Myocardial Infarction in Rats Fed Diets Containing High Fat, Cholesterol, Thiouracil, and Sodium Cholate

Transcription

1 Myocardial Infarction in Rats Fed Diets Containing High Fat, Cholesterol, Thiouracil, and Sodium Cholate By WILBUR A. THOMAS, M.D., AND W. STANLEY HARTROFT, M.D., PH.D. Investigators have produced arteriosclerosis in experimental animals by dietary means for almost half a century. In contrast to the situation in man, however, all previous reports indicate that complicating thrombosis and infarction have been extremely rare in experimental animals. The current report presents dietary regimens by means of which the authors have produced significant numbers of myocardial and renal infarets in rats (6 of 1 in one group and 4 of 1 in each of 2 others). It is notable that thrombosis occurred before the appearance of severe structural changes in the arterial walls, although abundant fat could often be demonstrated histochemically. FOR half a century experimental pathologists have known that arterial damage followed the feeding of cholesterol to rabbits. Within the last decade similar lesions have been produced in rats by a variety of methods, all more complex than that which suffices for the rabbit. A few reports1-3 of isolated examples of myocardial infarction developing in animals subjected to these procedures have recently appeared. But we are unaware of any established method for the dietary production of myocardial infarction in significant percentages of experimental animals. The foregoing suggests that factors responsible for atheromatous changes in both experimental animals and man, may not be identical with those responsible for the complications of the disease-coronary arterial thrombosis and myocardial infarction.4 For several years we have investigated factors influencing the clotting mechanism, particularly those concerned with Clot lysis.5-8 Also, we have emphasized9 the need for an experimental model for studying mechanisms not only for changes in arterial walls but also occlusive thromboses. With this object, an attempt was made to combine a number of conditions which together might produce arterial damage and occlusion with infarction. Downloaded from by on October 5, 218 From the Department of Pathology, Washington University School of Medicine, St. Louis, Mo. These investigations were supported by U.S. Public Health Grant No. H-182. Presented in part at the A.M.A. Symposium on Fats in Human Nutrition, New Orleans, La., March 15, 1957 (J.A.M.A.). 65 Although the experiments were not adequately controlled in all respects, results sufficiently approximate the objective to report them here. METHODS Seventy male rats (Wistar albinos) initially weighing 12 Gm. were placed in 7 groups. They were housed in individual, wire-bottom cages and offered water ad libitum. All were weighed twice weekly (table 1). Each day food not consumed was weighed and discarded, and measured portions of fresh diet were placed in sterilized glass containers. Records of daily food consumption of each animal were thus obtained. Pair feeding on either a food-weight or body-weight basis was not carried out, all rats being offered more than they would eat. Animals that died were refrigerated until autopsied, usually less than 12 hours after death. After 18 weeks, all survivors were killed under ether anesthesia and autopsied. Diets. Seven semisynthetic diets were prepared according to table 2. Dry ingredients were thoroughly blended in a Hobart food mixer. Various supplements as listed were mixed in a mortar with small portions of dry ingredients and blended with the remainder in the mixer. Fats were added last; and if solid, they were warmed at room temperature until liquid. Diets were kept in tightly stoppered plastic containers and refrigerated at 4 C. for no longer than 2 weeks. Under these conditions, even high-fat diets did not become rancid. Microscopic Studies. Sections of major viscera and vessels were placed in either cobalt-formalin' (1 to 2 weeks) or Bouin solutiont (24 hours). *Cobalt nitrate, 1 Gm.; calcium chloride, 1 Gm.; concentrated formaldehyde (37 per cent), 1 ml.; distilled water, 9 ml. tsaturated aqueous picric acid, 75 ml.; concentrated formaldehyde (37 per cent), 25 ml.; acetic acid (glacial), 5 ml. Circulation, Volume XIX, January, 1959

2 Downloaded from by on October 5, TABLE 1.-Average Weights of Rats at Biweekly Intervals* Week Group No I II III IV V VI VII *All rats were weighed twice each week, but the biweekly weight record 1above provides a general view of the weight curves. Hearts, kidneys, and aortas (fixed in formalin) were sectioned with a freezing microtome a nd stained to demonstrate abnormal deposits of fat by Wilson's modification of Lillie's supersaturated isopropanol oil red technic.1 Selected paraffinembedded blocks of hearts and kidneys were seetioned serially and stained by a combined aldehyde-fuchsin-van Giesen technic for demonstration of elastic tissue and collagen. Other special stains were employed as needed, including those demonstrating ceroid and iron. Levels of cholesterol in the plasma of most animals that survived until the end of the experiment were measured by the method of Pearson, Stern, and MeGavack"1 and are reported in table 3. RESULTS Within the first 3 or 4 weeks most animals lost weight (table 1) and several died (table 3) presumably from thiouracil intoxication. Three rats (1 each from groups I, I1, and V) lost most of the hair from their backs and flanks, suggesting an essential fatty acid deficiency state. Only one of these had a myocardial infarct. The total number of rats that developed cardiac and renal infarcts or both are listed in table 3. Other findings at autopsy included fatty livers, bronchopneumonia and, in 2 cases, pyelonephritis with multiple abeesses in liver and mediastinum. Hemorrhagic renal necrosis12 was responsible for early deaths in animals of group VI (high fat, low protein, low choline). Cardiac Infarcts. When present, cardiac infarcts were grossly visible on anterior aspects and apices of left ventricles. They were yellow or white and sometimes outlined THOMAS, HARTROFT by hyperemic borders between normal and damaged muscle. Infareted areas were thin and frequently covered by attached mural thrombi that sometimes nearly filled the entire left ventricular chambers (fig. 1). Dissections of coronary arteries of these tiny hearts were not attempted lest recent thrombi be dislodged and lost. ReWal Infarcts. They were easily seen in situ and were yellow or yellow-white areas situated on lateral margins, extending anteriorly and posteriorly toward hilar regions, sometimes involving entire poles. On cut surfaces they were classically wedge-shaped with apices toward the hilum and bases at outer surfaces. Splenic infarets were not found in any animal. Brains were not examined. Fatty livers were regularly encountered. Microscopic Studies. Cardiac infarets found in these rats were morphologic duplicates of those regularly encountered in man (fig. 2). Characteristic subepicardial layers of muscle in infarcted regions are often normal. Junctions between dead and normal muscle were sharply defined by varying degrees of infiltration by inflammatory cells (fig. 3). Mural thrombi were invaded by endothelial cells, mononuclear cells, and young fibroblasts. Extent of organization of intraventricular thrombi was variable but always sufficient to indicate clearly their antemortem character (fig. 4). Deposits of neutral fat and cholesterol were abundant in subendocardial positions. Fatty degeneration was evident throughout all portions of infarets (fig. 5). In frozen sections, abnormal deposition of large amounts of stainable fat was often present throughout all layers of walls of coronary arteries (fig. 6). Deposition of abnormal lipid was demonstrable in almost every section of arteries in rats of groups, V, VI, and VII, and to lesser degrees in other groups. Fatty deposits in intimal, subintimal, arid inner medial zones of aortas paralleled in extent those in coronary arteries. Mural plaques, fibrous intimnal thickenhig, medial fibrosis or neerois were not demonstrated in any arteries of rats. Absence of structural

3 MYOCARDIAL INFARCTION IN RATS (as distinguished from histochemical) changes deserves emphasis. Serial sections were made of hearts in which infarets were discovered; in some we could demonstrate short segments (.2 to.5 mm.) of coronary arteries occluded by antemortem thrombi. They were composed of masses of disintegrated red cells, fibrin, and platelets invaded by large plump cells (of endothelial origin?) exhibiting many characteristics of young fibroblasts (fig. 7). At sites of thromboses vessel walls were not abnormal but for extensive deposits of stainable fat as previously described. Sections of renal infarets closely resemble those of man. Coagulative necrosis (fig. 8) of tubules and glomeruli left only typical "ghost" outlines of their originals. At zones of demarcation between dead and normal tissue, congestion and inflammatory cell infiltration (neutrophils) were present. Occlusive thrombi were found in major branches of renal arteries that supplied some of the infareted regions. But in these sites structural alterations again were not evidenced and the only abnormality was deposition of stainable fat (fig. 9). Livers contained extensive deposits of stainable fat including cholesterol. In those rats that died early in the experiment, lipid distribution was periportal, but in those that survived longer, every portion of every lobule contained fat. In animals killed at the end of 14 weeks, early periportal fibrosis was present Ṫhyroid glands had undergone typical hy - perplasia with papillary formation of epithelium and increased cellularity characteristic of thiouracil administration. Other organs, including adrenal glands and spleens, were unremarkable (except for excess lipid). Downloaded from by on October 5, 218 DISCUSSION The factors responsible for myocardial infarction in rats were not apparent from the experimental design employed. It is not even clear why cardiac and renal infarcts developed at all, in view of reports of similar experiments, previously conducted by others, in TABLE 2.-Ingqredients in Diets (Percentage by Weight) Group No. I II Ill IV V VI VII Casein ' Alpha soya protein Sucrose Salt mix* Alphancel Vitamin mixt Propylthiouracil Choline chloride Sodium cholate Corn oil Butter Lard Crisco Cholesterol Totals *This salt mixture is the Wessoii modification of the Osborne and Mendel salt niixture.13 teach kilogram of the vitamin mixture contained the following triturated in dextrose: Vitamin A concentrate (2,(iunits/Gim.)... Vitamin D concentrate (4, unlits/gimi.).. Alpha tocopherol... Ascorbic acid... Illositol... Menadione... acid... Niacin..... Riboflavin... Pyridoxine (hydrochloride)... Thiamine (hydrochloride)... Calcium panthothenate... Biotin... Folic acid... P aiminobenzoie Gm which only pathologic changes in arterial walls were found and no occlusion by thrombi. Protein levels in the diets were not significant as infarcts developed in as many rats in groups fed food mixtures containing 2 per cent protein as those fed diets containing 12 per cent. Variations in carbohydrate content of the 7 diets cannot be correlated with results. Of probable significance were quantity and type of fat, and supplements of choline, thiouracil, cholesterol, and sodium cholate. 6)7

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7 MYOCARDIAL INFARCTION IN RATS The greatest number of infarcts were developed in animals fed large quantities of either butter or lard along with the cholesterol-elevating regimen (sodium cholate, etc.). But an equally high level of less saturated fat (2 per cent corn oil and 2 per cent Crisco) did not have the same effect and may even have afforded protection against the thiouracil, cholesterol, and bile salt. Thus, high levels of butter or lard potentiated the tendency of the diets to produce infarcts. The effects of lipotropic supplements is less clear. Although only.2 per cent choline was added to the diet of group V (high in butter) in which almost as many infarets developed as in group VI (high in lard) given 1 per cent choline, the total available lipotropic content of the 2 diets was probably comparable. Adding.2 per cent choline to a diet containing 2 per cent casein makes its lipotropic activity not less than that of a diet containing only 6 per cent casein and 6 per cent soya protein supplemented with 1 per cent choline. Thus it appears from the results with groups V and VI that the lipotropic supplements did not protect the animals from myoeardial injury and may even have potentiated Downloaded from by on October 5, 218 development of lesions in some manner. The supplement of choline chloride was varied in groups I, II, III (all receiving 1 per cent corn oil as their only fat) from. to.2 to 1.. Four of the 3 rats in these groups developed infarcts; all were in group III receiving a 1. per cent choline supplement. However, the numbers are probably insufficient to warrant further conclusions regarding the effect of lipotropic substances. Essential fatty-acid deficiency may be a factor in the production of myocardial infarction in our rats. The fact that a few rats developed signs strongly suggesting essential fatty-acid deficiency may indicate that others were deficient to a lesser degree. We are further investigating this aspect in other experiments, but at present no conclusions are warranted. Experiments are in progress for attaining a clearer evaluation of all factors. Until their results are available, the only conclusion perniissible is that adequate levels of protein, TABLE 3.-Incidence of Myocardial and Renal Infarcts I II III IV V VI VII c4n > > + z :, z = W 1225(7) 83 (4) 243(5) 2 98 (3) 336 (2) 4 _- 3-1 Totals w 4(4%) 6(6%/c ) 4 (4%clo ) 1(1% ) *Initially, each group contained 1 male rats. tall surviving rats were killed after 131 days oa specified diets. tcholesterol levels in the plasma were determined at the end of 131 days on the number of rats indicated in parentheses. minerals, and vitamins including choline, plus generous amounts of saturated fats of animal origin incorporated in diets containing thiouracil, cholesterol, and cholic acid, will, when fed to rats, produce in many of them myocardial infarets. The absence of structural changes in arterial walls at sites of thrombosis is of considerable significance. It reinforces our earlier suspicions that arterial plaques may not be essential in the pathogenesis of coronary occlusion, although their formation may play a part in determining sites of intravascular clotting and may enhance their formation. Experiments previously reported by us5-8 establish that butter( and probably other saturated fats of animal origin) may affect profoundly the fibrinolytic properties of blood in vivo and in vitro. In the experiments reported herein, both coagulation and fibrinolysis of the animals' blood may have been altered significantly by the various dietary regimens, so that formation and persistence of intravascular clots were favored. The method outlined may prove useful in studying, in experimental animals, the problem of prevention or treatment of heart disease in man. The resulting investigations 71

8 Downloaded from by on October 5, may provide at a later date information of more practical value, but at the present time our results have no application to this major problem. SUMMARY Cardiac and renal infarcts resulting from intravascular thromboses in fatty, but otherwise normal, arteries occurred in as many as 6 per cent of rats fed various dietary regimens in groups of 1. The infarets closely resembled cardiac and renal infarets in man. Several dietary factors were employed and inadequately controlled in these experiments, so that evaluation of their roles in production of myoeardial infaretion is difficult. But diets containing thiouracil, cholesterol, cholic acid, and 4 per cent butter or lard when fed to rats produced infarets within 4 to 14 weeks. The method should prove useful for further study of diet in relation to cardiovascular disease. SUMMARIO IN INTERLINGUA Infaretos cardiac e renal resultante ab thromboses intravaseular in grassiose sed alteremente normal arterias occurreva in usque a 6 pro cento del rattos in gruppos decadie mantenite a varie regimes dietari. Le infarctos resimilava fortemente infaretos cardiac e renal in humanos. Plure factores dietari esseva introdueite in iste experimentos. Lor regulation esseva inadequate de maniera que le evaluation de lor rolo in le production de infarcimento mnocardial es difficile. Sed dietas contininte thiouracil, cholesterol, acido cholic, e 4 pro cento butyro o grassia de poreo produceva infaretos in le rattos intra inter 4 e 14 septimanas. I.e methodo promitte esser de utilitate ini studios additional del dieta in su relation a morbo cardiovascular. THOMAS, HARTROFT REFERENCES 1. FILLIos, L. C., ANDRUS, S. B., MANN, G. V., AND STARE, F. J.: Experimental production of gross atheroselerosis in the rat. J. Exper. Med. 14: 539, WISSLER, R. W., EILERT, M. L., SCHROEDER, M. A., AND COHEN, L.: Production of lipomatous and atheromatous arterial lesions in albino rat. A.M.A. Arch. Path. 57: WILGRAM, G. F., Calcified atheromnatosis in the rat. Fed. Proc. 16: 27, HARTROFT, W. S., AND THOMAS, W. A.: Pathological lesions related to disturbances of falt and cholesterol metabolism in man. J.A.M.A. 164: 1899, THOMAS, W. A., O'NEAL, R. M., AND LEE, K. T.: Thromboembolism, pulmonary arteriosclerosis and fatty meals. A.M.A. Arch. Path. 61: 38, ,,-, AND KoNIKOV, N.: Saturated versus unsaturated fats in experimental arteriosclerosis, A.M.A. Arch. Path. 63: 571, RABIN, R. E., THOMAS, W. A., LEE, K. T., KoNIKOV, N., AND SCOTT, R. F.: Thromboembolism and experimental systemic arteriosclerosis. A.M.A. Arch. Path. 64: 75, SCOTT, R. F., AND THOMAS, W. A.: Methods for comparising effects of various fats on fibrinolysis. Proc. Soc. Exper. Biol. & Med. 96: 24, HARTROFT, W. S.: Pathogenesis of atherosclerosis. Proc. Food and Nutrition Board, Nat. Res. Council 16: 52, WILSON, W.: A trichrome method for staining fat with oil red in frozen sections. Internat. A. M. Museums Bull. 31: 216, PEARSON, S., STERN, S., AND MCGAVACK, T. H.: Determination of total cholesterol in serum. Anal. Chem. 25: 813, HARTROFT, W. S.: Pathogenesis of renal lesions in weanling rats and young adult rats fed choline-deficient diets. Brit. J. Exper. Path. 29: 483, OSBORNE, T. B., AND MENDEL, L. B.: Scientific apparatus and laboratory methods. Science 75: 339,