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Permanent gross demonstration of the conduction tissue in the dog heart with palladium iodide.

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Permanent Gross Demonstration of the Conduction
Tissue in the Dog Heart with Palladium Iodide'
RICHARD I). BAERG AND DAVID L. BASSETT
Department of Anatomy, University of Washington School of Medicine,
Seattle, Washington
ABSTRACT
A permanent gross demonstration of dog cardiac conduction tissue
was attempted unsuccessfully using standard glycogen and f a t stains, experimentally
induced lipogenesis, iodine, and the iodides of fluorescein, silver, and lead. Staining
with insoluble sulfides was also unsuccessful. Permanent gross differentiation of the
tissue can be accomplished with palladium iodide. The best fixative for minimum
tissue distortion which still permits good differentiation is neutralized formol-saline.
The opened and fixed heart is dipped in alcohol for 15 minutes prior to staining, then
placed in a n iodine-potassium iodide solution for two to five minutes. It is then
rinsed in running water for one minute and transferred to a solution containing
PdCln and HC1 and left for ten minutes. Following the staining reaction, a differentiation lasting several hours is necessary. By this method, the entire sub-endo.
cardial ramification of the specialized tissue can be permanently demonstrated. Alternatively, the above process may be replaced by painting the endocardium with the
various solutions, the results being equivalent but more localized.
The conducting system of the heart,
especially its sub-endocardial ramifications, has been studied in detail using
histological methods and wax reconstruction. In recent years biochemical and
histochemical observations have led to a
considerable body of knowledge concerning such aspects as lipid concentrations
(Mallov et al., '53), enzyme activities
(Carbonell, '56), potassium and glycogen
concentrations (Poppen et al., '53), and
experimental lipogenic abilities (Kuwabara and Cogan, '59). That these contributions are important to an understanding
of the essentials of the system and its role
in cardiac conduction is obvious. However, the gross characteristics of the cardiac conduction system are seldom appreciated, especially in the human and in the
dog. It is difficult to dissect and cannot
be demonstrated by India ink injection of
the surrounding sheath, as is the case in
bovine hearts.
The purpose of the present work was
to develop a stain for the system that
would be easy to use, require a minimum
of time, have permanence, and be applicable to fixed material.
MATERIALS AND METHODS
Approximately 120 dog hearts were
used in the study. All of the dog hearts,
with one exception, were either studied or
placed in the fixative within one hour after
death.
Standard f a t and glycogen staining.
Gross differentiation of the Purkinje system based on its alleged lower native neutral fat content was largely unsuccessful
with Sudan IV, Sudan Black, or oil red 0
staining. Nor did standard glycogen stains
such as PAS and Best's carmine stain give
a good gross demonstration of the specialized tissue.
Experiment a 1 lipogenesis . At tempts
were made to synthesize oleic acid esters
in the septa1 Purkinje system of dogs
and humans by the method described
in a series of papers (Cogan and Kuwabara, '57a; Kuwabara and Cogan, '57;
Cogan and Kuwabara, '57b; Hill et al.,
'59; Ciccarelli and Kuwabara, '59; Kuwabara and Cogan, '59). The procedure
used was the following: Dog heart septa
were incubated in a 37°C water bath for
24 hours. The incubation medium was
citrated cow plasma that had been refrigerated at 4°C for periods of time not in
excess of one week and, prior to the incubation, had been fortified with oleic acid.
Best results were obtained when the oleic
1 This investigation was supported in part by Public
Health Semce Medmal Student Research Trainin
Program 2R-22(C5) MSRT. Division of General M e t
ical Sciences.
313
314
RICHARD D. BAERG AND DAVID L. BASSETT
acid concentration was 3 mg/ml plasma,
and the pH adjusted to 7.0 with KOH.
The septa1 blocks were fixed in 10% formalin and stained with Sudan IV, Sudan
Black, or oil red 0. With this method
there was not enough contrast for the process to be used as a gross demonstration.
There was, however, evidence of sudanophilic tissue in the region where Purkinje
tissue is normally observed.
Iodine stain for glycogen. An iodine
method (Uhley and Rivkin, '59; Allen
et al., '59) was employed using a 2% KI
solution saturated with resublimed iodine.
For a minimum of distortion and a maximum of stain differentiation, the best fixative was 10% neutralized formol-saline.
The entire hearts were fixed a minimum
of 24 hours and then placed in the iodine
solution for approximately five minutes.
The iodine formed an intense brown compound in the Purkinje cells which disappeared from the tissue in approximately
one-half hour.
Apparently it is impossible to keep
iodine in the cells of the conduction system for long periods of time if the heart
is kept in any kind of a preserving fluid.
Optimum results were obtained on a heart
fixed and stained simultaneously in a
solution of Bouin's fluid (four parts),
dioxane (two parts), absolute alcohol
(one part), and iodine (2% ). The heart
stained by this method had distinguishable Purkinje tissue for several weeks.
The remainder of the heart was, however,
very much discolored and distorted.
Iodide precipitate methods. If iodine
staining is quickly followed by impregnation with a chemical that will react with
iodine to form an insoluble salt, the possibilities of permanent staining are increased. Lead nitrate, lead acetate, and
silver nitrate impregnations yield pale
colored iodides which are unsuitable for
gross work. Precipitation of the lead as
a sulfide by rinsing the tissue repeatedly
with water and then impregnating the tissue with sodium sulfide yields a tissue
too dark to be of any value. Differentiation of this tissue in strong acid does not
lighten the tissue sufficiently. Reduction
of the silver iodide with light or potassium
hydroxide in the presence of magnesium
salts results in a poorly differentiated tissue.
Fluorescein reacts with iodine to yield
tetraiodofluorescein (iodoscein) , orange in
color. However, attempts at the impregnanation of the iodine-stained tissue with
fluorescein did not yield hearts with well
differentiated conduction tissue.
Palladium ions react with iodine to form
a black precipitate which is insoluble in
most solvents. Therefore, it is a logical
ion with which to impregnate the iodinesoaked tissue. For this reaction, the opened
heart is taken from the formol-saline and
placed in 95% alcohol for 15 minutes. It
is then immersed in the iodine solution for
two to five minutes and rinsed in water
for one minute, after which time it is
transferred to a 0.18% solution of PdC1,
to which has been added 6 ml of 37% HCl
per 100 ml of solution. The entire endocardial surface darkens quickly, but the
tissue should remain in the solution for at
least ten minutes. For the stain differentiation, the heart is transferred to a solution
containing 13% of 37% HC1 and 13% of
30% HzO, and left for several hours. This
appears to be the most critical part of the
procedure. If the solution is made more
concentrated, air pockets form under the
endocardium and ruin the preparation.
The length of time necessary for this procedure varies with the individual heart but
usually two to six hours are sufficient. The
degree of differentiation should be checked
every half hour, and the heart should be
removed just before the desired contrast
is obtained. The entire heart will be
bleached, including the conduction system, if it is left in the solution for too long
a time. Finally, the heart should be
washed repeatedly in water and stored in
70% alcohol. Alternatively, the above solutions can be painted on the endocardia1
surface, with equivalent but more localized results.
The tissue was sectioned and stained
with hematoxylin and eosin in an attempt
to localize the palladium iodide microscopically.
OBSERVATIONS
The palladium iodide method demonstrates the conduction tissue in the interventricular septum, moderator band, papillary muscles, and in the free walls of the
CARDIAC CONDUCTION T I S S U E
ventricles. The left main bundle may be
seen emerging from under the aortic cusps
and dividing into anterior, middle, and
posterior branches (figs. 1, 2). In the
right ventricle, the right bundle can be
traced easily from beneath the septa1 leaf
of the tricuspid valve through the moderator band to the anterior papillary muscle
and onto the ventricular wall where it
ramifies extensively.
Microscopically, the palladium iodide
precipitate was localized almost exclusively in the conduction tissue (fig. 3).
Although penetration of the palladium was
less than 1 mm, this was quite sufficient
to stain the sub-endocardial part of the
system.
DISCUSSION
Iodine supposedly reacts reversibly with
glycogen to form a black compound. Since
this reaction depends on the availability
of glycogen, the fixing agent should be one
conducive to glycogen preservation. For
this reason, the pH of the formol-saline
fixative was adjusted with calcium carbonate. Placing the previously fixed tissue in 95% alcohol increased the contrast
in the tissue, presumably by making the
glycogen insoluble.
The reason for the success of the iodine
method rather than the standard glycogen
stains lies in the fact that iodine is not
as specific a stain for glycogen as PAS
or Best’s carmine. These stains appear to
be more sensitive to smaller concentrations of glycogen and therefore give little
color contrast between specialized tissue
and ordinary myocardium. The difference
between the concentration of glycogen in
the conduction tissue and the rest of the
heart muscle is ideal for the application
of the iodine-iodide concentration that was
used. With this concentration, the contrast between the tissues was optimal.
315
Acidified hydrogen peroxide is a very
strong oxidizing agent which oxidizes the
tissue proteins and the palladium ions.
The palladium iodide formed in the final
reaction is PdL or a higher salt of the palladium ion. It is insoluble in water, alcohol, or formalin, and soluble in excess
potassium iodide. The acidified hydrogen
peroxide is also used to oxidize any palladium metal that is deposited and therefore makes it available for the reaction
with iodine. The endocardial bleaching
is a product of this reaction.
LITERATURE CITED
Allen, P., J. J. Lederman and G. J. Pearl 1959
Prevention of surgical heart block by the use
of supravital stain. J. Thor. and Cardiovas.
Surg., 38: 57-61.
Carbonell, L. M. 1956 Esterases of the conductive system of the heart. J. Histochem. and
Cytochem., 4: 87-95.
Ciccarelli, E. C., and T. Kuwabara 1959 Experimental aberrant lipogenesis: VI. Biochemical characterization of the sudanophilic material produced. A.M.A. Arch. of Ophthalm., 62:
125-129.
Cogan, D. G., and T. Kuwabara 1957a Experimental aberrant lipogenesis: I. Serum factor.
A.M.A. Arch. of Path., 63: 381-386.
1957b Experimental aberrant lipogenesis: 111. Tissue factor. Ibid., 64: 23-33.
Hill, K., J. H. Kinoshita and T. Kuwabara 1959
Experimental aberrant lipogenesis : V. Biochemical evidence of oleate-induced lipid formation in the cornea. Ibid., 61: 361-365.
Kuwabara, T., and D. G. Cogan 1957 Experimental aberrant lipogenesis: 11. Substrate factor. A.M.A. Arch. of Path., 63: 496-501.
1959 Experimental lipogenesis by Purkinje fibers. J. Biophysic. and Biochem. Cytol.,
6: 519-522.
Mallov, S., J. M. McKibbin and J. S. Robb 1953
The distribution of some of the essential lipides
in beef heart muscle and conducting tissue.
J. Biol. Chem., 201: 825-838.
Poppen, K. J., D. M. Green and H. T. Wrenn
1953 The histochemical localization of potassium and glycogen. J. Histochem, and Cytochem., I: 160-173.
Uhley, H. N., and L. M. Rivkin 1959 Visualization of the left branch of the human atrioventricular bundle. Circulation, 20: 419-421.
PLATE 1
EXPLANATION O F FIGURES
316
1
Left ventricle of a dog heart with part of the ventricular wall removed. The heart has been stained by the palladium iodide method.
The left bundle can be seen emerging from under the posterior aortic
cusp, dividing into major branches on the septum, and ramifying
extensively on the septum and ventricular wall.
2
Enlargement of small area outlined on figure 1. The interlacing black
bands of specialized tissue are especially prominent in the lower part
of the septum in both ventricles and the ventricular walls. Grossly,
a l l are directly or indirectly continuous with the main bundle
branches.
3
Microscopic section of the left ventricular septum of the same heart.
The section was counterstained with hematoxylin and eosin and
shows a dense precipitate of palladium iodide in specialized subendocardia1 tissue. The endothelium is at the top of the figure. X 200.
CARDIAC CONDUCTION TISSUE
Richard D. Baerg and David L. Bassett
PLATE 1
317
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