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Studies of the atrioventricular bundle with polarized light.

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AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, OCTOBER 1
STUDIES O F T H E ATRIOVENTRICULAR BUNDLE
W I T H POLARIZED LIGHT
PAUL N. JOHNSTONE
Anatomical Laboratories of the University of Georgia
This investigation has t o do with the appearance of certain
of the constituent parts of the atrioventricular bundle when
studied with the polarizing microscope. Such a study is of
interest a t this time as it might serve to aid in clarifying the
unsettled question of whether or not this structure represents
contractile tissue. Attempts to study its contractility directly,
as Erlanger has done, have led to uncertain results, so that
the only evidence that we have has come from histological
studies .
Since Tawara’s investigations it has been known that there
are parts of the Purkinje tissue in the mammalian heart that
fundamentally resemble the two well-known types of striated
muscle, voluntary and cardiac, in that they are arranged in
the form of fibrils which are cross striated. I n the calf’s
heart these cross-striated fibrils are present in all of the regions of the atrioventricular bundle. I n the terminal expansions and in the distal parts of the right and left limbs they
are grouped more to the periphery of the Purkinje fibers,
while in the proximal parts of the limbs and in the trunk they
are t o be found not only toward the periphery, but also in the
more central parts. It is the purpose of this study to determine whether these cross-striated fibrils further resemble
the striated muscles by possessing alternating dark and bright
segments when placed between crossed Nichol prisms.
Though Ellis has shown that in certain embryonic hearts
double refraction does not develop until long after contractil145
THE ANATOMICAL RECORD, VOL. 26. NO. 2
146
PAUL N. JOHNSTONE
ity has become established, so f a r as is known all adult
muscle tissue possesses this property. The separate fibrils
of striated muscle consist of alternating dim and light disks,
or segments, which give the cross striations that are so characteristic. These two disks are obviously different in physical
structure as seen by ordinary light; but when examined by
polarized light this difference becomes much more evident,
f o r the dim substance possesses the property of double refraction. When the muscle fiber is examined with the polarizing microscope, the dim bands are bright, while the light bands
are dark. The material of the light bands is spoken of as
isotropic and that in the dim bands as anisotropic. The
anisotropic material in the dim band in ordinary light, or what
is the same thing, the bright band in polarized light, is said
t o consist of doubly refracting positive uniaxial particles
wliich are responsible for its doubly refracting properties.
Smooth muscle also possesses the property of double refraction, though the particles responsible for it are not arranged
in the form of alternating dark and bright bands.
The atrioventricular bundle of the calf’s heart has been the
subject of many studies. This has resulted in perhaps a
greater accumulation of information about its structure than
about that of any other animal. Particularly does this seem
t o be true about its nature in respect t o contractility. It seems
to be particularly adapted for anatomical studies-especially
for such a study as this. Material from cow and calf hearts
has been used exclusively in this investigation. Tissue has
been studied from the atrioventricular node and the trunk,
from the proximal undivided part, distal divided part, moderator bands, and other portions of the terminal expansions of
the left limb, as well as from the right limb and its terminal
expansions.
Observations have been made on both fresh and fixed preparations. The fresh preparations have not been as satisfactory, especially in the study of frozen sections, as the fixed,
for diffusion of the protoplasm of the atrioventricular bundle
P U R K I N J E FIBERS I N POLARIZED LIGHT
147
occurs readily. The kind of fixation does not seem to be of
great importance. Ten per cent formalin o r 70 per cent
alcohol gives good results. Formalin fixation is perhaps the
simplest, as the entire heart may be fixed and portions of the
atrioventricular bundle studied at leisure.
The studies were made on frozen sections and teased
preparations. Where the fibers are of good size, as in the
distal divided part and terminal expansions of the left limb,
they may be quite satisfactorily teased, but where they are
smaller and branch and anastomose more extensively, as in
the trunk, it is difficult to isolate Purkinje tissue with certainty
by teasing so that in this region the method of frozen sections
is much superior. Prepared material may be mounted directly
on a cover-glass which is inverted over a glass cell, previously
fixed to a slide by vaseline. If more permanent mounts are
desired, vaseline may be replaced by Gerlach’s mixture (beeswax 2 parts, lump resin 3 parts). Gerlach’s mixture should
not be used if the specimen is later to be stained. Evaporation is further counteracted by placing in the cell a small bolus
of cotton saturated with the fixing solution. I n such a chamber material can be kept for considerable lengths of time.
Specimens may be studied in the cell, but more accurate observations may be made if the cover-glass with the attached
specimen is lifted off, wiped free from grease, and mounted
in the usual manner on an ordinary slide. This permits of
staining with methylene blue without moving the field after
examination of the section has been made with the polarizing
microscope .
A single specimen injected in the manner described in an
earlier paper has been studied with the polarizing microscope.
This preparation was fixed in Carnoy’s fluid, imbedded in
paraffin, sectioned, and cleared in xylol. The adjacent section
was stained with hematoxylin and eosin. The injection was
used f o r determining the location of the Purkinje fibers.
Teased material from the terminal expansions of the left
limb that has later proved to be Purkinje tissue after staining
148
P A U L N. JOHNSTONE
with methylene blue, when examined with the low-power lens
(ob. 16, oc. 10) of a microscope equipped with the polarizing
attachment manufactured by the Bausch & Lomb Optical Company (style C polarizer and style 9analyzer), seems to possess doubly refractile properties of an obscure nature. It is
not sharply active. A number of times preparations have
failed to show any optical activity. If the material optically
active under the low power be examined under the high-power
lens (ob. 4,oc. 10) of the polarizing microscope there may be
seen in favorable spots indications of the cross-striated Purkinje fibrillae. It is better to examine the edge of the material,
as fibrillae projecting from the tissue may quite frequently
be found. If the tissue is optically active, when examined
under the oil-immersion lens of the polarizing microscope, it
is seen to be arranged in the form of alternate dim and bright
bands. Considerable care must be exercised to make sure that
connective tissue is not beneath or on top of the Purkinje
tissue examined, or that heart muscle does not also become a
source of confusion.
Though many preparations have proved unsatisfactory,
alternate dark and bright bands i n the presence of polarized
light have been seen in the Purkinje fibrillae in all of the
regions of the atrioventricular bundle where teasing has
proved feasible. I n experiments with both striated and heart
muscle treated in exactly the same manner as the Purkinje
tissue, specimens have frequently been seen that had apparently lost their property of double refraction in the presence
of polarized light.
I n sections of the atrioventricular bundle stained with hematoxylin and eosin there a r e seen to be apparently two types
of connective tissue surrounding the Purkinje fibers. One of
these types is extremely heavily nucleated. I n paraffin sections fixed in Carnoy’s fluid its fibers seem to be contracted,
so that i n longitudinal sections its nuclei are arranged in
waves. The other is less dense and less nucleated, and is in
closer relation with the Purkinje fibers. The denser connec-
P U R K I N J E F I B E R S I N POLARIZED L I G H T
149
tive tissue, in frozen sections, is seen to be the most optically
active constituent of the atrioventricular bundle. When
viewed in polarized light, it glistens almost as much as a silk
thread. I n the injected specimen cut in paraffin this tissue
retained its optical activity. Double refraction does not seem
to be entirely absent in the other type of connective tissue,
though it is very much less marked.
I n frozen sections Purkinje tissue retaining the property of
double refraction has quite frequently been observed. Sections of Purkinje tissue entirely free from connective tissue,
except on its periphery, may be made by cutting specimens of
the terminal expansions o r distal divided part of the left limb
at a thickness of 2 5 ~ . Only the fibrillae seem to possess
doubly refractile qualities. Examination of a single fibrilla
with the oil-immersion lens of the polarizing microscope, in
these sections, shows alternate dark and bright bands. This
eliminates the possibility of error due t o connective-tissue
interference. It is more difficult t o become assured that connective tissue is not a factor in producing optical activity of
Purkinje tissue in the regions of the trunk of the atrioventricular bundle where the fibers are very much smaller. It
is necessary f o r the sections t o be much thinner and after
examination with the polarizing microscope t o be stained and
carefully examined in optically active spots for connective
tissue on top or beneath the Purkinje fibers.
It has not been found possible in this study to determine
whether the dark band with the crossed Nichol prisms is the
light band with the uncrossed.
The evidence added pertinent to the question at issue, i.e.,
the contractility of the Purkinje fibers, may be summarized
in the statement: the Purkinje fibrillae possess isotropic and
anisotropic bands. It is beyond the scope of this report to discuss Englemann’s theory of contractility, its drawbacks or
merits, or to attempt to interpret this evidence in the light
of that hypothesis.
150
PAUL N. J O H N S T O N E
The significant histological facts that seem to have a bearing on whether or not the atrioventricular bundle is contractile are :
1. The Purkinje fibrillae are not only cross striated, but
further resemble muscle in that they possess alternate dark
and bright bands in the presence of polarized light.
2. The vascular supply of the Purkinje fibers is very scanty
in comparison to heart muscle.
3. The presence of a sheath, thought to be a bursa by Curran, but interpreted t o be a sheath by Lhamon and others,
which isolates the atrioventricular bundle from the rest of
the heart, even to its terminations.
The finding that the cross striations of the Purkinje fibrillae
are alternately iso- and anisotropic, like the cross striations
of voluntary and cardiac muscle, strengthens the view, based
on the presence of the cross striations, that the Purkinje fibers
must possess the property of contractility.
On the other hand, the strikingly scanty vascularity, in
contrast to the extremely rich supply of capillaries in cardiac
muscle indicates that, if these fibers are contractile, their contractility is either much less than o r of a different type from
that of cardiac muscle.
Furthermore, the considerable amount of connective tissue
which is found both between and around the Purkinje fibers
would seem to argue for a modified type of contractility-if
contractility exists-as connective tissue is present in no such
abundance around cardiac muscle cells.
It would seem entirely possible that Purkinje fibers may
possess a moderate power of contractility and that the power
of this system to transmit the contraction wave from atrium
t o ventricle may be associated with slight contraction of these
fibrillae, individual elements of which can be traced for long
distances in the bundle, and which possess alternately iso- and
anisotropic cross striations.
I n conclusion, I wish to express my appreciation t o Dr. E.
R. Clark f o r the assistance that he has rendered in the carrying out of this study.
P U R K I N J E FIBERS I N POLARIZED L I G H T
151
BIBLIOGRAPHY
CURRAN,
E. J. 1909 A constant bursa in relation with the bundle of His; with
studies of the auricular connection of the bundle. Anat. Fkc., vol. 3.
ELLIS, F. W. 1912 The development of double refraction in the muscles of fish
embryos. Am. Jour. of Physiol., vol. 31.
ENGLEMANN,T. W. 1878 Neue Untersuchungen iiber die mikroskopischen
Vorgiinge bei der Muskelcontraktion. Archiv fur die Gesammte
Physiologie., Bd. 18.
ERLANGER,
JOS. 1912 Observations on the physiology of Purkinje tissue. Am.
Jour. of Physiol., vol. 30.
RAYCRAFT,
J. B. 1891 On the minute structure of striped muscle. Proc. Roy.
SOC., vol. 49.
JOHNSTONE,
P. N., WAKEFIELD,
F. H., AND CURREY, H. M. 1922 On the comparative vascularity of heart muscle and of the Purkinje fibers. Anat.
Rec., vol. 24.
JOHNSTONE,
P. N., AND WAKEFIELD,
F. H. 1922 On the character of the Purkinje fibers in various regions of the atrioventricular bundle. Anat.
Rec., vol. 24.
LHAMON,
R. M. 1912 The sheath of the sino-ventricular bundle. Am. Jour.
Anat., vol. 13.
STEWART,
M. J . 1920 On the use of polarized light in the detection and investigation of suture materials embedded in the tissues. British Med.
Jour., vol. 1.
STUMP,
D. M. 1922 A n application of polarized light to resolution with the
compound microscope. Jour. Royal Microscop. Soe., September.
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