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Visualization of the purkinje network of the beef heart.

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VISUALIZATION O F THE PURKIXJE XET\\'OBK
O F THE BEEF H E A R T 1
R. C. TRUEX, J. L. C C R R P A S D M. Q. SMYTI-IE
Departnients o f A n a t o m y and Radiology, Hahneniann Xedicul College
and Hospital, Plialadelphaa, Pennsylvania
SEVEN FIGURES
The left and right branches of the bundle of His and their
terminal ramifications within the interveiitricular septum and
ventricles of the beef heart have been demonstrated by numerous investigators. A variety of colloidal suspensioiis have
been injected into the space whicli surrounds the large and
small fascicles of Purkinje fibers as they form a subendocardial network within the left and riglit ventricles. The
most widely used iiijection mass was diluted India ink for
its fine particles remained in suspension and flowed readily
through even the smallest spaces. Hearts prepared by this
method provide good teaching specimens and were illustrated beautifully in the detailed studies of Cardwell and
Abramson ( '31) and Abramsoii and Margolin ( '36). The
reader is referred to the abovc authors for a review of the
pertinent literature and a complete description of the endocardial network as observed in specimens injected with India
ink.
The present study was an attempt to demonstrate this
unique systeni of cardiac fibers by a radiographic method.
Tlie studies of Cardwell and Abramson ('31) and Abranison
and Margolin ( '36) had demonstrated that the injection niass
often extended along the Purkinje fibers a s they left the enclocardium to penetrate into the myocardium. I n soine spcciThis inxestig:rtioa
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T H E A N 4 T 0 3 I I C A L RZCORD T'OTi
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by V. S. Puhlic Health Grant
724
It. C. TRCES, J. L. CUI:i:Y
A S D JI. Q . SllITTHE
nieiis they noted tliat bruiiches of the left liundle could be
filled following tlie injcctioii of the terminal branches of tlle
right bundle aiid rice z'erstr. Tliey believed such aiiastonioses
traversed tlic septum for tlie proximal portions of the left
and right bundles coiitaiiicd 110 injection material. l y e liopod
t o denionstrate these finer myocardial raniifications of tlie
Pui~liiiijeiietwork by the iiijectioii of a suitable mdio-opclue
suhstaiice aiid subsequent exposure to I*adiograpliic tcclinic.
A total of 48 uiicut adult beef 1ieai.ts obtaiiiecl f r o m the
abattoir were stoiwiI iii a refrigerator 24 hours prior to use.
The cliillcci hearts were always placed iii warm water for a
few minutes before they were opelied and injection was
a t t ciii1)t ed.
Several excellent specimens resulted f roiii the iii jcc tioii of
liitlia iiik. Hearts also were injected with different coiiceiitrations of thorium dioxide, barium 111-oniidc,loariuni iodide,
sodium iodide, Diodrast L' and a suspensioii of barium sulfate
in Diodrast. The best x-ray films were obtained from hearts
injected with tlie latter substances (i.e. 25 gin barium sulfate suspeiitlcci in 50 c m ' $ of 70% Dioclrast mixed thoroughly
f o r 10 minutes in a liigli speed hlciidel*). A warnicd syi.inge
and a No. 25 hypodermic needle were essential in order to
prcrcnt the early precipitation of the barium sulfate.
After injection the opened hearts were placed flat oil the
x-ray film lioldcr and exposed by a modified extimnity techriic (i.e. 35-40 K v ; 1/10 see. 011 100 K.2 at a clistaiice of 36
inches). Tissue blocks were taken from tlie injected areas of
the interventricular septum and both ventricles. Following
fixation aiid paraffin embedding, serial sections of these areas
were- stained with hematoxylin and eosiii, Van Gieson ' s and
Mallory 's connective tissue, o r Weigert 's elastic technics.
Foot's silver inetliocl was used to demonstrate the reticular
Diodrast - A
New York.
rcgistcrcd trade name of Wiiithrop-Stearns h e . , New York,
P U R l I I h ' J E SETWORII
725
fibers. Microscopic slides were made also of the dissected
conimon crus and its branches.
RESUI,? S
Specinleiis injected witli diluted India ink (1: 25) were easy
to prepare and the Purkinje network within both ventricles
was well demonstrated after three or 4 injections. 9 study
of these preparations corroborated the detailed report of
Cardwell and Abramson ( '31). W e also were able to observe
the injection material 8 mm beneath the endocardium and
within the myocardium of the septum and ventricles. I n only
one specimen did we observe a filling of tlie rami of the left
buridle branch while injecting into a distal network of the
right bundle branch.
Formaldehyde fixation bleaches the myocardium and the injected black network is then prominent against a white hackground. Such hearts can be preserved as teaching material
for long periods of time, and can be used a s a source for microscopic preparations if desired.
The inorganic chemicals (i.e. barium bromide and iodide ;
sodium iodide and thorium dioxide) had been selected because of their molecular weights, solubilities and radiographic
properties. However, we did not have a single satisfactory
specimen after repeated experiments with these injection
substances. They flowed readily from the needle, but seeped
through the connective tissue sheath and discolored the adjacent endocardium. These chemicals were not only skin irritants, but lacked sufficient density in the small amounts
used, to produce good x-ray films.
Although barium sulfate possesses a low solubility in water
and alcohol, we found that small particles would remain in
suspension in heated Diodrast for longer periods than at
room temperature. This injection mass flowed readily through
a small caliber needle and resulted in sharper x-ray films
a s shown in figure 1. After injection was completed this
heart was closed and the radiographic film made. I n this figure one can observe clearly the relationships of the left bun-
726
I:.
C. T R U E S , J. L. CUI:l:Y
A N D AT. Q . S M Y T H E
dle branch (LBB), atid right bundle branch (RBR) to tllc
ventricles (LV, RV) and interventricular septum (IS).
The gross aiid radiologic appearances of the right aiid left
bundle braticlies (figs. 2, 3, 4, 5) are more dramatic atid instructive. As in India ink preparations one is anlazed by
the fiiie network of Purkiiije elements about the lmse of the
papillary muscles in the right (figs. 2, 3) and left ventricles
(figs. 4, 5). Sonic of thc fincr ramifications of this endocardial iictworlr ofteii iwiiaiii u~ifillcd. For esaniplc, wc could
Fig. 1 Radiographic film of iiijccted beef heart demonstrating tlic intact rehtionsliips of the left (LBB) aiid riglit (RBR) branches of the common hundle of His within the interventricular septum ( I S ) , left (I,\') and right (ItV)
ventricles.
P U R I C I N J E NETWORK
727
see macroscopically the lacy network a t the junction of tlie
right ventricular wall and interventricular septum (below the
moderator band in figs. 2 and 3 ) , but these spaces defied
our injection attempts. I n no single specimen were we able
to inject tlie bariuni sulfate suspension into every fine branch
which was visible 011 the endocardial surfaces of both ventricles. We were disappointed particularly by our inability
to demonstrate tlie fine ramifications of this network within
the myocardium. The small pel-i-Purkinje-fiber space limited tlie aniount of injection mass, so that insufficient densities prevented clear radiographic films.
Histologic sections demonstrated the injection mass within
the thin connective tissue sheath which surrounds the Purkinje fibers (fig. 6). The space about several Pui.kiiije fibers was larger than that which surrounded r? fcw, or a single Purkinje fiber. The sheaths and spaces of the endocardial
network were continuous and afforded free egress to the
injection mass if the needle tip was properly placed. However, the thin connective tissue sheath was more closely related to the strands of Purkinje fibers a s they entered the
myocardium so that the potential iiijcctioii space was reduced o r absent (fig. 7). Distally tlie sheath loses its identity
by becoming continuous with the supporting connective tissues of the niyocardiuni.
It is interesting to note that we could not inject the coinmon crus of tlie bundle of His. The reason was obvious after
study of this area in injected specimens. Although there
were some variations in the proximal extent of the connective tissue sheath and its subjacent space, both were absent
in all specimens in the proximal three-fourths of the commoil bundle. As in the distal myocardium of the septum and
ventricles, the sheath becomes continuous with the connective tissue stroma of the Purkinje elements and cardiac
muscle.
With the use of differential connective tissue stains the
wall of the sheath was found to consist of a n interworen
admixture of fine elastic and reticular fibers which may h a r e
728
R. C.
THEEX,
J. L. C U R R Y A N D M. Q. SMYTHE
a discontinuous inner layer of small elongated connective tissue cells. The outer wall of tlie sheath consists of larger
elastic and collagenous fibers which a r e arranged parallel to
the loiigitudirial plaiic of the eiisheathed Purkinje fibers.
COhlMESTT
The teaching films from such preparations (fig. 1) were
most useful in conveying to the student the intact relatioiiships of tlie buridle of His and its branches. The student
can also better visualize the oblique position of the muscular
interventricular septum of the heart.
W e believe this method offers more permaimit and useful
specimeiis than do those which a r e iiijected with fluorescent
dyes and visualized by ultraviolet light (Radice, Lloveras,
and Kneiszl, '48). The method may prove useful to others
who desire ail injection substance with adequate x-ray density for use in small vessels. The ingredients of the injection material a r e inexpensive, procured easily, aiid a r e relatively non-toxic.
I n each of tlie common bundle areas we observed an enormous number of nerve fibers. Also in two cases we observed
discrete ganglion cells in tlie coriiiective tissue between tlie
Purkinje fibers in this area. The functional significance of
neural elernieiits, observed previously by Truex and Copenliaver ('47), are now being investigated in this laboratory.
CONCLUSIONS
Barium sulfate suspended in Diodrast was injected into tlie
space that surrounds the Purkinje network in the endocardium of the beef heart. The left arid right branches of the
bundle of His and their finer fascicles were well visualized
on films by the use of a modified radiographic extremity
technic. The conimori bundle and the fine myocardial tcrminals of this system were not visualized. The peri-Purkiiijefiher sheath is absent in the proximal portion of the common bundle and tlie small caliber of the space about the
Purkinje fibers within the myocardium limited the flow of
P U R K I N J E KETWORK
729
the suspensioii used. The histologic structure of the coiiiiective tissue sheath and the advantages of the method are presented.
I.ITERA4TIJ11E CITED
ABRAXSON,
D. I., A N D S. MARGOLIN1936 A Purkinje couduction network in
the myocardiuin of the niamnialian ventricles. J . Anat., 7 0 : 251-259.
('ARDWELL,
J. C., AND D. I. ABRAMSON 1931 The atrioventricular conduction
system of the beef heart. Am. J. Anat., 4 9 : 167-192.
RADICE,J. C., J. H. LLOVERAS
AND J. J. KNEISZL 1948 Visualizacinones de
las terininaciones del fasciculo de His por medio de la fluoresceina.
Arch. Soc. Argent. Anat. N.y. Pat., 10: 172-175.
TRIiEX, R. C., A N D W. M. COPENHAVER1947 Histology of the moderator band
in man and other mammals with special reference t o the conduction
system. Am. J. Anat., SO: 173-202.
2
Pliotograpli of injected be(+ 1ic:irt deiiionstr:itiiig tlic relations a n d distt.il,utioii of t h e I’urkiiije iict\vork within the eiidoc:trdium of the riglit ventricle.
3
Radiographic film of the slwcimeii slio\ril :ibove
730
ill
figure 2 .
PURKIXJE SETWORK
R. C. TRUES, J. L. C C R R Y A S D 31. Q. S Y Y T H E
731
PLATE 2
ESPLA4NATIOPi OF F I G U R E S
4
Photograph of injected beef heart demonstrating the relations and distribution of the Purkinje network within the endocardiuni of the l e f t ventricle.
5
Radiographic film of the specimen sliown above in figure 4.
-.,32
P U R K I S J E NETT%-ORK
R. C . TRUEX, 3 . 1,. CUIIIIT A X D X. (2. S h l l T I f b
PLATE 2
PLATE 3
EXPLANATION OF’ FI G U RE S
6
Transverse section of the interventricular septum showing the subendocardial
rrlatioiis of a portion of the right hrancli of the bundle of His. The black
injection niass is observed in the space between the fine connective tissue
sheath and the Purkinje fibers. Beef heart. Hcmatoxglin and eosin. X 100.
7
Purkinje fibers and their slientli within tlie myocardium of the right ventricle. The fine connective tissue sheath is closely adherent t o the enclosed
Purkinje fibers, so that the area of the injection space is reduced or absent
withiu the deeper portions of the myocardium. Beef heart. Hematoxylin and
eosin. X 350.
734
P U R I i I S J E SETWORK
R . C . THCES, J. 11. C U R R Y A N D >I. (3. S3IYTHE
I’LhTE 3
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