”iri'" .. . —_ m... .‘- 7 M f ‘83". f I"; -:::¢‘.:::~‘,¢x$é ‘ eff"? i%?%?.‘*‘”" ‘ V THE INFLUENCE or mm mm A on ammsw m MPANESE QUAIL Thesis for the Degree of Pa D. MICHIGAN STATEUNWERSWY ROBERT CLARK BAYER A 7‘ 1 1972 ‘ r vvvv :1 .- ‘ ..'.- .......................... N: ' LIBRARY pHE$.r Michigan State University my tzwtrgfij \ a ABSTRACT THE INFLUENCE OF DIETARY VITAMIN A ON ATHEROGENESIS IN JAPANESE QUAIL BY Robert Clark Bayer Experiments were conducted to study the influence of vitamin A on atherosclerosis. In one experiment quail chicks (Coturnix coturnix japonica) were hatched and imme- diately placed on diets deficient in vitamin A (880 U.S.P. units/kg. of feed), normal in vitamin A (4,400 U.S.P. units/ kg. of feed) or excessive in vitamin A (22,000 or 220,000 U.S.P. units/kg. of feed). In another experiment, mature male quail were placed on the same diets as in the preceding experiment. At the end of 10 weeks on experimental rations, blood pressure was measured by cannulation of the carotid artery. A scoring system was devised to quantitate aortic lipid. The results indicated that birds fed low levels of vitamin A had the highest incidence of coronary atheroscle- rosis and the most severe aortic atherosclerosis. It was determined that a minimum of approximately 15,000 U.S.P. units of the vitamin were needed to minimize atherosclerosis in the thoracic aorta in growing quail chicks. Blood pres- sure and heart rate and plasma cholesterol were not signifi- <:ant1y altered by varying vitamin A in the diet. w ‘F_ [a that adeq: few 1 cells proje intac minim that was me min A baseme mine P endoth endoth the bi. CYtic ; dothel: degenex Examination under the electron microscope revealed that the endothelium from the aortas of birds fed a ration adequate in vitamin A was generally closely adherent with few lipid vacuoles. By comparison, the aortic endothelial cells from birds deficient in the vitamin were detached and projecting into the lumen of the vessel. The percent of intact endothelial cells was negatively correlated with minimal aortic lipid infiltration. A modification of a silver methenamine electron stain, that is specific for mucopolysaccharides and mucoprotein, was made. Observations using this stain revealed that vita- min A deficiency resulted in apparent broken or absent basement membranes and increased numbers of silver methena- mine positive particles in the endothelium. Myocardial capillary ultrastructure was studied. The endothelium of the vitamin A deficient animals had numerous endothelial phagocytic flaps, which were seldom seen in the birds fed sufficient vitamin A. The presence of phago- cytic flaps is an indication of overload of the reticuloen- dothelial system, probably as the result of liver phagocyte degeneration from vitamin A deficiency. THE INFLUENCE OF DIETARY VITAMIN A ON ATHEROGENESIS IN JAPANESE QUAIL BY Robert Clark Bayer A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Poultry Science 1972 for 9: this I Theo C during author and ”f during Pittma: Asher, and Ms Howard for gin UniVers C troh mi F Clarenc doCtOra MEghan' '\ ACKNOWLEDGMENTS The author is most grateful to Dr. Robert K. Ringer for guidance during his graduate program and direction of this research project. The assistance and advice of Dr. Theo Coleman in the preparation of this manuscript and during the graduate program was certainly appreciated. The author would like to thank Dr. Donald Polin for his counsel and "feedback" on some of the nutritional problems encountered during the course of research. The assistance of Dr. Robert Pittman, Mr. Sulo Hulkonen, Mr. Edward Cogger, Ms. Susan Asher, Ms. June Mack, Mr. Robert Glick, Ms. Joyce Ingalls and Ms. Virginia Ross was greatly appreciated. The author wishes to express his appreciation to Dr. Howard C. Zindel, Chairman of the Poultry Science Department, for giving him the opportunity to study at Michigan State University. Dr. Gordon C. Spink's interest and the use of his elec- tron microsc0pe facility was gratefully appreciated. Finally, the author wishes to thank: his grandfather, Clarence F. Bayer, for financial assistance during his doctoral program, and his wife, Juanita, and daughter, Meghan, for their moral support. Acknowledgments . . Table of Contents . List of Tables . . List of Figures . . Introduction . . . Literature Review . TABLE OF Mechanical Factors Turbulence . Hypertension Lipid Role in Atherosclerosis Atherosclerosis and Mucopolysaccharides Vitamin A and Mucopolysaccharide Synthesis Vitamin A and Cell Membranes Vitamin A and Cholesterol Metabolism Vitamin A and Cholesterol Absorption in Atherosclerosis CONTENTS Vitamin A and Cholesterol Synthesis . The Influence of Vitamin A on Atherosclerosis Spontaneous Atherosclerosis in the Aorta and Coronary Arteries of Domestic Birds Spontaneous Atherosclerosis in Wild Birds Function of Vitamin A in Cytodifferentiation . Influence of Vitamin A on Gut and Its Epithelium iii Page ii iii vi vii 10 11 ll l3 13 15 Resu E) Influence of Behavior on Birds 0 O O O O O C O 0 Objectives . . . . . . . . Experimental Procedure . . Experiment One . . . . . Experiment Two . . . . . Experiment Three . . . . Experiment Four . . . . Experiment Five . . . . Experiment Six . . . . . Experiment Seven . . . . Experiment Eight . . . . Results . . . . . . . . . . Experiment One . . . . . Experiment Two . . . . . Experiment Three . . . . Experiment Four . . . . Experiment Five . . . . Experiment Six . . . . . Experiment Seven . . . . Experiment Eight . . . . Discussion . . . . . . . . Coronary Atherosclerosis Aortic Atherosclerosis . Blood Pressure . . . . . summary 0 O O C O O O O O O Atherosclerosis iv in Page 16 18 19 19 22 23 24 28 29 31 32 33 33 43 52 56 68 79 82 82 95 95 98 105 106 Concl Sugge Liter Appen= Page Conclusions . . . . . . . . . . . . . . . . . . . . . 108 Suggestions for Future Study . . . . . . . . . . . . . 110 Literature Cited . . . . . . . . . . . . . . . . . . . 111 Appendices . . . . . . . . . . . . . . . . . . . . . . 120 Table Table l. 2. LIST OF TABLES Basal ration . . . . . . . . . . . . . . . . The effect of dietary vitamin A on the percent male Japanese quail exhibiting coronary atherosclerosis . . . . . . . . . . The effect of dietary vitamin A on aorta score in male Japanese quail . . . . . . . . The effect of dietary vitamin A on blood pressure in male Japanese quail . . . . . . The effect of dietary vitamin A on heart rate in male Japanese quail . . . . . . . . The effect of dietary vitamin A on aorta score, intact endothelial cells and quantity of silver methenamine positive material . . vi Page 20 40 42 44 45 81 Figure l. 10. ll. 12. 13. LIST OF FIGURES Photograph of a Japanese quail showing signs of vitamin A deficiency . . . . . Photomicrograph of a coronary artery from a vitamin A deficient Japanese quail . Photomicrograph of a coronary artery from a vitamin A deficient Japanese quail . Photomicrograph of a coronary artery from a vitamin A deficient Japanese quail . Graph of the linear regression of aorta score on log vitamin A concentration in experiment one . . . . . . . . . . . . Graph of the linear regression of aorta score on log vitamin A concentration in experiment two . . . . . . . . . . . . Graph of the linear regression of aorta score on log vitamin A concentration in experiment three . . . . . . . . Electron micrograph of aorta from Japanese quail . . . . . . . . . Electron micrograph of aorta from Japanese quail . . . . . . . . . Electron micrograph from aorta of A deficient Japanese quail . . . Electron micrograph from aorta of A deficient Japanese quail . . . Electron micrograph from aorta of A deficient Japanese quail . . . Electron micrograph from aorta of A deficient Japanese quail . . . vii a normal normal vitamin vitamin vitamin vitamin Page 27 35 37 39 47 51 55 58 60 63 65 67 70 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Electron micrograph of aorta from a Japanese quail with hypervitaminosis A . . . . . . . . Electron micrograph of aorta from a normal Japanese quail . . . . . . . . . . . . . . . . Electron micrograph of aorta from a normal Japanese quail . . . . . . . . . . . . . . . . Electron micrograph of aorta from a vitamin A deficient Japanese quail . . . . . . . . . . Electron micrograph of myocardial capillary from a normal Japanese quail . . . . . . . . . Electron micrograph of a myocardial capillary from a normal Japanese quail . . . . . . . . . Electron micrograph of myocardial capillary endothelium from a vitamin A deficient Japanese quail . . . . . . . . . . . . . . . . Electron micrograph of myocardial capillary from a vitamin A deficient Japanese quail . . Electron micrograph of myocardial capillary from a vitamin A deficient Japanese quail . . Electron micrograph of myocardial capillary from a vitamin A deficient Japanese quail . . Diagram demonstrating the probable origin of intimal thickening . . . . . . . . . . . . Graph of the regression line of aorta score and the vitamin A concentration used to pre- dict the amount of vitamin A required for minimal atherosclerosis . . . . . . . . . . . viii Page 72 74 76 78 84 86 88 90 92 94 97 101 INTRODUCT I ON Nutrition is one of many factors that influences athero- genesis. Vitamin A is an essential nutrient for growth and development, that plays a role in many physiological func- tions including vision, lipid metabolism and mucopolysac- charide synthesis. Either extremely high or extremely low levels of vitamin A in the diet tend to cause morphological and physiological changes in the animal body. Vitamin A in large doses may cause mucous metaplasia in squamous epithelium, skin lesions, oversecretion of the lacrimal glands, plus other physiological aberrations. Deficiencies of vitamin A may result in weak animals with scaled skin. The epithelium tends to keratinize and there may be nerve tissue degeneration. At the cellular level vitamin A affects the brush border of the cells of the small intestine and is necessary for maintenance of most cell membranes. Atherosclerosis, a disease that involves thickening of the intima and lipid infiltration of the vascular wall, is present throughout much of the animal kingdom. Changes in lipid metabolism and intercellular mucopolysaccharide have been associated with the development of atherosclerosis. Variation in vitamin A status, such as in the vitamin A deficient state, results in metabolic and permeability changes that could be conducive to atherogenesis. The present study was designed to investigate the effects of vitamin A intake on resistance to spontaneous atherosclerosis in the Japanese quail, a bird which is subject to spontaneous atherosclerosis in the thoracic aorta and coronary arteries. ta r2 ges Ath‘ bifL hemo curv. on t1 lifti turn the d. turbuj in dOg fin -««E£E LITERATURE REVIEW Mechanical Factors in Atherosclerosis Turbulence Physical stress within the vasculature has been sug- gested as one of the mechanisms that enhances atherogenesis. Atherosclerosis is often seen at arterial curvatures and bifurcations, places of turbulent flow or where changes in hemodynamic pressure occur (Texon, 1957). The flow around a curvature has been shown to cause low hemodynamic pressure on the surface of the lesser curvature causing a physical lifting and stretching of the endothelial cells which in turn increases permeability of the endothelial lamina in the dog carotid artery (Texon 33 31., 1960). By creating turbulence surgically, Sako (1962) has demonstrated that in dogs bifurcation turbulence increases atherogenesis. Hypertension Hypertension has been indicated as enhancing the athero- genic process in many species. The primary effect of hyper- tension is apparently mediated through permeability changes. Even in plaque-free areas, hypertensive rats showed elevated cholesterol content in their aortic tissue. Duncan (1963) suggested that lipid entrance is a secondary effect as the result of stretching the arterial wall from elevated systemic 3 press: to coi (Wiene with J betwe] parti: inter: partit the s: Since (Schoé I be re! that pressure. In hypertensive rats arteries were more permeable to colloidal particles than arteries in normotensive animals (Wiener st 31., 1969). In the hypertensive rat, injected with colloidal carbon, arterial endothelial gaps were seen between the endothelial cells. Between the gaps were carbon particles indicating permeability through this space. The intercellular space is not normally permeable to colloidal particles (Buck, 1958). Carbon particles were also seen in the subendothelium, suggesting breaks in the basement membrane since basement membrane is normally a colloidal filter (Schoefl, 1963). Permeability changes brought about by hypertension may be reversible. A study by Kojimahara st 31. (1971) indicated that in rats permeability of the arterial wall to colloidal gold may be reduced to a normal level if the hypertensive animals are given anti-hypertensive drugs. This may be an indication that permeability changes associated with hyper- tension are the result of passive stretch of the arterial wall which separates the endothelial cells. When the pressure was relieved, the intercellular space probably closed and became impermeable. Further evidence of the possible im- portance of blood pressure on atherosclerosis was seen when comparing the high incidence of atherosclerosis in avian species with a relatively high blood pressure, and the low incidence of atherosclerosis in reptilian species with a low blood pressure (Finlayson EE.§l°' 1962). Turbulence and hypertension are two factors that contribute toward athero- genesis. has be Using strate lipid found was pe plasma have t PGITReaII increaI diet wl Ii feedin theliu: Engele feedin C rabbit cellule Lipid Role in Atherosclerosis The source of lipid seen in the atherosclerotic plaque has been demonstrated to be primarily from blood plasma. Using an isotope tracer technique, Christensen (1962) demon- strated endothelial permeability to cholesterol and phospho- lipid in cholesterol-fed cockerels. Duncan and Buck (1959) found that endothelium of aortas from cholesterol-fed dogs was permeable to labeled cholesterol in proportion to its plasma concentration. Atherogenic high cholesterol diets have been indicated as a cause of increased endothelial permeability. This has been shown in rats as indicated by increased colloidal iron accumulation when an atherogenic diet was fed (Veress 32 31., 1970). In parakeets hyperlipidemia associated with cholesterol feeding caused pinocytosis vessicles to appear in the endo- thelium (Hess and Staubli, 1969). Parakeet vascular smooth muscle cells became lipophagic as a result of cholesterol feeding. Colloidal material has been shown to pass through rabbit endothelial cells rather than permeating the inter- cellular spaces (Buck, 1958). Vacuoles containing cytoplasmic material (as indicated by the presence of a tracer substance) were seen in the endothelium (Buck, 1958). This indicated that vacuolar material, probably including that found in foam cells of atherosclerotic lesions, was of plasma origin. Leary (1941) suggested that the characteristic lipid laden foam cells were of reticuloendothelial origin. Hess and Staubli (1969) observed no evidence of blood borne cells in atherogenesis. Simonton and Gofman (1951) found no labeled foam cells in atherogenic rabbits with labeled reticuloen- dothelial cells. From most indications, it appears that the primary source of lipid in the atherosclerotic lesion is blood plasma. Atherosclerosis and Mucopolysaccharides Changes in vascular ground substance, mucopolysaccha- rides, have been implicated in the development of atheroscle- rosis. The direction of change of mucopolysaccharide is controversial among the workers in this field. In studies on humans (Zugibe and Brown, 1960; Curran and Crane, 1962; Sanwald st 31., 1971; and Hollander et_§l., 1968) increases in various mucopolysaccharides associated with atherogenesis were indicated. The increase in mucopolysaccharide content in atherosclerosis may be the result of an inflammation re- pair mechanism (Curran and Crane, 1962; Gillman and Hathorn, 1957). Botcher and Klynstra (1965) and Klynstra §£_al. (1967) found no correlation of total mucopolysaccharides with atherosclerosis in human aortas. Manley and Hawksworth (1965) found reduction of very specific mucopolysaccharides in areas of the human vascula- ture prone to atherosclerosis but not showing the disease. Manley and Mullinger (1967) observed reductions of one mucopolysaccharide in a human aortic atherosclerotic area compared to an uninvolved locus on the same vessel. Berensen (1961) found a general drOp in aortic mucopolysaccharide accompanying atherosclerosis. In animals different species vary in mucopolysaccharide changes associated with atherosclerosis. Old swine, animals that are subject to spontaneous atherosclerosis, had increased mucopolysaccharide atherosclerotic areas (Luginbuhl and Jones, 1965). Accumulation of cholesterol in the dog aorta induced by dietary manipulation resulted in a corresponding decrease in aortic mucopolysaccharide. Feeding cholesterol to cock- erels to induce atherosclerosis increased the synthesis of aortic sulfated mucopolysaccharides (Kowelewski, 1960). The effect of atherogenesis on vascular mucopolysaccharide con- tent is yet to be established, although variation with atherosclerosis has been observed. Vitamin A and Mucopolysaccharide Synthesis Mucopolysaccharide synthesis also appears to be influ- enced by the absence or presence of vitamin A. WOlf and Varandani (1960) showed that mucopolysaccharide synthesis was reduced by one-half in rat colon homogenates from vita- min A deficient rats. Synthesis of mucopolysaccharide was reduced in the liver (Sundaresan, 1966), kidney, uterus and trachea of vitamin A deficient rats (Ushimi, 1965). In rats Thomas and Pasternak (1969) found no change in synthesis rate of mucopolysaccharides but an increased turnover rate which could account for an overall decrease. There is a re- duction in synthesis of some glycoproteins in vitamin A deficiency (DeLuca 33 21., 1970). Brown gt El' (1963) theorized that cell permeability changes, associated with hypervitaminosis A, were the result of a change in sulpha- tion of polysaccharides needed for cell membrane structure. Permeability changes would also be likely as a result of vitamin A deficiency. Vitamin A and Cell Membranes Vitamin A has been shown to be a component of plasma membranes (Lui and Roels, 1969; Mack gt 31., 1970) and is necessary for proper membrane stability and permeability. Excess of vitamin A was found by Dingle SE.E$° (1962) to decrease the stability of lipoprotein membranes. Studies with erythrocytes, under phase contrast and electron micro- scopes, revealed that after the addition of vitamin A to the medium a pinocytosis-like process occurred which led even- tually to hemolysis. When erythrocytes from rabbit, ox, rat or human were placed in a medium rich in vitamin A, the cells lysed. The effect was described as similar to hemolysis caused by detergents, that is, disorganization of the membrane (Dingle and Lucy, 1962). The effect of excess vitamin A is not limited to red blood cell membranes. Addition of vitamin A to cultured fibroblasts caused distension and disintegration of cell and nuclear membranes (Dingle gt'al., 1962). Mitochondria, highly membranous organelles, have been shown to swell in a.concentrated vitamin A medium (Lucy'gt;al., 1963; Dingle g£;§1., 1962) probably as the result of a similar mechanism that caused cell membrane lysis. Dingle and Lucy (1962) suggested that vitamin A had its effect on the lipoprotein membrane of the cells and their component organelles by linking lipid to protein. Vitamin A methyl groups bind to cholesterol via Van der Walls forces and hydroxyl groups attach to protein by hydrogen bonding. Although it has been shown that vitamin A is a constituent of the cell membrane, its precise function has only been theorized. If vitamin A status does affect membrane integrity, lipid permeability could be altered, thus varying atherogenic potential. Vitamin A and Cholesterol Metabolism Vitamin A and Cholesterol Absorption WOod and Topliff (1961) observed that when they fed vitamin A together with cholesterol to chickens the vitamin A reduced the hypercholesterolemia produced by feeding cholesterol alone. March and Beily (1963) found that in the chicken vitamin A was effective in reducing serum cho- lesterol only in the presence of dietary cholesterol. When birds were fed a low cholesterol diet, the vitamin had no effect. Vitamin A was ineffective when the cholesterol level was elevated with diethylstylbestrol administration. In rats fed cholesterol Bring st 31. (1965) found an inverse relation- ship between dietary vitamin A and serum cholesterol as well as liver cholesterol. Krause gt 2l° (1968) observed that in beagles given atherogenic stress (ACTH, thiouracil and a high fat diet) along with a high level of dietary vitamin A and cholesterol there was an increase in serum lipids except cholesterol which was decreased. terol fed tc (1969) ducing terol terol min A terol A was the h: inter Vi w & Cl‘eas Of vi 1965) 10 Vitamin A appeared to have little influence on choles- terol metabolism unless the vitamin and cholesterol were fed together (Horner and Morton, 1960). Lakshmanan gt 31. (1969) found that although vitamin A was effective in re- ducing serum cholesterol in rats when vitamin A and choles- terol were fed simultaneously, there was no effect on choles- terol turnover rate. Beeler 33 El. (1962) found that vita- min A in chickens interfered with the bile salts in choles- terol absorption. The hypocholesterolemic effect of vitamin A was overcome by adding bile salts to the diet. Apparently, the hypocholesterolemic effect of vitamin A is mediated by interference with cholesterol absorption. Vitamin A and Cholesterol Synthesis In some cases vitamin A has been shown to cause in- creased serum cholesterol levels. Intramuscular injection of vitamin A increased liver cholesterol in rats (Misra, 1965). Van Bruggen and Straumford (1948) found that a massive oral dose of vitamin A given to normal human subjects increased serum cholesterol. In atherosclerotic subjects serum cholesterol was depressed by vitamin A administration (Kinley and Krause, 1959). High serum levels of vitamin A have been shown to be associated with elevated serum choles- terol (Wilcox gt 31., 1954; Bring 33 31., 1955). This in- crease in serum and liver cholesterol is due to the fact that vitamin A is essential for cholesterol synthesis (Phillips, 1961; Gloor and Wiss, 1959a; Gloor and Wiss, 1959b; Rao and Olson, 1967). scl rat and ant: cho] lest Int: crea 1967 on a anti 11 The Influence of Vitamin A on Atherosclerosis There are few studies relating vitamin A to athero- sclerosis. Krause 33 31. (1968) observed that vitamin A retarded cholesterol deposition in the arteries of beagles given atherogenic stress. In chickens (Beeler SE.El-v 1962) and rats (Weitzel and Buddecke, 1965) vitamin A demonstrates anti-atherogenic properties in cholesterol-fed animals. In cholesterol-fed rabbits vitamin A did not affect the cho- lesterol content of the aorta (Oppenheim and Bruger, 1952). Intraperitoneal injection of vitamin A in rats caused in- creased calcification of coronary arteries (Strebel gt 31., 1967). Relatively little is known of the role of vitamin A on atherosclerosis, but indications are that vitamin A has anti-atherogenic properties. Spontaneous Atherosclerosis in the Aorta and Coronary Arteries of Domestic Birds In chickens spontaneous lesions have been described by Siller (1965) and others in the thoracic aorta of both male and female birds. These early lesions were not seen upon gross examination. Histological sections revealed lipid, which reacted positive for cholesterol, in the medial layers. There was relatively little intimal lipid in early lesions. Studies by Dauber (1944) and Grollman gt 31. (1963) suggested that thoracic aorta lesions only occurred in hens, but these studies were based only on surface exami- nation. Lipid infiltration has been observed in the aorta of day-old chicks. However, this early lipid which is not cho- lesterol disappears with advancing age (Nichols gt 31., 1961). teriz- 1965)‘ thick- worke; genes. with . poult: lipid inter: smoot} HOWarc two t: elast lipid the m 12 In chickens early coronary lesions, which were charac- terized by diffuse intimal thickening, had no lipid (Siller, 1965). well-developed lesions may later show intimal thickening with lipid (Siller, 1965). Spontaneous atherosclerosis has been observed by many workers. Middleton (1965) described spontaneous athero- genesis in the aorta of turkeys as a process that begins with intimal lipid accumulation in day-old to two-week-old poults. In older birds the lesion becomes larger with more lipid and fibrous tissue (Ringer, 1960) with a fenestrated internal elastic membrane (Middleton, 1965). A block of smooth muscle is usually seen in the lesion (Gresham and Howard, 1963). The turkey coronary arteries are subject to two types of lesions. One involves an intact internal elastic membrane with intimal fibrosis and extracellular lipid; the other was characterized as lipid deposition in the media (Middleton, 1965). Different breeds of pigeons have been shown to vary in their resistance or susceptibility to spontaneous athero- sclerosis. White carneau and silver kings showed a high incidence of atherosclerosis. Racing homers and show racers appeared to be relatively resistant to the disease (Lofland and Clarkson, 1959). There was no sex difference in re- sistance to atherosclerosis observed among the pigeons (Prichard 33 31., 1964). Lesions have been seen throughout the aorta in all breeds (Lofland and Clarkson, 1959). When a resistant and susceptible breed (show racer and white 13 carneau) of pigeon was compared, Clarkson 33 31. (1962) ‘found that the aortic lesions were histologically the same, showing thickened intima with fibrocytic cells and intra- cellular lipid. Although the lesions are not histologically identical, numerous domestic birds are subject to spontaneous atherosclerosis. Spontaneous Atherosclerosis in Wild Birds Stenosed intramural coronary arteries resulting from non-lipid containing intimal thickening have been observed in the bald eagle (Aronson, 1962). In ducks spontaneous atherosclerosis is much less common in wild ducks as com— pared to domestic ducks (Wolf 33 31., 1949). The duck lesion as seen in zoo ducks is a fibrous lipid containing structure (Ratcliffe gt 31., 1960). Similar lesions have been observed in geese, pheasants, quail, hawks, birds of paradise and parrots (Finlayson 33 31., 1962; Ratcliffe 3£_31., 1960). Spontaneous lesions are not uncommon in domestic and wild species of birds. Function of Vitamin A in Cytodifferentiation The basal layer of most epithelial cells is capable of differentiation in two directions. Under the influence of vitamin A, it may become mucous secreting or it may become squamous in the absence of the vitamin (WOlf and DeLuca, 1969). Fell and Mellanby (1953) observed that different tissues appeared to have different thresholds of vitamin A sensitivity. The tissues that were more refactory to vitamin A were squamous under normal physiological conditions. Some 14 tissues that had squamous epithelium could be made to become secretory with the administration of a high dose of vita- min A (Fell and Mellanby, 1953; Fell, 1957; Kahn, 1954; Lawrence and Bern, 1960). 13 31552 mucous metaplasia was observed in chick embryo ectoderm by Fell and Mellanby (1953). Removal of the ectoderm from the media that con- tained the high level of vitamin A and returning it to a medium with a normal level of the vitamin resulted in keratinization of the basal cells which had not yet dif- ferentiated. Normal squamous cells were seen beneath the metaplastic secretory epithelium. This demonstrated that the effect of the vitamin was on the basal layer of dif- ferentiating cells. 13_X1££3 keratinization of vaginal epithelium associated with estrogen presence was suppressed with large doses of vitamin A (Kahn, 1954). 13 vivo mucous metaplasia of squamous epithelium was shown in hamsters by Lawrence and Bern (1960). Placement of a vitamin A pellet in the hamster cheek pouch caused mucous metaplasia. In rats New (1963) observed that excess vitamin A had no effect on epithelium that had hair follicles. Virus induced, keratinized tumors in rabbits have been seen to atrophy or slough off as the result of intraperi- toneal vitamin A injection (Chu and Malmgren, 1965). In hamsters metaplasia of the columnar secretory epithelium in the lung was seen as the result of benzo (a) pyrene hematite dust inhalation. Tumor formation normally followed the benzo (a) pyrene administration. The carcinogen usually resul level secre (Saff secre oping shown (Wolb. sensii trach¢ metapj that Seen a and H< metap: the pa the u: dec warka: naSal (Jungj 89331 In Not 15 resulted in 100 percent carcinoma incidence. Feeding high levels of vitamin A suppressed squamous metaplasia of the secretory epithelium and subsequent carcinoma formation (Saffioti 32 31., 1967). Deficiency of vitamin A caused squamous metaplasia of secretory epithelium in many tissues. In rats, with devel— oping vitamin A deficiency, trachea and bronchi have been shown to keratinize before the urinary tract epithelium (WOlbach and Howe, 1925). This probably indicated a greater sensitivity of urinary tract epithelium than that of the trachea or bronchi to vitamin A. In developing squamous metaplasia from vitamin A deficiency, mucous secreting cells, that had differentiated before the deficiency occurred, were seen above the newly differentiated squamous cells (W01bach and Howe, 1925). Nielsen 33 31. (1966) observed squamous metaplastic changes in the normally secretory epithelium of the parotid duct of Holstein calves. In utero metaplasia of the urethral epithelium of rat fetuses from vitamin A deficient mothers has been demonstrated by Wilson and warkany (1947). In chickens fed a vitamin A deficient ration nasal secretory epithelium shows distinct squamous metaplasia (Jungherr, 1943). In summary, epithelia differ in vitamin A sensitivity, but most are subject to metaplasia. Influence of Vitamin A on Gut and Its Epithelium Metaplasia due to excessive vitamin A application does not occur in all tissues. Fell et‘al. (1954) showed the in in in (ii; an th to Q~ 16 that embryonic chick gut grew normally and differentiated in a medium which had enough vitamin A to cause metaplasia in tissues of ectodermal origin. Weisman (1961) observed that in Xenopus laevus hypervitaminosis A resulted in mucinous diarrhea with large and numerous goblet cells in the proximal gut and colon. Vitamin A deficiency resulted in a heavier and longer intestine in the chicken (Donovan, 1965; Bayer 33 31., 1968). Associated with the increased weight and length of the intestines from vitamin A deficient birds was an increase in lymphoid tissue (Bayer, 1968). The gut epi- thelium does not appear to be subject to metaplasia from vitamin A influence, but other areas of the gut are subject to vitamin A influence. Influence of Behavior on Atherosclerosis in Birds Chickens have been used in numerous studies to observe the effect of behavior on atherosclerosis because they are susceptible to the disease and also exhibit a social order. Pick and Katz (1961) found that disturbing the social order by shifting cockerels from one group to another did not in- fluence atherosclerosis. During the same study, Pick and Katz (1961) found that placing cages of 10 cockerels per cage in isolation rooms increased atherosclerosis when compared with birds that were not isolated. McKissick‘gE 31. (1961) studied the rate and incidence of atherosclerosis comparing individually caged, paired, and grouped chickens. They found that although lesions were more pronounced in male pairs, particularly after sexual maturity, the rate of development was the same as grouped males Snyde slowe and S actio cage behav it is behav —_—_—— 17 males. Studies by McKissick 33 31. (1961) and Ratcliffe and Snyder (1964) showed that individually caged birds showed the slowest development of coronary atherosclerosis. Ratcliffe and Snyder (1964) found that the intensity of social inter- action that is a result of increased numbers of birds per cage resulted in increased incidence of atherosclerosis. Since behavior has been demonstrated to be a factor in atherogenesis, it is essential that environmental factors that influence behavior be considered in studying atherosclerosis. ather ather the IT :1: 01 H; rn_ OBJECTIVES 1. To study the effect of dietary vitamin A on atherogenesis in coronary arteries of male Japanese quail. 2. To study the effect of vitamin A in the diet on atherogenesis in the aorta of the male Japanese quail. 3. To evaluate the influence of vitamin A status on the myocardial capillaries. 4. To further understand the means by which vitamin A affects aorta lipid content. 18 7"“ E35284: that ' sever: EXPERIMENTAL P ROCEDURE Experiment One Experiment one was designed to test the hypothesis that variation in dietary vitamin A could influence the severity and/or incidence of atherosclerosis. Two hundred newly-hatched, random—bred Japanese quail were divided into four equal groups and fed a basal ration (Table l) supplemented with different levels of vitamin A palmitate. The chicks were placed on diets deficient in vitamin A (880 U.S.P. units/kg. of feed), sufficient in vitamin A (4,400 U.S.P. units/kg. of feed) or excessive in vitamin A (22,000 or 220,000 U.S.P. units/kg. of feed). At four weeks of age, when sex could be determined by feather pattern dimorphism, all female birds were removed from the experiment to eliminate sex as a variable. An attempt was made to keep environmental factors the same for the birds fed the different treatments in all experiments. Birds were reared in a Petersime model 2-SD brood-unit. Temperature was set at 99° F., light was provided for 14 hours per day and feed and water were provided 33 libitum. At 10 weeks of age blood pressure was measured after cannulation of the carotid artery. Blood pressure was measured using a Statham model P23DC pres- sure transducer connected to the bird via PElO Intramedic 19 20 TABLE 1 Composition of the basal diet Ingredient Percent of ration Ground wheat 40.5 Soybean oil meal 50.0 Cotton seed oil 1.5 Dicalcium phosphate 2.0 Salt .5 Limestone 1.5 Vitamin premix* 4.0 *Premix contains the following ingredients per 45.4 kg. of ration: riboflavin--200 mg.; calcium pantothenate-—500 mg.; niacin--l,250 mg.; choline chloride--2,000 mg.; vitamin B12--.5 mg.; vitamin K—-24 mg.; vitamin D3--55,000 ICU. 21 tubing and recorded through a Grass model 7 polygraph. A three lead electrocardiogram was made from each bird with the Grass model 7 polygraph. The birds were killed by cervi— cal dislocation after blood pressure and EKG measurement. Tissue specimens were taken near its apex and across the ventricles of the heart and from the thoracic aorta for examination under the light microscope. These specimens were fixed in neutral buffered formalin, dehydrated in alcohol and xylene, and embedded in paraffin. Histological sections were cut at four microns thickness and stained with orcein elastic tissue stain (Appendix I) or periodic acid Schiff's reagent (Humason, 1967). In order to study lipid distribution in the arteries, frozen sections of the specimens were cut at a thickness of 10 microns from pieces of thoracic aorta and ventricles that had been fixed in neutral buffered formalin and were stained with oil red O and hematoxylin (Appendix II). Intramural coronary arteries were examined for signs of the presence or absence of atherogenesis. These data were subjected to chi square analysis. The frozen sections made from aortas were evaluated for lipid content. A scoring sys- tem was designed to quantitate the degree of lipid infiltra- tion. One point was assigned for each lamina that had more than 50 percent of its area infiltrated with lipid. These data were analyzed by analysis of variance and Duncan's new multiple range test. Photomicrographs were taken through a model C4T6-HW American Optical Company binocular microscope on panatomic-X black and white 35 mm. panchromatic film. 22 Experiment Two Experiment one was designed to test the hypothesis that vitamin A influences atherosclerosis and to develop appropriate technique to demonstrate and test the hypothesis. Experiment two was designed to confirm the findings of experiment one. Approximately 200 coturnix quail chicks were hatched and immediately divided into 4 equal groups. Each group was placed on a diet which differed from the other diets only in the concentration of vitamin A palmitate supple- ment. The diet lowest in vitamin A, which was designed to be deficient, contained 880 U.S.P. units of vitamin A per kg. of feed. A diet containing 4,400 U.S.P. units of vita- min A per kg. of feed, within the range for normal growth (Georgis, 1966; Shellenberger and Lee, 1966; Vohra, 1971), was given to another group as a control diet. Two groups of birds were given diets which exceeded the vitamin A re- quirement. These two diets contained 22,000 and 220,000 U.S.P. units per kg. of feed, respectively. At four weeks of age, the time at which sex could be determined by feather pattern dimorphism, all female birds were removed from the experiment. After 10 weeks on experiment, direct blood pressures were measured on all surviving birds by cannulation of the carotid artery with a polyethylene Intramedic cannula (PElO) connected to a Statham Model P23DC pressure transducer. Re- cordings were made on a Grass model 7 polygraph. 23 Immediately after blood pressure measurements, all birds were killed, the heart and thoracic aorta removed, and the tissues fixed in neutral buffered formalin for frozen and paraffin sections. Tissue samples were included from those birds that died during the last seven weeks of the experiment. Paraffin sections were stained with Orcein connective tissue stain and counterstained with Van Geisen's stain. Frozen sections were stained with oil red O and hematoxylin. Thoracic aortas were scored for lipid deposition on the basis of one point for each lamina that stained posi- tive for lipid and two points for each lamina that con- tained lipid in more than 50 percent of its area. Coronary arteries were evaluated for the presence of atherosclerotic signs. Since it was not feasible to make objective scores based on the extent of coronary atherosclerosis, a judgment of presence or absence of the disease was made. Cholesterol analysis was done on plasma samples using the procedure of Zlatkis 32 31. (1953) (Appendix III). Experiment Three The level of dietary vitamin A appeared to influence atherogenesis in growing Japanese quail. Experiment three was designed to test the initial hypothesis, that vitamin A influences atherogenesis, using mature birds. All quail used in this experiment were grown to 10 weeks of age on a ration that contained 3,300 U.S.P. units 24 of vitamin A per kg. of feed. At 10 weeks of age, 4 groups of 10 male quail were placed on diets containing 880, 4,400, 22,000 or 220,000 U.S.P. units of vitamin A per kg. of feed. The experiment was terminated when the birds reached 20 weeks of age. Direct blood pressure was measured by cannulation of the carotid artery, as described in ex- periment one. Histological samples were taken from the anterior portion of the ventricles and were stained with orcein elastic tissue stain and counterstained with Van Geisen's stain. Frozen aorta sections were stained with oil red O and hematoxylin. Thoracic aortas were scored for the presence of lipid deposition on the basis of one point for each lamina that contained lipid and two points for each lamina that con- tained lipid in more than 50 percent of its area. Coronary arteries were evaluated for the presence of atherosclerosis. Since it was not feasible to make objective scores based on the extent of coronary atherosclerosis, a judgment of presence or absence of the disease was made. Experiment Four The results of replicate experiments demonstrated that vitamin A influenced the severity of aortic athero- sclerosis. The objective of this experiment was to find how the lipid infiltrated the aorta and the role of vita- min A in preventing this infiltration when fed in adequate amounts. 25 In experiment four newly hatched coturnix quail chicks were placed on rations that were deficient in vitamin A (200 U.S.P. units/kg. of feed), normal in vitamin A (4,400 U.S.P. units/kg. of feed) or excessive in vitamin A (22,000 or 220,000 U.S.P. units/kg. of feed). Birds were killed by cervical dislocation at four weeks of age when the birds fed the vitamin A deficient diets showed severe symptoms of vitamin A deficiency (ruffled feathers, squatting posture, general weakness, exudate around the eyes, as seen in Figure 1). Samples of thoracic aorta were immediately removed and fixed in an iced 6.25 percent glutaraldehyde so- lution made up in Sorensen's buffer (see Appendices IV and V) pH 7.2. The tissues were post-fixed in osmium tetroxide solution (see Appendix V) in an ice bath to improve membrane fixation. Tissues were washed in Sorensen's buffer pH 7.2, dehydrated in alcohol, then placed in propylene oxide to improve their permeability in the embedding medium. The tissues were embedded in Epon 812 (see Appendix VI). Specimens were sectioned on a Sorval model Mt-2 ultra- microtome using glass knives made daily on an LKB model- 7801A knife breaker. Sections showing gold and silver interference (600-900 Angstroms thickness) colors were saved on copper grids. The grid-mounted specimens were stained with uranyl acetate and lead citrate (see Appendix VII). The stained specimens were examined with a Philips model EM100 transmission electron microscope. Electron Figure l. 26 Photograph of a Japanese quail showing signs of vitamin A deficiency. 27 Figure 1 28 micrographs were made on 35 mm. Kodak FRP-473 fine grain release positive film and developed to a negative. Experiment Five Changes in mucopolysaccharide content and/or synthesis have been associated with atherogenesis in many species. Vitamin A has been shown to have a role in muc0polysaccharide synthesis or turnover. In experiment five the mucopolysac- charide content of the aorta ultrastructure was studied to test the hypothesis that dietary vitamin A level might affect the mucopolysaccharide content of the aorta. In the fifth experiment day-old chicks were placed on rations that contained either 200 U.S.P. units of vitamin A per kg. of feed or 15,000 U.S.P. units of vitamin A per kg. of feed. The control ration contained 15,000 U.S.P. units of vitamin A per kg. of feed as this was the level of vitamin A calculated to yield minimal atherosclerosis in the thoracic aorta. After four weeks on the experimental rations,the birds were killed by cervical dislocation. Sections of thoracic aorta were fixed in 6.25 percent glutaraldehyde in Sorensen's buffer pH 7.2, washed in Sorensen's buffer pH 7.2, dehydrated in alcohol, placed in propylene oxide and embedded in Epon 812 (Luft, 1961). Osmium tetroxide was not used for post-fixation, as was done in experiment four, due to its strong oxidizing properties, which would interfere with the histochemical test for mucoprotein and mucopolysaccharide. 29 Sections were cut using freshly broken glass knives. Sections showing gold and silver interference colors were mounted on titanium, stainless steel or copper grids. The method of silver methenamine staining described by Hayat (1970) was tried and found to be unsuccessful. Modifica- tions were made on the silver methenamine staining pro- cedure. The staining procedure (Appendix VIII) involved oxidation of the grid-mounted specimen with periodic acid and exposing the oxidized specimens to a silver methena- mine solution in an oven at 600 C. To determine if mate- rial being stained was either mucopolysaccharide or muco- protein, or was an unrelated substance, one grid was stained from each tissue with the procedure described in Appendix VIII omitting the oxidation with periodic acid. If the structure in question stained without oxidation with periodic acid, the substance was not mucoprotein or muco- polysaccharide. Grid-mounted specimens were examined with a Philips EM100 transmission electron microscope. Electron micrographs were taken using 35 mm. Kodak fine grain re- lease positive film. Experiment Six In experiment five, differences in the silver methena- mine content of the aorta endothelial ultrastructure were observed. The results of experiment four indicated that in the aortas from birds fed a vitamin A deficient ration many of the endothelial cells were becoming detached. It had 30 also been previously demonstrated that the birds fed the ration lowest in vitamin A had the highest level of lipid in their thoracic aortas. The objective of experiment six was to see if there was an interrelationship between any of the following: vitamin A status, the presence of methenamine silver positive material, detached cells in the endothelium, and aorta score. In the sixth experiment day-old chicks were placed on rations that contained either 200 U.S.P. units of vitamin A per kg. of feed or 15,000 U.S.P. units of vitamin A per kg. of feed. At four weeks of age the birds were sacri- ficed and samples of thoracic aorta from each bird were fixed in neutral buffered formalin or glutaraldehyde. Frozen sections were made from the formalin-fixed specimens. The frozen sections were stained with oil red O and hema- toxylin for the presence of lipid. The formalin-fixed specimens were viewed with a light microscope. The sec- tions stained for lipid were scored by assigning one point for each lipid containing lamina and an additional point for each lamina that contained lipid in more than half its area. Some of the glutaraldehyde-fixed specimens were post-fixed in osmium tetroxide. The post-fixed specimens were used to study the ultrastructure of the detached cells. Other glutaraldehyde-fixed specimens were to be used to test for mucopolysaccharide and mucoprotein; they were not post-fixed. All glutaraldehyde specimens were acetone dehydrated and embedded in Spurr's low viscosity embedding 31 medium (see Appendix IX). Embedded thoracic aortas were sectioned using freshly broken glass knives. Gold and silver sections were mounted on copper grids if they had been post-fixed with osmium tetroxide or mounted on titanium grids to be stained with methenamine silver. The osmium tetroxide post-fixed specimens were stained with uranyl acetate and lead citrate. Glutaraldehyde-fixed specimens were examined on a Philips EM300 high resolution electron microscope. Electron micrographs were taken using Kodak 70 mm. fine grain release positive film. Experiment Seven Effects of vitamin A status had been observed on elastic arteries (aortas) and muscular arteries (coronary arteries). In experiment seven the myocardial capillaries were examined for possible changes as the result of vitamin A deficiency. Sections of myocardium from the left ventricle were removed from the birds raised for experiment six. Sections of eight cubic mm. of myocardium were fixed and embedded at the same time using the same procedure as for the aortas. Thin sections were made from the myocardial specimens. Sections showing gold and silver interference colors were grid-mounted and stained with uranyl acetate and lead citrate or Spurr's stain (see Appendix X). Specimens were examined with a Philips EM300 transmission electron microscope. 32 Experiment Eight The results of experiment seven demonstrated that capillary endothelial flaps were phagocytizing unidentified material from the blood. It was the objective of the eighth experiment to identify the material that was being phagocytized. Blood samples were taken from a wing vein of five- week-old male Japanese quail and placed in a beaker. The blood was stirred with an etched glass rod until a clot formed. The clot was then emersed in a 6.25 percent glutaraldehyde solution in Sorensen's buffer pH 7.2 then post-fixed in osmium tetroxide, dehydrated in acetone and embedded in Spurr's embedding medium. Thin sections show- ing gold and silver interference colors were mounted on copper grids and stained with uranyl acetate and lead citrate or Spurr's stain. The specimens were examined with a Philips EM300 transmission electron microscope. RE SULTS Experiment One Histological examination for the presence or absence of coronary atherosclerosis revealed that the Japanese quail fed the ration lowest in vitamin A (880 U.S.P. units/ kg. of feed) had the greatest incidence of coronary athero- sclerosis (66.7%). The lowest incidence of atherogenesis was seen in the coronaries of birds fed the recommended dose of vitamin A (4,400 U.S.P. units/kg. of feed). In the diets excessive in vitamin A the incidence of coronary atherogenesis was 33.3 percent and 20.0 percent for the birds fed 22,000 U.S.P. units and 220,000 U.S.P. units of vitamin A per kg. of feed, respectively. Statistical analysis of the coronary atherosclerosis data indicated that there was a significant interaction(P < .005) between coronary atherosclerosis and the vitamin A level in the diet in experiment one. These data are summarized in Table 2. None of the developing atherosclerotic lesions showed the presence of lipid. The coronary arteries that showed histological signs of atherogenesis were characterized by intimal thickening, as seen in Figures 2 and 3, or duplication of the internal elastic membrane, as seen in Figure 4. Fenestration of the 33 ‘P’ ‘ I LE Figure 2. 34 Photomicrograph of a coronary artery from a vitamin A deficient Japanese quail stained with orcein elastic tissue stain. Note intimal thickening (A) and fenestration of the internal elastic membrane (C). 35 Figure 2 Figure 3. 36 Photomicrograph of a coronary artery from a vitamin A deficient Japanese quail stained with orcein elastic tissue stain. Note dif- fuse intimal thickening (A). Internal elastic membrane (D) is identified. 37 Figure 3 Figure 4. 38 Photomicrograph of a coronary artery from a vitamin A deficient Japanese quail stained with orcein elastic tissue stain. Note duplication of the internal elastic membrane (D) which is an indication of developing atherosclerosis. 39 Figure 4 40 .§ . v Q99. .mcsszmso 3 335:: «5 «.5 8355.8 5 38:52 as s .3; 8 2 .2; 2 an 6an $32313 .3me 3m .3 SN :1 as E 981 a . 2% 3m 2: as .2 n: E 9.2 3: EN 98 .3 nm a: 1.: a. as :N. in a a a a 82m 898 83 8» is s .53.. < 522; .0 £2. .a .m .3 am £83383...» races 8:3:on :23 1.358335 3 .582. 05 S < 552; but... a not» 2: m HES. 41 internal elastic membrane was often observed together with intimal thickening (Figure 2), which indicated that cells of the thickened intima probably passed through the fenestration. Staining with periodic acid Schiff's reagent revealed no difference in mucopolysaccharide content of the coronary arteries from birds fed different levels of vitamin A. Examination and scoring for the presence of aortic lipid showed a significant effect of vitamin A in decreas- ing the quantity of lipid (P < .01). The aortic lipid stained by oil red O appeared to be present in the endo- thelium and in the cells between the elastic lamina. The data on aorta scores are summarized in Table 3. The aortas from birds fed the vitamin A deficient ration had the highest mean aorta score (9.4 i 2.0). This value was not significantly different from the aorta scores from birds fed the control ration considered normal in vitamin A (5.4 t 1.8). However, the aorta scores from the birds fed the rations excessive in vitamin A were significantly lower (P < .05) than the aorta scores from the birds fed the vitamin A deficient ration. The aorta scores for the birds fed rations containing 4,400, 22,000 or 220,000 U.S.P. units of vitamin A were not significantly different in scores with 5.4 i 1.8, 2.5 i 0.9 and 2.9 i 0.8, respectively. Simple linear regression of aorta score versus the log of vitamin A concentration in the diet demonstrated an inverse relationship between the log of the concentration of 42 .m8. Vases .8. V9... 5:35:38 Lo :85: 2: 2m 33:29.3 :_ 285:2 .33. em :5 E 222:: 2.53.22: 95 $259.33 222:: 5:: 28 ESEtoaxm as s .3; 8 2 .2; 2 an m as s .3; 2218 SEN .m_3._o_u§u£: 3 8:8: :38; a 3.865 2:2 3:9: 5 3 “an a. as.” 5 a. 3 M as e. S H E 3: mm use“ 2.: a; ”as a. a; H 3 a. $41.23. 3% sacéfis $33.: a» 1.. 2: as T; a s. :m .u .m u ~88: atom :32 98.5 §.- 83‘ as as s .m .22 5:5; 3 £5. .a as am =3: 33:32 22. :. 28m «to: :o < See; but... 3 «8:0 2.: m HES 43 vitamin A and aorta score. This was a highly significant regression (P < .001) as was revealed by the analysis of variance on the regression. The regression line is shown in Figure 5. Blood pressure was examined because pressure changes can influence the rate of atherogenesis; also atheroscle- rosis in advanced stages can elevate blood pressure. Blood pressures (Table 4) of the birds fed the vitamin A deficient ration (880 U.S.P. units/kg. of feed), the control ration (4,400 U.S.P. units/kg. of feed and the ration slightly excessive (22,000 units/kg. of feed) in vitamin A were not significantly different. The birds fed the ration highly excessive in vitamin A (220,000 U.S.P. units/kg. of feed) had significantly elevated blood pressure (P < .05). However, in subsequent experiments this was not confirmed, leading to the conclusion that blood pressure was not in- fluenced by feeding excess vitamin A (see Table 4). No significant difference in the electrocardiogram pattern or heart rate (Table 5) was observed between those birds fed the four different levels of vitamin A. The presence of normal electrocardiograms in all birds indi- cated that atherosclerotic lesions in the coronary arteries were not of sufficient severity to result in myocardial infarction. Experiment Two In experiment two the incidence of coronary athero— sclerosis followed a trend similar to that observed in 44 .53. .R :5 S East: b28553 8: $389.83... 232:: :3; 2:: 3:55:25 .8. v a. 22523:: :0 38:5: :5 3: 8858.8 :_ £8532 2.: a as 8 s 3.: 2 an N as s .3: 2 s L a: an: an H 8.8 B a: m 8.8 s. a...“ M :32 a 8.: H e a: a. mm 3.: u 8.3 a. 3.2 .u as: .3 2 e H as: a. m: .H :22 3: sets H as: a «an L. 3.3 a. an: H 8.3 a. .3: M as: is H H .1 a .m H a... .55. 238:: :83 :82 95.8w 96.“ 8:6 :3 as 3 .342 < 522: s as: .a .m .2 am :3: 08:2! 32: E 2:32: :83 :o < £55; .53.: .8 tea 2: v 32: ' . :yrIbvaibfllnHZE‘ 45 52523:: 5 33:5: 8: 83:58:: :_ 23:52 as s 3.: :N s .3: 2 :9: do: 5 .32, S 2 2 5: gen N H an H can 5 3: fl :5: 2 :5 + 3:: 5 on L. Se 5 mm twigs: 223.925 92:98:: 3:92:12 LN 24:98:: 5:39:35 nsflsmsl 332:2: 2 .m. .m w 55:2: .5: £85 2:: :8: :82 c8.o- 8o.~m 8:6 oww J3 8331.9. :3 < 522;: 2:: an: slal :5: «3:82. 2:2 :_ £2 :8: :o < :25; 232: 5 28:: 2: m HES. Figure 5. 46 Graph of the linear regression of aorta score on the log of vitamin A concentration in the diet in experiment one. 47 XS“ -35 . > :ozgcoocou < £52; :3 m m muumwm N S 2 NH 81038 BlJOV 48 .experiment one (Table 2). The birds fed the ration de- ficient in vitamin A (880 U.S.P. units/kg. of feed) had the greatest incidence of coronary atherogenesis. In 70.0 percent of the deficient animals there were signs of coronary atherosclerosis. The birds fed the diet normal in vitamin A content showed signs of coronary atherosclerosis in the smallest number of animals (14.3%). The birds fed the high levels of dietary vitamin A (22,000 or 220,000 U.S.P. units/kg. of feed) showed signs of atherogenesis in 42.9 percent and 54.5 percent of the birds, respectively. The birds fed the ration containing 220,000 units of vitamin A per kg. of feed showed a large increase in their incidence of coronary atherosclerosis compared to the incidence seen in the birds which received this level of vitamin A in ex- periment one (54.5% compared to 20.0%). There was no indication why this occurred. Statistical analysis of the coronary atherosclerosis data indicated that, although most of the data followed the trend of experiment one, there was no significant interaction between the level of dietary vitamin A and the incidence of coronary athero- sclerosis. When statistical analysis was done on combined coronary atherosclerosis data from experiments one and two, a significant interaction (P < .05) of vitamin A with coronary atherosclerosis was observed. On the basis of the pooled data vitamin A appeared to influence the inci- dence of coronary atherosclerosis. 49 Examination of the aortas and scoring for the pre- sence of lipid (Table 3) showed that vitamin A had a sig- nificant effect on the quantity of lipid infiltrated into the thoracic aorta (P < .005). This confirmed the findings of experiment one. The aortas from birds fed the vitamin A deficient diet had the most lipid with a mean aorta score of 10.4 i 1.1. This score was significantly greater (P < .05) than the aorta scores of all other treatments. The birds fed the control ration had a mean aorta score of 4.3 t 0.9, which was not significantly greater than the mean aorta score of 3.8 i 0.9 from the birds fed the ration containing 22,000 U.S.P. units of vitamin A per kg. of feed. The aorta score of the control birds was significantly greater (P < .05) than that of the birds fed the ration con- taining 220,000 U.S.P. units of vitamin A per kg. of feed (2.7 i 0.5). The aortas from birds fed the two rations that were excessive in vitamin A were not significantly different in lipid content. Since there appeared to be an inverse relationship between the level of vitamin A and aorta score for lipid a linear regression analysis was done. Simple linear re- gression of the aorta scores versus the log of vitamin A concentration indicated a highly significant regression (P < .001) of the log of vitamin A concentration versus the amount of aortic lipid (Figure 6). Blood pressure and heart rate (see Tables 4 and 5) were not significantly affected by the level of vitamin A in the diet. 50 Figure 6. Graph of the linear regression of aorta score on the log of vitamin A concentration in the diet in experiment two. 51 838.588 < SEE; as x3.~ -32 . > m m musowm N S 3 2 2 91033 puov 52 Experiment Three Experiment three was conducted using mature Japanese quail. In this experiment a highly significant (P < .005) interaction was seen between the level of vitamin A in the diet and the incidence of coronary atherosclerosis (Table 2). The birds fed the vitamin A deficient ration (880 U.S.P. units of vitamin A per kg. of feed) showed signs of athero- genesis in all birds examined microscopically. The birds fed the control ration (4,400 U.S.P. units per kg. of feed) showed signs of atherosclerosis in 42.8 percent of those examined. The birds fed rations excessive in vitamin A (22,000 or 220,000 U.S.P. units of vitamin A per kg. of feed) showed signs of atherosclerosis in 22 percent and 37.5 percent of the birds. This concurs with previous findings with growing birds and indicates that even in developed coronary arteries of mature birds, atherosclerosis can be induced with vitamin A deficiency. In the mature birds the vitamin A deficient diet yielded the greatest aorta score, 6.5 i 1.7, compared with 3.0 t 0.7 for the birds fed the control ration and 3.1 i 0.9 and 3.6 i 1.1 for the birds fed rations excessive in vita- min A (Table 3). Although the results followed an aorta score trend similar to experiments one and two, the treatments did not yield significantly different re- sults. Simple linear regression of aorta score versus the log of vitamin A concentration in the diet showed a non- significant regression, indicating that in mature birds, 53 as opposed to growing birds in experiments one and two, there was no apparent relationship between the level of dietary vitamin A and aortic lipid content. The regression line is shown in Figure 7. As in previous experiments, heart rate (Table 5) was not affected by vitamin A level in the diet. Blood pressure (Table 4) was not significantly altered by dietary vitamin A. The blood pressure findings agreed with those of experiment two, but not with the find- ings of experiment one. The effect of vitamin A on blood pressure in experiment one was probably a chance occurrence and the vitamin did not affect blood pressure. Immature birds in experiment one which were fed ra- tions low in vitamin A concentration showed a significantly greater incidence of coronary atherosclerosis than the birds fed sufficient or excess vitamin A. In mature birds in experiment three there was a significant increase in athero- sclerosis observed among the birds fed rations containing low levels of vitamin A. However, in experiment two with growing birds the data followed a similar trend but were statistically non-significant. Pooling the data from experiments one and two revealed a significant interaction (P < .05) between the level of dietary vitamin A and the incidence of coronary atherosclerosis. Though the data from experiments one, two and three were not conclusive, it appeared that the lowest level of vitamin A fed in this study was conducive to coronary atherosclerosis in Japanese quail. 54 Figure 7. Graph of the linear regression of aorta score on the log of vitamin A concentration in the diet in experiment three. 55 x8.uom.m.> :ozgcmocou < 552; moo m h ousmflm m S S 2 91033 euov 56 In experiments one and two, with growing birds, there appeared to be an inverse relationship between the level of vitamin A in the diet and lipid content in the thoracic aorta. With mature birds in the third experiment vitamin A did not significantly alter lipid content. Sufficient vitamin A was essential in order to keep aortic lipid con- tent to a minimum. The vitamin apparently is functional in the development of the aorta which is relatively impermeable to lipid. Since vitamin A does not significantly affect aorta lipid content in the mature bird, probably its func- tion is on developing aorta. The apparent mode of action of the vitamin in increasing lipid infiltration was ex- plored by electron microscopy in subsequent experiments. Experiment Four The aorta endothelium was generally flattened in those birds fed the ration that met the normal requirement of vita- min A (4,400 U.S.P. units per kg. of feed). The endothelial cells were in some cases tightly packed together with a few spaces of separation (Figure 8) or flattened and tapered at each end (Figure 9). The normal endothelium has occa- sional vacuoles, scattered endoplasmic reticulum, and some ribosomes. Some apparently normal surface activity was seen on the endothelial surface facing the lumen in the form of what appeared to be phagocytosis flaps. The endothelium from the birds fed 22,000 U.S.P. units of vitamin A per kg. of feed appeared to show the same morphology as the controls. There appeared to be no difference between the Figure 8. 57 Electron micrograph of aorta from a normal Japanese quail (x8,900) stained with uranyl acetate and lead citrate. The aorta lumen (LU), an endothelial cell nucleus (N) and an elastic fiber (EL) are labeled. Endothelial cells are generally packed together but there is one space to the left of the photo. 58 Figure 8 Figure 9. 59 Electron micrograph of aorta from a normal Japanese quail (x10,300) stained with uranyl acetate and lead citrate. The endothelial cells have few lipid vacuoles (V). An endothelial cell nucleus (N), the lumen of the aorta (LU) and an elastic fiber (EL) are labeled. Note the flattened endothelial cells. 60 Figure 9 61 endothelial cells of the birds fed 4,400 or 22,000 U.S.P. units of vitamin A per kg. of feed. The birds fed 220,000 U.S.P. units of vitamin A per kg. of feed appeared to have a slight increase in the numbers of vacuoles in their aortic endothelium when compared to the birds fed 22,000 or 4,400 U.S.P. units of vitamin A per kg. of feed, but fewer vacuoles than the vitamin A deficient animals. These vacuoles indi- cated the presence of lipid. The morphology of the endothelium of the birds fed the vitamin A deficient ration was grossly different from the endothelium from the birds fed any other level of vitamin A tested. The most striking change from the normal endothelium was the apparent detachment of the endothelial cells from the subendothelium as seen in Figure 10. The endothelial cells appeared to project into the lumen. Endothelial de- tachment probably increased permeability of the endothelial layer yielding greater ease of permeation of the subendo- thelial tissue. The cells were highly vacuolated and the vacuoles appeared to be membrane enclosed. Some of the endothelial vacuoles appeared to be ruptured and expelling their contents into the lumen (Figure 11). Vacuolar rupture probably has no apparent function in atherogenesis. There were also large quantities of what seemed to be free ribosomes (Figure 12). This indicated active protein synthesis within the endothelial cells. The site of lipid, which had been demonstrated pre- viously in the media, appeared to be part of the smooth Figure 10. 62 Electron micrograph of aorta from a vitamin A deficient Japanese quail (x6,800) stained with uranyl acetate and lead citrate. Note the endothelial cells, which have numerous vacuoles (V) and project into the lumen (LU) of the aorta. Elastic fibers (EL) are also labeled. 63 '1 . -.. A Figure 10 Figure 11. 64 Electron micrograph of aorta from a vitamin A deficient Japanese quail (xl4,900) stained with uranyl acetate and lead citrate. Note several lipid vacuoles (V) in the endothelial cell that projects into the lumen (LU). The arrow points to material that appears to be expelled from a vacuole. Elastic fibers (EL) are seen beneath the endothelium. Figure 11 Figure 12. 66 Electron micrograph of aorta from vitamin A deficient Japanese quail (x15,300) stained with uranyl acetate and lead citrate. Note arrow pointing to some of numerous ribosomes. Several vacuoles (V) are seen on the endothelial cell, which projects into the lumen (LU). 67 Figure 12 68 muscle cell. Smooth muscle cells, particularly those from the vitamin A deficient animals with high aorta scores, contained numerous vacuoles, probably containing lipid (Figure 13). In the aortas of the birds fed the highest level of vitamin A many of the fibroblast cells were seen in a state of active secretion of collagen fibrils (Figure 14). Collagen secretion appeared to be stimulated by feeding an elevated level of vitamin A. Increased collagen might re— sult in increased strength of the aorta. Experiment Five Silver methenamine stain was used to test for the presence of mucopolysaccharide and mucoprotein. In order to determine if a substance stained with methenamine silver was mucopolysaccharide or mucoprotein or an unrelated com- pound, it was necessary to stain the material in question without previously oxidizing it with periodic acid. Stained, unoxidized material is not mucopolysaccharide or mucoprotein. Non-specific staining of collagen and marginated chromatin was observed in the unoxidized aorta specimens as seen in Figure 15. In the oxidized specimens methenamine silver stained the basement membrane of the endothelium and what appeared to be secretory particles in the endothelial cells (Figure 16). The basement membrane appeared to be broken or sometimes absent from the vitamin A deficient animals (Figure 17). The broken basement membrane, as seen in vitamin A deficiency, was probably more permeable than the 69 Figure 13. Electron micrograph of aorta (x6,900) from vitamin A deficient Japanese quail. Numerous lipid vacuoles (V) are seen in medial smooth muscle cells. A smooth muscle cell nucleus (N) and an elastic fiber (EL) are identified. 70 Figure 13 Figure 14. 71 Electron micrograph of aorta from Japanese quail suffering from hypervitaminosis A (x18,000), stained with uranyl acetate and lead citrate. Note the proliferation of collagen from the fibroblast cell. The fibro- blast cell nucleus is identified. 72 Figure 14 73 Figure 15. Electron micrograph of aorta from a normal Japanese quail (x21,000) stained with methena- mine silver but not oxidized with periodic achL Note non-specific staining of collagen (CO). The elastic fibers (EL) do not stain. 74 Figure 15 Figure 16. 75 Electron micrograph of aorta (x9,600) from nonmfl Japanese quail oxidized with periodic acid and stained with methenamine silver. Note stained secretory granules (SE) and basement membrane (BM). Collagen (CO) and elastic fibers (EL) are also identified. 76 Figure 16 77 Figure 17. Electron micrograph of aorta (x8,400) from a Japanese quail fed vitamin A deficient ration. Specimen was oxidized with periodic acid Schiff and stained with methenamine silver. Note stained secretory granules (SE) and broken basement membrane (BM). A smooth muscle cell nucleus (S) and elastic fiber are identi- fied. 78 Figure 17 79 intact membrane of the animal fed sufficient vitamin A. Positive staining indicated that the secretory particles and basement membrane structures were probably mucopolysac- charide or muc0protein. Neither of these structures were previously observed using standard electron microscopy technique as had been used in the previous experiment. The method for staining with methenamine silver as, described by Hayat (1970) was not successful when used with aortic tissue. Therefore, modifications were made. Copper grids, which are normally used, were not satisfactory be- cause of a coarse precipitate that resulted. Staining of grid-mounted specimens proved satisfactory when titanium or stainless steel grids were used. Counting the number of silver methenamine positive particles in several cells from five animals on each treat- ment revealed that the vitamin A deficient animals had a greater number of these particles in their endothelial cells than did the controls. A t-test confirmed this appar- ent difference (P < .05). The importance of the difference in silver methenamine content in developing atherosclerosis was investigated in experiment six. Experiment‘Six Aorta scores from the birds fed the vitamin A de- ficient ration, containing 200 U.S.P. units of vitamin A per kg. of feed, were significantly greater (P < .05) than the aorta scores from birds given the ration containing 15,000 U.S.P. units of vitamin A per kg. of feed, designed 80 to cause the minimal amount of atherosclerosis. These data are summarized in Table 6. When examined under the transmission electron micro- scope, the aortas from birds fed the ration sufficient in vitamin A had significantly greater (P < .05) numbers of intact endothelial cells than the aortas from birds fed the vitamin A deficient diet (Table 6). There was a significant correlation (r = -.631) between the aorta score and the arc sin of the percent of intact cells, indicating that an intact endothelium is probably not conducive to lipid in- filtration. The number of silver methenamine positive particles in the aortas from birds fed the vitamin A deficient diet, was significantly greater (P < .05) than the number of par- ticles in aortas from birds fed the ration designed to minimize lipid infiltration. There was a significant correlation (r = .633) observed between aorta score and the number of silver methenamine positive particles, which indicates that muc0polysaccharides or muc0protein probably had a role in atherogenesis. Arc sin of the percent of intact cells showed a significant correlation (r = -.726) with the count of silver methenamine particles in the aorta endothelium. This is an indication that the presence of muc0protein or mucopolysaccharide in the form described could have been related to the phenomenon of endothelial detachment. 81 .8. V: 282:: 22828;. m.:+:.m 3.3x: 3:: 5 SEE: 8.38:5 3 +. 2.2 3 M 9:: S H S E 32:: .._.m.: :8 o .m M .58 25:8 .8 .m u .1 .m H 2. 88.28%: < 5:5; :Eemcmcse :22: 8:8 685 E E... 92 .88... «to: :82 :82 3:38. 228: 8558852. :22... .: £28: 3: 2.8 3:85:28 825 IQ, E: B: .28: 2:8 :: E: 85 :_ 5.5.285: < £58; 8 .82: :5 o HES 82 Experiment Seven The endothelial cells of the capillaries (Figures 18 and 19) from birds fed sufficient vitamin A were characterized as generally having a large nucleus, endoplasmic reticulum with ribosomes and a few pinocytosis vacuoles. Some devia- tion from the normal was seen in capillaries from vitamin A deficient animals. The capillaries from the vitamin A de- ficient animals had numerous large vacuoles in their endo- thelium as seen in Figure 20. The vacuoles observed in the capillary wall were probably the results of endothelial phagocytosis. Numerous endothelial phagocytic flaps were observed (Figures 21 and 22). In some instances blood constituents could be seen entrapped by these flaps, as seen in Figure 21, which indicated that the flaps were functional in phagocytosis. Also observed were apparent thickening on the capillary wall which might cause stenosis of the capil- lary and restriction of blood flow (Figure 23). This change in the capillary endothelium probably indicated that vitamin A is essential for normal capillary endothelial maintenance. Experiment Eight In experiment eight an attempt was made to identify the blood constituent that was being phagocytized by the capillary endothelium. Nothing similar to the phagocytized particles was seen in ultra-structural examination of fibrin clots. These phagocytized structures were probably cellular debris. Figure 18. 83 Electron micrograph of myocardial capillary (x17,500) from a normal Japanese quail. The capillary endothelial cell nucleus (N), a mitochondrion (Mi) and cardiac muscle (CM) are labeled. 84 Figure 18 85 Figure 19. Electron micrograph of a myocardial capillary (xll,700) from a normal Japanese quail. Tissue was stained with uranyl acetate and lead citrate. 86 VI M ‘;> 39:12"? ‘ Figure 19 Figure 20. 87 Electron micrograph of myocardial capillary (x44,300) endothelium from a vitamin A deficient Japanese quail stained with uranyl acetate and lead citrate. Note numerous vacuoles (V) and the endothelial cell junction (EJ). Pino- cytosis vesicles are pointed out with arrows. 88 Figure 20 Figure 21. 89 Electron micrograph of a myocardial capillary from a vitamin A deficient Japanese quail (x38,000) stained with uranyl acetate and lead citrate. Note the endothelial phagocytosis flap (EF) which is enlarged in the inserted micrograph (x58,500). A mitochondrion (Mi) is also labeled. 90 Figure 21 91 Figure 22. Electron micrograph of a myocardial capillary (x17,500) from a vitamin A deficient Japanese quail stained with uranyl acetate and lead citrate. Note arrows pointing to endothelial phagocytosis flaps, which are enlarged (x54,500) in inserted micrograph. Numerous vacuoles (V) are seen in the capillary wall. A red blood cell (RBC) is seen in the lumen of the capillary. 92 Figure 22 Figure 23. 93 Electron micrograph of a myocardial capillary from a vitamin A deficient Japanese quail (xl7,500) stained with uranyl acetate and lead citrate. Note the endothelial thickening (ET) and endothelial flap (EF). 94 Figure 23 DISCUSSION Coronary Atherosclerosis The Japanese quail is susceptible to spontaneous atherosclerosis as are numerous other birds, both wild and domestic. Increased incidence of coronary atherosclerosis was observed in the vitamin A deficient Japanese quail. Histological examination of coronary arteries revealed diffuse intimal thickening and herneation through the in- ternal elastic membrane of what appeared to be smooth muscle cells. The cells of the thickened intima had the same morphology as the medial smooth muscle cells. The coronary atherosclerotic lesion observed in the quail ap- peared to be histologically the same as the chicken lesion described by Siller (1965). Although fenestration of the internal elastic membrane was not always seen at the site of intimal thickening, it seems probable that fenestrations were, in some cases, too small to be seen with the light microsc0pe. The section examined may not have included the site of fenestration but merely the extruded media. An example of how the medial cells migrate to the intima is shown in Figure 24. Extrusion of smooth muscle cells through fenestrae of the internal elastic membrane had been 95 96 Figure 24. Diagram showing possible means of intimal thickening. Arrows indicate the probable path of cell migration. 97 Figure 24 98 seen previously by Pease and Paul (1960) and Robertson (1960). There is no known direct relationship between vitamin A andoelastin, which would explain the apparent breakage of the internal elastic membrane that occurred frequently in vitamin A deficiency. Vitamin A deficiency could have caused fenestration of the internal elastic membrane in- directly, by the associated decrease in arterial collagen content from the deficiency. Herrmann and WOodward (1969) along with observations in the present study indicated that vitamin A stimulated collagen fibroplasia. Thus, the vita- min A deficient coronary artery should have less collagen making the artery more distensible. If the artery was per- mitted to stretch more as a result of less collagen, this could cause fenestration of the internal elastic membrane which would be followed by the movement of medial cells to the intima as was observed in the coronary arteries of the vitamin A deficient Japanese quail in the present study. In the examination of coronary arteries in this study only intimal thickening was observed with no lipid contain- ing or more advanced lesions. Intimal thickening would probably lead, in time, to more advanced forms of athero- sclerosis (Prior and Jones, 1952; Wilens, 1951). Aortic Atherosclerosis A theoretical dietary level of vitamin A was calcula- ted which was optimal for minimum aortic lipid. The 99 regression line on the aorta scores from birds fed the three rations lowest in vitamin A, using the pooled data from experiments one and two, is seen in Figure 25. The aorta scores from birds fed the two diets highest in vitamin A were used to indicate an approximate baseline for athero- sclerosis in the population. The point where the regression line intersects the 95 percent confidence interval about the mean of the aorta score baseline shows an approximate minimum requirement for least lipid infiltration. This requirement is 15,000 U.S.P. units of vitamin A per kg. of feed. This is greater than the reported requirement of 4,400 U.S.P. units per kg. of feed for optimum growth (Georgis, 1966) and 3,300 U.S.P. units per kg. of feed for growth and reproduction as found by Shellenberger and Lee (1966) . Lung carcinoma can be inhibited in hamsters by ad- ministration of a dose of vitamin A which is in excess of the normal physiological requirement (Saffioti gt_gl., 1967). The present study along with the findings of Saffioti EE.E£° (1967) provides evidence that the minimal requirement for this nutrient is not the same for "normal physiological functions" as it is for disease resistance. There are several probable causes for elevated lipid content in the aortas from birds fed a vitamin A deficient diet. The probable causes of lipid elevation could be a result of detachment of aorta endothelial cells, broken base- ment membrane or poor cell membrane stability. Any one of Figure 25. 100 The regression line on the aorta score of the birds fed the three rations lowest in vitamin A using pooled data from experiment one and two. The aorta score from the birds fed the two diets highest in vitamin A were used to indi- cate an approximate base line for atherosclerosis in the population. The point where the regres- sion line intersects the 95 percent confidence internal about the mean of the aorta score base line shows the theoretical minimum require- ment of vitamin A for prevention of atherosclerosis. 101 PV- uhcfil sv.v~u> mm magmas < sass m3 m ~ h _ 4.0 $8 P—u— away .1035 these ml could a' N of the number was fou could i the res of deta in the vitami detach: could ] ferent (1969) underg rat (w 1966), mOdifi the 11 quail genes There of th 102 these mechanisms or an interaction of several mechanisms could account for increased lipid infiltration. Normal intact endothelium is a barrier to permeation of the vascular wall (Bjorkerud and Bonjers, 1971). The number of detached endothelial cells in the present study was found to be correlated with increased aortic lipid, which could indicate that increased aortic lipid permeability is the result of removal of the endothelial barrier. The site of detachment probably is a locus of increased permeability in the vascular wall. The modified appearance of the aorta endothelium from vitamin A deficient animals, which was characterized by detachment from the basal layer and vacuolated appearance, could be a function of the vitamin influence on cytodif- ferentiation, which has been described by Wolf and Deluca (1969). Although the aorta endothelium did not appear to undergo absolute metaplasia, as was seen previously in the rat (WOlbach and Howe, 1925) and in calves (Nielsen gt 31., 1966), its morphology and probable function were certainly modified. The presence of endothelial cells that protrude into the lumen of the aorta in vitamin A deficient Japanese quail should be a source of blood flow turbulence. Athero- genesis is facilitated by turbulent flow (Texon‘gg.gl., 1957). Therefore, atherosclerotic lesions would be expected in areas of the thoracic aorta that have detached endothelial cells as previously described. 103 In the normal Japanese quail aorta there appears to be a basement membrane. Basement membrane even in the ab- sence of endothelial cover has been shown to be an excellent colloidal filter (Majno and Palade, 1961). In the vitamin A deficient quail the basement membrane appeared fragmented, which could account for greater ease of penetration of lipid or lipoprotein molecules. Sufficient vitamin A is probably required to synthesize or maintain the mucopolysaccharide basement membrane. Since vitamin A appears to be necessary for proper membrane stability (Dingle and Lucy, 1962), then vitamin A deficiency should cause the cell membrane to become less stable. Instability, particularly in the endothelial cells, could increase the permeability of the vascular wall. This appears to be the least likely cause of increased lipid in- filtration as it would probably require an extreme deficiency to cause depletion of the small quantity of vitamin A in the cell membrane. The quantity of methenamine silver positive material in the aorta endothelium shows an inverse relationship to lipid infiltration and to the number of detached cells. The increase in methenamine silver positive particles may be evidence for increased muc0polysaccharide turnover as found by Thomas and Pasternak (1969) in vitamin A deficiency. Since significantly greater numbers of silver methenamine positive particles were found in vitamin A deficient animals, which had little or no basement membrane present, the 104 increase in silver methenamine positive material may be a condensation product of basement membrane. The quantity of silver methenamine positive material also showed a negative correlation with the number of intact endothelial cells. According to Ham (1969) basement membrane is an adhesive material which is functional in attachment of endothelial cells. If the basement membrane was condensing to form the particles of silver methenamine positive material, then this could account for the observed relationship of the de- tached cells, observed in vitamin A deficiency, with the silver methenamine positive particles. The absence of basement membrane probably resulted in endothelial cell detachment. Numerous endothelial phagocytic flaps were seen in vitamin A deficient animals. Some flaps encompassed unidenti- fied blood fragments while others appeared to contain blood plasma. Large vacuoles were seen in the capillary walls probably containing the phagocytized material. Using colloidal carbon as an electron dense tracer, Cotran (1965) has demonstrated that capillary vacuoles similar to those ob- served in the quail, contained material that was seen in the endothelial flaps. Capillary endothelial phagocytosis is not a normal occurrence, but is seen as a result of over- load of the reticuloendothelial system (Majno, 1965; Cotran, 1965). In the vitamin A deficient animals, Kupffer cells in the liver showed signs of degeneration (Uotila and Simola, 1938; DeRuyter, 1934). Assuming that the Kupffer cells were not functioning in the vitamin A deficient Japanese quail, 105 overload of the remaining functional reticuloendothelial system would occur, causing phagocytosis in the capillaries. Blood Pressure It appears that the blood pressure increase observed in the first experiment was a chance occurrence. This phenomenon could not be confirmed in follow—up studies. In most cases, mean blood pressure approximated the mean blood pressure previously measured by Ringer (1968). Blood pressure elevation might have been expected in the birds that showed intimal thickening. Probably either the intimal thickening did not disturb the normal distensibility of the arteries or there was a compensatory mechanism operating. The heart rates measured in the present study were slightly less than those previously observed by Ringer (1968). SUMMARY Experiments were conducted to study the influence of vitamin A on atherosclerosis. In one experiment quail chicks (Coturnix coturnix japonica) were hatched and immediately placed on diets deficient in vitamin A (880 U.S.P. units/kg. of feed), normal in vitamin A (4,400 U.S.P. units/kg. of feed) or excessive in vitamin A (22,000 or 220,000 U.S.P. units/kg. of feed). In another experiment, mature male quail were placed on the same diets as in the preceding ex- periment. At the end of 10 weeks on experimental rations, blood pressure was measured by cannulation of the carotid artery. A scoring system was devised to quantitate aortic lipid. The results indicate that birds fed low levels of vitamin A had the highest incidence of coronary atheroscle- rosis and most severe aortic atherosclerosis. It was deter- mined that a minimum of approximately 15,000 U.S.P. units of the vitamin were needed to minimize atherosclerosis in the thoracic aorta in growing quail chicks. Blood pressure and heart rate were not significantly altered by varying vitamin A in the diet. Plasma cholesterol levels were not influenced by feeding diets that varied in vitamin A content. 106 107 Examination under the electron microscope revealed that the endothelium from the aortas of birds fed a ration adequate in vitamin A was generally tightly packed with few lipid vacuoles. By comparison, the aortic endothelial cells from birds deficient in the vitamin were detached and projecting into the lumen of the vessel. The percent of intact endothelial cells was negatively correlated with minimal aortic lipid infiltration. A modification of a silver methenamine electron stain, that is specific for mucopolysaccharides and mucoprotein, is outlined. Observations using this stain revealed that vitamin A deficiency resulted in apparent broken or absent basement membranes and increased numbers of silver methena- mine positive particles in the endothelium. Myocardial capillary ultrastructure was studied. The endothelium of the vitamin A deficient animals had numerous endothelial phagocytic flaps, which were seldom seen in the birds fed sufficient vitamin A. The presence of phagocytic flaps is an indication of overload of the reticuloendothelial system, probably as the result of liver phagocyte degenera- tion from vitamin A deficiency. CONCLUSIONS Experiments conducted feeding male Japanese quail rations that differed only in vitamin A content indicated that the dietary level of vitamin A influenced atherogenesis. The incidence of coronary atherosclerosis, characterized by diffuse intimal thickening and fenestration of the in- ternal elastic membrane, was increased in vitamin A de- ficiency. It was concluded that the influence of vitamin A on coronary atherosclerosis was indirect by increasing vessel distensibility, probably through decreased collagen content from vitamin A deficiency. The results of this study indicated that feeding low levels of vitamin A enhanced lipid infiltration in the thoracic aorta. The level of dietary vitamin A that previous studies suggested was optimum for growth and reproduction was not adequate to result in minimal lipid content in the thoracic aorta. Observation under the electron microscope revealed a probable mechanism for enhanced lipid permeability in the thoracic aortas of vitamin A deficient quail. The endothelial cells in the aortas of the vitamin A deficient Japanese quail appeared to be detached from the basal layer facilitating permeation into the subendothelial layers. Staining with a histochemical electron stain which was specific 108 109 for mucopolysaccharide and mucoprotein revealed increased numbers of silver methenamine positive particles in the endo- thelium of the aortas of vitamin A deficient Japanese quail when compared with the aortas from quail fed sufficient vita- min A. The silver methenamine positive material in the endo- thelium could have been the result of condensation of the broken basement membrane which was observed in the vitamin A deficient quail. Vitamin A deficiency resulted in endothelial phago- cytosis in the endothelial cells of the coronary capillaries. It was concluded that endothelial phagocytosis was a function of overload of the reticuloendothelial system. Overload of the reticuloendothelial system was probably the result of degeneration of the Kupffer cells of the liver as a result of vitamin A deficiency. Thickening of the endothelial wall of the vitamin A deficient animal was another result of vitamin A deficiency in the quail. This degenerative change probably results in reduced blood flow in the effected capillaries. Thus, vitamin A appears to have a role in main- tenance of capillary endothelium. SUGGESTIONS FOR FUTURE STUDY 1. Test the influence of vitamin A on atherogenesis in turkeys to see if feeding a slight excess of vitamin A will reduce the severity and/or incidence of atheroma and dissecting aneurism which is associated with atherosclerosis. 2. See if there are permeability changes that can be demonstrated to be related to the vascular endothelial changes in morphology associated with vitamin A deficiency. This could be done with a Ferretin injection technique. 3. 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Histochemical studies in atherogenesis: Human aortas. Circ. Res. 8: 287-295. APPENDICES APPENDIX I Procedure for Orcein Elastic Tissue Stain Modified from McManus and Mowry (1960) 1. Acid Orcein SolutiOn Orcein 1 gm. 70% Alcohol 100 m1. Concentrated Hydrochloric Acid 0.6 m1. Dissolve orcein in alcohol. When solution is complete, add 0.6 ml. concentrated hydrochloric acid. 2. Ten Drops Hydrochloric Acid in 100 ml. Absolute Alcohol 3. Van Gieson Solution 1% Aqueous Acid Fuchsin 15 m1. Saturated Aqueous Picric Acid 50 ml. Distilled Water 50 ml. Procedure 1. Bring paraffin sections to water. 2. Place for 45 minutes in acid orcein. 3. Rinse in distilled water for 3 minutes. 4. Wipe off excess water and dip into 95% alcohol to remove excess dye. 120 10. 121 Transfer to absolute alcohol for decolorization for 10 minutes. This should be followed both visually and with the microscope and when the section first becomes a pale brown, it should be examined in the low power of the microscope, where the elastic fibers stand out as clear purple units. Complete the decolorization of the background in 10 drops of hydrochloric acid in 100 m1. of absolute alco- hol. This requires 4 minutes. Wash in tap water, at least 5 minutes. Counterstain with Van Gieson solution 2 minutes. Differentiate by dipping in picric acid solution until only collagen is red. Dehydrate in graded alcohols, clear in xylene, and mount. Results red. Elastica is deep dark brown or black. The collagen is The cytoplasm and muscle are pale yellow to yellow- brown and nuclei are faintly colored with the orcein. APPEND IX I I Oil Red 0 Lipid Staining Procedure Oil‘Red‘O'Solution Oil Red 0 5 gm. Propylene Glycol 100 ml. Add a small amount of propylene glycol to the oil red O and mix thoroughly. Add the remainder of the propylene glycol, stirring periodically. Heat solution to 950 C., but do not allow it to go over 1000 C. Let stand 12 hours, then filter. Refilter if solution becomes turbid. Staining‘Procedure 1. Collect frozen sections in distilled water. 2. Dip in 70% ethyl alcohol for 5 to 10 seconds. 3. Stain in oil red O for 4 minutes. 4. Rinse quickly in 70% alcohol. 5. Wash in distilled water. 6. Stain in Harris hematoxylin 1 minute. 7. Wash in distilled water 5 minutes. 8. Wash in tap water 5 minutes. 9. Place sections in distilled water in a glass staining dish, removing the staining platform. 122 10. 11. 12. 123 Float sections onto clean slides using a dissecting needle. Allow section to dry to slide, then place in distilled water to rehydrate 30 minutes. Mount with warm glycerin jelly. APPENDIX I I I Procedure for Plasma Cholesterol Analysis Based on That of Zlatkis ‘_e_t_' 'e_1_1_., 1953 1. Standard solutions of 100, 200, 300 mg. % were made by adding 100, 200 and 300 mg. of pure, dry, ash-free cholesterol to 100 m1. 100% glacial acetic acid. Ferric Chloride--Solution was made by adding 10 gm. of reagent grade ferric chloride to 100 ml. of 100% glacial acetic acid. The color reagent was made by diluting 2.0 ml. of ferric chloride solution to 200 m1. H2804 (C.P. concentrated H2804) using a volumetric flask. Ferric chloride solu- tion should be added slowly while swirling flask. AnalysiS‘Procedure 1. Mix thawed plasma. Place 10 ml. of glacial acetic acid in 20 m1. test tubes. Pipette 0.1 ml. of sample to each tube. Add 7 m1. of color reagent and mix. Cover tubes with plastic stoppers and invert tubes three times (tubes will become hot). Let stand for one hour. Read on spectrophotometer at 560 mu. zeroed with a blank of 10 ml. glacial acetic acid and 7 ml. of color reagent. 124 APPENDIX IV 0.2 Sorensen's Phosphate Buffer Stock Solution A 0.2 solution monobasic NaHZPO4 27.8 gm./1000 m1. H o 2 Stock Solution B 0.2 M solution dibasic Na2HPO4 53.65 gm. Na HPO .7 HZO/IOOO ml. H 2 71.7 gm. NaZHPO 4 4 .12 H20/1000 m1. H Solution A Solution B 33 67 28 72 23 77 19 81 Keep refrigerated 125 2 2 O 0 pH 7.1 7.2 7.3 7.4 APPENDIX V E. M. Fixation Procedure for Avian Tissue The following procedure gave optimal results fixing aorta and myocardium of Japanese quail. The procedure has been found successful with other avian tissue. 1. Fix in 6.25% glutaraldehyde in Sorensen's buffer (pH 7.2) for 2 hours. 2. Post-fix in 2% osmium tetroxide aqueous solution diluted to 1% with Sorensen's buffer (pH 7.2) for 1 hour.* 3. Wash twice with Sorensen's buffer 15 minutes each. 4. Dehydrate in 25%, 50%, 75%, 95% for 10 minutes in each and in 100% alcohol or acetone twice for 15 minutes each. *This step should be carried out under a ventilator hood as osmium tetroxide fixes lung and corneal tissue. 126 APPENDIX VI Embedding Procedure using Epon 812--Based on Luft's (1961) Procedure The Embedding Medium Formulation of Epon embedding medium varies from batch to batch depending on the weight per epoxide equivalent (WPE.) of epoxy resin in the batch. Variation in formulation is essential in order to obtain reproducible embedding that can be sectioned easily. Formulations are calculated in parts of reactant per 100 parts of epoxy resin (p.h.r.). The amount of anhydride required is calculated as follows: 100 p.h.r. anhydried = fi§§—-x anhydride equivalent x ratio (anhydride-epoxy) 100 = grams of epoxy resin WPE. (epoxy equivalent) = weight of epoxy resin con- taining one equivalent weight of epoxide Anhydride equivalent = molecular weight of anhydride Ratio canhydride - epoxy = ration of equivalent of epoxide equivalent (.7) Mixture‘A When the WPE. of Epon is 150, add 100 gm. of DDSA (dodecenyl 127 128 succinic anhydride) in to 80 gm. of Epon 812. Shake vigorously. p.h.r. = ‘80 x 226 x .7 = 99.3 Mixture B When the WPE. of Epon 812 is 150, add 84 gm. of NMA (nadic methyl anhydride) to 80 gm. of Epon 812. Shake vigorously. p.h.r. NMA = %g%-x 178 x .7 = 83.07 When WPE. of Epon 812 is not 150, use table below to deter- mine DDSA and NMA requirement: Epon 812 Mixture A* Mixture B** WPE. DDSA NMA Wt. in grams Wt. in grams 140 106 89 141-142 105 88 143-144 103 87 145-146 102 86 147 101 85 148-149 100 84 150-151 99 83 152-153 98 82 154 97 81 155-156 96 80 157-158 95 79 159-160 94 78 A mixture of A:B, 7:3 plus four drops of UMP-30 accelerator was optimal for the tissues examined. This combination pro- duced consistently good sections. 129 Mixtures A and B can be stored for six months. The refrigerated mixtures must be warmed to room temperature before combining in order to prevent water condensation. Water in Epon will result in soft blocks from which thin sections cannot be obtained. It is essential that the A, B and DMP-30 combination be mixed, using glass rod, for a full five minutes to insure homogeneity. The Embedding Process 1. Dehydrate as described previously. 2. Wash in propylene oxide twice 20 minutes each. 3. Place specimens in 1:1 mixture of propylene oxide and Epon for 12 hours (in dessicator). 4. Infiltrate with Epon 12 hours (in dessicator). 5. Harden in 600 C. oven for 48 hours. APPENDIX VII Electron Staining Procedure with Uranyl Acetate and Lead Citrate Uranyl Acetate Solution 1. Make a saturated solution of uranyl acetate (approximately 1%). 2. Filter the solution. Lead Citrate Solution (Reynolds, 1963) Lead nitrate 1.33 gm. Sodium Citrate 1.76 gm. Distilled Water 30 ml. Shake intermittently for 30 minutes. Add 8 ml. of IN. NaOH (carbonate free) to clean. Staining‘Procedure 1. Place drops of uranyl acetate on clean surface. 2. Place specimen grids, sample side down, on the uranyl acetate solution. 3. Cover with a petri dish and stain for 30 minutes. 4. Rinse specimens three times with distilled water. 5. Place drops of lead citrate on a clean surface. 6. Place specimen grids, sample side down, on the lead citrate solution. 130 131 Cover with petri dish under which NaOH pellets have been placed. The NaOH pellets are used to adsorb CO2 and pre- vent lead carbonate precipitation on the specimens. Rinse specimens in .02N. NaOH. Rinse in distilled water. APPENDIX VI I I Methenamine Silver Staining Procedure Silver Methenamine Solution Hexamethylene tetramine ( 3%) 18 m1. Silver nitrate (10%) 2 m1. Sodium borate ( 2%) _2_ml. 22 m1. Centrifuge the above solution at 3,000 rpm for 10 minutes and use the supernatant to stain with. The stain must be used the same day it is made. Staining Procedure The following procedure gave consistent optimal results. 1. Fix in 6.25% glutaraldehyde made up on Sorensen's buffer pH 7.4 for 12 hours. ‘No post-fixation. 2. Dehydrate in alcohol and embed in Epon 812 (Luft, 1961). 3. Section and place sections unsupported on either stain- less steel or titanium grids. 4. Oxidize specimens for 15 minutes in a freshly made 1% periodic acid solution. 5. wash 3 times with distilled water. 6. Place sections on grids in freshly made silver methenamine solution (see Table l) uncovered in a darkened oven at 60° c. for 45 minutes. 132 133 7. Wash three times with distilled water. 8. Submerge grids in 3% sodium thiosulfate solution for 5 minutes. 9. Wash three times with distilled water. To determine if the material being stained was either mucopolysaccharide or mucoprotein, or was an unrelated substance, one grid was stained from each tissue with the described procedure omitting the oxidation with periodic acid (step 4). If the structure in question stained with- out oxidation, then the substance was not a mucopolysac- charide or a mucoprotein. Centrifuge the silver methenamine solution at 3,000 rpm for 10 minutes and use the supernatant for staining. New staining solution must be prepared daily because of its instability. The usual procedure for staining is to use covered con- tainers to prevent contamination from dust in the air and reduce evaporation loss (Hayat, 1970: Pease, 1964). In preliminary studies in this laboratory covered containers used during silver methenamine exposure resulted in re- fluxing of the unstable silver solution which caused pre- cipitation of the silver solution and obscured the specimen, leaving the staining containers uncovered prevented the pre- cipitation problem. Staining on titanium or stainless steel grids proved to be satisfactory, whereas copper grids were not. In addition, grid mounting resulted in greater ease of handling than floating the sections, as was done by Rambourg and Leblond (1967) and Suzuki and Sekiyama (1961). APPENDIX IX Spurr Low Viscosity Embedding Medium (Spurr, 1969) Spurr Embedding Medium 1. Vinylcyclohexene diocide 10 gm. 2. Diglycidyl ether of polypropyleneglycol 6 gm. 3. Nonenyl succinic anhydride 26 mg. 4. Dimethylamino ethanol .4 gm. The mixture may be used immediately or stored in a freezer for 4 months. Procedure 1. Fix as described in Appendix V. 2. Dehydrate in acetone. 3. Infiltrate 4 hours with a mixture of 3:1.acetaon:Spurr mixture. 4. Infiltrate 12 hours in 1:1 acetone:Spurr mixture. 5. Infiltrate 12 hours in Spurr mixture. 6. Polymerize at 700 C. for 7 hours. 134 APPENDIX X Spurr Stain Procedure Spurr Stain 100% Methanol 25 ml. 70% Ethanol 75 ml. Uranyl Acetate .5 gm. Stir the solution and let stand. StainingiProcedure 1. Place drops of stain on clean surface. 2. Place specimen grids on stain solution. 3. Rinse three times in distilled water. 135 APPENDIX XI Analysis of variance of blood pressure Experiment One Source of variation df Mean square Treatment 3 1754.06* Error 11 440.13 Total 14 Experiment Two Source of variation df Mean square Treatment 3 367.15 Error 23 383.59 Total 26 Experiment Three Source of variation df Mean square Treatment 3 456.22 Error 20 218.75 Total 23 *Significant at P < .05. 136 APPENDIX XII Analysis of variance of aorta scores Experiment One Source of variation df Mean square Treatment 3 69.06** Error 17 9.90 Total 20 Experiment Two Source of variation df Mean square Treatment 3 96.73*** Error 27 6.01 Total 30 Experiment Three Source of variation df Mean square Treatment 3 24.82 Error 29 13.85 Total 32 **Significant at P < .01. ***Significant at P < .005. 137 APPENDIX XII I Analysis of variance of heart rate Experiment One Source of variation df Mean square Treatment 3 6145.38 Error 11 21932.87 Total 14 Experiment Two Source of variation df Mean square Treatment 3 6530.73 Error 23 5406.68 Total 26 Experiment Three Source of variation df Mean square Treatment 3 3087.60 Error 20 2815.26 23 Total 138 APPENDIX XIV Analysis of variance of plasma cholesterol values Source of variation df Mean square Treatment 3 14520.59 Error 21 6969.49 Total 24 139 APPENDIX XV Analysis of variance of regression of aorta score and log vitamin A concentration Experiment One Source of variation df Mean square Regression l l73.1** Deviation from regression 19 10.67 Total 20 Experiment Two Source of variation df Mean square Regression 1 l90.27** Deviation from regression 29 9.04 Total 30 Experiment Three Source of variation df Mean square Regression l 28.42 Deviation from regression 31 15.06 Total 32 **Significant at P < .01. 140