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Morphology of the atrioventricular node bundle and proximal bundle branchesA study employing computerized reconstruction.

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Morphology of the Atrioventricular Node, Bundle and
Proximal Bundle Branches: A Study Employing
C o mpute r ized Reconstruction
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
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.
REC. (1979) 195: 699-706.
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).
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
MS, Membranous septum
RB, Right bundle
RVC, Right ventricular
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.
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
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.
Grossly the most superficial sections-i.e.,
those from immediately beneath the right
endocardial surface show the right bundle and
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.
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
“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).
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
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.
Anderson, R. H., A. E. Becker, C. Brechenmacher, M. J.
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morphology, atrioventricular, branches, computerized, stud, node, employing, bundles, proximal, reconstruction
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