-exposed rabbits (Astrup et al., 1967). However, hemorrhages and infarct-like areas in the heart and greater aortic atheromatosis and aortic cholesterol seen in those rabbits were not seen in our control or CO-treated monkeys. Some of these cardiac changes might have occurred if longer continuous periods of exposure to CO had been used. It should be pointed out that focal myocytolysis, nonspecific focal lymphocytic aggregates and inflammatory reactions of numerous parasitic infections in the myocardium are often seen in the hearts of squirrel monkeys. These lesions may be confused with pathologic changes in the myocardium produced experimentally. A recent study (Wolf et al., 1969) indicated that rather severe experimental measureswere required to produce myocardial infarction in the Braziliantype squirrel monkey. Our studies provide no data on whether the mechanism of the coronary artery atherosclerosis involves hypoxia, enzyme inhibition, or other factors. Atherosclerotic coronary arteries are unable to increase blood tlow significantly and tissue hypoxia can only be prevented by increased oxygen extraction from the blood; even small amounts of carboxyhemoglobin could hinder such extraction (Ayres et al., 1965). Increases in coronary blood flow and decreasesin myocardial extraction ratios for oxygen have been observed in humans and in dogs following acute elevation of carboxyhemoglobin (Ayres et al., 1969). Early stages of coronary artery atherosclerosis could conceivably be aggravated by the hypoxia associated with carboxyhemoglobinemia. The numbers of coronary artery lesionsseenamong the CO-treated monkeys were not as strikingly increased as the size of the lesions (expressed as the percentage of the lumen obliterated). This observation would suggest that the CO effect was not oneof induction but’ rather one affecting rate of coronary artery plaque development. Factors which interfere with the tricarboxylic acid cycle, thus decreasing ATP formation, would be expected to possibly enhance atherogenesis. Succinic and lactic dehydrogenases, catalysts for the production of ATP, are inhibited by CO. ATP production, and this energy for normal lipid metabolism, would also be markedly reduced when oxidative phosphorylation is hindered by CO inhibition of cytochrome enzymes. Delays in ventricular conduction were present in the precordial leads of the electrocardiograms of several CO-exposed monkeys. It is perplexing that more striking changes were not seenin the standard leads of these monkeys. Several test monkeys

48

WEBSTER,

CLARKSON,

AND

LOFLAND

had terminal tracings strongly suggestive of complete right bundle block, while no control monkeys had similar tracings. While two of these test animals had tracings which indicated a ventricular conduction delay or incomplete right bundle branch block on base line tracing, the terminal electrocardiographic complexes are certainly changes from the base line towards a worsening delay, or complete bundle branch block. These electrocardiographic findings would be consistent with diffuse or patch) ventricular fibrosis or diffuse intramyocardial arterial atherosclerosis in man. As the squirrel monkey develops considerably more intramyocardial than proximal coronary disease, it would seem reasonable that this could account for the electrocardiographic changes especially since we were unable to relate these electrocardiographic changes with lesions of the myocardium. The greater frequency of such shifts in the CO-treated monkeys might also have been due to cardiac hypertrophy. The simplest explanation would be cardiac hypertrophy produced by the CO-induced hypoxemia. Definitive electrocardiographic evidence of left ventricular hypertrophy or myocardial ischemia in the CO-treated animals is lacking, however. Marked S-T segment elevations were related to hypertrophied ventricular muscle, in a recent study of cebus monkeys (Bullock et al., 1969). The comparatively heavier hearts of the test animals could not be related to an increased packed cell volume, for no consistent increases in this characteristic were seen in any CO-exposed monkey. The test animals did not develop striking polycythemia for two possible reasons: (1) they had only intermittent exposure to CO and thus lacked a more prolonged stimulation of the bone marrow, and (2) blood samples were drawn at frequent intervals, thus reducing the product of any marked erythropoiesis which may have occurred. A 2-ml blood loss, assuming a blood volume of 4045 ml/500 gm monkey, would represent approximately a 5 % blood loss, three times per month. The squirrel monkey may never develop a profound polycythemia in response to CO, as had been suggested is the case with the rhesus monkey (van Bogaert et al., 1938) and man (Grant and Root, 1952). Clotting times and clot retraction times apparently were not affected by the low levels of CO. There was a positive correlation (r = 0.68) between heart weight:body weight ratios and the percentage of coronary arteries with lesions in the test monkeys whereas there was none in the control animals. As expected, there was significant positive relationship between total serum cholesterol and coronary artery atherosclerosisin both groups. It is unfortunate that monkeys of more similar age were not available for study. Thymic weight and ovarian histologic data suggested that the monkeys varied widely in age. If there was a greater number of older monkeys in the control group, perhaps their susceptibility to induced atherosclerosis change was increased, as compared to the test animals. More advanced age could account, in part, for the somewhat greater aortic atherosclerosis,both on a macroscopic and tissue cholesterol basis, in the control monkey group. However; there is recent indication from experiments now in progressin our laboratories that age has no effect on serum cholesterol levels in squirrel monkeys. ACKNOWLEDGMENTS

The authors gratefully acknowledgethe helpful advice of Drs. Bill Bullock, Noel Lehner, andRichard St. Clair andthe excellenttechnicalassistance of Carolyn Pulliam andJeanLewis.

SQUIRREL

The advice of Dr. J. Stanton acknowledged.

MONKEY

49

ATHEROSCLEROSIS

King in the preparation

of the manuscript

is also gratefully

REFERENCES P., KJELDSEN, K., and WANSTRUP, J. (1967). Enhancing influence of carbon monoxide on the development of atheromatosis in cholesterol-fed rabbits. J. Atheroscler. Res.

ASTRUP,

7, 343-354. ASTRUP, P. (1967). Carbon monoxide and peripheral Invest. Suppl. 99 19, 193-197. AYRES, S. M., GIANNELLI, S., and ARMSTRONG, R. G.

arterial

disease. &and.

J. Clin. Lab.

(1965). Carboxyhemoglobin: hemodynamic and respiratory responses to small concentrations. Science 149, 193-194. AYRES, S. M., MUELLER, H. S., GREGORY, J. J., GIANNELLI, S., and PENNY, J. L. (1969). Systemic and myocardial hemodynamic responses to relatively small concentrations of carboxyhemoglobin (COHb). Arch. Environ. Health 18,699-709. BARTLETT, D. (1968). Pathophysiology of exposure to low concentrations of carbon monoxide. Arch. Environ. Health 16, 719-727. BULLOCK, B. C., CLARKSON, T. B., LEHNER, N. D. M., LOFLAND, H. B., and ST. CLAIR, R. W. (19.69). Atherosclerosis in Cebus albifrons monkeys. III. Clinical and pathological studies. Exp. Mol. CHARGAFF,

Pathol.

10, 39-fj2.

E., and DAVIDSON, Academic Press, New York.

J. N. (eds.)

(1955).

“The

Nucleic

Acids,”

Vol. 1 and 2.

T. B., PRICHARD, R. W., LOFLAND, H. B., AND GOODMAN, H. 0. (1962). tions among dietary fat, protein, and cholesterol in atherosclerosis-susceptible Effects on coronary atherosclerosis. Cir. Res. 11,40&404. DEBRUIN, A. (1967). Carboxyhemoglobin levels due to traffic exhaust. Arch. CLARKSON,

Health. FILLIOS,

Interacpigeons. Environ.

15, 384-489. L. C., ANDRUS,

S. B., and NAITO, C. (1961). Coronary lipid deposition during chronic anemia or high altitude exposure. J. AppZ. Physiol. 16, 103-106. GRANT, W. C., and ROOT, W. S. (1952). Fundamental stimulus for erythropoiesis. Physiol. Rev. 32, 449-498. HELLUNG-LARSEN,

P., LAURSEN, T., KJELDSEN, K., and ASTRUP, P. (1968). drogenase isoenzymes of aortic tissue in rabbits exposed to carbon monoxide.

Res. Hwu,

Lactate

dehy-

J. Atheroscler.

8, 343-349.

J. (1959). Carbon monoxide and cyanide poisoning. I. Changes in cytochrome oxidase activity in carbon monoxide and cyanide poisoning. Kokumin Eisei 28,636-643. JAFFE, N. (1965). Cardiac injury and carbon monoxide poisoning. S. Afr. Med. J. 39, 611615. KJELDSEN, K., and DAMGAARD, F. (1968). Influence of prolonged carbon monoxide exposure and altitude hypoxia on serum lipids in man. Stand. J. CZin. Lab. Invest. Supp. 109 22,16-19. KJELDSEN, K., WANSTRUP, J., and ASTRUP, P. (1968). Enhancing influence of arterial hypoxia on the development of atheromatosis in cholesterol-fed rabbits. J. Atheroscler. Res. 8, 835-845. KRITCHEVSKY,

D., TEPPER, S. A., ALAUPOVIC, P., and FURMAN, R. H. (1963). Cholesterol content of human serum lipoproteins obtained by dextran sulfate precipitation and by preparative ultracentrifugation. Proc. Sot. Exp. BioZ. Med. 112, 259-626. LORENZEN, I., and HELIN, P. (1967). Arteriosclerosis induced by hypoxia. Acta Pathol. Microbial. Stand. 69, 158-159. MACNINTCH, J. E., ST. CLAIR, R. W., LEHNER, N. D. M., CLARKSON, T. B., and LOFLAND, H. B. (1967). Cholesterol metabolism and atherosclerosis in cebus monkeys in relation to age. Lab. Invest. 16, 444-452. MALINOW, M. R., MARUFFO, C. A., and PERLEY, A. ‘M. (1966). Experimental atherosclerosis in squirrel monkeys (Saimiri sciureus). J. Pathol. Bact. 92, 491-510. MIDDLETON, C. C., CLARKSON, T. B., LOFLAND, H. B, and PRICHARD, R. W. (1967). Diet and atherosclerosis of squirrel monkeys. Arch. Pathol. 83, 145-153. MYASNIKOV, A. L. (1958). Influence of some factors on development of experimental cholesterol atherosclerosis. Circulation 17, 99-113. NANIKAWA, R., HAMAOKA, H., and AWATA, Y. (1969). Succinic dehydrogenase of cardiac

50

WEBSTER,

CLARKSON,

AND

LOFLAND

muscle. Chemical and histochemicd findings in CO poisoning death from cold, and hypoxic respiration. Nagoya Med. J. 6, 2740. OMURA, T., SATO, R., COOPER, D. p., ROSENTHAL, O., and ESTABROOK, R. W. (1965). Function of cytochrome P-450 of microsomes. Fed. Proc. 24,1181-1189. QUICK, A. J. (1945). Determination of prothrombin. Amer. J. Clin. Pathol. 15, 569-566. RAMSEY, J. M. (1967). Carboxyhemoglobinemia in parking garage employees. Arch. Environ. Health. 15, 580-583. VAN BOGAERT, L., DALLEMAGNE, M. J., and WEGRIA, R. (1938). Chronic and acute oxygen want in Macacus rhesus. Absence of experimental lesions of nerve centers after intoxication by carbon monoxide, sodium nitrite and oxygen impoverishment of the air. Arch. Int. Med. Exp. 13, 335-378. WARBURG, 0. (1926). “ijber die Wirkung des Kohlenoxyds auf den Stoffwechsel der Hefe.” Biochem. 2. 177, 471486. WHITEHEAD, T. P., and WORTHINGTON, S. (1961). The determination of carboxyhemoglobin. Clin. WOLF,

Chim. Acta 6, 356-359. R. H., LEHNER, N. D.

cardiogram

of the squirrel

M., MILLER, E. C., and CLARKSON, T. B. (1969). Electromonkey, Saimiri sciureus. J. Appl. Physiol. 26, 346-351.