Septation of the respiratory and digestive tracts in human embryosCrucial role of the tracheoesophageal sulcus.код для вставкиСкачать
THE ANATOMICAL RECORD 238:237-247 (1994) Septation of the Respiratory and Digestive Tracts in Human Embryos: Crucial Role of the Tracheoesophageal Sulcus KATHARINE S. SUTLIFF AND GROVER M. HUTCHINS Departments of Art as Applied to Medicine and Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland ABSTRACT Esophageal atresia and tracheoesophageal fistula, common malformations of the respiratory and digestive tracts, are of unsettled pathogenesis. Part of the difficulty in understanding these abnormalities arises from the uncertainties about the normal developmental processes in the region. This study examined the development and fate of the tracheoesophageal septum. Normal human embryos from the Carnegie Embryological Collection and fetuses from the Hopkins Pathology Collection were examined, and reconstructions of selected specimens were made from photomicrographs of serial histologic sections. The results show that the lung bud appears in Carnegie stage 12, rapidly enlarges, and bends caudally, thereby producing a sulcus between the foregut and the respiratory system on its caudal aspect. The cranial aspect of this tracheoesophageal sulcus remains fixed at the levels of the first cervical vertebra throughout subsequent embryonic and fetal development. At the same time the trachea and esophagus elongate to bring those parts of the respiratory and digestive systems into their definitive anatomic positions. Examination of the tracheoesophageal sulcus shows that its growth-limiting properties may be explained by its catenoidal configuration. Catenoidal, or saddle-shape, sulci have been shown to have similar regional growth-limiting properties in the embryonic heart. These regions contrast with outwardly convex regions in both the developing heart and lung where growth of the tissues occurs. The observations made here suggest that the origin of the tracheoesophageal malformations must be sought in a configurational abnormality in the area of the developing lung bud in Carnegie stage 12. 0 1994 Wiley-Liss, Inc. Key words: Embryonic development, Esophagus, Growth, Trachea During Carnegie stage 12 and 13 in the development of the human embryo, the respiratory system appears as a ventral outgrowth of the foregut, generally referred to as the lung bud, and acquires its relationship to the foregut and other mediastinal structures. The mesenchyme that separates the respiratory and digestive tubes is known as the tracheoesophageal septum. The development of the human respiratory and digestive tracts has been described. The classical account of the development of the human trachea and esophagus appears in The Manual ofHuman Embryology (Keibel and Mall, 1912).A later publication by Smith (1957)had the advantage of being based on staged embryos. More recent compilations based on staged embryos have been prepared by O’Rahilly and Boyden (19731, ORahilly (19781, and O’Rahilly and Muller (1984). Despite these previous studies, the manner in which the mesenchyma1 septum forms and thus separates the trachea and esophagus during the critical period of stages 12 and 13 has not been fully understood. The present study was undertaken to reexamine the septation of the respiratory and digestive tracts in Carnegie stages 12 and 13. 0 1994 WILEY-LISS, INC MATERIALS AND METHODS Materials for this study were obtained from the Carnegie Embryological Collection through the courtesy of Dr. Ronan ORahilly, director of the Carnegie Laboratories (University of California, Davis). The 351 Carnegie embryos (CE) in good condition and prepared as serial histologic sections of Carnegie stages 9 through 23 (Streeter, 1951; Heuser and Corner, 1957; O’Rahilly, 1973) were reviewed and photomicrographs prepared. In this study, particular attention was directed to the 34 embryos in stages 12 and 13, which span the period during which the tracheoesophageal septum appears. From these embryos, five were chosen for reconstruction based on their relevance to this study and on the amount and condition of material available. The following are the embryos selected for reconstruction. Received June 4, 1993; accepted September 2, 1993. Address reprint requests to Dr. Grover M. Hutchins, Department of Pathology, The Johns Hopkins Hospital, 600 N. Wolfe St., Baltimore, MD 21287. 238 K.S. SUTLIFF AND G.M. HUTCHINS Stage 12 Early Stage 12 (Fig. 1) CE 8506, 3.7 mm crown-rump length (CRL), sectioned in the sagittal plane at 8 pm; CE 7852, 3.7 mm CRL, sectioned transversely a t 10 pm; and CE 5923, 4.0 mm CRL, sectioned transversely at 10 pm. A 3.7 mm CRL embryo (CE 85061, sectioned sagittally, was selected to illustrate the developing lung bud at an early stage of septation. The lung bud consists of a knoblike thickening of the endodermal wall of the foregut and is growing outward in a ventral direction. The ventral epithelium of the lung bud is three times as thick as the dorsal epithelium of the foregut. Mesenchyme surrounds the lung bud and the other developing structures of the foregut. Inferior to the lung bud is the developing liver diverticulum. The heart occupies the space ventral to the lung bud. The lung bud is in the mesenchyme of the venous end of the dorsal mesocardium (Bliss and Hutchins, 1992). The notochord is continuous with the endoderm of the pharyngeal region and is separated by intervening mesenchyme elsewhere. The notochord is also surrounded by a column of sclerotomic cells, which are a trail of proliferating cells left behind as the somites grow dorsolaterally. Three pairs of occipital somites are present. The first occipital somite is not visible because it has begun to undergo involution. Consequently, the rostralmost pair seen is actually the second pair (Arey, 1920). The lung bud is at the level of the fifth pair of somites. The normal relationship of the digestive tube to the whole embryo is shown in the upper right of Figure 1. The embryonic body curvature reflects the beginning of ventral flexion of the thoracolumbar region. The previous stage is characterized by a relatively straight linear axis. A frontal plane reconstruction of the lung bud area of CE 7852 is shown in the lower left image of Figure 1. The lung bud is also growing outward in a lateral direction. The level a t which the foregut and primitive respiratory tract have a common lumen is within the lower half of the foregut. The lumen of the foregut is very narrow below the lung bud. It widens as it ascends until it forms an oval at the level of the primitive pharynx just above the lung bud. A groove, the longitudinal sulcus, is apparent along the outside lateral wall and marks the transition point between the thick epithelium of the lung bud and the thin epithelium of the foregut. It runs inferior-ventrally from just above the lung bud to just below it. The transition is gradual toward the primitive pharynx and more abrupt at the base of the lung bud. The groove is not apparent along the inside lateral wall, which appears smooth and projects slightly medially. Stage 13 CE 9297, 4.5 mm CRL, sectioned sagittally at 8 pm; and CE 5874, 4.8 mm CRL, sectioned transversely at 10 pm. The foregut region was reconstructed to demonstrate the developing structures of the digestive and respiratory systems. These structures included the lung bud, trachea, esophagus, liver, and stomach. Surrounding structures were also reconstructed to show their anatomic relationships. These structures included the neural tube, notochord, somites, heart, and mesenchymal structures. The reconstructions were in the form of illustrations indicating development in left lateral, frontal, and median views. Histologic sections for CE 8506, CE 9297, and CE 5923 had been photomicrographed for reconstruction. Details of the photography have been described by Magovern et al. (1986). The negatives of each section were projected through a Dokumater DL-2 Microfilm Reader, and the specimens were traced. The developing vertebral column and the body contour were utilized for registration. Every section was traced in the area of interest and approximately every other section on either side. The tracings were then stacked and superimposed for study with illumination from below. Composite drawings were made from the tracings to provide left lateral views of the whole embryo. For a more detailed study of the developing respiratory and digestive tracts, composite drawings were made of the foregut region and surrounding structures. Structures that overlay and obscured the composite image were traced on separate sheets of paper while maintaining careful registration (Gaunt, 1971). For concise understanding of the growth and septation of the trachea and esophagus, median and frontal plane projections were made from the tracings of CE 5923 and from xeroxed bromides of CE 7852 and CE 5874 (Payne and Hutchins, 1973). These projections consisted of only the area of the foregut where the respiratory system develops. Photomicrographs of the serial histologic sections of embryos of stages 14 through 23, sectioned in the sagittal plane, from the Carnegie Collection and four fetuses from the Hopkins Pathology Collection, cut in the median plane, were examined to determine the anatomic relationship of the tracheoesophageal septum to the vertebral column. The four fetal specimens were of 80.0 mm, 125.0 mm, 185.0 mm, and 350.0 mm CRL. RESULTS The stage of appearance of selected anatomic features in 351 Carnegie embryos of stages 9 through 23 are listed in Table I. Most of these data have been previously published (Moore and Hutchins, 1981). The lung bud and its primary branches, the right and left mainstem bronchi, appear in stage 12. Lafe Stage 12 (Fig. 2) A 4.0 mm CRL embryo (CE 59231, sectioned transversely, was selected as being intermediate between the first appearance of the lung bud observed in the earlier stage 12 and the more advanced septation of the trachea and esophagus seen in Stage 13. The lung bud is more prominent and separation has progressed. Two ridges of endodermal cells on the inside lateral wall join in the midline and take the form of a saddle-shape fold, the tracheoesophageal sulcus. Furthermore, the lung bud continues to grow ventrally and bends downward into the mesenchyme ventral to the foregut. The part of this mesenchyme that comes to lie between the respiratory and digestive tubes consti- 239 TRACHEOESOPHAGEAL SULCUS TABLE I. Stage of appearance of selected anatomic features in 351 normal human embryos' Carneaie staae Number of embryos Crown-rump length* Lung Lung bud Primary bronchi Tracheal cartilage Pulmonary vasculature Pulmonary capillaries One pulmonary vein Pulmonary arteries Two or more pulmonary veins Heart Cardiac asymmetry Septum primum Ostium primum Ostium secundum Cardiac vasculature Coronary capillaries Coronary veins Coronary arteries Left superior vena cava obliterated Other Parietal pericardium Ribs Sternum Liver Omental bursa Spleen Mesoneuhrosis Metanephrosis 9 10 11 12 25 3 12 19 3.1 3.8 1.8 2.2 20.6 20.8 20.8 10.7 13 25 4.9 20.7 8 / 2 5 + 2125+ 21125 17/25 14 45 6.5 10.9 15 16 17 31 38 29 9.7 12.4 7.8 21.1 11.5 21.2 18 32 15.0 21.5 + + + + + + + + + + + + + + + + + + + + 35/45 + + + + 19 20 19 17 18.4 20.8 11.5 21.4 + + + 5132 14119 10112+ + + 7 / 2 5 + 7/25 + + + + + + + + + + + 33138 7/29 17130 30138 28129 + + 5/45 24131 1/31 12138 27/29 + + + + + + + + 1119 10117 + + + + + + + + + + + + + + + + + 16132 + + 21 22 17 18 22.7 25.2 11.7 2 1.3 + + 23 21 28.7 22.3 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + c 2117 10118 18121 1/38 20129 31132 + 1138 24129 6119+ 13/24 19125 + + + + + + 1/23 37145 + + + + + + + + + + + + 2129 + + + + 2/12 8/11 + + + + + + + + 27/32 + + + + + + + + + + Feature present in all embryos examined; a blank space indicates that the feature was absent in all embryos examined. * Mean + standard deviation. tutes the tracheoesophageal septum. The superior mar- somite at which point it divides into the right and left gin of the septum is at the level between the fifth and lung buds. The esophagus elongates and the distance sixth somite. The anterior concavity of the thoracolum- between the lung bud and liver increases further. The bar region has progressed further. The mesenchyme stomach is evident as a fusiform dilatation of the between the foregut and the neural tube has increased. foregut. A 4.8 mm CRL embryo (CE 5874), sectioned transThe distance between the lung bud and liver diverticversely and reconstructed in the frontal plane, was seulum has also increased. The longitudinal sulcus, which was present on the lected to show the state of the developing lung bud. The lateral outside wall of the 3.7 mm embryo, is evident. lung bud has lengthened caudally and has bifurcated However, it is now also apparent on the inside lateral into the right and left lung buds. The left mainstem bronchus is more in line with the trachea and is longer. wall as a ridge of endodermal cells. The longitudinal sulcus persists but now has an infeStage 13 (Fig. 3) rior-posterior direction. A 4.5 mm CRL embryo (CE 92971, sectioned sagitConfiguration of the Tracheoesophageal Sulcus (Fig. 4) tally, was selected to show the separation of the traThree embryos (CE 7852, CE 5923, and CE 5874) are chea and esophagus by the tracheoesophageal septum. The esophagus and trachea become clearly evident in presented in a manner to show the configuration and stage 13. The tracheoesophageal septum is 0.33 mm rapid growth and septation of the tracheoesophageal in length (ORahilly and Tucker, 1973). Below the sep- area. The dotted line indicates the level of the top of the tation point, the lumen of the esophagus narrows sig- tracheoesophageal sulcus, which remains constant relnificantly, almost to the point of occlusion. There is ative to vertebral structures as the lung bud grows also marked narrowing directly above the septation caudally. Figure 4A shows left lateral reconstructions of three point until reaching the pharynx where the lumen widens. Figure 3 also shows the septation point to be at the embryos in which the longitudinal sulcus described level between the fifth and sixth somite. The lung bud above is clearly evident. In Figure 4B, left lateral rehas descended to the level of the seventh and eighth constructions cut in the median plane are shown. The - 240 K.S. SUTLIFF AND G.M. HUTCHINS \ Lung bud ml 'Neural t u b e Notochord Mesenchyme FRONTAL VIEW ' 0.1 mm' Fig. 1. Two Carnegie embryos show the developing lung bud as a ventral outgrowth of the foregut. The left lateral median plane reconstruction of the foregut and adjacent structures are from CE 8506, sections 41215 through 4 / 3 4 . The dorsal aorta is not depicted in order to obtain a n unobstructed view of the foregut. The normal relationship of the digestive tube (indicated by a dotted line) to the whole embryo is shown in the upper right. The image in the lower left is a frontal plane reconstruction of the lung bud region from CE 7852, sections 2/1/13through 21317. The level at which the foregut and primitive respiratory tract have a common lumen is marked by ( + ). Liv, liver diverticulum; LA, left atrium, LV, left ventricle; SV, sinus venosus. TRACHEOESOPHAGEAL SULCUS 24 1 - FRONTAL VIEW 0.1 mm Tracheoesophageal sulcus Fig. 2. Carnegie embryo 5923 shows the progression of the developing lung bud and the septation of the respiratory and digestive tracts. The left lateral median plane reconstructions (upper right and center) are based in part on reconstruction drawings in Streeter (1942, Fig. 2, xii). The upper right image shows the normal relationship of the digestive and early respiratory tract (indicated by a dotted line) to the whole embryo. Within the center image, the cut median plane view through the lung bud region is a lateral reconstruction of sections 11615 through 211112. A saddle-shape fold, the tracheoesophageal sulcus (*), is evident. In addition, the tracheoesophageal septum (Te sept) makes its first appearance. The dorsal aorta is not depicted in order to obtain an unobstructed view of the foregut. The image in the lower left is a frontal plane reconstruction from sections 11615 through 211112 of the lung bud region. The level at which the digestive and respiratory tubes have a common lumen is marked by ( + 1. Te sept, tracheoesophageal septum; Gb, gallbladder. 242 K.S. SUTLIFF AND G.M. HUTCHINS Stage 13 \ Dorsal aorta Esophagus '0.1 mm I I Fig. 3. Two Carnegie stage 13 embryos show the progressive septation of the trachea and esophagus. The left lateral median plane reconstructions are from CE 9297, sections 31111 through 51315. The normal relationship of the digestive and respiratory tube (indicated by the dotted line) to the whole embryo is shown in the upper right. The image in the lower left is a frontal plane reconstruction of the lung bud region from CE 5874,sections 51314 through 61411.The level at which the trachea and esophagus have a common lumen is marked by ( + ). Dp, dorsal pancreas; Gb, gallbladder; St, stomach. 243 TRACHEOESOPHAGEAL SULCUS Early Stage 12 Late Stage 12 Stage 13 Fig. 4. Reconstructions of the lung bud and foregut from three Carnegie embryos summarize the rapid progression of the tracheoesophageal area in stages 12 and 13.The arrows describe the configuration of the tracheoesophageal sulcus by convex or negative (B) and concave or positive (C) curvatures. The early stage 12 column is based on a reconstruction of CE 7852, sections 211113 through.21317. The late stage 12 column is based on a reconstruction of CE 5923, sections 11615 through 211112 and the stage 13 column is based on a reconstruction of CE 5874, sections 51314 through 61411. The dotted line through each stage indicates the level between somites 5 and 6. formation of the tracheoesophageal sulcus is evident. It begins as a slight convex curve in a dorsal-ventral direction when the lung bud begins to protrude in early stage 12. It progresses to a curvature that is convex cranially in late stage 12 and becomes more accentu- ated in stage 13. In Figure 4C, frontal reconstructions, cut just anterior to the foregut in stage 12 and the esophagus in stage 13, are shown. In this view, the tracheoesophageal sulcus has the form of a curved surface, which in the coronal plane is concave in the cra- 244 K.S. SUTLIFF AND G.M. HUTCHINS Fig. 6. The static position of the tracheoesophageal sulcus. A hemisected 350.00 mm CRL fetus of the Hopkins Pathology Collection shows the top of the tracheoesophageal septum to be a t the level of the first cervical vertebra, as indicated by the line. Fig. 5.The static position of the tracheoesophageal sulcus. A sagittally sectioned normal human embryo of the Carnegie Collection. Section 26/112 of a stage 23 embryo (CE 5422) shows the top of the tracheoesophageal septum to be at the level of the first cervical vertebra, as indicated by the line. nial direction and in the sagittal plane is convex in the cranial direction. ure 6 show that the top of the tracheoesophageal septum remains at the level of the first cervical vertebra throughout prenatal life. DISCUSSION An understanding of the normal septation of the human respiratory and digestive tracts, which begins in Static Position of the Tracheoesophageal Sulcus Carnegie stage 12, may help to suggest the cause of (Figs. 5 and 6) abnormal septation leading to the malformations Examination of sections from the approximate me- esophageal atresia and tracheoesophageal fistula. In dian plane of embryos of stages 14 through 23 showed normal septation a mesenchymal structure, the trachethe progressive increase in length of the tracheoesoph- oesophageal septum, separates the digestive and respiageal septum. The right and left lung buds, now re- ratory tubes shortly after the appearance of the lung ferred to as primary bronchi, curve dorsally around the bud (O’Rahilly and Muller, 1984). In the pathologic esophagus. The first indication of the primitive larynx condition, tracheoesophageal fistula, a persistent comis visible, as an inlet, with the appearance of the hy- munication connects the trachea and esophagus and is popharyngeal eminence, arytenoid swellings, and epi- usually associated with esophageal atresia. These abthelial lamina. The epithelial lamina is a t the level of normalities occur in several forms and a t present there somite 5 (ORahilly and Muller, 1984). The atlas ((31) is no agreement as to their cause (Smith, 1957; O’Racan be used to identify the site of somite 5 since scle- hilly and Muller, 1984). Understanding the pathogenrotome 5 gives rise to the neural arch of the atlas. The esis of esophageal atresia and tracheoesophageal fispoint at which the trachea and esophagus separate re- tula requires an understanding of the mechanics of mains at the levels of somites 5 and 6, which corre- normal development of these structures. The morphologic observations presented here in responds to the tip and base of the dens of the second cervical vertebra and the centrum of the first cervical gard to the septation of the respiratory and digestive vertebra (Muller and O’Rahilly, 1986). The stage 23 tracts are consistent with those of previous studies. The embryo in Figure 5 and the 350 mm CRL fetus in Fig- respiratory system makes its first appearance at stage 245 TRACHEOESOPHAGEAL SULCUS Catem 1 Catenoid: surface of revolution of a R, = -R, Net zero curvature Catenoidal tulcus Lung bud Fig. 7.Diagram illustrating the generation of a catenoid from rotation of a catenary and the catenoidal configuration of the tracheoesophageal sulcus. The orthogonal radii (R) of curvature at every point are equal but opposite in direction (R, = -RJ. Thus every point on a catenoid has net zero curvature. This property of zero curvature determines that pressure differences between the two sides of the surface will not affect tension since the curvature is zero (P = T (l/ri, + l/rout).Similarly, tension in the surface will have no effect on pressure in the surroundings. 10 as a widened terminal portion of the laryngotracheal sulcus known as the respiratory primordium. By the end of stage 11,the respiratory primordium is characterized by a knoblike thickening of the endodermal wall, which will become the lung bud. During stage 12, the lung bud appears and separation of the respiratory and digestive tracts begins. With growth, the lung bud becomes more prominent and bends downward into the mesenchyme ventral to the foregut. The part of this mesenchyme that comes to lie between the respiratory and digestive tubes constitutes the tracheoesophageal septum. Smith (1957) describes septation by the union of proliferating endodermal ridges from the lateral wall of the foregut. Later studies by O’Rahilly and Boyden (1973), O’Rahilly and Tucker (1973), and O’Rahilly (1978) support these findings. A more recent study by O’Rahilly and Muller (1984) support and enhance the earlier work. They describe a longitudinal sulcus (the Grenzfurche), where thin epithelium runs into thicker epithelium and is visible externally. The longitudinal sulcus apparently indicates the subsequent line of separation between the respiratory and digestive tracts (Davis, 1923).These observations are supported by this study. However, previous studies have not made it clear that the endodermal ridges described by Smith (1957) and the longitudinal groove described by O’Rahilly and Muller (1984) take the form of a saddle- shape fold, the tracheoesophageal SU~CUS, as they unite caudally to divide the foregut into respiratory and digestive portions. This sulcus appears to be crucial to the formation of the tracheoesophageal septum and, therefore, to the normal septation of the trachea and esophagus. As in the present study, the study by ORahilly and Muller (1984) also found that the cranial end of the septum remains static relative to vertebral structures while the lung bud and trachea descend. The location of somites and vertebrae remain relatively stable during embryonic development. Therefore, as emphasized by Muller and ORahilly (19861,failure to use the vertebral column as a reference has resulted in the commonly held, but incorrect, view that the tracheal bifurcation remains a t a constant level in the embryo, while the septation point ascends. The septation point is the summit of the mesenchymal septum, and it is this area that remains at the level between somites 5 and 6 , the future first cervical vertebra. The configuration of the tracheoesophageal sulcus appears to account for both the development of the tracheoesophageal septum and the static position of its top. As shown diagrammatically in Figure 7, the sulcus has a catenoidal configuration. Catenoidal structures remain static during development in contrast to spherical or tubular structures which grow, expand, and 246 K.S. SUTLIFF AND G.M. HUTCHINS Sphere: surface of revolution of a semi-circle R, = R, Two positive curvatures Spherically 0 1 I " N w Fig. 8. Diagram illustrating the generation of a sy :re from rotation of a semicircle and the spherical configuration of the lung bud. The orthogonal radii (R) of curvature are equal and of the same direction (R, = RJ. Every point of a sphere has a net curvature. This property of net curvz ire determines that a pressure difference between the two sides of the surface will change tension in the surface. Similarly, tension development in the surface will exert pressure on the surroundings. lengthen (Fig. 8). A catenoidal configuration has been suggested to be of importance in studies of sulci and saddle-shape folds in the developing heart (Magovern et al., 1986;Hutchins et al., 1979;Meredith et al., 1979; Kundmueller and Hutchins, 1990). The growth-limiting properties of the atrioventricular, interventricular, outflow tract, and interatrial sulci appear crucial in determining cardiac septation and the correct alignment of the components of the developing heart. A catenoid is the surface of rotation of a catenary, which is a natural curvature that can be represented by the curve of repose of a suspended chain. The surface of a catenoid has the property of having orthogonal radii of curvature a t every point that are equal but opposite in direction and thus sum to zero (Thompson, 1942).As a result of its net zero curvature, the catenoid surface may have a pressure differential between its two sides, but this pressure difference does not change tension in the surface. This property is a reflection of the Laplace relationship where pressure equals tension times curvature. Similarly, tension produced within a catenoidal surface will not affect the pressure of its surroundings. However, surfaces that do not have a net zero curvature are subject to expansion or extension as a result of pressure differences between the two sides of the surface. In other words, a pressure difference between the two sides of a curved surface will produce tension in the structure and tension development in the structure produces a pressure difference in the surroundings. In the development of the respiratory system, a sulcus with a catenoidal configuration may have different potential for growth and expansion than adjacent areas that have outwardly convex curvatures such as the spherically shaped lung bud shown in Figure 5. Whereas catenoidal properties would maintain the position of the sulcus, the adjacent convex or positive curvatures of the lung bud would permit expansion and growth. In conclusion, the relative lack of growth in the saddle-shape fold of the tracheoesophageal sulcus, in contrast to the rapid growth of the convex components of the lung bud, is attributable to the different mechanical properties of the two configurations. The normal development of the tracheoesophageal septum appears to depend on this saddle-shape fold. The observations made here suggest that the origin of tracheoesophageal malformations should be sought in a configurational abnormality arising in Carnegie stage 12. LITERATURE CITED 1. 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