Morphology of the atrioventricular node bundle and proximal bundle branchesA study employing computerized reconstruction.код для вставкиСкачать
Morphology of the Atrioventricular Node, Bundle and Proximal Bundle Branches: A Study Employing C o mpute r ized Reconstruction NIGEL K. ROBERTS AND KENNETH R. CASTLEMAN Departments of Pediatrics and Medicine, Center for Health Sciences, UCLA, Los Angeles, California 90024 and California institute of Technology, Jet Propulsion Laboratory, 4800 Oak Groue Driue, Pasadena, California 91103 ABSTRACT The morphology of the human atrioventricular node, atrioventricular bundle and bundle branches is described. A block of tissue bounded by the ostium of the coronary sinus, the pars membranacea, the septa1 leaflet of the tricuspid valve and the atrial and ventricular septa is removed. This block is then sectioned serially from the right endocardia1 surface in the frontal plane of the heart. Sectioning in this way produces fewer sections than from techniques previously described. Outlines of the atrioventricular node, atrioventricular bundle and proximal bundle branches are digitally registered and stored in a computer. Three dimensional reconstructions of the structures are then generated by computer and displayed on an oscilloscope so that the entire three dimensional image can be rotated in any plane. Stereoscopic image pairs are produced to assist perception of the shape of the atrioventricular node, bundle and branching patterns of the bundles. This technique is unique in that it describes a method from which a relatively small number of histologic sections are generated permitting not only a complete histologic examination, but also a study of the morphology of the area. Although dissection of the atrioventricular node and bundle is possible, it is a tedious and relatively unreliable way to study these tissues (Curran, '09; Walls, '45). In 1951 Lev et al., reported a histologic study of the atrioventricular node bundle and branches. Since then there have been various minor modifications to his technique (James, '61; Titus e t al., '63; Hudson, '63; Truex and Smythe, '67; Anderson and Latham, '71; Davies, '71; Ferris and MacLennan, '73; Becker and Anderson, '76). In this communication we wish to present an alternative method for an examination which requires fewer histologic sections. This method allows us to visualize the atrioventricular junctional and subjunctional tissues microscopically and permits study of the shape of the atrioventricular node and bundle and branching characteristics of the proximal subjunctional branches. This latter examination, at the present time, is generally omitted from an examination of the atrioventricular node, bundle and bundle branches. ANAT. REC. (1979) 195: 699-706. MATERIALS Hearts for this study came from autopsies performed a t the Center for the Health Sciences, UCLA, after the appropriate conditions required by the Human Subjects Protection Committee, had been met. Twenty hearts from ten male and ten female patients who had died as a result of a process which did not involve the heart were used in this study. The patients were between 20 and 30 years of age. The computer hardware, a PDP-11 minicomputer and the Evans and Sutherland Picture SystemTM were in the image processing laboratory, a Division of the Jet Propulsion Laboratory, California Institute of Technology. The software was developed in these laboratorie~.~ Received Mar. 3, '79. Accepted June 13, '79. 'This work was supported by a grant from the Greater Lo8 Angeles Affiliate of the American Heart Association. %Addressfor reprints: Nigel K. Roberts, Department of Medicme, Division of Cardiology, UCLA, Los Angeles California 90024. Evans and Sutherland, Picture SystemvM, Santa Monica, California. 'Interested investigators may obtain information related to the software upon application to the senior author (NKR). 699 700 NIGEL K. ROBERTS AND KENNETH R. CASTLEMAN A bbreuiations A, Atrial muscle AS, Atrial septum AVB, Atrioventricular bundle AVN, Atrioventricular node CS, Coronary sinus FO, Foramen ovale HB, His bundle LB, Left bundle LVC, Left ventricular cavity MS, Membranous septum RB, Right bundle RVC, Right ventricular cavity V, Ventricular muscle Fig. 1 Figure 1A shows the right side of the heart exposed to demonstrate the foramen ovale and the coronary sinus in relationship to the conducting system. The atrioventricular node and His bundle are drawn over existing structures for reference. The area of the membranous septum is marked. The box outlines the block taken for histologic section and is equivalent to the figure below (fig. 1B). Also see text for further details. (Modified from Roberts and Pepin, '77, with permission). Figure 1B is a cleared section of the outlined area of figure 1A. The atrial septum and ventricular septum surround t h e AVN and HB. MORPHOLOGY OF THE CARDIAC CONDUCTING SYSTEM METHODS The right atrium is opened in the conventional fashion by cutting from the inferior vena cava up to the tip of the atrial appendage. The right ventricle is subsequently opened down the lateral border cutting through the tricuspid valve ring and extending the incision down to the apex. The heart is then fixed for three days in a solution of Kaiserling 1 (1.5%potassium nitrate W/V and 3.0%potassium acetate W/V in 2% formalin). Later the atrioventricular node and bundle and proximal subjunctional structures are excised in an oblong block of tissue which on the right side includes the ostium of the coronary sinus, the pars membranacea, the septal leaflet of the tricuspid valve, and appropriate amounts of interatrial and interventricular septa (fig. 1A).To remove this block the septal leaflet of the mitral valve and the noncoronary cusp of the aortic valve are detached. This tissue is flattened and preserved for 2-3 days in 10% neutral buffered formalin. It is then dehydrated and cleared in methyl salicylate. A more detailed description of the technical aspects of this technique have been published elsewhere (Roberts and Pepin, '77). Clearing in methyl salicylate makes the atrioventricular node and bundle stand out as a brown streak against the transparent background of fat and connective tissue. The atrioventricular bundle and bundle branches can be seen emerging from the node and running down close to the ventricular muscle (fig. 1B). The tissue is then taken through two changes of xylene and embedded in paraffin. Three days later it is clamped on the microtome, for sectioning, with the right endocardial surface exposed. With a small drill (DremelTM) six holes are placed a t right angles to the external plane of sectioning. In order that subsequent realignment of sections can be performed, into each hole a nerve fiber bundle is inserted (Burston and Thurley, '57). The bundles are derived from previously stretched fibers and each is coated in paraffin wax. Just before insertion, the paraffin coated nerve fiber bundle is dipped into melted wax which fixes and seals the markers in position. All six nerve fiber bundles are thus inserted into the paraffin block a t right angles to the plane of sectioning to provide markers for alignment of serial sections. Histologic sectioning then begins a t the right endocardial surface and proceeds through the septa and pars membranacea toward the left side. Sections of 5 701 p m thickness are cut and every fifth section is placed side by side on glass microscope slides generating a range of 75-90 sections mounted three to a slide. The cut tissue, on microscope slides, is stained with hematoxylin, phloxine and tartrosine and serial photographs made as transparencies on 35-mm color film, or as negatives on 35-mm high contrast black and white film. Tracings are made of the outlines of the atrioventricular node, bundle and subjunctional system from each photograph. These sections are oriented with respect to each other by the nerve fiber bundle markers. Generally each heart provides 80 sections mounted three to a slide. Every third stained section is traced representing nearly 30 outlines a t 75-pm intervals. The resulting outlines are manually registered with an x-y coordinate digitizer and stored on a disc in a PDP11 minocomputer. The position of the nerve fiber bundle markers is also indicated. The computer program for storage and subsequent display of the outlines is written after the standard techniques of Newman and Sproule ('75). The traced outlines are displayed on an Evans and Sutherland Picture SystemTM which enables them to be rotated in any plane. The usual planes from which photographs are generated from the Picture System are: (1)The plane of sectioning. (2) The plane parallel to the ventricular septum. (3) Planes a t oblique angles in order to separate the right and left bundle components. After each view has been selected and photographed from the display the outline is rotated 6" in the horizontal plane so as to obtain stereoscopic image pairs of each projection (Weinstein and Castleman, '72). An additional group of similar hearts were processed after the classical technique of Lev et al. ('51). The number of 5-pm histologic sections in these studies ranged from 200-350 sections mounted from three to six on each slide. In a similar fashion to the method described above, outlines of the conducting system are manually registered with an x-y coordinate digitizer. One section on each microscope slide is so processed, thereby generating about 60 outlines. Stereoscopic image pairs are then generated. RESULTS Grossly the most superficial sections-i.e., those from immediately beneath the right endocardial surface show the right bundle and 702 NIGEL K. ROBERTS AND KENNETH R. CASTLEMAN 703 MORPHOLOGY OF THE CARDIAC CONDUCTING SYSTEM M ." E '5 e ."" 704 NIGEL K. ROBERTS AND KENNETH R. CASTLEMAN Fig. 4 (Top, middle and bottom) These line drawings are stereoscopic-imagepairs generated by compiling the outlines of the conducting system. Each illustration represents a different conducting system. The images are mounted similarly and from left-to-right represent the atrioventricular node, common bundle and bundle branches (for details consult text). Top figure. From the left can be seen the atrioventricular node and in a superior and backward position are the fibers of the right bundle. The lower and more forward thicker lines to the right of the figure are the massed fascicles of the left bundle. Middle figure. In a similar fashion to the top figure the atrioventricular node can be seen to the left and from it arises posteriorly and superiorly the right bundle which in this study curls around the right ventricular cavity. The left bundle again arises from the atrioventricular bundle. Bottom figure. This stereoscopic-imagepair is generated by outlines derived from sections obtained in the classic manner of Lev et al. (‘51). Although the detail is not as fine as the top and middle images, the left side of the image is the atrioventricular node and bundle and left bundle can be seen anteriorly. The right bundle in this rotation coalesces with the superior part of the left bundle. MORPHOLOGY OF THE CARDIAC CONDUCTING SYSTEM the superficial surface of the atrioventricular node (figs. 2A,B). As the sections progress through the specimen block, the outline of the right bundle diminishes, the proximal portion of the atrioventricular node enlarges (fig. 2C) and the left bundle appears (figs. 2C,D). The atrioventricular node attains its largest size about half-way through the series of sections. At this point it is 3-5 mm long (figs. 3A,B). In deeper sections (i.e., farther left) the proximal portion of the atrioventricular node diminishes in size and eventually disappears; the atrioventricular bundle and left bundle branches become more evident (fig. 3C). The deepest series of sections display the wide posterior division of the left bundle (fig. 3D). The stereoscopic-image pairs reveal that the node is a rather flattened structure in which the outline is pear-shaped and in the illustration (figs. 4A,B,C), the left end of each reconstruction is the broadest and most proximal portion of the atrioventricular node. Figure 4A is the representation of the atrioventricular node, bundle and bundle branches illustrated in figures 2 and 3. The relationship of the atrial musculature and proximal portion of the atrioventricular node can be seen in the mid sections (figs. 2C,D, 3A,B). The right ventricular cavity is seen to diminish and the left ventricular cavity is seen to enlarge as the sections are viewed in sequence. This corresponds to the three dimensional appearance of the right bundle being above the fan-shaped left bundle in figure 4A. Stereo image pairs were similarly generated from the specimens sectioned in the manner of Lev et al. (’51).These images, in three dimensions, were essentially in agreement with those generated in the previously described study, after they were rotated to comparable planes of orientation. Figure 4C is such a representation. The overall outline is very similar to the other illustrations generated by our method (figs. 4A,B) although it can be seen that more and smaller outlines are required. In figure 4C, there is a slight clockwise rotation in the long axis and thus the branching of the right bundle is not as clear as in the other illustrations. DISCUSSION “The morphology and architecture of the human atrioventricular node and its adjacent junctional areas must still be considered a controversial subject.” (Anderson et al., ’75). 705 These authors went on to comment that controversy reflects the differences existing between individual hearts, growth of the tissues, differences in histologic techniques and differences in nomenclature (Becker and Anderson, ’76). We felt that we could reduce the area of controversy by using hearts from patients who died from causes which primarily did not involve the heart. We also used a rather narrow age range, namely 20 to 30 years, thus avoiding the effects of ischemic heart disease. The majority of reported studies on the conducting system, have concentrated mainly upon the microscopic appearance of the atrioventricular node, bundle and bundle branches. We propose a method which will allow observation upon the shape and form of these structures and which a t the same time offers a procedure which may be more cost-effective than current methods. Since the atrioventricular node and bundle can be as long as 15 mm and is only 2-3 mm thick, it was our hypothesis that so long as we could be sure of obtaining the entire system, we should be able to process the tissue in many fewer sections if we cut parallel rather than a t right angles to the plane of the conducting system. This proved to be true. “In order to comprehend fully the exact arrangements of the atrioventricular junctional area, reconstructions are essential,” wrote Truex and Smythe (’67). Despite this provocative comment only rarely have the atrioventricular node, bundle and bundle branches been reconstructed in such a fashion (Truex and Smythe, ’67; Anderson et al., ’75; Becker and Anderson, ‘76). As part of our study the three-dimensional registration of the atrioventricular node, bundle and bundle branches was important, moreover since we are suggesting a novel approach, a comparison of data from our method and those from conventional techniques was appropriate. After processing the conducting tissue we compared the reconstruction model with those from the traditional technique. We observed that the reconstructions are comparable. The overall shape of the atrioventricular node is similar in both techniques. In the example of the conventional technique (fig. 4C) the rotation, looking proximally, is slightly clockwise in the long axis of the A-V bundle and AV node, and so the branching portion of the right bundle is obscured by the fan-shaped left fascicles. We 706 NIGEL K. ROBERTS AND KENNETH R. CASTLEMAN again point out that many fewer outlines are required for our technique which has generated the upper two figures (figs. 4A,B). A clear understanding of the normal shape and form of the conducting system may be important in order to achieve insight into the mechanisms by which pathologic processes can cause functional disturbances. From the surface electrocardiogram we can recognize impaired physiologic function of the human cardiac conducting system. Indeed, even mild slowing of the conduction velocity a t the level of the atrioventricular node can now be recognized with the His bundle electrogram. Complete impairment of conduction at or below the junctional area, in the two major divisions of the left bundle and in the right bundle are all associated with typical electrocardiographic findings. The classic work of Lev has concentrated upon the microscopic anatomy of the conducting system, and indeed, he has been able to associate certain fibrotic or inflammatory lesions with electrocardiographic data (Lev, '64). He has not, however, used reconstruction methods to demonstrate a complete lack of communication between one area of the conducting system and another. Some other workers, have been unable to correlate so accurately the relationship between microscopic fibrosis and conduction impairment on the electrocardiogram (Demoulin and Kulbertus, '72). We believe that more emphasis should be placed upon the shape and morphometry of the atrioventricular node, bundle and bundle branches in order to understand more fully the normal growth and development patterns and disease states. The morphology may then be integrated with the specific microscopic anatomy of the individual components. In conclusion, we have described a different method of studying the human atrioventricular node, bundle and bundle branches and have provided some normal data. We have demonstrated that the technique requires fewer sections to obtain a complete study and thus may be more economical in terms of both time and material than the more traditional studies. We have also proposed a way of displaying the conducting system as an adjunct to the microscopic examination. LITERATURE CITED Anderson, R. H., A. E. Becker, C. Brechenmacher, M. J. Davies and L. Rossi 1975 The human atrioventricular junctional area. A morphological study of the A-V node and bundle. Europ. J. Cardiol., 3(1): 11-25. Anderson, R. H., R. A. Latham 1971 The cellular architecture of the human atrioventricular node, with a note on its morphology in t h e presence of a left superior vena cava. J. Anat., 209: 443-445. Becker, A. E., and R. H. Anderson 1976 Morphology of the human atrioventricular junctional area. In: The Conduction System of the Heart, Structure, Function and Clinical Implications. H. J. J. Wellens, K. I. Lie and M. J. Janse, eds. Lea and Febiger, Philadelphia, pp. 263-286. Burston, W. R., and K. Thurley 1957 A technique for the orientation of serial histologic sections. J. Anat. (London), 91: 409-412. Curran, E. J. 1909 A constant bursa in relation with the bundle of His; with studies of the auricular connections of the bundle. Anat. Rec., 3: 618-631. Davies, M. J. 1971 Pathology of Conducting Tissues of the Heart. Appleton-Century-Crofts, New York. Demoulin, J. C., and H. E. Kulbertus 1972 Histopathological examination of concept of left hemiblock. Brit. Heart J., 34: 807-814. Ferris, J. A. J., and J. R. MacLennan 1973 A simplified method for examining the conduction tissues of the heart. Med. Sci. Law, 13: 285-288. Hudson, R. E. B. 1963 The human conducting system and its examination. J. Clin. Path., 16: 492-498. James, T. N. 1961 Morphology of the human atrioventricular node with remarks pertinent to its electrophysiology. Am. Heart J., 62: 756-761. Lev, M. 1964 Anatomic basis for atrioventricular block. Amer. J. Med., 37: 742-756. Lev, M., J . Widran and E. E. Erickson 1951 A method for t h e histopathologic study of the atrioventricular node, bundle and branches. Arch. Path., 52: 78-83. Newman, W. M., and R. F. Sproule 1975 Interactive Computer Graphics. John Wiley, New York. Roberts, N. K., and D. W. J. Pepin 1977 The atrioventricular node, His bundle and bundle branches. A new histologic technique. Stain Tech., 52: 131-135. Titus, J. L., G. W. Daugherty and J. E. Edwards 1963 Anatomy of t h e normal human atrioventricular conduction systems. Am. J. Anat., 113: 407-415. Truex. R. C., and M. Q. Smythe 1967 Reconstruction of the human atrioventricular node. Anat. Rec., 58: 11-20. Walls, E. W. 1945 Dissection of the atrioventricular node and bundle in the human heart. J. Anat.. 9: 45-47. Weinstein, M., and K. R. Castleman 1972 Reconstructing 3-D specimens from 2-Dsection images. Proc. S.P.I.E.,26: 131-137.