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The use of stereoscopic roentgenograms in studying the circulatory system of vertebrates.

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Resumen por 10s autores, Dean L. Gamble y
Raymond 0. Hitchcock,
Universidad Cornell, Tthaca.
El empleo de roentgenogramas estereosc6picos en el estudio del
sistema circulatorio de 10s vertebrados.
El sistema circulatorio de formas tales como Necturus, la
rana combn, la tortuga o el gat0 puede estudiarse ventajosamente por medio de roentgenogramas estereosc6picos. Los vasos
sanguineos del ejemplar que se desea estudiar se inyectan con
una mezcla con sulfatto barico en suspensi6n y despubs se aplican
10s rayos X. Se puedcn inyectar varios ejemplares, demostrando en cada uno de ellos una parte diferente del sistema circulatorio. Pueden inyectarse las arterias de un ejemplar, la
vena portahephtica de otro y las restantes venas en un tercer
ejemplar. Las exposiciones a la acci6n de 10s rayos X se efectuaron utilizando un tub0 auto-rectificador de Coolidge capaz
de adrnitir corrientes hasta de 10 ma. Para un animal como
Necturus se emple6 el siguiente dispositivo: La corriente de 8
miliamperios, longitud de chispa de 5 pulgadas, distancia focal
de 20 pulgadas; exposici6n, 6 segundos. Para animales mas
grandes es necesario emplear una exposici6n mas larga. Para
obtener roentgenogramas estereosc6picos es necesario hacer dos
exposiciones del niismo ejemplar. Se hace una exposici6n, se
mueve el tub0 23 pulgadas hacia 1e derecha o izquierda y se repite la misma operaci6n. Cuando se reducen las placas a un
tamafio apropiado, se obtienen las positivas y se montan estas
tiltimas dejando entre ellas unn distancia de 2 ; pulgadas, 10s
vasos sanguineos y otras partes aparecen en su posici6n relativa caracteristica cuando se examinan con un esteri6scopo ordinario. Las diapositivas son mas satisfactorias que las obtenidas
sobre papel. Las primeras se obtienen utilizando placas Seed,
las cuales se montan entre placas de vidrio, mantenihdolas en
posici6n por niedio de cinta de encuademar, empleitndolas del
mismo mod0 que las positivas hechas sobre papel. Los roentgenogramas preparados de este mod0 no solo daii excelentes datos
para 10s estudiantes que disecan el sistema circulatorio, sin0 que
susniinistran un medio para el exitmen rapid0 y exacto de un
graii nGmero de especies sin necesidad de disccci6n.
Transhtion by JosB F Nonidez
Carnegie Institution of Wxshiiigton
AUTHOR'S ABETRAW
OF THIS P A P E R I S R U E D
BY T E E BIBLIOGRAPHIC SERVICE. FEBRUhRY
2
T H E USE OF STEREOSCOPIC ROENTGENOGRAMS I N
STUDYING THE CIRCULATORY SYSTEM
O F VERTEBRATES'
D. L. GAMBLE
.WL)
It. 0 . HITCHCOCK
Zoological Laboratory, Cornell University
TWO TEXT F I G U R E S A N D TWO PLATES
The use of the Roentgen-ray in studying blood-vessels injected
with some opaque material was first described by H. Brause ('96).
He used metallic mercury in filling the blood-vessels and gives a
Roentgenogram of a human hand prepared in this way.
G. H. Parker ('13) described a method of preparing small
animals for studying variations in the blood system. The vessels were filled with an injection mass so compounded as to be
opaque t o the Roentgen-rays and yet firm enough t o make subsequent dissection possible.
The purpose of the present paper is to outline the method of
procedure in preparing specimens for the study of the circulatory
system by means of the Roentgen-ray and in obtaining stereoscopic Roentgenograms. Stercvxcopic exposures enable one to
study the relations of blood-vessels without subsequent dissection, thus saving a great deal of time. An added advantage is
that they afford excellent references for students in comparative
anatomy while dissecting in the laboratory.
1 The writers wish to express their indebtedness to Dr. H. D. Reed for his
suggestions and cooperation throughout the progress of this work. and to Mr.
L. P. Larkin, of the Department of Physics, for his invaluable help i n the work of
x-raying specimens. Thanks are also due Mr. R. E. Gove for his enthusiastic
assistance.
We are especially indebted to the Department of Physics for the use of their
excellent laboratory facilities and x-ray apparatus.
1'5
TRE ANATOMICAL RECORD, VOL.
18, NO. 2
126
D. L. GAMBLE AXD It. 0. HITCHCOCK
INJECTION MASS
There is need of accurate knowledge of the origin and distribution of blood-vessels, and to make the best of such studies
toward true morphology the method employed must be one t>hat
will take into account all vessels, both large and small.
It is obvious, therefore, that the material used in filling the
blood-vessels must, possess the following characteristics : first, it
must be opaque to the Roentgen-ray; second, it must be composed of particles fine enough to pass through the smallest vessels without, clogging, and, third, it must be easy of manipulation. A suspension mixture of barium sulphate meets all these
requirements.
There is one objection to barium sulphate. When it is thin
enough to fill the smallest arterioles or venules, it will not give a
clear Roent,genogram of the large vessels. This difficulty, however, is easily overcome by injecting first a small amount, of thin
barium sulphate and following this by as thick a mixture as can
be forced into the blood-vessels. If this is done, every vessel
will cast a clear shadow.
By using BaS04 as an injection mass, satisfactory Roentgenograms have been obtained of the blood systems of Necturus, frog,
spotted salamander, turtle, and cat. This method will probably prove equally serviceable in studying and demonstrating
the circulatory syst,em of the sharks and bony fishes.
METHODS OF INJECTION
As the blood system of Necturus was the first one to be studied
by us in this way, the method of procedure in making Roentgenograms of this animal will be outlined. The same procedure, with
few exceptions, may be used in working with any other form.
The specimens chosen for this work should be those which have
not been kept in captivity long enough to admit of injury to the
external gills. Animals which have been killed twenty-four
hours before they are to be used give much more satisfactory
results. This allows time for the relaxation of the small arteries
connecting the afferent and efferent branchial vessels, thus facilitating the passage of the injection mass into the dorsal aorta.
USE OF X-RAY I N COMPARATIVE ANATOMY
127
If all the arteries and veins of a single specimen are injected,
coilfusion of detail will result and the plates will be very difficult
to interpret. To obviate this difficulty, several specimens are
used, each one injected to show a certain portion of the circulatory system. For example, o m specimen might be used to show
arterics only; another to show the veins niaking up the hepatic
portal system, and still another to show the branches of the
postcava.
For forcing the barium-sulphate injection mass into the bloodvessels, an all-metal veterinarian's hypodeiniic syringe of 30-cc.
capacity and ametal cannula were found to be more satisfactory
than either gravity or air p r e w m .
The following is the method of proccdure to be used in preparing Necturus for the inakirig of a Roentgenogram. ;1midventral
incision is made through the body wall just anterior to the coramid portion of the pectoral girdle. This opcns the pericardial
cavity and exposes the heart. 147th fine forceps pass a thread
around the truncus nrteriosus as far rephalad as possible and tie it
loosely. With fine scissors make an oblique cut in the truncus at
the point 11here it leaves the ventricle, insert a steel cannula of
suitable size and draw the knot down tightly so that the cannula
iq held firmly in the ve-;sel. The syringe diould now be filled
I\ ith the injection inacs and attached to the cannula. By applying
a light, steady pressure to the plunger of the syringe, the barium
sulphate will be forced through the afferent braiichial arteries,
fill the capillaries of the external gills, then pa:s through the
communicating branches into the efferent branchial veFsels filling
both the carotids, the pulmonary arteries, the dorsal aorta and
its branches. Pressure should be applied until all vessels are
coinpletel y filled.
To inject the hepatic portal system, a second anirnal should
be used. -4ventral incision in the abdominal \$all about 1 cm.
to the right of the midventral line is madc. Pull out a coil
of the wiall intestine and locate the mesenteric vein, arid into
it inject thc barium sulphate both cephnlad and caudad.
-1third specimen may lie used to hhou the reninining'veins.
The truncus arteriohus is ligtttured ho that 110 injection matcrial
128
D. L. GAMBLE A N D R. 0 . HITCHCOCK
will pass through the heart, into the arteries. An oblique cut
should then be made in the postcava just cephalad of the point
where it leaves t,he middorsal line to extend to the liver. The
barium sulphate is then injected into the postcava in both
directions from the incision.
X-RAY APPARATUS
The x-ray photographs were taken with the usual apparatus.
-1detailed description is unnecessary, since any machine which
is in working order would answer the purpose. The choice of a
tube, however, is a matter of some consequence. As much detail
as possible is the aim in making x-ray pictures, and it is, therefore, desirable to employ a fine-focus tube. X-ray negatives are
shadow pictures. Just as when dealing with the shadows cast by
ordinary light, the smaller the source of illumination the sharper
the shadow so in x-ray work the smaller the area on the target
of the tube from which the x-rays emanate the sharper the
shadow cast on the photographic plate. X-rays are set up by
the sudden stopping of the rapidly moving electrons by the
t,arget. If these electrons are focused by a concave mirror so
that they all strike the target at the same spot, the x-rays
would emanate from this place as a point source. This is an
ideal focus for a tube and cannot be attained in practice because of the great amount of heat generated when the electrons
are stopped. A fine-focus tube, however, approaches this ideal
focus and gives good detailed shadows.
I n making these experiments a self-rectifying Coolidge tube
capable of taking currents up to 10 MA. was found very satisfactory. The advantage of using a Coolidge tube lies in the
fact that it is easier to control than the gas tube. The Coolidge
tube differs from the old gas tube in that the source of electrons
is different. ,4n x-ray tube will not take any current if there
are no electrons present, since these are the ‘carriers.’ I n the
gas tube, the high electric stress applied to the tube breaks up the
gas into electrons and a positive residue. On the other hand, a
Coolidge tube is as near a perfect vacuum as is now possible. At
USE O F X-RAT I N COMPARATIVE ANATOMY
129
the cathode there is a small filament (similar to that used in an
ordinary flash-light) which is heated by an auxiliary circuit supplied by a small step-down transformer. In this tube there is no
source of electrons except the heated filament. The hotter this
filament becomes the more iiunierou~are the electrons given off
and consequently the gi-eater the amount of current taken by the
tube. By installing a rheostat in the auxiliary filament circuit
it is very easy t o regulate the temperature of the filament and
in this way control the amount of current that the tube will
take. In the gas tube it is 1-er.y difficult to control the electron
supply, hince the amount of gas in the tube (the source of electrons) must be increased or decreased. This is accomplished
by ti device called :t ‘softener,’ which only the experienced
opcrator can employ 11 ith good results.
PROC‘EDI-RE I N ~ I . U i I N G ISSPOSURES
For Necturus it was found by trial that the following setting,
using a self-rectifying Coolidge tube, gax-e the proper exposurecurreiit of a 8 A M . , 5-inch spark gap, 20-inch target-plate distance, and 6 seconds’ exposure. The choice of a proper targetplate distance (distalire of target in tube from the photographic
plate) (fig. 1) is of considerable importance in this work, as this
factor in the setting has a direct bearing on the detail obtained
on the plate. If the plate is too near the tube, the shadow will
be greater in size than the object and will lack detail. If the
plate is a t the proper distance, the shadow will be nearly the
same size as the object and will be sharp.
The preceding paragraph gives only the approximate setting,
as it was found necessary to make slight changes constantly in
order to obtain the proper exposure for the varying density and
thickness of the specimens. Only the rays which penetrate the
specimen and strike the plate have any effect on the emulsion.
All t,hose that are absorbed in the tissue are lost. Absorption of
x-ray radiation depends on, first, the density of the absorbing
material (the specimen being photographed) ; second, the thickness of the absorbing material, and, third, the quality of the
radiation, i.e., the degree of penetration possessed by the x-rays.
130
D. L. GAMBLE AND R. 0. HITCHCOCK
All materials do not have the same density or, in other words,
they do not absorb the radiation equally. That is, each particular kind of material has its own absorption factor which depends
Fig. 1 Diagram of x-ray tube and plate-slide tunnel. A. Plate holders
taking 8 X lo+ inch plate. B. Glass or celluloid plate. C. Specimen. D. Lead
plates. F. Tatget. G. Target-plate distance 20 in.
on the nature of the substance itself. This absorption factor
holds only for a given quality of radiation. Each tissue has its
own absorption factor or density. Bone absorbs more radiation
than muscle under similar conditions. Two different masses of
USE OF X-RAY I N COMPARATIVE ANATOMY
131
muscle may have different absorption factors. Thus it was
found that of two Necturi, equal in size, one would absorb more
of the incident rays than the other, with the result that one plate
would be properly exposed and the other would not. To obviate
this variation in the density of the specimens, the time of exposure must be increased or decreased by an amount which must
be determined by trial.
Of two specimens one thicker than the other, the thicker
specimen will obviously possess a higher absorption factor.
Thus a cat will absorb more radiation than a Necturus not so
much because the tissue of a cat has a greater density, but because the cat is a larger animal than a Necturus and the rays
must pass through more tissue before reaching the plate. An
increase in the time of exposure will also obviate this increase in
thickness, unless the part is very thick and dense when an increase in the applied voltage is necessary. The higher the
voltage, the more penetrating are the rays, and they are said
to be ‘harder’ in quality. Obviously, high-penetration rays
will not be absorbed as much as those of low penetrating power.
-1very important consideration is the choice of a setting which
n-ill give enough soft-tissue detail to outline the organs, thus
rendering the plate more intelligible. If too high penetration is
used, the plate lacks contrast and does not show the outline of the
organs adequately. Better results are obtained by using as low
a spark gap as is possible. X good method of determining the
proper setting is that of exposing four plates, using a different
spark gap for each. When the plates are developed, i t is usually easy to choose the one that most nearly approximates the
correct setting, Then, by a slight correction of this approximate setting, it is possible to obtain one which will give the
desired result.
After setting the machine, the specimen is arranged on the
plate (which is protected from ordinary light by two light-proof
envelopes) according to the aspect to be photographed and the
particular structures desired. The usual method is that of
placing the animal upon its back and carefully spreading out the
gills and viscera so as to obviate unnecessary superposition.
132
D. L. GAMBLE AND R. 0. HITCHCOCK
The tube must then be placed at the proper distance from the
plate and accurately centered so as to cast the shadow in the
center of the plate and so that the whole plate will be exposed.
The central ray should strike the center of the plate at a right
angle to the surface of the plate. This is followed by the simple
operation of exposing the plate.
STEREOSCOPIC EXPOSURES
Plat,es secured in the way mentioned above offered a good
picture of the blood system of Necturus, but with the serious objection that the parts photographed were all in one plane. This
renders interpretation very difficult for those unskilled in reading x-ray plates and leaves one in doubt, often, as to the accuracy
of his observations. The difficulties encountered are such as an
attempt to determine the relative position of two vessels which
cross or the location of a vessel in its relation to the body walls
or viscera. This difficulty is somewhat obviated by photographing an animal in two different aspects representing two different
planes which would give some idea of the depth of the vessels.
This method was found rather clumsy and inaccurate for any
except the larger vessels. With a view to obviating such difficulties, some experiments were made with stereoscopic exposures, thus bringing into the picture the element of the third dimension. This shows the blood-vessels as well as other parts
in their proper relative positions.
I n brief, this method consists of taking an exposure of the
specimen, shifting the tube 2; inches and making another exposure on another plate. The specimen must not move between the
exposures. Each plate represents what each eye would see if the
eyes were at the positions the x-ray tube is during the two exposures. When these plates are viewed through the stereoscope
their virtual images are superimposed and we see the object in
three dimensions, and the plates are said to show stereoscopic
relief.
For taking these stereoscopic exposures a special piece of
apparatus is used, a stereoscopic plate-slide tunnel (fig .1). The
photographic plate-holder which accommodates an 8 x 10 inch
USE O F X-RAY I N C O M P A R A T I V E ANATOMY
133
plate (A) slides underneath a glass or celluloid plate (fig. 1, B) on
which the specimen is spread out for exposure. Thus the rays
pass through the specimen (fig. 1, C), then through the glass
plate, and then strike the photographic plate. The glass or
celluloid plate is one-half the size of the photographic plate. On
each of two opposite sides of this glass there is a piece of sheet
lead (fig. 1 , D) equal in size t o one-half of the photographic plate.
When the plate-holder is pushed half way in, one half the photographic plate is covered with lead, thus protecting it from the
rays, and the other half is under celluloid plate and is therefore
in position for exposure (fig. 1, B). Then, when the plate-holder
slide is pushed in full length, the part that has been exposed is
now covered with lead, hence being protected, the other half
that was covered with lead is now under the glass on which the
specimen lies and is in position for the second exposure. The
specimen has not moved, since it rests upon the glass plate and,
therefore, is not in direct contact with the plate-holder.
If a makeshift apparatus is set up (which can easily be done),
it is well to have the distance between the specimen and the plate
as small as possible so as not t o lose any detail. Direct contact
between the specimen and the plate is always desirable when
possible, because it gives better detail.
The specimen is spread out on the glass plate referred to in the
description of the plate-slide tunnel. It should be placed as
nearly in the center of the glass as possible. Since the stereoscopic exposures give the relative position of organs, it is better
not to spread out the viscera, but to leave them in their normal
position. The gills should be spread out as before.
The next operation is to accurately ' center' the specimen
under the tube (fig. 1) by moving either the tube or the plateslide tunnel. The specimen must remain as placed on the glass
plate. The plate-slide tunnel is then moved 1: inches to the left
in a line at right angles to the long axis of the specimen. The
plate is placed in the plate-holder in such a position that half
the plate will be exposed. After the first exposure is taken the
plate-holder is pusheq in so that t,he unexposed half of the plate
will be under the specimen, the whole slide tunnel is shifted 2+
inches to the right, and the second exposure made.
134
D. L. GAMBLE AND R. 0. HITCHCOCK
STEREOGRAPHS
Prints were made from the plates thus obtained upon F hard X
Glossy Azo or as transparencies on Seed Process Plates. The
latter gave far more satisfactory results, for when the transparency is placed in the stereoscope it is as though bhe eyes were a t
the target of the x-ray tube and looking through the specimen.
A much better stereoscopic effect, is obtained by looking at a
transparency than at a n ordinary print.
A
B
I
C
C
Fig. 2 Diagram of stereoscopic transparency. A and I3. The two stereoscopic exposures placed so t h a t their centers are 2: inches apart. C. Glass
plates between which are placed the transparencies. The plates are held together by means of lantern-slide binding type.
When prints were made upon paper they were mounted upon
sheets of cardboard 7 inches long and 4 inches wide. The same
parts in each exposure should be on the same level or the images
will not superimpose and the stereoscopic effect will be lost.
The prints should be so mounted that their centers will be from
23 to 3 inches apart.
The transparencies are placed the proper distance apart (2;
inches), and mounted between 4x7 inch glass plates (fig. 2). To
hold the transparencies in their proper' position, the coverplates are fastened together with lantern-slide binding tape.
USE O F X-RAY IN C O M P l R h T I V E ANATOMY
135
LITERATURE C I T E D
RARDEEN,
C. R. 1918 The value of the Rocntgen-ray and t h e living model in
teaching and researches in human anatomy. Anat. Rec., vol. 14, pp.
337-340.
IJRAUS,TI. 1896 [Jeber Photogramme von Metallinjectionen mittelst Rontgenstrahlen. Anat. Anz., Bd. 11, S. 6254329, Taf. 1.
DESCOMPS,
P., DE FALLETANS, G., ET DE L ~ L A U B IG.
E , 1910 Technique pratique
pour injcctions e t radiographies de pieces anatomique. Bull. e t Mem.
SOC.Anat. Paris, a m . 85, pp. 493-496.
FREDET,
P. 1900 Les arteres de I’uterus etudiees a u moyen de la radiographie.
Compt. rend. 13 Congress internat. med., Paris, sect. anat., pp. 103-108.
HASCHEK, E., UND LINDENTHAL,
0. T. 1896 Ein Beitrag zur praktischen Verwertung der Photographie nnch Rontgen. Wiener klin. Wochenschrift,
Jahrg. 9, S. 63434.
PARKER,
G. H. 1913 Notes on Roentgen-ray injection masses. Anat. Rec.,
VOI. 7, pp. 247-249.
Arteries injected with barium sulphate a n d exposures made steicoscopic.
To bet,t.er illustrat,e the possibilities of this method in studying the circulat.ory system of lower vertebrates remove the plate hy cutting along the dotted
line and place it, in an ordinary stereoscope
EXPLANATION OF FIGURES
PLATE 1
D. L. GAMBLE A N D R . 0 . HITCHCOCK
USE OF X-RAY IN]COMPAR.\TIVE A N.ATOWY
PLATE 1
PLATE 2
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