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EXPERIMENTAL PRODUCTION OF AORTIC STENOSIS
EXPERIMENTAL P R O D U C T I O N OF AORTIC STENOSIS Anthony
J. Munoz, M.D. (by invitation),
(by invitation),
Nashville,
and Sam E. Stephenson,
Jr., M.D.
Tenn.
T
of lesions simulating acquired human cardiac lesions pre sents a major problem in laboratory animals. Previous work directed at the production of experimental aortic stenosis has utilized the application of a cannula, 1 metal clips,2 or sutures 3 in the commissures. These experiments have afforded unpredictable and inconstant degrees of aortic stenosis. In an attempt to produce coronary arteriosclerosis in the experimental animal it was noted that the first site of plaque formation in the animals was one encircling the aortic annulus and, in some cases, fusion of the commissures developed. This then represents the early lesion of aortic stenosis. HE PRODUCTION
METHOD
Unselected mongrel male dogs, weighing from 11 to 23 kilograms, were utilized. After a baseline electrocardiogram and serum cholesterol levels were obtained, the animals were given 2 millicuries per pound of I 131 to render the dogs euthyroid or hypothyroid. (In a second group of animals [Group B], propylthiouracil, 750 to 1,000 mg. per day, was substituted for the radioactive iodine.) After a 2-week delay the animals were started on 100,000 I.U. of vitamin D daily and placed on a special diet (Table I ) . Fresh meat was added at frequent intervals. TABLE
A.
I*
93 grams 5 grams 2 grams
Commercial dog meal Commercial lard Crystalline cholesterol
B.
73.0 grams/100 Cooking oil 20.0 Cholesterol 5.0 Cholic acid 2.0 Thiouracil ♦Diet A was utilized in combination with IM* and diet B when thiourea derivative was substituted. Prom the S. R. Light Laboratory for Surgical Research, Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tenn. Aided in Part by U. S. Public Health Service Grant 3047. Read a t the Fortieth Annual Meeting of The American Association for Thoracic Surgery a t Miami Beach, Fla., May 11-13, 1960. 568
Vol. 40, No. S November, 1960
AOETIC STENOSIS
569
The vitamin D was continued until the dogs demonstrated the toxic mani festations of weight loss and bleeding from the gums and it was then reduced by one half. The special diet was continued until sacrifice. Monthly serum cholesterol levels were obtained (Table II). At the time of sacrifice some animals had coronary arteriograms and others had left heart catheterization for the gradient across the aortic valve. TABLE
A. I i s i Preop. 1 mo. 2 mo. 3 mo.
II*
+ Diet Avg. Mg./% 154 833 965 1,094
+ Vitamin D
+ + Diet Avg. Mg./% 171 Preop. 159 1 mo. 2 mo. 303 3 mo. 371 638 4 mo. 5 mo. 605 940 6 mo. ♦The group A animals show the more rapid rise in serum cholesterol this method and the lesions were more prominent in this particular group. B. Thiouracil
125-180 450-1,120 620-1,170 775-1,360 Vitamin D Bange 115-235 105-195 225-355 235-490 575-740 500-770 660-1,450 levels achieved with
The excised heart and great vessels were examined, photographed, and preserved in formalin solution. In some cases the entire heart and great vessels were stained with Sudan I I I for localization of fatty deposits not grossly visible. Typical areas of plaque formation were submitted for micro scopic examination. RESULTS
Six animals were prepared, using radioactive iodine, vitamin D, and the high cholesterol diet. All animals were sacrificed at 3 months and all showed marked atherosclerosis encircling the aortic annulus (Fig. 1). In some ani mals there were also marked changes in the pulmonary artery, arch of the aorta, descending aorta, and abdominal aorta. These areas of involvement grossly resembled human plaques, both in their appearance and location. The location of these plaques was primarily on the superior wall of the thoracic aorta, around the orifices of the branch vessels, and on the posterior wall of the abdominal aorta. Unfortunately pressure gradients were not obtained on these animals as the finding of commissure fusion was unexpected. Four additional animals were prepared, but propylthiouracil rather than I 131 was given to suppress thyroid function. Although these animals also responded with a high serum cholesterol level, the findings were not as con sistent and plaque formation not as prominent. These animals were sacrificed at the end of 6 months and demonstrated an average gradient of 49 mm. Hg across the aortic valve.
570
MUNOZ AND S T E P H E N S O N
J. Thoracic and Cardiovas. Surg.
The serum cholesterol values on these animals prepared with radioactive iodine averaged 1,094 mg. % at 3 months. It is possible, however, with this regimen to obtain cholesterol values in excess of 1,800 mg. %. In reviewing these groups of animals it seems that levels in excess of 600 to 800 mg. % need to be maintained for 3 consecutive months and then satisfactory plaque formation will have occurred. It must be stated that the lesions observed in these animals have not progressed as yet to the full blown "pile of rocks" so classically seen in clinical calcific aortic stenosis. These lesions have been early lesions sug gestive of aortic stenosis, both to us and to the pathologist. The gross lesions have appeared as deposits with or without calcification encircling the annulus, fusion of the commissures, and fatty deposition on the leaflets, the latter demonstrated by supravital staining of the gross specimen with Sudan III.
Fig. 1.—Photograph of typical specimen from animal sacrificed a t 3 months. Com missure fusion is not as marked in this specimen as in some but it also demonstrates the lesions in the arch, pulmonary artery, and coronary arteries.
Attempts to demonstrate this degree of aortic stenosis by artériographie means have not been satisfactory. Demonstration of the nicking from plaque formation in the main and branch coronary arteries has been possible by radiographie study prior to sacrifice. Although the gross examination of these specimens resembles the plaques seen in human arteriosclerosis, the microscopic appearance is somewhat differ ent and thus not completely analogous. We would like to reproduce the human counterpart of the disease but these experimental lesions offer a means heretofore unavailable for the study of the early changes of aortic stenosis, coronary sclerosis, and partial occlusion (by plaque formation and narrowing) of the small branch vessels.
Vol. 40, No. S November, 1960
AOBTIC STENOSIS
571 ° ' x
The microscopic picture is primarily one of subintimal hemorrhage and fatty deposits in the early stages. These changes are soon followed by an increase in fibrous tissue, large fatty deposits, and, later, marked calcification of these areas (Fig. 2) with elevation of the intima, progressing in some cases to actual intimai ulcération. For the most part these changes are similar to the lesion in medial sclerosis. DISCUSSION
The early work of Creech and associates4 pointed out the feasibility of the development of arteriosclerotic-like plaques in dogs. Up until that time this lesion had been extremely difficult to produce. Soon after the reported
Fig. 2.—Photomicrograph of lesion at the aortic annulus. This animal had been treated for 90 days. One can see the marked calcification and numerous cholesterol clefts. ( X 2 5 ; reduced %.)
work of Creech, we set up a long-term study, using the Tulane method of producing arteriosclerotic disease, in an attempt to assess mechanical factors concerned in the selective deposition of the material. Through a period of 3 years we have been able to produce some lesions. These, however, have not been of the magnitude expected and the long period of treatment is not only exhausting to the animal and the research group but the cost is exorbitant. It is possible, however, to produce changes in the abdominal aorta by this method and this process is enhanced by mechanical irritation of the vessels.
572
MUNOZ AND STEPHENSON
J. Thoracic and Cardiovas. Surg.
Blake, 5 using a variant of this method, was able to develop similar lesions in dogs. In these studies, coronary lesions developed in somewhat over 24 months of treatment. Crisp 6 in 1958 reported the fairly rapid development of lesions in dogs by another method. Utilizing regular dog chow as a diet the animals were given 100,000 I.U. of vitamin D daily. After about 90 days, lesions in the media of major vessels were evident. The procedure utilized in this report is essentially a combination of these two methods. The vitamin D in such large doses produces multiple subintimal hemorrhages. These areas of injury then predispose to the deposition of the excess lipids in these particular areas. Although the lesions produced by this method are not completely analogous to the human lesions of arteriosclerosis, the sites of deposition are the same. The experimental lesions select, in sites of decreasing frequency, the aortic annulus, the superior wall of the ascending aorta, the pulmonary artery, the lumbar portion of the abdominal aorta, and, last, the coronary arteries. It would be preferable to be able to develop lesions in animals which are microscopically the same as those in human beings. As yet this is not possible. Experiments are now in progress using primates with the same technique in an effort to evaluate this type of experimental animal. Until it is possible to develop the human type lesions, the method herein described appears to represent more closely the human counterpart than the lesions from previously described methods. The utilization of propylthiouracil instead of radioactive iodine, for prep aration of the animals before vitamin D and cholesterol are started, produces similar lesions but these lesions are not as marked, and the degree of fusion of the aortic commissures is inconstant. At the present time we consider that I 131 should be used to produce the more marked results. It must be remem bered, however, that if the animals are kept under observation for longer than 6 months, repeat treatment with I 131 may be necessary. It is hoped that animals prepared over a longer length of time will show a more complete and advanced aortic stenosis. Many other aspects of this over-all problem need to be evaluated and will form the basis of a subsequent report. SUMMARY
1. A method is described to produce arteriosclerotic-like lesions in male dogs. These lesions develop in less than 90 days. 2. Among the earliest lesions are those encircling the aortic annulus and, if permitted to progress, cause fusion of the aortic commissures and the early lesions of aortic stenosis. 3. The use of I131, 2 millicuries per pound of animal weight, to suppress thyroid function is superior to the utilization of antithyroid drugs for the consistent development of the lesions in this particular experiment.
Vol. 40, No. 5 November, 1960
AORTIC STENOSIS
573
REFERENCES 1. Meerson, P . Z., and Kobozev, G. W.: O methodike sozdanica eksperimeiitalnogo stenoza aorty, Bulletin Eksperimental'noi Biologic i meditsiny (Moskow) 39: 50-52, 1955. 2. Salerno, E. A.: The Experimental Production of Valvular Aortic Stenosis, Ann. Surg. 149: 368, 1959. 3. Boerma, J. E., Germs, J., and Blickman, J. E.: Artificial Stenosis of the Aorta, Chirurgicum Neerlandicum 3: 123, 1951. 4. Creech, O. J., J o r d a n , G. L., Jr., De Bakey, M. E., Overton, E. C , and Halpert, B.: The Effect of Chronic Hypercholesterolemia on Canine Aortic Transplants, Surg. Gynec. & Obst. 101: 607, 1955. 5. Blake, T. M. : Personal communication, 1960. 6. Crisp, W. W., Jr., Eoot, H. D., Jenson, C. B., Imamagler, K., and Yonehiro, E. G. : Experimental Production of Arteriosclerosis in the Dog, S. Eorum 9: 304, 1958. (For Discussion,
see page 631)
J. Munoz, M.D. (by invitation),
(by invitation),
Nashville,
and Sam E. Stephenson,
Jr., M.D.
Tenn.
T
of lesions simulating acquired human cardiac lesions pre sents a major problem in laboratory animals. Previous work directed at the production of experimental aortic stenosis has utilized the application of a cannula, 1 metal clips,2 or sutures 3 in the commissures. These experiments have afforded unpredictable and inconstant degrees of aortic stenosis. In an attempt to produce coronary arteriosclerosis in the experimental animal it was noted that the first site of plaque formation in the animals was one encircling the aortic annulus and, in some cases, fusion of the commissures developed. This then represents the early lesion of aortic stenosis. HE PRODUCTION
METHOD
Unselected mongrel male dogs, weighing from 11 to 23 kilograms, were utilized. After a baseline electrocardiogram and serum cholesterol levels were obtained, the animals were given 2 millicuries per pound of I 131 to render the dogs euthyroid or hypothyroid. (In a second group of animals [Group B], propylthiouracil, 750 to 1,000 mg. per day, was substituted for the radioactive iodine.) After a 2-week delay the animals were started on 100,000 I.U. of vitamin D daily and placed on a special diet (Table I ) . Fresh meat was added at frequent intervals. TABLE
A.
I*
93 grams 5 grams 2 grams
Commercial dog meal Commercial lard Crystalline cholesterol
B.
73.0 grams/100 Cooking oil 20.0 Cholesterol 5.0 Cholic acid 2.0 Thiouracil ♦Diet A was utilized in combination with IM* and diet B when thiourea derivative was substituted. Prom the S. R. Light Laboratory for Surgical Research, Department of Surgery, Vanderbilt University School of Medicine, Nashville, Tenn. Aided in Part by U. S. Public Health Service Grant 3047. Read a t the Fortieth Annual Meeting of The American Association for Thoracic Surgery a t Miami Beach, Fla., May 11-13, 1960. 568
Vol. 40, No. S November, 1960
AOETIC STENOSIS
569
The vitamin D was continued until the dogs demonstrated the toxic mani festations of weight loss and bleeding from the gums and it was then reduced by one half. The special diet was continued until sacrifice. Monthly serum cholesterol levels were obtained (Table II). At the time of sacrifice some animals had coronary arteriograms and others had left heart catheterization for the gradient across the aortic valve. TABLE
A. I i s i Preop. 1 mo. 2 mo. 3 mo.
II*
+ Diet Avg. Mg./% 154 833 965 1,094
+ Vitamin D
+ + Diet Avg. Mg./% 171 Preop. 159 1 mo. 2 mo. 303 3 mo. 371 638 4 mo. 5 mo. 605 940 6 mo. ♦The group A animals show the more rapid rise in serum cholesterol this method and the lesions were more prominent in this particular group. B. Thiouracil
125-180 450-1,120 620-1,170 775-1,360 Vitamin D Bange 115-235 105-195 225-355 235-490 575-740 500-770 660-1,450 levels achieved with
The excised heart and great vessels were examined, photographed, and preserved in formalin solution. In some cases the entire heart and great vessels were stained with Sudan I I I for localization of fatty deposits not grossly visible. Typical areas of plaque formation were submitted for micro scopic examination. RESULTS
Six animals were prepared, using radioactive iodine, vitamin D, and the high cholesterol diet. All animals were sacrificed at 3 months and all showed marked atherosclerosis encircling the aortic annulus (Fig. 1). In some ani mals there were also marked changes in the pulmonary artery, arch of the aorta, descending aorta, and abdominal aorta. These areas of involvement grossly resembled human plaques, both in their appearance and location. The location of these plaques was primarily on the superior wall of the thoracic aorta, around the orifices of the branch vessels, and on the posterior wall of the abdominal aorta. Unfortunately pressure gradients were not obtained on these animals as the finding of commissure fusion was unexpected. Four additional animals were prepared, but propylthiouracil rather than I 131 was given to suppress thyroid function. Although these animals also responded with a high serum cholesterol level, the findings were not as con sistent and plaque formation not as prominent. These animals were sacrificed at the end of 6 months and demonstrated an average gradient of 49 mm. Hg across the aortic valve.
570
MUNOZ AND S T E P H E N S O N
J. Thoracic and Cardiovas. Surg.
The serum cholesterol values on these animals prepared with radioactive iodine averaged 1,094 mg. % at 3 months. It is possible, however, with this regimen to obtain cholesterol values in excess of 1,800 mg. %. In reviewing these groups of animals it seems that levels in excess of 600 to 800 mg. % need to be maintained for 3 consecutive months and then satisfactory plaque formation will have occurred. It must be stated that the lesions observed in these animals have not progressed as yet to the full blown "pile of rocks" so classically seen in clinical calcific aortic stenosis. These lesions have been early lesions sug gestive of aortic stenosis, both to us and to the pathologist. The gross lesions have appeared as deposits with or without calcification encircling the annulus, fusion of the commissures, and fatty deposition on the leaflets, the latter demonstrated by supravital staining of the gross specimen with Sudan III.
Fig. 1.—Photograph of typical specimen from animal sacrificed a t 3 months. Com missure fusion is not as marked in this specimen as in some but it also demonstrates the lesions in the arch, pulmonary artery, and coronary arteries.
Attempts to demonstrate this degree of aortic stenosis by artériographie means have not been satisfactory. Demonstration of the nicking from plaque formation in the main and branch coronary arteries has been possible by radiographie study prior to sacrifice. Although the gross examination of these specimens resembles the plaques seen in human arteriosclerosis, the microscopic appearance is somewhat differ ent and thus not completely analogous. We would like to reproduce the human counterpart of the disease but these experimental lesions offer a means heretofore unavailable for the study of the early changes of aortic stenosis, coronary sclerosis, and partial occlusion (by plaque formation and narrowing) of the small branch vessels.
Vol. 40, No. S November, 1960
AOBTIC STENOSIS
571 ° ' x
The microscopic picture is primarily one of subintimal hemorrhage and fatty deposits in the early stages. These changes are soon followed by an increase in fibrous tissue, large fatty deposits, and, later, marked calcification of these areas (Fig. 2) with elevation of the intima, progressing in some cases to actual intimai ulcération. For the most part these changes are similar to the lesion in medial sclerosis. DISCUSSION
The early work of Creech and associates4 pointed out the feasibility of the development of arteriosclerotic-like plaques in dogs. Up until that time this lesion had been extremely difficult to produce. Soon after the reported
Fig. 2.—Photomicrograph of lesion at the aortic annulus. This animal had been treated for 90 days. One can see the marked calcification and numerous cholesterol clefts. ( X 2 5 ; reduced %.)
work of Creech, we set up a long-term study, using the Tulane method of producing arteriosclerotic disease, in an attempt to assess mechanical factors concerned in the selective deposition of the material. Through a period of 3 years we have been able to produce some lesions. These, however, have not been of the magnitude expected and the long period of treatment is not only exhausting to the animal and the research group but the cost is exorbitant. It is possible, however, to produce changes in the abdominal aorta by this method and this process is enhanced by mechanical irritation of the vessels.
572
MUNOZ AND STEPHENSON
J. Thoracic and Cardiovas. Surg.
Blake, 5 using a variant of this method, was able to develop similar lesions in dogs. In these studies, coronary lesions developed in somewhat over 24 months of treatment. Crisp 6 in 1958 reported the fairly rapid development of lesions in dogs by another method. Utilizing regular dog chow as a diet the animals were given 100,000 I.U. of vitamin D daily. After about 90 days, lesions in the media of major vessels were evident. The procedure utilized in this report is essentially a combination of these two methods. The vitamin D in such large doses produces multiple subintimal hemorrhages. These areas of injury then predispose to the deposition of the excess lipids in these particular areas. Although the lesions produced by this method are not completely analogous to the human lesions of arteriosclerosis, the sites of deposition are the same. The experimental lesions select, in sites of decreasing frequency, the aortic annulus, the superior wall of the ascending aorta, the pulmonary artery, the lumbar portion of the abdominal aorta, and, last, the coronary arteries. It would be preferable to be able to develop lesions in animals which are microscopically the same as those in human beings. As yet this is not possible. Experiments are now in progress using primates with the same technique in an effort to evaluate this type of experimental animal. Until it is possible to develop the human type lesions, the method herein described appears to represent more closely the human counterpart than the lesions from previously described methods. The utilization of propylthiouracil instead of radioactive iodine, for prep aration of the animals before vitamin D and cholesterol are started, produces similar lesions but these lesions are not as marked, and the degree of fusion of the aortic commissures is inconstant. At the present time we consider that I 131 should be used to produce the more marked results. It must be remem bered, however, that if the animals are kept under observation for longer than 6 months, repeat treatment with I 131 may be necessary. It is hoped that animals prepared over a longer length of time will show a more complete and advanced aortic stenosis. Many other aspects of this over-all problem need to be evaluated and will form the basis of a subsequent report. SUMMARY
1. A method is described to produce arteriosclerotic-like lesions in male dogs. These lesions develop in less than 90 days. 2. Among the earliest lesions are those encircling the aortic annulus and, if permitted to progress, cause fusion of the aortic commissures and the early lesions of aortic stenosis. 3. The use of I131, 2 millicuries per pound of animal weight, to suppress thyroid function is superior to the utilization of antithyroid drugs for the consistent development of the lesions in this particular experiment.
Vol. 40, No. 5 November, 1960
AORTIC STENOSIS
573
REFERENCES 1. Meerson, P . Z., and Kobozev, G. W.: O methodike sozdanica eksperimeiitalnogo stenoza aorty, Bulletin Eksperimental'noi Biologic i meditsiny (Moskow) 39: 50-52, 1955. 2. Salerno, E. A.: The Experimental Production of Valvular Aortic Stenosis, Ann. Surg. 149: 368, 1959. 3. Boerma, J. E., Germs, J., and Blickman, J. E.: Artificial Stenosis of the Aorta, Chirurgicum Neerlandicum 3: 123, 1951. 4. Creech, O. J., J o r d a n , G. L., Jr., De Bakey, M. E., Overton, E. C , and Halpert, B.: The Effect of Chronic Hypercholesterolemia on Canine Aortic Transplants, Surg. Gynec. & Obst. 101: 607, 1955. 5. Blake, T. M. : Personal communication, 1960. 6. Crisp, W. W., Jr., Eoot, H. D., Jenson, C. B., Imamagler, K., and Yonehiro, E. G. : Experimental Production of Arteriosclerosis in the Dog, S. Eorum 9: 304, 1958. (For Discussion,
see page 631)