NEW ANIMAL MODEL FOR ATHEROSCLEROSIS RESEARCH

Charles E. Day and Walter W. Stafford Diabetes and Atherosclerosis Research, The Upjohn Company, Kalamazoo, Michigan

SUMMARY Japanese quail were investigated for their utility as a model for the discovery and evaluation of anti-atherosclerosis compounds. Although they possessed suitable characteristics for a screening animal, their development of atherosclerosis was too variable to make them a practical model. A search was conducted to find a means to make quail uniformly atherosclerotic. To this end a line of quail susceptible to experimental atherosclerosis (SEA) were selectively bred. Thus, the SEA Japanese quail is a new animal model for atherosclerosis research.

INTRODUCTION Animal models suitable for use in pharmaceutical discovery and evaluation must meet more stringent requirements than models used for basic research. Most drug discoveries result from random screening of organic compounds in a suitable test system. Generally the success rate is quite low, so large numbers (thousands) of compounds must be screened. If a whole animal is used for screening, then several immediate considerations are apparent. The animal must be readily available, inexpensive, and small. In addition, it should be easy to maintain, dose, and handle routinely. Rats and especially mice fit these requirements almost ideally. This is in no small part why mice and rats are the number one and two animals used in biomedical research in the pharmaceutical industry. 339

D. Kritchevsky et al. (eds.), Lipids, Lipoproteins, and Drugs © Plenum Press, New York 1975

340

C.E. DAY AND W.W. STAFFORD

Mice can be mass produced quite inexpensively. For example, The Upjohn Company alone produces over one half million per year. If 10 mice, 30g each are placed on a test compound for 1 week at a screening dose of 50 mg/kg/day, then approximately 100 mg of compound is needed for the test. Housing space and labor for mouse maintenance are minimal. This example serves to illustrate the good economic sense that an animal with the characteristics of a mouse makes for drug screening operations. Unfortunately, both rats and mice are very poor models for atherosclerosis. They generally have low serum cholesterol and extremely diminuitive low density lipoprotein (LDL). They also are quite resistant to the development of atherosclerotic lesions. Animals that do develop atherosclerosis, such as rabbits, chickens, monkeys, and pigeons are too big and/ or too expensive to use for screening of anti-atherosclerotic agents. About 2 years ago atherosclerosis development in Japanese quail was reported (1), and subsequently confirmed by other investigators (2, 3). Japanese quail (Coturnix coturnix japonica) are small (approximately 100g), hardy animals that can be rapidly and inexpensively mass produced. In addition they can be easily handled, dosed, bled, and maintained. For these reasons we turned our attention to the utility of these animals for a screening model for anti-atherosclerotic agents over 2 years ago. This report deals largely with our experience with development of coturnix as an atherosclerosis screening model.

MATERIALS AND METHODS Japanese quail were obtained from a closed stock colony maintained at The Upjohn Company. This colony was derived from stock obtained from 4 separate university colonies maintained in the U. S. A. We originally fed a standard chicken mash diet £4). The standard maintenance is now Purina Game Bird Startena or Game Bird Layena R . Data in this report are from animals fed the chicken mash diet. For cholesterol feeding experiments crystalline cholesterol is normally fed at a level of 2% in the ground diet. To induce atherosclerosis birds are commonly placed on cholesterol diet for 15 weeks. At the end of each experimental period birds are bled via the right jugular vein. Up to 2 ml can be drawn without apparent injury to the animal. The maximum amount that can be

341

ANIMAL MODEL

obtained is 4 to 5 ml from each bird. At this level the mortality rate is fairly high. The animal is decapitated, and the thoracic aorta and brachiocephalic and subclavian arteries (just after the first branch point) are removed as one piece. The arteries are placed in physiological saline, cleaned, opened longitudinally, and scored for gros s atherosclerotic lesions by three independent observers on a scale of 0 to 100. Mter blotting, the arteries are weighed and then homogenized in a combination tissue grinder and screw cap test tube with O. 5 ml redistilled isopropanol. Mter grinding, 5. 0 ml isopropanol is added. Tubes are capped, shaken, and allowed to extract overnight at room temperature. Cholesterol concentration is then determined in the isopropanol extract by an automated FeC1 3 method (5). Male British Range, English White, Tuxedo, Manchurian Golden, and Pharaoh Dl strains of Japanese quail were purchased from Marsh Farms, Garden Grove, California 92643. All quail rearing and breeding equipment was purchased from either Marsh Farms or C. Q. F. Manufacturing Company, Savannah, Georgia 31402. Both cockerel and porcine aortic elastins were prepared according to the method of Kramsch, Franzblau, and Hollander (6). Elastin was suspended in complete Freund's adjuvant and injected subcutaneously at 5 separate sites (nape of neck, under each wing, and on both sides of cloaca) with a total of 3 injections given over a period of 6 weeks.

RESULTS AND DISCUSSION From our initial studies with coturnix one fact was quite apparent. Fewer than half of a given population from. our stock colony developed atherosclerotic lesions in response to dietary cholesterol (For example see FO data in Table V). In birds in which gross atherosclerosis was detectable, the extent of lesions was highly variable, ranging from < 1% surface area involvement to virtually 100%. A few male quail (approximately 10%) developed severe atherosclerosis with almost total occlusion of major arteries exiting from the heart. Because of the high variance of lesions quail from our stock colony were not suitable for evaluating antiatherosclerosis agents. It was necessary then to develop a method to make the animals uniformly atherosclerotic.

342

C.E. DAY AND W.W. STAFFORD

Table I

EFFECT OF SEVERAL ATHEROSCLEROTIC COMPOUNDS IN JAPANESE QUAIL INCIDENCE OF ATHEROSCLEROSIS

AORTIC ATHEROSCLEROSIS SCORE

AORTIC CHOLESTEROL

SERUM CHOLESTEROL

WEIGHT CHANGE

(mc/cl

(mc/dl)

(c)

CONTROL

0/8

1.0

1.85

706

0.6

NEGATIVE CONTROL (NO CHOLESTEROL)

0/9

1.0

1.22

230*

1.0 5.4

TREATMENT

10% COCONUT OIL

1/8

1.2

2.04"

450

0.05% THIOURACIL

2/8

2.1

2.09

1221

3.2

0.5% CHOLIC ACID

5.17

13.2-

4.31

1528-

-0.3

0.05% THIOURACIL + 0.5 % CHOLIC ACID

417

3.7

2.40

2163-

3.0

0.05% DESOXYPYRIDOXINE

0/7

1.0

1.59

665

'4.4

0.2% SUDAN IV

7/9

11.3-

2.28

964

0.4

1% CHOLESTANOL

219

2.4

1.84

513

-1.3

1% CHOLESTANOL (NO CHOLESTEROL)

0/9

1.0

1.28

282-

-1.3

POOLED % STANDARD DEVIATION

122

62

53

-SIGNIFICANTLY DIHERENT FROM CONTROL (P~o.oS)

To accomplish the desired objective, we initiated a threepronged approach to the problem. One possible solution was enhancement of atherosclerosis by chemical induction. Ideally this method would be one by which the diet could be modified in such a way as to produce extensive and uniform lesions. Time consuming methodologies, such as surgical procedures, were ruled out since disease had to be produced in hundreds of animals. Results on a few of the compounds tried in cholesterol fed quail are listed in Tables I and II. Only cholic acid, at a level of O. 51. 0% in the diet, consistently produced a significant increase in the atherosclerosis score. This increase was primarily in the brachiocephalic artery (Table II). In one experiment quail immunized with porcine aortic elastin at 4 mg/kg had a significant elevation in their brachiocephic atherosclerosis score. No attempt was made to confirm this observation since the immunization procedure was too time consuming to make it suitable as a routine technique for inducing atherosclerosis in large numbers of animals.

343

ANIMAL MODEL

Table II EFFECT OF ATHEROSCLEROTIC COMPOUNDS IN JAPANESE QUAIL INCIDENCE OF ATHEROSCLEROSIS TREATMENT

AORTA

BRACHIOCEPHALIC

SUBCLAVIAN

ATHEROSCLEROSIS SCORE SUBBRACHIOAORTIC CEPHALIC CLAVIAN

ARTERIAL SERUM CHOLESTEROL CHOLESTEROL (mg/g)

(mg/dl)

CONTROL

4/13

4/13

3/13

3.1

3.3

2.7

2.46

653

0.5% CHOLIC ACID

5/14

14/14

12/14

2.6

20.0'

15.1'

2.49

907

1.0% CHOLIC ACID

9/15

14/15

13/15

7.2

27.8'

16.5'

4.15

1486'

0.2% SUDAN IV

9/13

11/13

II I 13

13.3

30.6'

22.7'

4.02

1204'

0.2% SUDAN IV

9/15

12115

II liS

3.9

8.2

7.2

2.46

470

COCKEREL ELASTIN S.C.

8/15

7/15

7/15

3.4

5.0

3.7

2.08

767

6/6

6/6

11.4

28.2'

18.0

3.60

1825'

4/7

0/7

1.2

2.3

1.0

3.06

628

12/18

7118

2.8

6.7

4.1

2.21

548

0.5% CHOLIC ACID

+

PORCINE AORTIC ELASTIN S.C. (4 mg/kg) 516 PORCINE AORTIC ELASTIN S.C.(40mg/kg) 1/7 1% PARAFFIN

7118

, SIGNIFICANTLY DIFFERENT FROM CONTROL (P ,,0.05)

A second approach to finding uniform atherosclerosis in quail was to examine the different strains or stocks of birds that were readily available. We placed 5 different strains - British Range (black), English White, Manchurian Golden, Tuxedo (black and white) and Pharaoh Dl - on a cholesterol diet and examined birds for gross atherosclerosis, arterial cholesterol, and serum cholesterol. No strain was more susceptible to atherosclerosis than were our stock colony birds (Table Ill). One strain, Tuxedo, appeared to be more resistant to atherosclerosis than our stock animals. Both gross atherosclerosis and arterial cholesterol were significantly reduced when compared to controls. No atherosclerosis has been seen in any animal that has not been on a cholesterol diet (Table IV). The investigation of different quail strains was not a fruitful approach.

344

C.E. DAY AND W.W. STAFFORD

Table III STRAIN DIFFERENCES IN ATHEROSCLEROSIS DATA IN CHOLESTEROL FED JAPANESE QUAIL INCIDENCE OF AORTIC ATHEROSCLEROSIS

AORTIC ATHEROSCLEROSIS SCORE

ARTERIAL CHOLESTEROL

SERUM CHOLESTEROL

(mg g)

(mg/dl)

967 874

STRAIN

(0.'0)

STOCK BRITISH RANGE

48 (10/41) 56 (10/18) 50 ( 9/18)

5.9 4.1 4.1

3.73 1.10 1.11

( 6/ II)

ENGLISH WHITE MANCHURIAN GOLDEN TUXEDO

54

774

1.8

1.08

584

PHAROAH 01

9 ( 1/11) 30 ( 3/10)

1.1' 1.6

1.54' 1.38

585 716

OHIO STATE

11

1.9

1.78*

669

( 3/14)

t DATA TRANSFORMED TO LOGARITHMS AND EXPRESSED AS ANTI-LOG MEANS • SIGNIFICANTLY DIFFERENT FROM STOCK COLONY (P< .05)

Table IV STRAIN DIFFERENCES IN CONTROL JAPANESE QUAIL INCIDENCE OF ATHEROSCLEROSIS

ARTERIAL CHOLESTEROL

(0/0)

ATHEROSCLEROSIS SCORE

BRITISH RANGE

0 (016)

0

1.17

159

ENGLISH WHITE

0 (017)

0

1.10

154

MANCHURIAN GOLDEN

0 (0/8)

0

1.19

163

TUXEDO

0 (0/4)

0

1.11

196

PHAROAH 01

0 (017)

0

1.37

314

STRAIN

(mg/g)

SERUM CHOLESTEROL (mg/dl)

ANIMAL MODEL

345

Table V SUMMAR Y OF ATHEROSCLEROSIS DATA ON JAPANESE QUAIL INCIDENCE OF AORTIC ATHEROSCLEROSIS

ARTERIAL CHOLESTEROL

(%)

AORTIC ATHEROSCLEROSIS SCORE

SERUM CHOLESTEROL

GROUP

GENERATION

STOCK

FO

44 (34/77)

21

5.45

886

SEA

FI

73 (45/62)

35

6.05

785

REA

FI

16 ( 4/25)

1.92

357

(mg/g)

(mg/dl)

Table VI SEX DIFFERENCES IN ATHEROSCLEROSIS DAT A FROM JAPANESE QUAIL GENEGROUP RATION

INCIDENCE OF AORTIC ATHEROSCLEROSIS SEX

(%)

AORTIC ATHEROSCLEROSIS SCORE

ARTERIAL CHOLESTEROL

SERUM CHOLESTEROL

(mg/g)

(mg/dl)

STOCK

FO

MALE

48 (20/42)

29

6.29

1205

STOCK

FO

FEMALE

40 (14/35)

12

4.43

505

SEA

FI

MALE

68 (21/31)

41

7.35

1195

SEA

FI

FEMALE

77 (24/31)

29

4.79

375

REA

FI

MALE

17 ( 2112)

4

1.63

488

REA

FI

FEMALE

15 ( 2113)

2.19

235

346

C.E. DAY AND W.W. STAFFORD

Our third approach to the problem of developing uniformly atherosclerotic quail was selective breeding. We randomly paired a colony of our stock animals, placed them on a cholesterol diet, and reared offspring from each pair. After 15 weeks on cholesterol the parents were sacrificed, and their aortas examined and graded for atherosclerosis. Offspring from parents with high atherosclerosis scores were selected for one line. The birds were designated as Susceptible to Experimental Atherosclerosis (SEA). One pair had no atherosclerosis and low arterial and serum cholesterol. Its offspring were selected for a second line designated Resistant to Experimental Atherosclerosis (REA). Brother-sister matings were made for each line, and the rearing procedure described above repeated. From a summary of data on the first generation of selective breeding (Table V), it can be seen that a segregation was achieved in this generation. There was a significant different between SEA and REA in atherosclerosis score, arterial cholesterol and serum cholesterol. Although the incidence of atherosclerosis for both male and female are about the same, the lesions in male animals tend to be more severe (Table VI). Serum cholesterol levels are also higher for males. It is interesti~ to note that the data for male REA is virtually identical to the male Tuxedo quail data (Table VII). We measured a number of serum chemistry values on SEA and REA quail. The only consistent difference was serum alkaline phosphatase levels. Alkaline phosphatase was significantly less in SEA when cOlnpared to REA quail. However, this finding may have no physiological significance since it is quite probable that aortic atherosclerosis and alkaline phosphatase were independently co- selected. Our breeding program with SEA and REA quail is continuing now into the fourth generation. Although there are still problems to be resolved, the SEA quail could prove to be a valuable tool for anti-atherosclerosis drug discovery.

347

ANIMAL MODEL

REFERENCES 1. 2. 3. 4.

5.

6.

A. D. Ojerio, G. J. Pucak, T. B. Clarkson and B. C. Bullock. Lab. Animal Sci. 22: 33-39, 1972. R. C. Bayer, R. K. Ringer and E. A. Cogger. Poultry Sci. 51: 925-929, 1972. R. L. Smith and D. M. Hilker. Atherosclerosis 17: 63-70, 1973. D. M. Tennent, H. Siegel, G. W. Kuron, W. H. Ott, and C. W. Mushett. Proc. Soc. ExptI. BioI. Med. 96: 679-683, 1957. W. D. Block, K. J. Jarrett, and J. B. Levine. In: Automation in Analytical Chemistry, ed. by L. T. Skeggs, Mediad ITlcorporated, New York, 1966, pp. 345-347. D. M. Kramsch, C. Franzblau and W. Hollander. J. CUn. Invest. 50: 1666-1677, 1971.

New animal model for atherosclerosis research.

Japanese quail were investigated for their utility as a model for the discovery and evaluation of anti-atherosclerosis compounds. Although they posses...
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