Transposition of the great vessels and other cardiovascular abnormalities in rat fetuses induced by trypan blue.код для вставкиСкачать
Transposition of the Great Vessels and Other Cardiovascular Abnormalities in R a t Fetuses Induced by Trypan Blue ’ I. W. MONIE, EVA TAKACS AND J. WARKANY Department of Anatomy, University of California Medical Center, San Francisco, California, and the Children’s Hospital Research Foundation and the Department of Pediatrics, University of Cincinnati, Cincinnati, Ohio ABSTRACT Complete and p a r t i d transposition of the great vessels, dextrocardia, and absence or stenosis of the tricuspid or mitral valves were encountered singly or in combination in rat fetuses from mothers injected with trypan blue solution subcutaneously on the 8th or 9th day of gestation. Hearts with transposition of the great vessels were usually characterized by shortness of the aorta and pulmonary trunk, cranial location of the aortic valve, displacement of the coronary arteries, and a channel or “track” extending from the cranial portion (infundibulum) of the right ventricle to the commencement of the transposed pulmonary trunk. Inadequate expansion of the atrioventricular ring about the 12th or 13th day of gestation possibly leads to deformity of the atrioventricular cushions and to absence or stenosis of the tricuspid or mitral valves. Sinuosity of the truncus arteriosus seen in many 13th day and older embryos of the trypan blue series is considered a significant factor in the development of transposition of the great vessels. Transposition of the great vessels occurs in about 12% of cases of human congenital heart disease which come to autopsy (Keith et al., ’58). In complete transposition the aorta and pulmonary trunk arise from the right and left ventricles respectively, and pass cranially more or less parallel to one another. Other forms of transposition include partial transposition in which both great arteries arise from the right ventricle, and “corrected” transposition in which the aorta and pulmonary trunk spring from the proper ventricles, but the former vessel lies ventrosinistrally to the latter. Transposition is often accompanied by ventricular or atrial septa1 defect or by patency of the ductus arteriosus; in addition, the coronary arteries are usually displaced and the ventricular septum lacks its membranous portion and is entirely muscular (Lev. ’53). In about 7% of human hearts with transposition the tricuspid valve is absent; on the other hand, almost 30% of cases of absence of the tricuspid valve are accompanied by transposition Various hypotheses have been advanced to account for this malformation. Thus, Rokitansky (1875) attributed it to abANAT. REC., 156: 175-190. normal convexity of the proximal truncobulbar septum and its incorrect fusion with the ventricular septum while Pernkopf and Wirtinger ( ’ 3 3 ) incriminated abnormal spiralling of the entire truncobulbar septum following faulty rotation of the developing heart tube. Shaner (’51), however, concluded from a study of pig embryos that tardy descent of the aorticopulmonic septum and its delayed fusion with the bulbar cushions (ridges) after they had undergone rotation was the key factor in the causation of transposition. Keith (’09), on the other hand, attributed transposition to abnormal absorption of the bulbus cordis whereas Lev and Saphir (’45) blamed maldevelopment of the bulbus cordis and the presence of an abnormal bulbar cushion. On somewhat similar lines, de la Cruz and da Rocha (’56) have stressed the importance of a combination of abnormality of the conoventricular flange (bulboventricular spur), the truncoconal ridges, and the primordia of the aortic and pulmonary valves as factors 1 Supported by U. S. Public Health Service grants HE-07029.HD-00419 and. HD-00502. The term “transposition” hereafter will refer to transposition of the great vessels unless otherwise stated. 2 175 176 I. W. MONIE, EVA TAKACS AND J. WARKANY producing this abnormality. Recently, the significance of the truncobulbar region in the development of transposition has been shown by Le Douarin ('61) who x-irradiated this part in the chick embryo and obtained abnormal spiralling of the contained septum while Rychter ('62) has produced transposition in the chick by temporarily applying a microclip around the bulbus cordis. Spitzer ( ' 2 3 ) , using a phylogenetic approach, attempted to explain transposition principally on the emergence of reptilian characteristics in the mammalian embryo. He suggested that a n aorta arose from the right ventricle as in certain reptiles while that from the left ventricle was suppressed; this, accompanied by altered location of the ventricular septum, supposedly resuIted in the pulmonary trunk springing from the left ventricle. Initially, this view received considerable support (Abbott, '36; Harris and Farber, '39) although it was known that mammals most likely evolved from a pre-reptilian ancestor lacking the cardiovascular peculiarites of modern reptiles (Goodrich, '30) ; also, no reptilian characteristics have ever been observed in developing mamalian hearts. Evidence against Spitzer's concept has been summarized recently by Shaner ('62). Bremer ('28) proposed that the blood streams ejected from the developing right and left ventricles influenced truncobulbar septation and, consequently, alteration in the course of one or both streams might result in transposition or other abnormality of the great vessels. The importance of such streams in normal and abnormal cardiac development in man (De Vries and Saunders, '62) and chick (Jaffee, '65) has been discussed recently. From a later study of a n early human embryo Bremer ('42) concluded that other factors, increased flexion of the heart tube and altered disposition of the sinusoids in the heart wall, might also be of importance in producing transposed great vessels. Difficulty i n determining the fundamental cause of transposition has been due largely to the lack of mammalian embryos which show this abnormality in the early stages of formation. However, it has been shown that x-irradiation (Wilson et al., '53) and trypan blue (Wilson, '55) can each produce transposition in rat young and consequently valuable tools have been provided for studying this malformation. Studies on trypan blue as a cardiovascular teratogen have been reported also by Fox and Goss ('56, '57, '58), Christie ('61), Wegener ('61), Smith ('63), and Inoue ('64). Since none of the hypotheses yet advanced to explain transposition of the great vessels seems entirely satisfactory, it was decided to study the development of this abnormality in rat fetuses from mothers injected with trypan blue solution during early pregnancy. In addition to obtaining transposition of the great vessels, dextrocardia, and abnormalities of the tricuspid and mitral valves were often eiicountered (fig. 1). The apparent causes of these different malformations and their possible interrelationships will be considered. MATERIALS AND METHODS Pregnant rats of a Long-Evans substrain were injected subcutaneously with 1 cm3 of 1 % trypan blue in 0.8% saline on either the 8th or 9th day of gestati0n.j Fetuses were removed from control and trypan blue injected mothers from the 11th to 22nd day of pregnancy and some newborns were collected i n addition. A total of 226 experimental and 48 control young were examined (table l ) , prepared for histological study, serially sectioned at 8-10 p, and stained with hematoxylin and eosin. Wax plate reconstructions were made of hearts from 40 experimental and 9 control fetuses. Since interest was focused mainly on transposition of the great vessels a n attempt was made to select for detailed study fetuses which on inspection appeared to have this abnormality; this bias, however, relates only to older fetuses in which the aorta and the pulmonary trunk were separate and readily distinguishable. 3Obtained from Rockland Farms. New Citv. N. Y. Both before and during gestation rats were fed Purina Laboratory Chow, Ralston Purina Company, St. Louis, Missouri. 4 Trypan blue (Direct blue 14) CI 23850; Matheson, Coleman and Bell. 5Day of finding sperm in the vagina is considered the first day of pregnancy. 177 TRANSPOSITION O F GREAT VESSELS NORMAL T R A N S POSIT!ON LV RV T R l C U S P l D VALVE ABSENCE 0 OF GREAT VESSELS RV \ LV @ .__- P Fig. 1 Normal and abnormal hearts from rat fetuses late in pregnancy. The left anterior vena cava terminates in the right atrium in the same manner as, the coronary sinus in man. The ductus arteriosus connects the pulmonary trunk with the aorta. Key: A, aorta; L, left anterior vena cava; LA, left atrium; LV, left ventricle; P, pulmonary trunk; PC, posterior vena cava; R, right anterior vena cava; RA, right atrium; RV, right ventricle. TABLE 1 Rat embryos sectioned Trypan blue injected on 8th day Trypan blue injected on 9th day 11 11 15 31 7 7 0 0 0 0 0 5 5 6 5 18 11 10 16 16 21 16 7 10 9 17 20 49 18 17 16 16 21 16 7 15 14 81 145 226 12 13 14 15 16 17 18 19 20 21 22 1 Total 1 Total Day of gestation EgEi Controls 5 4 4 4 6 4 3 3 4 3 4 4 48 Includes some newborn. RESULTS Embryos from trypan blue injected mothers were generally smaller than those Of Of the age and Of the 226 examined, 76 (30.0% ) showed abnormalities of the heart and great vessels,e sepa- rately or in Combination; the principal cardiovascular abnormalities observed in embryos from the 13th to 22nd day are 6 The types of cardiovascular abnormalities obtained with injection of trypan blue on !he Sth da,y of gestation were similar to those following lnjectlon on the 9th day. 178 I. W. MONIE, EVA TAKACS AND J. WARKANY 0 .* r= %.i xl a“$ M shown in table 2. Of the anomalies encountered i n other systems, anophthalmia and microphthalmia (left eye - 11; right eye - 7) were the most frequent. Exencephaly and diaphragmatic hernia each occurred twice while spina bifida, hydrocephaly, absence of the left lobe of the thyroid gland, and esophageal atresia were each observed once. These malformations showed no particular relationship to any of the accompanying cardiovascular abnormalities except esophageal atresia which was associated with a double aorta. No instance of absence of the spleen was encountered. Control embryos. In 11th day control embryos the heart was a n S-shaped tube consisting of sinus venosus, primitive atrium, primitive ventricle, bulbus cordis (conus arteriosus) and truncus arteriosus. The bulbus ’ lay to the right of and usually somewhat dorsal to the ventricle while the truncus swept craniodorsally in a sinuous manner to give origin to the first. and occasionally to the second, branchial (aortic) arch arteries. On the 12th day the ventricle was more caudally situated with the primitive atrium expanding to the right. The truncus was generally similar to that of the previous day but, in some embryos, the two truncobulbar cushions were beginning to form; the latter spiralled through about 90”. The second, third and fourth branchial arch arteries were now discernible, and the common pulmonary vein usually entered the left horn of the sinus venosus. With the establishment of the septum primum and the interventricular septum on the 13th day, the four heart chambers became recognizable, the primitive ventricle forming the left ventricle and the bulbus cordis the right ventricle. The dorsal and ventral atrioventricular cushions were now apparent (fig. 5c) although not always in contact and each atrium communicated either with the left ventricle only or with the ventricle of its own side. The truncus now ascended obliquely from the heart (fig. 2a) and the truncobulbar cushions showed increased spiralling which in one embryo amounted to about 225”. The third, four, and sixth branchial arch 7The term “bulbus” or “bulb” refers to the bulbus cardis; likewise “truncus” refers to truncus artenosus. TRANSPOSITION O F GREAT VESSELS NORMAL EXF E R I M E N T A L 179 E XPERl MENTA L I ZTU DAY I 4TH DAY 15'" DAY Fig. 2 Wax-plate reconstructions of normal and abnormal rat embryo hearts from the 13th, 14th and 15th day of gestation. Each is shown from the front (left) and from the left side (right). The controls were normal in appearance. 13th day: ( a ) control; ( b ) and ( c ) sinuous truncus. 14th day: ( d ) control; ( e ) and ( f ) sinuous undivided truncus accompanied by absence of the tricuspid valve. In ( e ) the left atrium is small and lies dorsal to the right atrium; i n ( f ) the right ventricle is cranially located. 15th day: ( g ) control; ( h ) the right ventricle is cranially situated, the pulmonary trunk lies dorsal to the aorta, and the right and left atria open into the left ventricle; ( i ) absence of tricuspid valve, cranial location of right ventricle, and dextrocardia. In ( c ) , ( e ) and ( f ) spiralling of the truncobulbar cushions was slight or absent. Key: B, bulbus cordis; D, ductus arteriosus; T, truncus arteriosus; V, primitive ventricle. Rest of key as in figure 1. arteries were present and the pulmonary arteries appearing. By the 14th day the foramen secundum was apparent in the septum primum, the pulmonary trunk was separating from the aorta, and portions of the branchial arch arteries were regressing; also, the right umbilical vein was disappearing and the left umbilical vein enlarging. The aorta now ascended almost vertically from the heart (fig. 2d) while the partially separated pulmonary trunk swept directly dorsally to be continuous with the left ductus arteriosus. On the 15th day the atrioventricular cushions were fused and the pulmonary trunk, almost completely separated, passed around the left side of the aorta (fig. 2g). The interventricular foramen was still patent and the ventricles had become pyriform in shape. The septum secundum was recognizable. By the 16th day, part of the dorsal atrioventricular cushions had advanced into the interventricular foramen and the ascending aorta, communicating with the left ventricle through the former, increasing in caliber. The aortic and pulmonary valves 180 I. W . MONIE, EVA TAKACS AND J. WARKANY were now distinguishable, the former lying caudal to the latter; the left coronary artery was usually discernible. On the 17th day the margins of the interventricular foramen were opposed, the right coronary artery recognizable, and the right aorta and right ductus arteriosus disappearing. The configuration of the heart and great vessels remained generally the same for the rest of gestation (fig. 3a). The left umbilical artery disappeared about the 19th day. Embryos f r o m t r y p a n blue injected mothers 11th a n d 12th d a y embryos. Generally, the hearts of 11th day embryos resembled those of the corresponding controls. Cardiac abnormality was difficult to assess as even the controls vaned considerably in NORMAL shape, nevertheless, in one embryo the position of the ventricle suggested beginning dextrocardia. In 12th day embryos the hearts were usually similar to those of the controls but in one the atrioventricular canal appeared narrow while in another the common pulmonary vein terminated in the right horn of the sinus venosus indicating possible inversion of the atria (fig. 5a). In several embryos the appearance of the branchial arch arteries was delayed. 13th, 14th a n d 15th d a y embryos. In 8 of the 11 wax-plate reconstructions made of hearts from the 84 embryos of this group the truncus still followed a sinuous path especially where the right or left atrioventricular channel was absent. In the latter instance, the bulbus or the right ventricle usually lay cranially and one or TRANSPOSITION RA LV 0 Fig. 3 ( a ) Normal rat fetal heart on 21st day. ( b - f ) Complete transposition of the great vessels in hearts from the 18th to 22nd day of gestation accompanied by: persistent interventricular foramen ( b ) ; closed interventricular foramen ( c ) ; absence of tricuspid valve ( d ) ; and absence of tricuspid valve and dextrocardia (e). In ( f ) there is partial transposition in which both aorta and pulmonary trunk arise from the right ventricle; also, the aorta lies ventral and sinistral to the pulmonary trunk. The picture is ahnost that of “corrected” transposition. Figures in parentheses indicate the distance from the aortic valve to the aortic arch expressed as a percentage of the distance from the posterior caval opening to the aortic arch. Key as in figure 1. TRANSPOSITION O F GREAT VESSELS 181 Fig. 4 Portions of wax-plate reconstructions of ( a ) normal and ( b ) abnormal hearts of 14th day embryos. Each shows the relationship of the truncobulbar outflow tract to the interventricular septum and to the ventricles. The truncobulbar cushions are shown (coarse stipple) and septation is incomplete. In ( a ) the entrance to the aorta ( A ) lies dorsally and that to the pulmonary trunk ventrally; the latter overlies the right ventricle. Only a small crescentic portion of the left ventricle (LV) is visible between the upper edge of the ventricular septum (IVS) and the left truncobulbar cushion. In ( b ) the tricuspid valve is absent and the truncus so sinistrally placed that its lumen straddles the free edge of the ventricular septum. Truncobulbar septation is retarded and there is only slight spiralling of the cushions. The pulmonary trunk occupies the dorsal portion of the truncus and, consequently, it almost entirely overlies the left ventricle; the aorta is ventrally 10cated and mostly overlies the right ventricle. The bifid ventral atriovenlricular cushion is shown in black in (b). A portion of the dorsal atrioventricular cushion ( D ) is seen in contact with the free edge of the ventricular septum i n both (a) and (b). Rest of key as in figure 1. other atrium was enlarged and displaced (fig. 2b,c,e,f). In three embryos the region of the truncobulbar junction was so sinistrally placed that it communicated with both the left and right ventricles (fig. 4b). In several 15th day embryos separation of the aortic and pulmonary trunks was delayed and in these the truncobulbar cushions showed only minimal spiralling or none at all; in such embryos, the pulmonary trunk lay mostly dorsal and somewhat caudal to the aorta (fig. 2h). Absence of the tricuspid valve was observed in 12 embryos while in two embryos with inversion of the ventricles the tricuspid valve proper was absent in one and the mitral valve proper was missing i n the other; in addition, one embryo showed tricuspid stenosis. Where the tricuspid or mitral valve was absent the dorsal and ventral atrioventricular cushions were usually large and misshapen; the latter cushion was especially affected and frequently was bipartite or horse-shoe-shaped so that it partly encircled its dorsal counterpart (fig. 5d). Where the tricuspid or mitral valve was absent the corresponding atrium was usually distended but occasionally it was small and the opposite atrium enlarged; in some instances one atrium lay cranial to the other and the ventricles were displaced (fig. 2h,i). In two 14th day embryos both the tricuspid and mitral valves opened into the left ventricle as in some 12th day control embryos. Dextrocardia was noted in eight embryos and in one of these it was accompanied by inversion of the ventricles and absence of the mitral valve proper; in four others the tricuspid valve was absent. The three remaining embryos of this group were examples of: isolated dextrocardia, 8 In this paper “absence of the tricuspid (or mitral) valve” means that neither the ostium nor the valve leaflets were pmesent. I. W . MONIE, EVA TAKACS AND J. WARKANY Fig. 5 ( a ) Beginning inversion of the atria i n a 12th day experimental embryo common pulmonary vein opens into the right horn of the sinus veaosus. x 65. 111 which the ( b ) Inversion of the ventricles in a 13th day experimental embryo; the atria are normally placed. Cushions are visible within the truncus. X 35. x ( c ) 13th day control embryo showing disposition of chambers and atrioventricular cushions. 42. ( d ) 13th day experimental embryo with absence of the tricuspid valve, displaced deformation of the ventral atrioventricular cushion. X 42. atria and Key; AT, primitive atrium; D, dorsal atrioventricular cushion; LA, left atrium; LV, left ventricle; PV, common pulmonary vein; RA, right atrium; RV, right ventricle; T, truncus artenosus; V, ventral atrioventricular cushion; X, anatomical left ventricle; Y, anatomical right ventricle (bulbus cordis). TRANSPOSITION O F GREAT VESSELS complete situs inversus, and partial situs inversus. 16th a n d 17th d a y embryos, Of 32 embryos, 14 showed delayed separation of the aorta and pulmonary trunk accompanied by patency of the interventricular foramen. In one 16th day embryo the aortic and pulmonary valves lay in the same transverse plane, and a similar arrangement was found in a 17th day embryo in which the right ventricle was displaced cranially and the aorta and the pulmonary trunk were shorter than in the controls. 18th to 2 2 n d d a y embryos a n d n e w borns. Excluding patency of the interventricular foramen, the most frequent cardiovascular abnormalities in the 73 embryos of this age group were: transposition of the great vessels ( 2 0 ) , dextrocardia ( 7 ) , and absence of the tricuspid valve (7). Of the 20 embryos with transposition (table 2 ) , the aorta arose from the right ventricle and the pulmonary trunk from the left ventricle, in ten. In the remainder, either the aorta ( 2 ) or the pulmonary trunk (5) overrode the ventricular septum, while in three both the aorta and the pulmonary trunk arose from the right ventricle, the former vessel being ventral and sinistral to the latter (fig. 3f). Where the pulmonary trunk overrode the ventricular septum it always lay dorsal to the aorta. In 17 of the 20 embryos with transposition the interventricular foramen was patent, sometimes widely so, and in two there was a ventricular septal defect in addition; the latter abnormality also occurred in one embryo i n which the interventricular foramen was closed. A n atrial septal defect was seen twice in association with transposition being accompanied in one embryo by pulmonary stenosis and in the other by absence of the tricuspid valve. Transposition of the great vessels was associated with absence of the tricuspid valve in four embryos, once with tricuspid stenosis, twice with absence of the mitral valve, and once with mitral stenosis. Dextrocardia accompanied transposition in three embryos in which there was also absence of the tricuspid valve ( 2 ) or tricuspid stenosis. In one 18th day and one 20th day fetus with transposition, both the tricuspid and mitral valves opened into the left ventricle 183 and the right ventricle was reduced i n size. I n many fetuses, both those with and without transposition, the time of appearance of the coronary arteries was delayed. I n the presence of transposition the coronary arteries were often abnormally located although usually retaining the same general relationship to the pulmonary trunk as in the controls; in some instances, however, they arose normally then followed a bizarre course. In one 22nd day fetus with transposition a single coronary artery was present. In all fetuses with transposition the aortic valve lay either on the same level as the pulmonary valve or cranial to it and the normal relationship (aortic valve caudal to pulmonary valve) was not seen; in such embryos both the ascending aorta and pulmonary trunk seemed shortened (fig. 3b-f). Also, where the great vessels were transposed the heart was often markedly rotated to the left or right and the atria displaced i n a n extreme fashion. In fetuses with both transposition and absence or stenosis of the tricuspid valve, cranial displacement of the right ventricle was marked (fig. 3d, e ) . Several embryos with transposition showed a channel or “ t r a c k extending from the cranial end of the right ventricle towards the origin of the pulmonary trunk (figs. 6c and 7b, e ) ; in some instances, it communicated with the left ventricle. The path of this “ t r a c k and the course of the pulmonary trunk together formed a curving line which swept around the ascending aorta in the same manner as the pulmonary trunk in the controls. Absence of the tricuspid valve occurred in 7 of the 73 fetuses of this group (18 to 22 days fetal age) and in four was associated with transposition; in two of the fetuses with the latter abnormality there was also dextrocardia. Absence of the mitral valve occurred twice and in both instances was associated with transposition. Tricuspid stenosis was also encountered twice and was accompanied by transposition and dextrocardia once; mitral stenosis occurred twice and was associated with transposition once. Dextrocardia also occurred in 7 of the 73 fetuses of this group. In two, it was 184 Fig. 6 X 16. 1. W. MONIE, EVA TAKACS AND J. WARKANY ( a ) Great vessels and heart of normal 22nd day embryo; pulmonary valves are shown. ( b ) A more caudal section of the previous embryo showing the aortic valves and infundibulum of the right ventricle. X 16. ( c ) Transposition of great vessels in a 19th day experimental embryo. The pulmonary trunk lies dorsal to the aortic valve two cusps of which are seen. X 24. ( d ) A more caudal section of the heart of the previous embryo showing the pulmonary trunk arising mostly from the left ventricle. The interventricular foramen is open. x 24. Key: A, aorta; C , “track” or channel; I, infundibulum; P, pulmonray trunk. Rest of key as in figure 5. T R A N S P O S I T I O N O F GREAT VESSELS 185 Following their study of 18% and 19% accompanied by absence of the tricuspid valve alone, and in one by the same ab- day rat fetuses obtained from mothers innormality and transposition. Dextrocardia jected with trypan blue, Fox and Goss ('57) was also encountered as part of complete considered that the primary cause of carsitus inversus in one embryo and as an diovascular malformations induced by this isolated event in another. In two fetuses agent was abnormal looping of the heart with dextrocardia the left, and in three the tube which eventually led to displacement and rotation of the primitive atrium; this right, descending aorta was present; in suggestion has received support from both two others there was a double aorta. Christie ('61) and Smith ('63). Other cardiovascular abnormalities obAbnormal looping of the heart tube, served in this group of fetuses, singly or except for that associated with beginning in combination, were : persistent truncus dextrocardia, was difficult to determine in arteriosus (5) ; pulmonary stenosis (3); 1l t h day experimental embryos because atrial septa1 defect (2); and single coro- of the variations in heart shape observed nary artery ( 2 ) . Pulmonary stenosis was in controls of similar age. However, apalways accompanied by stenosis or absence parent inversion of the atria and narrowof the ductus arteriosus. A single pulmo- ness of the atrioventricular canal were nary artery, an aortic valve with four distinguishable on the 12th day, and by cusps, stenosis of the left duct of Cuvier the 13th day abnormalities of the atrio(left common cardinal vein), and stenosis ventricular channels and truncal sinuosity of the right anterior vena cava each OC- were readily recognized. Although the lastcurred once. mentioned was seen in some 11th and Cardiovascular abnormalities observed 12th day control embryos, it was never in litter mates were dissimilar except in observed in those of 13 days and older. the case of three pairs of embryos. Abnormal looping of the heart tube reHowever, even then the malformations sulting in inversion of the ventricles, how(inversion of ventricles, both atria com- ever, was recognized in some 13th day municating with the left ventricle, and and older embryos from trypan blue transposition of the great vessels) although treated mothers. similar were not identical; frequently, abDextrocardia was noted in 15 embryos normal embryos were accompanied by one from the 13th to 22nd day of gestation or more apparently normal litter mates. and in one was associated with inverOnly a few embryos in each litter, how- sion of the ventricles; mirror-image dexever, were subjected to detailed examina- trocardia was seen in only two fetuses. tion. In seven of the fetuses dextrocardia was accompanied by absence of the tricuspid COMMENTS valve and in one transposition was presEmbryos from mothers injected with ent in addition. The frequent association trypan blue were usually smaller than con- of absence of the tricuspid valve with trol embryos of corresponding age indicat- dextrocardia suggests that hearts with the ing that this teratogen, like many others, former malformation may be inclined to retards fetal growth in general; next to rotate to the right possibly as a result of abnormalities of the heart and great ves- change in atrial or ventricular size. sels those of the eye and nervous system Absence or stenosis of the tricuspid or were the most frequent. mitral valves in many 13th, 14th and The commonest malformations of the 15th day embryos perhaps results from cardiovascular system (table 2) were trans- inadequate expansion of the atrioventricposition of the great vessels, absence or ular ring which leads, in turn, to deforstenosis of the tricuspid or mitral valves mation of the dorsal and ventral atrioaccompanied by atrial or ventricular dis- ventricular cushions. Fox and Goss ('57) placement, and dextrocardia, findings also observed similar valvular abnormaliwhich generally agree with those of others ties in rat fetuses as a result of trypan who have employed the same teratogen in blue while Haring ('60) has reported them as a teratogenic effect of carbon dioxide. rats. 186 I. W. MONIE, EVA TAKACS AND J. WARKANY In the present study the ventral atrioventricular cushion was usually thrust around its dorsal counterpart and often seemed bipartite (fig. 5d). Sometimes one or both atrioventricular cushions appeared larger than those in the corresponding controls, a finding also noted occasionally with respect to the truncobulbar cushions. Failure of the atrioventricular ring to expand adequately apparently results in both atria continuing to discharge into the left ventricle, a s in controls of the 12th day, or to absence or stenosis of the tricuspid or mitral valve. The former valve was more often affected than the latter and it seemed as if the valve on the same side as the bulbus was more prone to malformation. Indeed, it is possible that some examples of apparent mitral valve abnormality in the older fetuses may actually be instances of tricuspid valve abnormality associated with inverted ventricles; one such example was encountered in this study. While inversion of the ventricles is easily recognized when the bulbus is a distinct structure, it is less readily apparent in older fetuses. Absence or stenosis of the tricuspid or mitral valve leads eventually to reduction in size, or virtual absence, of the corrcsponding ventricle and, in older fetuses, only one ventricle may be recognizable. Shaner (’49) observed malformed atrioventricular cushions in young pig embryos which he related to abnormalities of the tricuspid and initral valves and to aortic and pulmonary stenosis in older fetuses. These findings are supported by those of Fox and Goss (’57) who also noted that atrioventricular valve abnormalities and pulmonary stenosis frequently occurred together in rat fetuses from mothers injected with trypan blue. In the present investigation, pulmonary stenosis was observed in only three fetuses and was unaccompanied by abnormality of either the tricuspid or mitral valve. In a study of rat young from PGA-deficient mothers (Monie and Nelson, ’63) pulmonary stenosis was observed in 8% and was not associated with abnormality of the atrioventricular cushions or valves; it is therefore probable that pulmonary stenosis may arise by more than one mechanism. The examples of transposition of the great vessels encountered showed some or all of the following features: ( 1 ) shortened ascending aorta and pulmonary trunk; ( 2 ) location of the aortic valve at a level similar or cranial to that of the pulmonary valve; ( 3 ) rotation or relocation of the coronary arteries; and ( 4 ) a channel or “track,” sometjmes incomplete, extending from the cranial portion of the right ventricle to the commencement of the transposed pulmonary trunk. An important factor in the causation of transposition in these fetuses may be retarded development of the truncus which leads to prolongation of its sinuous stage, and to reduction in its length as well as that of its derivatives, While it is probable that these disturbances may result from cellular damage by trypan blue directly or indirectly, it is also possible that retardation of thoracic growth limits the space available for the enlarging heart and delays change in truncal shape. Truncal sinuosity and shortening are probably responsible for the sinistral location of the truncus (and cranial portion of the bulbus) observed in some 14th and 15th day embryos i n which the lumen of that structure lay over the cranial edge of the ventricular septum (fig. 4b). As a consequence of this, eithcr from derotation or from altered bloodflow, spiralling of the truncobulbar cushions is minimal or absent which leads to the dorsally placed pulmonary trunk communicating directly with the left ventricle while the ventrally located aorta retains its connecFig. 7 (a,b, c ) Transposition of the great vessels in a 21st day experimental embryo in which the interventricular foramen is open. The sections are sequential and show the pulmonary trunk dorsal to the aorta and having a more caudal level of origin. In ( a ) the left coronary artery and portions of the aortic valve cusps are visible. In ( b ) a “track” or channel extends from the right to the left ventricle; it is also apparent in (c). X 20. (d, e, f ) Transposition of the great vessels in a 22nd day experimental embryo i n which the interventricular foramen is closed, The sections are sequential. In ( e ) a “track” extends from the right to the left ventricle. x 20. Key: DC, left duct of Cuvier; F, interventricular foramen; LCA, left coronary artery. Rest of key as in figures 5 and 6. TRANSPOSITION O F GREAT VESSELS 187 P- 188 I. W. MONIE, EVA TAKACS AND J. WARKANY tion with the right ventricle; the essential elements for complete transposition are now present. Conceivably, where truncal displacement is less marked partial transposition ensues. Apparently, a further consequence of the changes just described is that the cranial portion of the bulbus, which normally forms the infundibulum of the right ventricle, is drawn around the left side of the aorta and its lumen encroached on by the caudal end of the aorticopulmonic septum. In complete transposition traces of the infundibular channel may be seen as a “track passing from the right ventricle towards the commencement of the transposed pulmonary trunk [fig. 6c) ; sometimes, however, an actual channel is present which communicates with the left ventricle and i t is possible that should this persist a ventricular septa1 defect will result (fig. 7b, e). Undue persistence of the bulboventricular spur (conoventricular flange) has been suggested as a factor of importance in transposition and other cardiovascular abnormalities, yet, in the fetuses of this study, it did not appear to play a significant role in this regard. Frequently, it was much less evident in abnormal hearts than in those of corresponding controls and it is possible that undue sinistral location of the truncus hastens its disappearance. Such findings may seem to conflict with those of Rychter (’62) who considered that transposition, produced in chick embryos by application of a microclip to the bulbus, is due to restriction of the morphogenetic movements of that structure. However, the effect of trauma on a small portion of an otherwise normal heart cannot be compared with the action of a teratogen affecting the entire heart even if certain portions of it may be less severely disturbed than others; the situations are dissimilar even if the resulting malformations resemble one another. Thus, rather than being in conflict, these observations seem to suggest that similar abnormalities may arise through different mechanisms. No atavistic trends (Spitzer, ’23) were seen in any of the embryos examined and delayed descent of the truncal component Qf the truncobulbar septum, as described in Pig embryos (Shaner, ’51), was not observed; admittedly, in the latter, difference in species and in rate of development may play a significant role. Abnormal location of sinuses in the developing heart wall as described by Bremer (’42) in a young human embryo were not recognized, although the illustrations of that author suggest that the heart he described might have had some degree of truncal sinuosity. Of 20 fetuses with transposition of the great vessels, four (20% ) showed absence of the tricuspid valve; the same four embryos represented more than half of all the fetuses ( 7 ) with absence of the tricuspid valve in this particular age group. In man a similar relationship is usually found between these two anomalies. It is possible that in early development a sinuous truncus remaining closely applied to the heart may limit expansion of the atrioventricular ring and lead to abnormality of the tricuspid or mitral valves; the appearance of some wax-plate reconstructions of abnormal hearts suggests such an effect. On the other hand, trypan blue may directly damage cells of the atrioventricular region and disturb their development. The relatively high frequency of transposition associated with absence of the tricuspid valve may result from the smaller and often cranially located right ventricle in the latter instance predisposing to sinistral location of the truncus and to the events apparently ensuing from this. In fetuses where both transposition and absence of the tricuspid valve were present, the aorta appeared shorter and the aortic valve more cranially placed than in transposition alone. In regard to the sex incidence of transposition of the great vessels, the findings of the present study resemble those in man; out of 35 rat fetuses with this malformation 20 (57% ) were males and 15 (43%) were females; no reason for this distribution was apparent. In addition to complete and partial transposition, instances of the aorta overriding the dorsal portion of the ventric9 Includes dissected but unsectioned fetuses TRANSPOSITION O F GREAT VESSELS Ular septum in association with a normally arising pulmonary trunk were encountered. It is possible that this abnormality arose from inhibition of the remodelling of the ventral atrioventricular cushion which normally occurs about the 15th or 16th day of gestation and which permits the aorta to communicate directly with the left ventricle. Although many teratogens are known to affect cardiovascular development, only x-irradiation and trypan blue have been shown to produce transposition of the great vessels in quantity and consistently. In rat young from PGA-deficient mothers (Baird et al., '54; Monie and Nelson, '63) transposition is rarely encountered, although this may be partly explained by the frequency of incomplete aorticopulmoiiic septation and the fact that transposition cannot be diagnosed with certainty unless this septum is virtually complete. On the other hand, abnonnalities of the branchial arch arteries and their derivatives resulting from trypan blue generally resemble those produced by other teratogens. The frequency of transposition with trypan blue and to a lesser extent with x-irradiation has suggested some degree of specificity of action in the case of these teratogens. Trypan blue has been observed to interfere with cardiogenesis and cellular activity in the somatic mesoderm (Mulherkar, '60; Stkphan and Sutter, '61) of the chick embryo, and to cause damage to the septal myocardium (Wegener, '61) in fetal rats. Smith ('63), on the other hand, has described changes in the myoepicardial cells of rat embryos following use of the same teratogen, which he considers lead to increased glycogen storage, myocardial thinning, and to reduction in the amount of cardiac jelly. In the present study, decrease in the quantity of cardiac jelly was not apparent nor was thinning of the myocardium a marked feature except in resorbing embryos. Such differences may be due to the influence of genetic factors, to the chemical nature of the teratogen, and to variation in dose and time of administration. The type of abnormality resulting from teratogenic action must also be related to genetic background as transposition of the 189 great vessels and other cardiovascular abnormalities apparently are more readily produced in rats of the Long-Evans strain than in others, Genetic factors most likely account for the different types of abnormalities seen in litter mates following subjection of the mother to a teratogenic agent although conceivably slightly different rates of development and perhaps variations in uterine blood supply also play a role. All of the cardiovascular abnormalities encountered in the rat fetuses of this study have their counterpart in man and conceivably the mechanisms involved in their formation may be similar. ACKNOWLEDGMENTS Gratitude is expressed to Dr. R. C . Armstrong and Mr. J. Morgan for assistance with the histological aspects of this study and to Mr. J. Maeno for his outstanding skill in the making of wax-plate reconstructions. LITERATURE CITED Abbott, M. E. 1936 Atlas of Congenital Cardiac Disease. Amer. Heart Assoc. Baird, C . D. C . , M. M. Nelson, I. W. Monie aiid H. M. Evans 1954 Congenital cardiovascular anomalies induced by pteroylglutamic acid deficiency during gestation in the rat. Circ.Res., 2: 544-554. Breiner, J. L. 1928 An interpretation of the development of the heart. Am. J. Anat., 42: 307-369. 1942 Transposition of the aorta and the pulmonary artery. Arch. Path., 34: 10161030. Christie, G . A. 1961 A n embryological analysis of certain cardiac abnormalities produced in rats by the injection of trypan blue. Scot. Med. J., 6: 465476. De la Cruz, M. V., and J. P. da Rocha 1956 A n autogenetic theory for the explanation of congenital malformations involving the truncus and conus. Amer. Heart J., 51: 782-805. De Vries, P. A., and J. B. deC. M. Saunders 1962 Development of the ventricles and spiral outflow tract in the human heart. Contrib. Embryol. Carnegie Inst., Wash., 37: 87-114. Fox, M. H., and C. M. Goss 1956 Experimental production of a syndrome of congenital cardiovascular defects in rats. Anat. Rec., 124: 189-208. 1957 Experimentally produced malformations of the heart and great vessels in rat fetuses. Atrial and caval abnormalities. Anat. Rec., 129: 309-332. 190 -- I. W. MONIE, EVA TAKACS AND J. WARKANY 1958 Experimentally produced malformations of the heart and great vessels in rat fetuses. Transposition complexes and aortic arch abnormalities. Am. J. Anat., 102: 65-92. Goodrich, E. S. 1930 Studies on the Structure and Development of Vertebrates. Macmillan, London. Haring, 0. 1960 Cardiac malformations in rats induced by exposure of the mother to carbon dioxide during pregnancy. Circ. Res., 8 : 1218-1227. Harris, J. S., and S. Farber 1939 Transposition of the great cardiac vessels with special reference to the phylogenetic theory of Spitzer. Arch. Path., 28: 427-502. Inoue, A. 1964 Congenital cardiovascular anomalies induced by trypan blue in rats. Nagasaki Univ. J., 39: 640-650. Jaffee, 0. C. 1965 Hemodynamic factors in the development of the chick embryo heart. Anat. Rec., 151: 69-76. Keith, A. 1909 Malformations of the Heart. II. Lancet, I I : 433-435. Keith, J. D., R. D. Rowe and P. Vlad 1958 Heart Disease in Infancy and Childhood. Macmjllan, New York. Le Douarin, G. 1961 Malformations resultant d’irradiations 1ocalisCes de diffkrentes parties de l’bbauche cardiaque chez l’embryon de poulet. J. Embryol. Exp. Morph., 9 : 556-562. Lev, M. 1953 Autopsy diagnosis of congenitally malformed hearts. Thomas, Springfield. Lev, M., and 0. Saphir 1945 A theory of transposition of the arterial trunks based on the phylogenetic and ontogenetic development of the heart. Arch. Path., 39: 172-183. Monie, I. W., and M. M. Nelson 1963 Abnormalities of pulmonary and other vessels from maternal pteroylglutamic acid deficiency. Anat. Rec., 147: 397405. Mulherkar, L. 1960 The effect of trypan blue on chick embryos cultured i n vitro. J. Emb. Exp. Momh., 8: 1-5. Pernkopf, E., and W. Wirtinger 1933 Die Transposition der Herzostien. Z. Anat. Entwickl., 100: 561-711. Rokitansky, C. F. 1875 Die Defekte der Scheidewande des Herzens. Wien. Rychter, 2. 1962 Experimental morphology of the aortic arches and the heart loop in chick embryos. Adv. Morph., 2: 333-371. Shaner, R. F. 1949 Malformations of theatrioventricular endocardia1 cushions of the embryo pig and its relation to defects of the conus and truncus arteriosus. Am. J. Anat., 84: 431456. 1951 Complete and corrected transposition of the aorta, pulmonary artery and the ventricles of pig embryos, and a case of corrected transposition i n a child. Am. J. Anat., 88: 35-62. 1962 Comparative development of the bulbus and ventricles of the vertebrate heart with special reference to Spitzer’s theory of heart malformations. Anat. Rec., 142: 519529. Smith, W. N. A. 1963 The site of action of trypan blue in cardiac teratogenesis. Anat. Rec., 147: 507-523. Spitzer, A. 1923 Uber den Bauplan des normalen and missbildeten Herzens. Virchow’s Arch. Path. Anat., 243: 81-272. Stephan, F., and B. Sutter 1961 Reaction de I’embryon de poulet au bleu trypan. J. Emb. Exp. Morph., 9: 410-421. Wegener, K. 1961 tjber die experimentelk Erzeugung von Herzmissbildungeii durch Trypanblau. Arch. f . Kreislaufforschung, 34: 99-144. Wilson, J. G. 1955 Teratogenic activity of several azo dyes chemically related to trypan blue. Anat. Rec., 123: 313-326. Wilson, J. G., H. C. Jordan and R. L. Brent 1953 Effects of irradiation o n cmbryonic development. 11. X-ray4 on the ninth day of gestation on the rat. Am. J. Anat., 92: 153-187.