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American Association of Anatomists. Sixty-ninth annual session; Marquette University School of Medicine Milwaukee Wisconsin April 4 5 6 1956. Officers abstracts demonstrations (pp. 449 У500)

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ABSTRACTS OF PAPERS READ BY TITLE
449
406. An anatomical approach t o the problem of bronchial asthma. M. Wharton
YOUKG, Howard University, College of Medicine.
Bronchial asthma is a universal malady characterized by marked disturbance i n
respiratory rhythm The etiological concept of spasm of the bronchus” antedates
the microscopic revelation of the paucity of muscle in the bronchial wall. There
is no definite muscular layer encircling the lumen of the bronchus and the bulk
of cartilage which sustain the patency of this tube f a r exceeds the thin muscular
network. Bronchoscopist Chevalier Jackson has never observed a “spasm of the
bronchus. ”
Breathing is controlled and regarded by a hypersensitive respiratory center
located in the reticular formation in the hindbrain within which Magoun and
Pitts ( ’39) have delineated an anatomically distinct inspiratory and expiratory
division. I n asthma the derangement between inspiration and expiration could
result from disorder between the two divisions of the respiratory center. The
automaticity of respiration is revealed in sleep and anesthesia; the converse
activity of inspiration and expiration suggests a sympathetic-parasympathetic
control. The effreacy of adrenalin in asthmatic therapy may result from adrenergic
influences which reestablish the normal interrelationship between the inspiratory
and expiratory centers rather than to its presumed relaxation of visceral muscle
spasm. Surgical removal of the carotid body has proved beneficial in chronic cases
of asthma (Nakayama). The fact that asthmatic attacks may be initiated psychogenetically or by slight environmental changes further suggests its neurological
origin.
.
DEMONSTRATIONS
D $9. Quantitative data on lymphatic absorption of blood, yeast and tumor cells.’
Lane ALLEN and James MRACEK”, Department of Gross Anatomy, Medical College of Georgia.
suspensions of cells were injeoted immediately upon the inferior surface of the
diaphragm of adult rabbits. After a 10 minute interval the animals were exsanguinated, the thorax opened widely and lymph was obtained from nicked vessels
on the superior surface of the diaphragm and analyzed.
Under optimal conditions, as an average, for every 100 erythrocytes in the peritoneal cavity 85 erythrocytes appear in lymph, or 85% absorption. Leucocytes
show 22.8% absorption, Yoshida r a t sarcoma cells show 19.1% absorption and
yeast cells 16% absorption. Data, slides and photographs will be shown.
-
1
Supported in part by Lederle.
D 6 . A gold chloride-mercuric bromide impregnation.’ Rudolf ALTSCBUL, Department of Anatomy, University of Saskatchewan.
Deparaffinized sections from tissues, fixed in 95% ethanol, are treated €or 4-6-12
hours in a saturated aqueous solution of mercuric bromide, containing 0.01%
brown gold chloride. After rinsing, treatment with 2 4 % “hypo” and counterstaining with eosin, the sections are dehydrated and mounted. I n epithelial cells
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AMERICAN ASSOCIATIOX O F ANATOMISTS
of various types, in nerve cells, and in striated and smooth muscle, a black, finely
granular deposit is found. I t is absent in endothelium, i n goblet cells, in the lining
of pulmonary alveoli, in thyroid epithelium, in the collecting tubules of the kidney,
and in lymph follicles. Fibroblasts are not showing it, but activated histiocytes
are. Variations of intensity or lack of impregnation among elements of the same
cell type seem t o depend on ceIIular activity prior t o fixation.
Chick embryos are negative to the 5th day; but on the 6th day, liver, kidney
and spinal ganglia can be impregnated. A similar change is seen in mouse fetuses
between the 13th and 14th day.
A reaction of the metal conipound with phospholipids has t o be considered.
Although this is no final explanation, it is felt that these observations should be
reported, because the lack of straight parallelism with other histochemical procedures, whether metal impregnations or not, suggests that this method is a new
indicator of some metabolic processes.
~
1
Supported by a grant from the Medical Division, National Research Council of Canada.
D40. Evidence f r o m electron micrographs on the passage of material through
pores of the nuclear membrane. Everett ANDERSON* and H. W. BEAMS,
Department of Zoology, State University of Iowa.
Electron micrographs of nurse cells in the ovary of Rhodnius prolixus reveal
the nuclear membrane to contain pores of approximately 400 A i n diameter. Concentrated at certain regions near the nuclear membrane are masses of small graaules. Each granule measures about 200 A in diameter. Some micrographs reveal
an apparent extension of nuclear material (including granules) through the
pores into the cytoplasm where it is clustered in irregularly shaped bodies.
1 Supported by grant (B-301,C2)from the National Institutes of Health, U. S . Public
Health Service.
D 3 3 . d g e changes in nerve cells. Warren ANDREW, Department of Anatomy,
The Bowmaa Gray School of Medicine, Wake Forest College.’
Photomicrographs, presented as color transparencies, illustrate the degenerative
changes in the nucleus, cell body, and processes of nerve cells in old age, chiefly
in human material. The general alterations, such as loss of Nissl substance, accumulation of pigment, increasing basophilia of the nucleus, and actual death
and removal of cells are shown. The specific nature of alterations in particular
groups of cells is emphasized for the Purkinje cells, cells of the inferior olive,
and hypothalamus. The specificity of time of appearance and rate of progression of senile change in different parts of the brain, as presented in the conclusions of Oskar and CBcile Vogt, is confirmed and amplified. Illustrations
are given of peculiar changes which apparently represent ‘ reaction” to senile
change, a compensatory activity of nucleolus, nucleus, and cell body, as seen
both in the author’s material and that of the Vogts.
Preparation of this demonstration has been aided by the Gray Pharrnaccutical Company
and the Josiah M a w . Jr., Foundation.
ABSTRACTS O F DEMONSTRATIONS
451
D 2'. Skeletal changes correlated w i t h irradiation in radium and plutoniumintosicated dogs.' James S . AENOLD*, Radiobiology Laboratory, Department of Anatomy, Vniversity of Utah College of Meaicine. (Introduced by Thomas F. Dougherty)
Dogs were given radium and plutonium intravenously in amounts calculated
t o produce retention of 2-3 mc/kilo after one year. The skeletal changes involved massive death of both cortical and spongy bone with loss of osteocytes,
fibrosis of endosteal surfaces, and later reconstruction and proliferation of metaplastic bone. Pathologic fractures occurred at a time following bone death
but preceding extensive reconstruction. The bone marrow was all but destroyed
in areas of spongy bone but later regenerated following fibrosis of bone surfaces. There was early and persistent hyperplasia of marrow in long bone shafts.
I n spite of a considerable difference in the pattern of irradiation to bone between Ra and Pu, the morphologic pattern was quite similar, suggesting mechanisms other than direct irradiation involved in their pathogenesis.
1
Supported by A.E.C. Qrant dT' (11-1)-119.
D J 4 . Stereoscopic color photographs of the heart. David L. BASBETT, Department of Anatomy, Stanford University, California.
Normal internal structures o f the dissected heart are shown in three-dimensional
color transparencies.
D 8. Liver tumor cells in the anterior chamber of the eye. Sylvia H. BENSLES,
Department of Anatomy, University of Toronto.
A liver tumor, produced in a young white hybrid rat by feeding 4-dimethylaminoazobenzene, was found to be an adenocarcinoma. A suspension of cells
from this tumor was injwted into the anterior chamber of the eye of another
white hybrid control rat. The latter was sacrificed fourteen days later and
the eye was prepared for microscopic study.
Although there was considerable vascular and tissue reaction t o the tumor
implant, the tumor cells grew well and there was very little evidence of necrosis.
Where the tumor cells were free in the aqueous humor they were anaplastic; but
where they came in contact with the iris they became differentiated so as to
resemble intestinal epithelium, forming mucous glands and surface goblet and
absorptive cells. Moreover, the tissue of the iris developed the structure of a
lamina propria with papillary projeetions like villi, so that t h i s area resembled
the mucous membrane of a diminutive duodenum.
D I d . Sudan black staining of gross brain sections.' Harold BRODY* and John
E. WIRTH", Department of Anatomy, University of Buffalo, School of
Medicine. (Introduced by Oliver P. Jones)
Sudan black B has been used in staining frozen sections of many tissues
including nervous system. It has been found that it is particularly adaptable
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AMERICAN ASSOCIATION O F ANATOMISTS
for gross brain sections cut at any thickness. The differentiation between gray
and white matter and the rapidity of the staining technique makes the procedure useful both in the neuroanatomy and neuropathology laboratories. Specimens will be shown which may be stored in formalin or embedded in plastic.
1 This investigation. was supported in part by research grant B-645 ( C ) from the National
Institute of Neurological Diseaaea and Blindness of the National Instltutes of Health, Public
Health Service.
D 58. Hormonal dependence of transplanted adrenal cortical tumors of mice.
Henry C. BROWNING, Department of Anatomy, University of Texas
Dental Branch and Baylor Medical College, Houston.
The intraocular transplantation technic was applied to four adrenal cortical
tumors arising in castrate ce or C mice with the following results in autoIOgOUS hosts:a. One ce and two C tumors showed hormonal dependence. Suitability of
the host, in terms of latency after transfer and actual growth rate, increased in
the sequence; intact males, intact females, castrate females, castrate males.
b. I n castrate males transplants commenced growth between the 30th and
60th day and filled the anterior chambers by the 120th day. In intact males
the latency was approximately 120 days and the anterior chambers were not
filled until the 180th day.
c. Tumor growing i n intact hosts still showed, on retransplantation, increased
latency in intact as compared with castrate hosts.
d. Shortening of latency was only effective if castration was executed within
45 days of transfer.
e. I n male castrates testosterone inhibited growth and caused some regression
of growing transplants ; growth was resumed after cessation of administration.
1 Supported by a grant from ( C - 2 4 6 3 ) the National Cancer Institute. National Institutes of
Health.
D 52. Abnormalities produced in the chick embryo by radioactive phosphorus
(Paa).IArthur 0. CHAPMAN and John 8. LATTA, Department of Anatomy, University of Nebraska, College of Medicine.
Tissue necrosis in chick embryos resulting from ionizing irradiation has been
previously reported (Chapman and Latta, Anat. Rec., 129: 292, ’52; Chapman, J.
Morph., 95: 451-470, ’ 5 4 ) .
A few embryos have developed regional malformations of the central nervous
system, eyes, notochord and somites aa a result of Pn injected into the egg before starting incubation. Brain changes range from rosettes i n the wall t o severe
distortions. I n one 5-day embryo, part of the forebrain wall projects inside-out
through the unclosed anterior neuropore to form a large ‘‘encephalocele.” The
eyes tend to be retarded and sometimes malformed. One eye i n the 5-day embryo mentioned above has a very small optic cup which failed to enclose the
lens. In one ?-day embryo the optic CUPS show marked retardation and degeneration and the lenses failed to develop.
ABSTRACTS O F DEMONSTRATIONS
453
The spinal cord also may show rosettes in the wall, have partial duplication,
fail t o close dorsally, or be completely absent in some areas. Malformation or
absence of the notochord or somites is seen in parts of some embryos. There
is sometimes a regional correlation between spinal cord, notochord and somite
abnormalities.
Isupported by research grant No. B-534 (R) of the National Institute of Neurological
Diseases and Blindness, United States Public Health Service.
D 48. Neonatal differentiation of renal tubules in the mouse, studiea with the
electron microscope.' Sam L. CLARK, Jr., Department of Anatomy,
Washington University School of Medicine.
Kidneys from fetal, neonatal, and adult mice were fixed in 1%osmium tetroxide
in dichromate buffer at p H 7.2, embedded in methacrylate, and sectioned with
glass knives. Sections 2~ thick were examined by phase contrast microscopy and
after staining with periodic acid-Schiff technique, and sections approximately
200 A thick were observed by electron microscope. I n contrast to the proximal
and distal convoluted tubules of the adult, those of full-term fetal mice do not
possess rod-like mitochondria in parallel array, separated by invaginations of the
basal plasma membrane. Instead, the mitochondria are round or oval and the
basal plasma membrane is simple in contour. In addition, the proximal convoluted
tubules of fetal mice are distinguished by variable, rudimentary brush borders,
and the presence of electron-dense round bodies which stain with the periodic
.
micrographs will be
acid-Schiff technique and reach a diameter of 3 ~ Electron
shown to demonstrate the development of these tubules from the fetal to adult
form during the first few days of life.
1
Supported by grant RQ-3784 USPHS.
D 19. A method for implanting chronic electrodes in thc rat. B. Vaughn CRITCHLOW*, Department of Anatomy, University of California at Los Angeles
and Veterans Administration Hospital, Long Beach, California. (Introduced by Charles H. Sawyer)
A method developed by Green, Maxwell and Osborn for chronic implantation of
electrodes in cats and rabbits (to be published) has been modified for the rat.
The anesthetized animal is placed in a stereotaxic instrument, the skull is trephined,
and a pattern of concentric, bipolar electrodes, which are attached to earrier tubes
by a low-melting electronic cement, is lowered into a pre-determined position.
The exposed portions of the electrodes and three small anchoring screws in the
skull are then covered by a thin layer of dental cement. When the cement has
hardened thoroughly the carrier tubes are removed by heating them slightly, a
small electronic plug is soldered to the electrode leads, and a second layer of
dental cement is poured around the solder joints and the base of the plug. This
technique, which has proved quite satisfactory for electroencephalographic (EEG)
studies on unanesthetized rats, affords an easy, stable and long-lasting connection
to recording equipment ; and preparations with three sub-cortical and two cortical
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AMERICAN ASSOCIATION OF ANATOMISTS
electrodes have been maintained in good condition for as long as 5 months. The
only artifacts seen thus f a r have been those associated with gross movements, and
these show up in all EEG channels as easily recognizable, high-amplitude slow
waves.
Il 17. A method for reclaiming poorly impregnated silver nitrate nerve# preparations. J. David DECK*, Department of Anatomy, University of Virginia.
(Introduced by C. C. Speidel)
Sets of serial sections of larval urodele forelimbs were inadequately stained
three years ago using a technique for the differentiation of nerves. These sections
had been impregnated with silver nitrate, reduced in sodium bisulfite, toned with
gold chloride, and fixed i n sodium thiosulfate. Due to the capriciousness of the
technique, nerves were not selectively impregnated. Recently, sample sets of
these serial sections have been reclaimed with very good results by bleaching with
solutions of strong acids followed by reimpregnation.
Cover glasses were removed from chosen slides and the sections were hydrated.
A solution of acids (three parts cone. hydroch1oric:one part cone. nitric: 10 parts
water) was prepared and the slides were immersed i n the acid mixture for a
period of from one to 5 minutes, until the sections were bleached white. Sections
were not destroyed or distorted, though care was necessary t o keep from washing
them off the slides after the acid treatment. After prolonged washing, sections
were reimpregnated, toned with gold chloride, and counterstained with eosin.
Nerve fibers were differentiated quite as well as could be expected in an original
silver nitrate impregnation.
D d 6 . Some observations on neurosecretion in the rat. Jacob DE GROOT*, Department of Anatomy, School of Medicine, University of California a t Los
Angeles. (Introduced by John D. Green)
Female rats were subjected to various experimental procedures (dehydration
by thirst or administratioil of hypertonic saline, administration of colchicine,
pituitary stalk section, placement of electrolytic brain lesions) and their estrous
cycles were studied. The amount and distribution of Gomori-positive material and
its relation to nerve fibers were studied using the chrome-alum hematoxylin and
Bodian methods, in these rats as well as in the normal rat, cat, dog and monkey.
Material staining similarly to the neurosecretory substance in the supra-optic
and paraventricular nuclei was found in the habenular nuclei, in ependyma and
glia cells, and in macrophages near lesions.
D 44. Electron microscope observations on several major cell types in bone marrow
f r o m the human and the rat.' Quin B. DE MARSH and Jean KAUTZ*,
Department of Anatomy, University of Washington.
Human bone marrow was removed by sternal biopsy and fixed for two hours
in 0.25% buffered osmic acid. Rat marrow was dissected out of the femur intact
and similarly fixed. Electron microscope observations are compared and correlated with phase and polarization microscope observations.
ABSTRACTS O F DEMONSTRATIONS
455
Some previously unreported phases in the life cycle of plasma cells will be
pictured. These cells appear to have a “storage phase” during which the spaces
within endoplasmic reticulum sacs are filled and swollen with a moderately dense,
finely precipita€ing material. Except i n the case of plasma cells, endoplasmic
reticulum is rarely found in mature blood cells. Clusters of Golgi membranes are
often seen.
Using the presence of cristae as a criterion, mitochondria can be clearly distinguished from specific granules in the granulocytes and in megakaryocytes.
Details of the submicroscopic morphology of both megakaryocytes and platelets
will be presented, as well as findings related to the production of platelets by
megakaryocytes.
Lymphoblasts, myeloblasts and monoblasts from leukemic patients will be shown.
Marrow capillaries are often encountered, especially in the unbiopsied rat marrow. Possible morphological evidence for the way in which marrow cells may enter
the circulation will be discussed.
1
Supported by a grant (C-133245 ) from the United States Public Health Service.
D b 7 . The histology of the ovary of Thomomys, the western pocket gopher.’
Kenneth L. DUKE, Department of Anatomy, Duke University School of
Medicine.
The ovarian surface of Thornomys is covered by a germinal epithelium consisting
of flattened cells. I n some ovaries, near the hilus, the cells may be cuboidal in
shape. Epithelial invaginations into the cortex are uncommon.
Numerous primary oocytes are located near the ovarian surface and are characterized by large, spherical nucleoli. Such oocytes measure 25 p in diameter.
As the follicles grow in size, the most unique feature is the spectacular hypertrophy of the theca interna. Preovulatory follicles with a diameter of 8 0 0 p
may be surrounded by a strip of theca interna measuring 500 p in thickness. This
layer atrophies following ovulation. The cells of the theca interna are large
epithelioid cells, and the layer is extremely vascular.
There is a high rate of follicular degeneration. The theca interna of atretic
follicles usually gives rise to interstitial cells. The degeneration of many intraovarian ova is accompanied by the appearance of large cytoplasmic vacuoles.
Polar bodies are often formed in the ova of degenerating follicles, and the spindle
for the second maturation division may develop.
The ovarian stroma is well populated with argyrophilic fibers. Collagenous
fibers are most apparent in the tunica albuginea of the hilar region and in the
perivascular tissue of the medulla. Elastic fibers are confined to the vicinity of
blood vessels, and the subperitoneal layer of the mesovarium.
* Supported by
a grant from the Duke University Research Council,
D 9. Human liver carcinoma and vertebrate evolution? Hans ELIAS, Department
of Anatomy, The Chicago Medical School.
I f one studies the structure of many cases of primary carcinoma of the human
liver, a chaotic picture seems to unfold. However, a student of comparative
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AMERICAN ASSOCIATION O F ANATOMISTS
embryology will find that every form of such tumors has its counterpart in the
embryonic or adult liver of some vertebrate: muralia of many cells thick walls as
in the liver of turtle embryos; globular masses as in snake embryo livers; trabeculae as in pig embryo livers; tubules as i n livers of embryos of sharks, alligators
and song birds; duct-like structures as in chick embryo livers; spindle cell masses,
as in marsupial, rat and primate embryo livers.
Thus atavism manifests itself in the manifold varieties of hepatocellular tumors
of man. This phenomenon provides a new piece of evidence for the theory of
human evolution from lower vertebrate forms.
1
Supported by a grant from the U. 9. Public Health Service.
DS. Rat and mouse whole bones grown as isogencuus subcutaneous implants?
William J. L. FELTS, Departments of Anatomy, Tulane University and
University of Minnesota.
This demonstration will consist of line drawings and cleared specimens illustrating the following series. (1) Phalanges of 5-day rats, implanted i n littermates and allowed to remain for as long as 200 days. ( 2 ) Humeri of two-day
mice, implanted in the female parent, remaining for 28 days. ( 3 ) Humeri of mice
ranging i n age from three days prenatal t o 28 days postnatal, implanted in the
parent for 28 days. ( 4 ) Humeri of two-day mice implanted for 4 weeks but
held at constant room temperature (23-24°C.) for up to 72 hours before implantation.
This material will show that immature long bones will grow and mature remarkably well as nonfunctioning implants, and maintain size and shape after major
growth is completed. Data showing the effects of donor age upon subsequent
behavior of the immediate implant will be presented. I n addition, it will be shown
that the whole bone, even a t a relatively high storage temperature, retains a
potential for growth for many hours (e.g., cartilage growth results in an appreciable length increase during the 28 day period even after 36 hours storage).
However, an extended period of storage results in the orderly suppression of potentials for each of the several tissue regions.
Currently under investigation are the question of induction phenomena i n
implants and the effects of a wide range of temperatures and storage periods.
* The latter part of this study has been aided by a grant from the Minnesota Chapter of the
American Cancer Society.
D 60. Cutaneous and transcutaneous respiration in man. Laurence R. FITZGERALD, Division of Anatomy, Medical Units, University of Tennessee.
It has been shown by various workers that about 2% of the total oxygen taken
up by the entire human body is taken in through the skin surface. Slightly more
than 2% of the total carbon dioxide given off by the body passes out through this
surface. Since the magnitude of the cutaneous respiration is less than the inherent error of measurement of the total respiration, it has justifiably been neglected in studies of over-all metabolism.
Calculations will be presented which show that all or most of the epidermis of
the skin can receive sufficient oxygen by diffusion through the surface to main-
ABSTRACTS OF DEMONSTRATIONS
457
tain the observed metabolic rate. I n many regions of the body, oxygen can diffuse
into the level of the capillaries of the dermis at a concentration higher than that
in the blood. However, the small area of the capillaries in the skin prevents absorption of large amounts of oxygen through the skin into the blood vessels.
Thus transcutaneous respiration probably can never be a major factor in total
bodily economy in man. The role of cutaneous respiration in renewal of epidermal
cells and in wound-healing seems to deserve further study.
D 30. Abnormalities i n the pattern and relationships o f the major arterial trunks
and aortic arch derivatives of fetal rats following maternal trypan blue
injections? Marjorie H. FOX and Charles M. GOSS, Department of
Anatomy, Louisiana State University School of Medicine.
Approximately 30% of all fetuses recovered from dams injected with l m l of
a 1% aqueous solution of trypan blue either 73 or 83 days after insemination
exhibited some degree of transposition or rotation of the aorta and main pulmonary
artery. All 4 major types of human congenital transposition complexes (overriding aorta, partial transposition, Taussig-Bing, complete transposition) were
well represented. Complete transposition of the aorta was invariably accompanied
by a counter-clockwise rotation of the aortic valve and alterations in the distribution pattern of the coronary arteries.
Approximately one-half of the fetuses with aortic or pulmonary transposition
had additional abnormalities in the pattern of the aortic arch derivatives. The
tendency towards this concomitant aberration was more marked in the 83 day
series. Over 1 0 abnormal patterns have been identified, but distally arising
(retroesophageal) right subclavian artery, right aortic arch with mirror imaging
of the normal pattern, and double aortic arch formed by various combinations
of right and left 4th and 6th arches occurred most frequently.
Supported by grant from the National Heart Institute.
D 45. Electron microscopic study of normal human leukocytes? James A. FREEMAN"; Department of Anatomy, Louisiana State University School of
Medicine. (Introduced by Frank N. Low)
Normal leukocytes were recovered from human blood by a technic not utilizing
anticoagulants or chelating agents, and fixed in buffered OsO,. The morphology
of these cells is presented, with particular reference to ultrastructure i n mitochondria, specific granulations, nuclear membranes, and other organoids and
inclusions.
1 Aided by grants from the Public Health Service (H-1663) and The Idly Research Laboratories.
2 Public Health Training Fellow (HTS-5111).
D $3. Fused quartz rod demonstration of small blood vessels and circulating blood
cells of A m p h h m a ? Fann EARDING*, Hilda DEBACKERI and Melvin
H. KNISELY, Department of Anatomy, Medical College of South Carolina.
Amphiumae have the largest red cells known to man, about 7 0 long.
~
White
cells also are large. Relaxed or dilated capillaries are wide enough to permit
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AMERICAN ASSOCIATION O F ANATOMISTS
these large cells to pass undistorted. Capillaries may measure 100 i n diameter.
Kidney glomeruli are enormous, measuring up to 3.5 mm i n diameter. L. Warner
('49) recorded in motion pictures the plugging of individual Amphiuma glomeruli
with agglutinated blood cells.
Thus, microphysiological and micropathological studies can be conducted at
low magnifications. At any magnification, much more anatomical detail can be
observed in Amphiumae than in other experimental animals.
Therefore, many problems of physiology and pathology can be attacked easily
i n Amphiumae. For instance, we need to know each and every anabolite necessary
for nourishing the linings of small blood vessels sufficiently well so that they do
not leak blood plasma proteins. We need to know the responses of small vessel
linings to many kinds of specific toxins, so that we may use these responses to
test for the possible presence of toxins in blood drawn from patients with various
pathologic conditions and diseases. Amphiuma bladder mesentery is a n excellent
area for experimental manipulation and study. Cannulation of individual capillaries is possible.
A copy of a large bibliography on Amphiumae, and some sources of supply of
these animals, will be given to those interested.
Supported by USPHS Grant H-1683.
D 49. A preliminary study of the electron microscopy of renal juxtaglomerular
cells; correlation with light microscopy? Phyllis Merritt HARTROFT,,
Department of Pathology, Washington University Medical School. (Introduced by W. Stanley Hartroft)
I n an attempt t o elucidate further the nature of juxtaglomerular cells, a study
using the electron microscope is currently underway.
Renal tissue from normal, adrenalectomized, and salt-deficient rats was fixed
in Dalton's ffuid (buffered osmic acid) and embedded in methacrylate. Thin
sections were cut for examination with the electron microscope and thicker ones
(under la) were prepared for phase microscopy. I n addition, paraffin sections
of tissue from the same kidneys were stained by a special technique previously
described. These sections and those prepared f o r phase microscopy served to
correlate the appearance of juxtaglomerular cells as visualized with the light
microscope with their appearance in electron micrographs.
In paraffin sections, juxtaglomerular cells are most easily recognized by their
granules. I n electron micrographs, these granules are very conspicuous by virtue
of their osmophilia. They are spherical t o oval in shape, similar in appearance
to granules seen in other secretory cells. Mitochondria can be readily distinguished
as can the abundant ergastoplasm with its prominent ergastoplasmic granules.
These features provide a sharp contrast between juxtaglomerular cells and adjacent cells in the arteriolar media and support the concept that they may be
secretory in nature.
Supported by a grant from the Nutrition Foundation. Acknowledgment is also made to the
Departments of Anatomy and Radiology, Washington University Medical School, for facilities.
ABSTRACTS O F DEMONSTRATIONS
459
0 5 5 . A human ovum with a n estimated ovulation age of about nine days.'
Chester K. HEUSER, Department of Microscopic Anatomy, Medical College of Georgia.
I n the uterus of a 34-year-old white woman removed surgically by Dr. Richard
Torpin an intact ovum was found implanted in the crevice a t the junction of the
posterior and anterior walls. The ovum is partly covered in by the endometrial
epithelium. Reconstructions prepared from the serial sections reveal a symmetrical
germ disk (0.05 X 0.09 X 0.132 mm) placed normally in a chorion measuring
0.36 X 0.58 X 0.59 mm. Size of chorionic cavity, 0.1 X 0.1 X 0.3 mm. The primary
yolk sac is forming as is indicated by cells which have spread from the entodermal
layer of the germ disk. Numerous small communicating lacunae are present in
the thickened syncytium on the abembryonic pole of the ovum. A few lacunae
communicate with surrounding endometrial vessels. Some of the lacunae contain
small amounts of static blood. An unusual feature of the ovum - which may be
related to the method of formation of the lacunae -is the presence of large ghostlike spheres or nuclei entrapped in the syncytial spaces.
'This investigation was supported (in part) by a research grant R.G.4222 from the National Institutes of Health, Public Health Service.
D 35. Methods for studying regression of atheromata using intraocular transplants and implants? A. C. HIGGINBOTHAM, Department of Anatomy,
Medical College of South Carolina.
Atheromata contribute to the death of many persons today. Cholesterol is
present in all atheromata, and its removal may be essential for their regression.
Thus f a r no method has been developed for studying the regression of individual
atheromata. Green ( '38), Turner ( '39), Markee ( '40), Martinovitch ( ' 5 5 ) , and
Ramm ( ' 5 5 ) have successfully used anterior chamber transplantation for other
purposes. Atheromata or large cholesterol crystals placed in the anterior eye
chamber can be studied and their dimensions measured frequently. Experimental
atherosclerosis can be developed easily in rabbits, but dogs are resistant.
Homotransplants of two non-atheromatous and 4 atheromatous aortas were
made into the anterior eye chambers of 6 young male rabbits. Two homotransplants
of non-atheromatous aorta were made into young male dogs. Heterotransplants
of atheromatous human aorta were made into the anterior eye chambers of two
adult male rabbits. Large cholesterol crystals were implanted in one dog and
5 rabbits.
Gross characteristics of transplants and implants can be visualized and photographed through the corneas of host animals. In histologic sections, unstained
and following Oil Red 0 staining, birefringent substances, including cholesterol
and its esters, can be identified and their distribution studied with the polarizing
microscope. Other details of the transplants can be studied in variously stained
sections.
1
Supported by USPHS Grant H-1020.
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AMERICAN ASSOCIATION O F ANATOMISTS
D 3 8 . T h e fine structure of normal and leultemic marrow cells.' A. F . ROWATSON* and E. A. McCULLOCH*, Departments of Anatomy and Medicine,
University of Toronto. (Introduced by A. W. Ham)
This exhibition consists of electron micrographs of sections of cells from bone
marrow and peripheral blood, obtained from normal and leukemic patients. About
20 specimens of marrow and 4 of leukemic blood were examined. The fine structure o f cells from the lymphocytic, granulocytic, megakaryocytic, erythrocytic and
plasma cell series is illustrated. I n some instances parallel examples of normal
and leukemic cells are shown.
I n addition the appearance of cells from marrow and peripheral blood after
many months of continuous cultivation i n vitro is demonstrated.
Aided by a grant from the hational Cancer Institute of Canada.
D.20. Studies in dogs on absorption from the subarachnoid space and on the
ultrastructure of the choroid plexus. Guy M. HUNT and E. Harold
SHRYOCK, Department of Anatomy, College of Medical Evangelists.
The rate o f absorption of Ringer '8 solution introduced a t graded pressures
(from 100 to 600 mm of water) into the subarachnoid space at the cisterna magna
was found t o be mathematically related to the pressure. Graphic analysis o f these
data introduces the possibility of estimating rates of absorption at pressures less
than those utilized in the experiments. A simple apparatus for measuring absorption rates while maintaining constant pressure levels will be demonstrated.
Tissues for light microscopy were obtained after perfusing the anesthetized
animals through both common carotid arteries with Ringer's solution followed by
10% formalin. The perfusate was injected under a pressure calculated to slightly
exceed the animal's normal blood pressure. Sections were stained with hematoxylin
and triosin, Heidenhain '8 hematoxylin, and Krichesky '8 modification of Mallory 's
aniline blue collagen stain. Representative sections will be displayed on microscopes and by colored transparencies.
Tissues for electron microscopy were removed promptly after the death of the
animals, fixed in buffered osmium tetroxide and sectioned a t approximately 0.05 F .
Prints of electron photomicrographs will be displayed.
D99. Electron microscopy of growing oocytes of Fundulus? Norman E. KEMP
and Margaret D. ALLEN*, Department of Zoology, University of Michigan.
Small pieces of ovaries or single, mature eggs from specimens of Fundulus
collected between June and August a t Woods Hole, Massachusetts, were fixed in
1% osmic acid in buffered artificial sea water, embedded in methacrylate and
sectioned at .05 L./ with a Porter-Blum microtome. Young oocytes lacking yolk have
numerous mitochondria, concentrated in the peripheral cytoplasm, and possess
small granules of undetermined nature in the perinuclear cytoplasm. A few thick
strands of highly osmiophilic material (eosinophilic with routine staining for
light microscopy) may be present among the follicular cells. As growth continues,
ABSTRACTS OF DEMONSTRATIONS
461
the zona radiata begins to differentiate a t the surface of the oocytes, accompanied
by the appearance of lipid yolk and later of protein yolk and cortical alveoli i n
the underlying cytoplasm. Follicular cells pull away from the oocyte, thus leaving
spaces containing protoplasmic processes of the follicular cells. The thick osmiophilic strands among the follicular cells become abundant during early growth
but later diminish in number. Within the differentiated zona radiata coiled fibers
believed to be the distal ends of follicular cell processes extend between parallel
bars, apparently the walls of cylinders, comprising a structural framework. Mature
oocytes from which the chorion has been removed have microvilli at the surface.
Cortical alveoli burst through the surface after fertilization.
Supported by grants from the National Science Foundation ( 0 1 1 6 6 ) and the Michigan
Memorial-Phoenix Project.
D 59. Visualization under ultraviolet lighting of the inspiratory and exppiratory
pathways through the dog's nose. J. Edward KING*, Department of
Anatomy, Duke University. (Introduced by R. Frederick Becker)
Gross specimens of parasagittally sectioned dog heads will be shown under
ultraviolet light. The animals previously had inspired or exhaled an aerosol containing fluorescein. Inspiration occurred during quiet breathing or during a sniff.
One-way passage of air through the nasal chambers was controlled by a special
tracheal valve inserted by cannulization in the living animal and was also simulated in decapitated heads for comparison.
Diagrams and photographs taken under ultraviolet light will also be shown.
D 10. Radioautographs of young rat tissue after administration of C"-labelled
bicarbonate, glucose and mannose. Y . KUMAMOTO*, Department of Anatomy, McGill University. (Introduced by C. P. Lehlond)
One- and three-day-old rats were injected with C"-bicarbonate, -glucose or
-mannose and sacrificed 3-4, 24, and 72 hours later. Tissue sections, prepared after
fixation in alcohol-formalin, Orth, or Carnoy, were coated with Eastman Kodak
NTB2 and 3 emulsions, exposed for 3 to 9 months, and processed by standard
radioautographic procedure.
A similar, widespread distribution of radiocarbon followed injection of the
three labelled substances. The radioautographic reaction of soft tissues was maximal a t the earliest interval and decreased thereafter. This decrease was generally
gradual, except in a few cases-chondrocytes
and the mucus of the sublingual
gland - where it occurred promptly. I n contrast, the radioautographic intensity
observed in bone and dentin durieg the experimental period was permanent.
The distribution of the isotope originating from the three substances studied
differed in several aspects: both sugars, but not bicarbonate yielded an intense
reaction in the goblet cells of the intestinal epithelium; whereas mannose but
neither glucose nor bicarbonate elicited an intense reaction of enamel. It is
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AMERICAN ASSOCIATION O F ANATOMISTS
concluded from these results that mannose can transform into glucose or bicarbonate, and that glucose can transform into bicarbonate, but that transformations in the reverse direction do not occur to a sufficient extent t o be detectable
radioautographically.
D 22. Demonstration of spatial relationships of capillaries to neuronal elements
in specific parta of cat nerz)ous syste7n.l Isabel LOCKARD*, J. BARHAM*,
N. FORLIDAS* and R. MYERS*,Department of Anatomy, Medical College
of South Carolina. (Introduced by J. A. Gavan)
Many methods have been devised for demonstrating central nervous system
capillary beds; few have demonstrated nerve cells simultaneously (The Circulation of the Brain and Spinal Cord, '38). Our demonstration material contains
vessels injected by the Williams ('48) method, and cells stained with thionin,
and permits measurements, comparisons and contrasts of specific and different
parts of the nervous system.
Krogh ('29) showed that each capillary supplies a cylindrical zone of
tissue surrounding it. Flow through single opossum brain capillaries is necessary to maintain the surrounding neurones (Scharrer, '39). Equations describing the rates of supply of oxygen to parts of tissue cylinders have been
developed by Ingram Bloch ('41, '43), G. W. Schmidt ('52, '53a, '53b),
Opitz and Schneider ('50). Methods are available for the measurement of
capillary densities (Craigie, '38; Chalkley, '43). Bed cells circulate freely in
health but become agglutinated in many diseases, forming masses which resist
passage through terminal arterioles, and can plug capillaries (Knisely et al.,
'47 ; Edward Bloch, '55). Reduced flow through capillaries, or other localized
circulatory disturbances, may underlie many neurologic disorders.
TO calculate oxygen supply of cells between capillaries in health and disease
as a basis for contributing to our understanding of these phenomena, we need
to know such information as: distance between capillaries, numbers per cubic
millimeter, diameters, lengths, and relationships to neuronal elements.
Supported by USPIlS Grant B-457 ( C ) .
D 41. Electron Micrographs of protoplasmic
double membrane systems.' Frank
N. LOW, Department of Anatomy, Louisiana State University School of
Medicine.
Double membrane systems comparable to those of nuclear membranes, endoplasmic reticulum and mitochondrial cristae, but of lesser dimensions, are illustrated. They were observed in the striations and apical cell membranes of
intestinal mucosa cells, golgi apparatus, single walls of cisternae of endoplasmic reticulum and in unidentified cytoplasm inclusions. The overall dimensions of these double membrane systems, which consist of two dense parallel membranes enclosing a lucid interspace, range from about 150 to 6 0 A or
less. This implies a thickness of 20 A or less in the individual dense membranes
in the finer complexes.
1 Aided by grants from the Public Health Service (H-1663) and The Lilly Research Laboratories.
ABSTRACTS OF DEMONSTRATIONS
463
D 5 1 . A case of human cyolopia associated with rudimentary pinnae, absence
of the right radius, polydactyly and syndactyly. E. W. LOWRANCE, M.
D. OVERHOLSER, Stuart 0. LANDRY, Jr.* and F. E. DOENGESI,
Department of Anatomy, University of Missouri.
A human male premature (32 weeks prenatal, living 45 minutes following
birth) 40 em in length and weighing 1485 gm presents the following apparent
anomalies: classical cyclopia with a median single eyeball but with eyelids extending laterally; a soft, club-shaped proboscis, located above the eye, approximately 20mm long and 12mm in maximum diameter, with a single, offset,
patent opening measuring approximately 1mm in diameter. The specimen also
has very rudimentary right and left pinnae which are only slightly raised above
the surface of the side of the head; right and left sixth digits present on the
superior extremities ; syndactyly involving digits two, three and four on the
right lower extremity, and digits three and four on the left lower extremity;
the right wrist and hand deviating abruptly toward the thumb side.
Roentgenograms show the absence of a right radius and the presence of
five metacarpals on each side. However, the shaft of the left fifth metacarpal
is thickened and its middle third bears a medially directed process with which
the left sixth digit appears to articulate.
An extended report will follow detailed dissection and study.
D Z l . The relation of the cranial aura to the cavernous sinuses in the cow.
C. P. MARTIN and William Henry BOYD, Department of Anatomy,
McGill University.
The cavernous sinuses are described as irregularly shaped, endothelial lined
venous spaces between the inner and the outer laminae of the cranial dura at
the sides of the body of the sphenoid bone.
I n the cow, the cavernous sinuses are within the outer lamina of the cranial
dura mater.
The outer dura which forms the floor of the cavernous sinuses and the sella
turcica, enclose the optic and the maxillary nervw in the side walls of the
cavernous sinuses.
The outer dura on the internal surface of the parietal bones, is continuous
with the dura enclosing the nerves i n the side walls of the cavernous sinuses.
The outer dura courses medially above the cavernous sinuses, and beneath the
lamina of the inner dura. The inner and the outer laminae of the dura form
the roof of the cavernous sinuses, and fuse about the stalk of the hypophysis
in the formation of the diaphragma sella. The outer lamina of the dura encloses the hypophysis, forming its capsule and the medial walls of the cavernous
sinuses.
D 1 8 . The rat brain in stereotamic co-ordinates. Leo C . MASSOPUST, Jr.,
Laboratory of Neuroanatomical Sciences, National Institutes of Health.
A new stereotaxic instrument and scaled co-ordinate maps of parasagittal
and frontal sections of the rat brain to be used with it will be demonstrated.
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AMERICAN ASSOCIATION O F ANATOMISTS
Measurements were taken on 30 animals weighing 100 to 300gm. Due to
the increase in brain size within this weight range, it was necessary to make
separate co-ordinate maps for animals of 100, 200 and 300gm. Because of the
large number of photographs for each weight group and the limitation of space,
the demonstration will be limited to illustrations of the 200gm group. Coordinates are represented on uniformly enlarged photographs of frozen sections
(100 p ) which were stained by the Sudan Black B technique. This method practically eliminates dehydration shrinkage.
D5. S h i f t s in the lumbar plexus correlated w i t h the lumbarization of the last
thoracic vertebra in mice. Wallace MCNUTT and Eddie DEAN*, Department
of Anatomy, The University of Texas Medical Branch, Galveston.
The lumbar plexus of 80 mice from strains SEA-se (vertebral formula 7
cervical, 13 thoracic, 6 lumbar) and SEC-dse (vertebral formula 7/14/6) were
dissected. The pattern of each strain showed a constancy that was attributed t o
the uniform genetic make-up. The caudal portion of the plexus was alike i n the
two strains in that the obturator and femoral nerves were formed by the lst,
2nd, and 3rd lumbar nerves; the tibioperoneal by the 3rd, 4th, and 5th; and
the pudendal by the 5th and 6th lumbar nerves. However, there was a strain
difference in the cranial portion of the plexus, the subcostal and iliohypogastric
arising from the last thoracic (12T) and the ilioinguinal from the 1st lumbar in
the SEA-se mice and all three of these nerves from the last thoracic (13T) in
the SEC-dse mice. Each of these strains has been bred brother by sister with forced
heterozygosis for the short-ear gene, which has as one of its effects the suppression
of the last pair of ribs without changing the number of presacral vertebrae. This
has a secondary effect upon the pattern of the cranial portion of the lumbar plexus
consisting of the spreading of the origins of the subcostal, iliohypogastric and
ilioinguinal nerves ; besides, of course, the caudal shift by one lumber vertebra
of the remaining nerves of the lumbar plexus.
D 84. Occwrrence of nerve cells in the sciatic nerve of albino rats. George
METZ* and Robert JUDICE*, Department of Anatomy, The University of
Texas Medical Branch, Galveston. (Introduced by John C. Finerty)
Sciatic nerves of albino rats were removed intact with their main branches
and fixed in 10% formol saline solution. Some were sectioned longitudinally at
10 or 15I./ and stained with either Nissl stains or by various silver methods. I n
addition t o the sectioned material, whole mounts of the entire nerve and its
branches were prepared and stained with Cresyl echt Violet. Nerve cells were
found in 42 of the 46 nerves studied. The number of cells was variable,
ranging from 4 to 144 ganglion cells per nerve. The cells occurred singly or
in groups along the entire length of the sciatic nerve, but the majority were
located a t the bifurcation into tibia1 and common peroneal branches. Cell
bodies were round t o oval, 25-45 p in diameter, and encapsulated by satellite
cells. They showed the characteristic centrally located nucleus, prominent nucleolus and granular clumps of Nissl substance scattered throughout the cytoplasm.
ABSTRACTS O F DEMONSTRATIONS
465
U 61. Interpretation of the electromyogram of graded voluntary movements in
normal subjects.' Alice L. O'CONNELL* and 0. A. MORTENBEN,
Department of Anatomy, University of Wisconsin.
The conventional electromyogram of moderate or strong voluntary contraction
records the characteristic interference pattern of action potentials. The number
of active motor units during such a contraction is so great that the finer details
of the relative activity in different parts of the muscle are obscured. I n this
study an attempt was made to demonstrate such details during graded voluntary
movements performed by normal human subjecta
Action potentials were recorded by an 8 channel ink-writing electromyograph
from 8 pairs of small surface electrodes arranged longitudinally along the
tibialis anterior muscle. Intramuscular electrodes were used to supplement the
records obtained from surface leads.
Relative differences in activity were recorded from the several parts of the
muscle. The superior portion consistently showed a lower threshold and a
higher frequency. This pattern suggests a correlation with the internal structure
of this muscle. The electromyogram also demonstrated the recruitment of additional motor units and the increase i n frequency of the same unit during
increasing effort. The variation in distribution of action potentials, presumably
from the same motor unit, in several channels may be evidence of the spatial
distribution of the motor units.
Differences in activity were most apparent during minimal contractions but
also detectable during moderate effort. Maximum effort disclosed co-contraction of
the tibialis anterior in the movement of eversion, but not i n plantar flexion.
1
Supported by a grant from the Wisconsin Alumni Research Foundation.
D 50. Electron microscopic studv of absorption of particubte matter from the
peritoneal cavity.' D. Louise ODOR, Department of Anatomy, University
of Washington.
Rats were injected intraperitoneally with a solution of colloidal mercuric sulfide
or of thorotrast and were killed at intervals thereafter. Thin sections of mesentery
and diaphragm were examined with the electron microscope. As early as 4
hour after the injection the administered particles adhered t o the surf aces of
the microvilli and the plasma membrane of the mesothelium. At this and a t
subsequent times the particles were localized within the mesothelial cytoplasm,
confined either within clear vacuoles of varying sizes or aggregated in relatively
dense bodies. No particulate matter was noted within the intercellular spaces. Some
was found within the cytoplasm of macrophages of the subjacent connective
tissue as early as f hour after the injection. Twelve hours later relatively few
particles remained adherent to the mesothelial cell surf ace, but there was an
increase in the number within both mesothelial cells and macrophages. The
particles of mercuric sulfide in the mesothelial cytoplasm varied in size from
about 50 to 220 A. More of the particulate matter was observed within the cyto-
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AMERICAN ASSOCIATION O F ANATOMISTS
plasm of the diaphragmatic than of the mesenteric mesothelium a t all time
intervals studied. Many particles were located in macrophages in the connective
tissue between the muscle fibers of the diaphragm, but none were situated within
the sarcoplasm.
-~
1 Supported in part by grant RG-4296 from the Department of Health, Education and Welfare, and by the Life Insurance Medical Research Fund.
D 5 4 . Apposition and fusion of placentae in the hamster.' Margaret Ward
ORSINI, Department of Anatomy, University of Wisconsin.
Apposition and actual fusion of adjacent placentae is not rare in the hamster.
The degree of union varies. It has been found in animals bearing their first, second
and third litters, and in individual horns containing as few as 5 young, and as
many as 11. As many as three double gestation swellings have been found in
one horn.
The youngest found ( 9 days and 8 hours) appear grossly as abnormally large
gestation swellings which, after clearing, show two embryos, each in its individual
decidual cavity.
By 12 days and 10 hours the individual placentae are distinguishable from
the enveloping tissue. I n one horn of this age two placentae appear fused, in
an adjacent swelling the placentae are only i n apposition.
Both apposition and fusion increase in late gestation.
Sectioned material from the 16th day, one preparturition and one midparturition, shows union of maternal tissue, apposition, and also of the fetal trophospongium, true fusion. The labyrinth is not involved so there is no mixing of the fetal
circulations.
All material indicates each placenta, despite union, still receives its individual
maternal arterial blood supply.
This intimacy of adjacent placentae appears t o be caused by nearness of the
original implantation sites and subsequent crowding. High litter size favors this.
1
Supported by a grant from the National Science Foundation.
D 67. Strains of mycotic parasites an red blood corpuscles f r o m hospital patients.
James W. PAPEZ and B. Pearl PAPEZ, Columbus State Hospital, Ohio.
Four strains of mycotic spores were found in red blood corpuscles (rbc) of
hospital patients. Each strain began with single spore that entered rbc, dispersed
in stroma into a colloidal stage, fluoresced in purple (indole) or rose, and organized
into particulate globules. Under phase contrast, oil immersion microscopes,
each strain and its stages were identified and watched for several days (3-21).
Four strains are illustrated. Each strain ends in a definite form which we named
rosette, beaded, mucoid and cystic. 1. Rosette strain, spore disperses, colloidal stage
gives rise t o formative spots that grow protrusions from surface of rbc converting
it to a rosette of 9 to 13 mycotic cells which separate. 2. Beaded strain, colloidal
stage disperses as ring in periphery of rbc, purple fluorescence, globules form out
of ring, project from surface as beads on a string. 3. Mucoid strain (Anat. Rec.
V. 118, Suppl. P 403, 1954), colloidal stage is ring or dispersed, forms numerous
particles, grows spines to outside, organisms reduce rbc t o mucoid sac. 4. Cystic
ABSTRACTS OF DEMONSTRATIONS
467
strain, colloidal cyst forms blister inside of rbc which breaks open, cyst with sawteeth, organisms are globules on stems. I n contaminated blood (hemorrhages) a
capsular strain grows a stout, pulsatile, globular filament which lays a gray
capsule in remnant of rbc. Mycotic spores in sclerotic patches in walls of blood
vessels are illustrated.
D 28. Stereoscopic photographs and drawings f o r teaching anatomy and embryology. Anthony A. PEARSON, Luis A. TURNER* and William J.
MIKKELSEN*, Department of Anatomy, University of Oregon Medical
School.
No abstract submitted.
D 1 4 . Vital staining of nerve tissue with methylene blwe. E. H. POLLEY*,
Department of Anatomy, Hahnemann Medical College.
W. Angulo)
(Introduced by A.
The use of methylene blue as vital stain in neurohistological research has not
enjoyed great popularity recently because of the great variability in the resulting
preparations. We have succeeded in obtaining preparations that demonstrate
the value of this technic despite its inherent variability.
Animals were anesthetized and methylene blue was administered parenterally
by a modification of the method of Feindel, Sinclair, and Weddell ('47). The
animals were then sacrificed and tissues of the peripheral and central nervous system
were removed for oxidation and subsequent fixation after the method of Bethe and
Cajal. Paraffin and frozen sections as well as whole mount preparations will
be demonstrated.
D 16. A modified stain for degenerating terminals. Margaret M. POWERS*,
Paul A. YOUNG* and George CLARK, Departments of Anatomy and
Physiology, University of Buffalo Medical School.
Silver stains are generally considered erratic and the stains for degenerating
terminals, at least in our hands, have proved no exception. However such a stain
was needed badly to complement the Nauta and Gygax stain for degenerating
axons (Stain Tech., 2 9 : 91, 1954). On analysis of published methods there
appeared to be an overwhelming number of variables involved. The procedures
were varied as systematically as possible. On the basis of these experiments
it was possible to develop a method which has given surprisingly consistent
results. Test material has been mainly from cats with complete transection of the
spinal cord. The changing picture of terminal degeneration, as seen in such
preparations, will be shown after 3, 4 and 5 days postoperative survival.
D 46. A n eZectronmicroscopic study of the Pncinian corpwscle. T. Andrew QUILLIAM, Department of Anatomy, University of California at Los Angeles
and Veterans Administration Center, Los Angeles.
The lamellae of the outer zone of the corpuscle are extremely thin yet truly
cytoplasmic in nature, and they exhibit a radial assymetry about the central
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AMERICAN ASSOCIATION O F ANATOMISTS
longitudinal axis. They are arranged in two well-defined groups in the more
superficial of which the lamellae are comparatively widely spaced, while i n the
deeper group they lie closer together. Over the surfaces of the lamellae lie
many circularly oriented collagen fibers. A distinct layer of thicker cells separates
these outer lamellae from the inner core of the corpuscle. This latter consists
of twin sets of cytoplasmic sheets bilaterally enfolding the terminal part of the
central axon. The two halves of the core are separated from each other by a
radial cleft. The axon, which is somewhat oval in cross section with its long
axis coincident with that of the cleft, has a limiting membrane immediately deep
t o which is a striking pallisade-like arrangement of mitochondria. No myelin is
distinguished around the terminal axonal filament but, proximally, nearer the point
of exit of the axon from the corpuscle a myelin sheath is acquired, and here
a few centrally situated mitochondria are demonstrable. Vascular capillaries occur
in considerable number near the myelinated segment of the axon but are not found
further distally.
D 47. Electron microscopic studies of kidney f r o m rats maintained on normal
or low potassium diet dkring vital staining w i t h silver nitrate.' James F.
REGER", Department of Anatomy, University of Colorado Medical Center.
(Introduced by V. L. van Breemen)
Electron microscopie studies were made of renal tissue from two groups of
rats whieh were given 0.5% silver nitrate solution as drinking water for
periods of 6-12 months. One group was maintained on normal diet plus silver
nitrate; the second, on low potassium diet plus silver nitrate. Low potassium
diet produced no differences in location or amount of silver deposition i n the
kidney, though it did cause muscular atrophy. I n both groups of animals silver
deposits were found as follows : sparsely distributed through perivascular and
peritubular loose connective tissue ; rarely located within endothelial cells j precipitated in endothelial basement membranes ; precipitated between smooth muscle cells
of arterioles ; most heavily precipitated in basement membranes of the glomerulus
and convoluted tubules ; and sparsely precipitated in thin basement membranes of
Henle's loop and collecting tubules. No silver was identified in urine collected from
several animals of each group. Preliminary x-ray diffraction studies indicate that
the silver is present i n the tissue as silver sulfide. It is possible that free sulfur or
sulfhydryl groups in the basement membranes and ground substance precipitate
the silver, the size of the precipitated granules varying with the amount of silver
available and to a much less degree with the amount of sulfur available.
1 Supported by grants from the Muscular Dystrophy Associations of America, Incorporated,
and the U. S. Public Health Service.
D J 1 . T h e finer pulmonary vascular architecture in f e t a l and neonatal guinea
pigs. S. R. M. REYNOLDS, Department of Anatomy, University of Illinois,
College of Medicine.
Thick (25 p ) sections of fetal guinea pig lung will be shown, demonstrating in
the fetal and neonatal state ( a ) terminal arterioles; (b) convoluted arterial
capillaries lying along the alveolar ducts; (c) the true capillary plexus that
ABSTRACTS OF DEMONSTRATIONS
469
joins these convoluted arterial capillaries with each other; (d) venous drainage
of convoluted arterial capillaries ; (e) elastic tissue about the arterial capillaries.
Methods of preparation, sectioning and staining the tissues were described in
abstracts for the meeting of the American Association of Anatomists in 1955
(Anat. Rec., 121 : 414, S.R.M. Reynolds: Pulmonary vascular changes and
hemodynamic changes).
D 3 6 . Heart Pain: Mechanisms and relief. Joseph T. ROBERTS, Veterans Ad-
ministration Hospital and University of Buffalo School of Medicine.
The exhibit shows work done on the role and changes in the vasa nervorum
or blood vessels of the nerves, especially as affected by drugs or in experiments
showing that the vasa nervorum of the left arm contract after ligation of a
coronary artery. This supports our belief that cardiac referred pain is caused
by ischemia in somatic nerves, due to a reflex vasospasm. This lowers the threshold
of irritability for various sensory and motor changes in the nerves in the area of
referral. This is thought to be a mechanism for persistent shoulder-hand pain,
Dupuytren 's contracture and other sequels of cardiac infarction. Methods of
revascularizing the ischemic heart are shown, e.g. the author 's method of arteriolizing the coronary sinus or increasing the abundance and size of the Thebesian
vessels. The various anastamoses of the coronary system are demonstrated, and
also the lymphatics of the heart muscle.
D5.9. Weight changes associated wit71 pregnancy in the guinea pig.' James B.
ROGERS and Richard C. TAYLOR", Departments of Anatomy and
Pathology, University of Louisville School of Medicine.
Weight changes during pregnancy, post-partum weights, litter weights and
placental weights from 917 guinea pig pregnancies will be reported.
The observations are made on guinea pigs from a colony which has been
maintained for fifteen years under relatively constant conditions as to diet,
temperature and care.
Early i n pregnancy the weights were taken at weekly intervals. Late in
pregnancy the animals were weighed daily, a t the same time of day each time
and were handled by the same person. Post-partum weights were taken immediately
after delivery. Each offspring was weighed individually soon after birth.
Placentas were taken from the cage when ever possible.
By selective breeding a strain of guinea pigs susceptible to a toxemia of
pregnancy has been maintained. Toxemia of pregnancy characterized by sluggish behavior, convulsions and death, just before, during or immediately after
delivery, occurs in 15% of pregnant female guinea pigs which have an inherited
susceptibility to the disease. Another strain which does not develop the toxemia
is maintained.
Data will be presented in tabular form for three groups of pregnancies (1) nonsusceptible (2) susceptible to but not developing the toxemia and (3) susceptible
animals which died from toxemia.
Increased amount of amniotic fluid seems to be characteristic of toxemic animals.
1 Aided by a grant from the U. S. Public Health Service and by a grant to the University
of Louisville from the Kentucky State Medical Research Commission.
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AMERICAN ASSOCIATION O F ANATOMISTS
D 2 5 . A schematic presentation of thalamic morphology and connections together
with observations on details of thalamocortacal relationships.’ Glenn V.
RUSSELL, Department of Anatomy, The University of Texas Medical
Branch, Galveston.
A schema is presented illustratiiig succinctly but in a simplified form a large
part of the fundamental information on the morphology, connections, and
organization of the dorsal thalamus. Evolved over the last several years by
the author as an adjunct to teaching medical students the anatomy and connections
of the dorsal thalamus, it has proved t o be a valuable teaching aid t o the
lecturer and a worthwhile and instructive study guide and mnemonic device to
the student. Elaborating upon the schema, recent observations of some details
of thalaniic projections to the cerebral cortex of man are demonstrated.
1
Supported by USPHS-NINDB Grant B - 7 5 1 ( C ) .
D 43. Electron microscopy of the epiphyseal apparatus. Bronnetta L. SCOTT*,
Department of Anatomy, University of California a t Los Angeles and
Veterans Administration Center, Los Angeles. (Introduced by Daniel C.
Pease)
Electron micrographs of undecalcified sections of the epiphyseal apparatus
demonstrate some of the ultra-structural components and relationships inaccessible
by previous methods. For this study the proximal end of the kitten humerus was
used. I n the epiphyseal cartilage, the appearance of the endoplasmic reticlum
indicates the approximate stage of cytomorphosis. The chondrocytes of the
proliferating zone present a characteristic dehydrated appearance, and the
endoplasmic reticulum is oriented into tightly-packed, undulating layers. Progressing toward the diaphysis as the cells become increasingly hydrated, the
endoplasmic reticulum becomes more loosely disposed. The cartilagenous matrix
consists of a network of fine fibrils without demonstrable periodicity. The capsular
portion lacks these fibrils. Osteoblasts are separated from the calcified matrix
by a layer of loose collagenous fibers. This layer is of variable thickness, reflecting
the rate of deposition of the matrix, and its subsequent calcification. Calcification
is essentially the same in both cartilage and bone. Small islands of crystals are
deposited a t random and then coalesce. Individual crystals, at least in early
stages, are less than 50 A wide and about ten times as long. The “brush border”
of the osteoclast is an intricate infolding of the plasma membrane. I t s association with mineral resorption in cartilage and bone is suggested by the presence
of crystals between its folds and by associated cytoplasmic vacuoles.
D 4 . Some unusual perforations of Indian skulls. H. Alan SKINNER, Department
of Anatomy, University of Western Ontario.
Four Indian skulls were found in the same burial pit on an island in the
St. Clair River. The 4 skulls show identical trepan openings in the same position
in the vault of the skull about 4cm behind the coronal suture and crossing the
ABSTRACTS OF DEMONSTRATIONS
471
median sagittal suture. The openings i n the skulls are i n the form of deep grooves
and the inner margins of the openings are ellipsoidal in shape. They are unlike
any other openings of the skull i n the author's experience and no reference to
them has been discovered in the literature of trepanning.
D1. The influence of Vesalius' Fabrica on Ruini's Anatomia d d Cavallo
(1598). J. F. SMITHCORL%*,Department of Anatomy, Michigan State
Universitg. (Introduced by Miriam Scott Lucas)
Carlo Ruini 's magnificent Anatomia deZ cuvuZZo is the first comprehensive
anatomy of a single animal. Attempts have been made to discredit the Ruini
plates as a theft from the work of da Vinci, but da Vinci never did more than
the surface anatomy of the horse. Rather, the superb craftsmanship of the
unknown creator of the Ruini plates appears to be directly related to that
of Vesalius' Fabrica. A curious, but hardly credible suggestion i n a posthumous edition of Ruini, credits the plates t o Titian (died 1576), although Titian
did visit Bologna while Ruini resided there. Inasmuch as many of the Vesalius
plates are the work of students of Titian, it seems logical that other students
or followers of the Titian school may have been responsible for the Ruini
drawings being done in the Veealius tradition. The number of similarities
between the two works is more than coincidental; the posing of subjects, the
rendering of muscle masses, the stylized representation of organs and systems,
and the landscape backgrounds all point to this. With nothing in animal anatomy
as a precedent, Ruini's work required the artistic tradition established by the
Fabrica of Vesalius.
D 13. A method for fixing guinea p i g cochleae under "physiological conditionsJ'
and preparing ribbons of sections following double embedding? Richard
H. SWIGART, Arthur L. JlLJERS* and James B. ROGERS, Departments
of Anatomy and Otorhinolaryngology, University of Louisville School of
Medicine.
During studies on age changes in various organs of senile guinea pigs it
seemed advisable to correlate cytological changes with tested deafness. Standard
methods for preparing cochleae were first tested using young adult guinea pigs
from an inbred strain. These methods did not meet our requirements, for
good cytological preservation was not consistently attained, and serial sections
of cellodion embedded specimens were cumbersome to prepare.
The method presented here was modified after that of Koenig et al (Stain
Tech., ZU: 13-22) f o r brain. After perfusion a t 37°C. with 0.9% Naa-xcacia
solution followed by Heidenhain's Suss' '-acacia solution, cochleae were dissected
and immersed in the latter for 24 Iiours, decalcified in 570 trichloracetic acid i n
10% aqueous formalin, washed in 9570 alcohol for 12-24 hours, dehydrated in
100% alcohol for 1 hour, in a 1 : l ether-alcohol mixture for 1 hour, placed i n
276, 4%, and 6% parlodion for 2, 3 and 4 days successively, and embedded i n 6% parlodion hardened with chloroform vapor for 6-8 hours.
Blocks were trimmed, cleared in benzene and infiltrated i n three changes of 52"-54"
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AMERICAN ASSOCIATION O F ANATOMISTS
paraffin for one day each and embedded. Ribbons of 1 0 p sections could bc
efficiently cut on a rotary microtome.
Charts, pictures of the constant temperature perfusion apparatus and other
features of the method, and photomicrographs of the results attained will be
presented.
1 Supported by a grant from the American Otologieal Society and by a grant to the University of Louisville from the Kentucky State Medical Research Commission.
D
6 2 . Injected vessels in &eared tissues.
P. F. SWINDLE*, Department of
Physiology, Marquette University School of Medicine. (Introduced by
Walter Zeit)
The gross and microscopic specimens are from a collection of many which were
injected and cleared over a period of 32 years in studies of comparative physiology
and anatomy. The most common injection media were red cinnabar and India
ink. The cinnabar was used quite extensively because of its radiopacity, because
it shows up as white in the infrared print (if reflected light is used in exposing
the plate), and also because it can be urged into the very small arterioles and
arteriovenous anastomoses without driving it into the capillaries in non-pathologic
tissues. The clearing was done by using a Spalteholtz method which leaves the
tissues preserved in methyl salicylate.
The animals were obtained mainly from the Washington Park Zoo in Milwaukee,
Wisconsin.
D 5 6 . Variations in depth o f implantation o f the human ovum correlated with
placental anomalies, spontaneous abortion and premature separation. Richard TORPIN, Department of Obstetrics and Gynecology, Medical College
of Georgia.
See abstract of paper from platform No. 179.
D 3 2 . Conduction tissue of anomalous human hearts.'
R. C. TRUEX and
J. A. KAIN*, Department of Anatomy, Hahnemann Medical College.
This study was based on celloidin serial sections of nine human hearts ( 2 days
t o 3 years of age) with high interventricular septal defects. Histologic appearences
Qf the conductive elements in certain hearts, as well as some variations in the
atrial cardiac muscle fibers will be demonstrated. The relationship of the septal
defect to the common bundle and its left and right bundle branches are to be
illustrated.
1
Supported by U. S. Public Health Qrant H - l 5 9 1 ( C ) .
D 46. Electron microscopic studies of lung tissue from guinea pigs and pre,mature
infants.' V. L. van BREEMEN and H. B. NEUSTEINr2, Departments
of Anatomy and Pathology, University of Colorado Medical Center.
Electron microscopic studies were made of normal and pathological lung tissue
from guinea pigs and premature human infants. Observations of normal lung
ABSTRACTS OF DEMONSTRATIONS
473
corroborated Lowe ’s observations on alveolar epithelium. Attenuated epithelial
sheets overlay the capillaries, separated from endothelial cells by basement
membranes. Epithelium apparently covered septa1 surfaces except where macrophages rested within the septum and protruded into the alveolar space. Macrophages (alveolar cells) occurred itlso as apparently free cells.
Pulmonary hyaline membrane disease occurs a s the only pathological finding
in about 25% of premature infant deaths. Lung tissue was obtained from
premature infants who died in this hospital. For comparative study hyaline
membrane disease was produced in guinea pigs by exposure for 72 hours to
98% oxygen atmosphere with 95% humidity. Electron microscopic study indicated
that the hyaline membranes in the guinea pig lungs were essentially the same a s
those in the infant lungs. Hyaline membranes occurred in alveolar space overlying
the epithelium. Finely granular material (probably plasma proteins) was found
next to the capillaries in the alveolar space. Fibrils extended from this material
into a mass of fibrils and debris, identified as the hyaline membrane. I n our
interpretation the fibrils are fibrin and the hyaline membrane is essentially a
fibrin clot enclosing some cellular and other debris.
Supported in part by a research grant from the National Institutes of Health, Public
Health Service.
Maytag Fellow of the American Csiicer Society.
D 7. Tissue culture studies of the rat thymus.’ Mina Lee VERNON”, Department
of Anatomy and The Tissue Culture Laboratory, University of Oklahoma
School of Medicine. (Introduced by Kenneth M. Richter)
Roller tube cultures of the r a t thymus were studied i n a living state, and after
fixation and staining “in toto” by a modified Giemsa method. Time-lapse
cinephotomicrographic data was collected on some of the cultures. Comparative
studies were made on routine histologic preparations of the uncultured thymus.
These studies revealed firstly, that many of the several cell types distinguishable
in ordinary histologic preparations of the r a t thymus can also be found in cultures.
Secondly, most of these cell types actually grow and reproduce in tissue culture
including fibroblasts, stellate shaped reticulum forming cells, endothelial cells,
and epithelioid cells. Thirdly, certain cells of thymic explants undergo such
extreme pleomorphism that a t times free cells (thymocytes9) change into
epithelioid cells, and, conversely, epithelioid cells change into free cells (thymocytes?). This has been observed only by means of cinephotomicrography. Fourthly,
histological differentiation of special structures occurs in thymic tissue cultures
including ( a ) a reticulum formed by stellate shaped cells; ( b ) Hassall’s
corpuscles with central differential staining suggestive of hyalinization ; (c)
capillaries many of which were patent proximally and contained fragments which,
on the basis of their differential ietaining, were suggestive of blood cells; and
(d) conspicuous epithelioid sheets of undetermined origin.
Basis of thesis submitted to the Qraduste Faculty of the University of Oklahoma in partial
fulfillment of requirements for the degree of Master of Medical Science.
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A M E R I C A N ASSOCIATION O F A N A T O M I S T S
D 15, A Bielchowski-Marchi technique f o r parafin sections. Gin0 DI VIRGILIOI,
Monroe GREG0R.Y" and Joseph HOLLENBERG*, Department of Anatomy, Howard University. (Introduced by W. Montague Cobb)
The finely myelinated fibers which are a source of equivocation with the
Marchi technique show up well with a silver method, i n which sections are mounted
in the usual manner and passed through mordants, oxidizing and reducing agents;
toned with gold (Auer and Di Virgilio, '53) and intensified with a Bielchowski
formula (Nauta and Gygax, '51). The treatment of the tissue with the various
chemicals inhibits silver deposition on the normal tissue. Silver will be deposited
on the degenerating axons. The end result is an extended Marchi picture.
Demonstrations are provided from the brains of cats, rats, rabbits and mice
subjected to various types of lesions and from pathological human material from
the Armed Forces Institute of Pathology, Washington, D. C. The sections are from
serially cut brain tissue, selected at regular intervals, and stained for both normal
and degenerated fibers. Corresponding series, selected a t the same intervals were
stained for the degenerating axons only.
A limited number of specimen slides is available on request.
D 37. Akimesia, rigidity, tremor and associated phenomena i n reserpinized
monkeys and chimpanzees. William F. WINDLE, Jan CAMMERMEYER,
Jane T. JORALEMON", John 0. SMART*, Earl FERINGA" and Michael
McQUILLEN", Laboratory of Neuroanatomical Sciences, National I n stitutes of Health.
Reserpine in single doses of 2.5-3.0 mg/kg orally, intravenously or subcutaneously in Af rican green monkeys and chimpanzees produced phenomena
resembling those i n human parkinsonism. These were recognizable within 30
minutes and became maximal in 2-4 hours, full recovery requiring 24-48 hours.
All animals showed in varying degrees: (1) muscular rigidity of the "cogwheel"
type, better in the chimp than the monkey; ( 2 ) akinesia, torpor, catalepsy and
lethargy; ( 3 ) tremor which varied inversely with degree of lethargy and
disappeared during sleep (resting 3/sec. tremor, disappeared on voluntary
movement); (4) sialorrhea and some increased waxiness of facial skin; ( 5 )
changes in mood and ( 6 ) general appearance including fixed facial expression,
poverty of bodily movements, marked kyphosis with head bent forward, and
elbows and hands flexed. Phases of akinesia often alternbted with brief spontaneous
activity, changes to successive voluntary movements being impaired. Grasp and
other reflexes were present.
Reserpine administered to monkeys daily for 4 months in doses of 0.2-0.4
mg/kg subcutaneously induced hypokinesia, slight rigidity and marked tremors,
but no vegetative phenomena. The maximum effect occurred a t the third or
fourth hour after the drug was given.
D 11. Early stages of intraepidermal melanoblasts in A-egro fetuses. Arnold A.
ZIMMERMANN and S. W. BECKER, Jr.", Departments of Anatomy
and Dermatology, University of Illinois.
Split-skin preparations treated with Masson's ammoniated silver nitrate reveal
immature forms as well as fully differentiated dendritic cells in surface views.
ABSTRACTS O F DEMONSTRATIONS
475
Such propigment cells appear first in the 11th week and rapidly form extensive
intraepidermal distribution patterns.
At 12 weeks they are present in all skin areas of the body, including palm
and sole. Cell counts on such preparations are more reliable than those made on
sectioned material. The technique essentially illustrates the maturation process of
the propigment cells. The “mongolian spot” of a 4.8 month fetus, consisting of
dermal melanoblasts, is also demonstrated.
MOTION PICTURES
M P 12. Collateral ventilation in the lung of dog.’ A, W. ANGULO, Vincent
KOWNACKI* and Edmund HESSERT, Jr.*, Department of Anatomy,
Hahnemann Medical College.
This Kodachrome film summarizes the results obtained in a series of in vitro
experiments in which a colloidal suspension of carbon particles ( 2 7 mp in diameter) was injected into a single secondary brounchus at pressures that ranged
within physiological limits.
I n each preparation the injection mass was observed to fill first the pulmonary
segment directly attached t o the injection apparatus. The injection mass was
then seen to appear in adjacent pulmonary segments.
These results clearly indicate that there is ample communication between the
segments of each lobe. Therefore, the bronchopulmonary segments are not
discrete anatomical or physiological units in the lung of dog.
~
Supported by U. S. Public Health Grant H-1366Ca.
* Supplied by Columbian Carbon C!ompany through the courtesy of Doctor George S.
Samuelsen.
M P 6. Platelets in the periphe:ral circulation of the hamster (Melsocricetcs
auratus).’ Herbert J. BERMAN*, George P. FULTON and Hrenton R.
LUTZ, Department of Biology, Boston University.
Platelets were cinephotomicrogrrzphed in civo in eddies, columns of plasma, and
in plasma spaces in vessels with flowing blood. I n wityo, filamentous and dendritic
processes, lysis, adhesiveness, and agglutination of platelets were recorded with
phase microscopy. Freshly drawn platelets appeared disc-shaped in surf ace view
and lenticular in side view. Later they became filamentous and dendritic.
Granules appeared to extend into the filamentous processes. Lysed platelets
appeared pale, smaller, and less granular with some loss of content. They were
observed adhering to glass. The agglutinated and nonagglutinated platelets in
a 24-hour slide preparation were lysed. Platelets observed in living blood vessels
were also disc-like on surface view and lenticular on side view. Filamentous and
dendritic processes were not observed in v i v a Platelets were photographed plugging
a hole made in a blood vessel wall with a microrod. Adhesion of platelets followed
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AMERICAN ASSOCIATION O F ANATOMISTS
mechanical, electrical, or chemical injury of a vessel wall. Some of the coagulation factors such as commercial thrombin and invasive thromboplastin (Russell’s
viper venom), caused platelet thrombo-embolism.
ISupported by grant H-902 ( e 4 ) , National Heart Institute, PHS, and the Department of
the Army, Office of the Surgeon General.
M P 5 . Color tebvision of microcircidation. Edward H. BLOCH, Louise WARNER,
Murray C. BROWN* and John W. CHRISTENSEN*, Department of
Anatomy, Western Reserve University; Department of Anatomy, Georgetown University ; The Clinical Center, National Institutes of Health and
Columbia Broadcasting System Laboratories.
The film depicts a pilot exploring some possibilities of electronic image
processing by color television.
Frogs were anaesthetized, laparotomized and their mesentery or liver quartz-rod
transilluminated. The tissue was observed with a stereobinocular microscope
( x 32-150), an area selected and a CBS color television camera swung over the
ocular. Subsequently the images were observed and recorded from a monitor.
The images were studied and recorded first at normal color balance, light
intensity and Kelvin temperature and then the following adjustments were
made : light intensity reduced, contrast varied for each color, colors removed,
‘ ‘crispening” circuits added t o vary apparent contrast and phase shifts produced
between camera color wheel and the monitor and recorder color wheels.
The images produced by this field sequential system had good resolution and
the color compared favorably with standard recording methods. I n general,
surface details were better and the images recordable at lower light intensities.
Since it was possible t o balance the colors of the image, variations i n Kelvin
temperature was not important. When the colors of the image were modified
individual cellular components were made either more or less prominent than
normal. Thus with this method it is possible to rapidly vary contrast by removing
or adding colors without affecting the tissue.
MP 10. Reduction and substitzition experiments on the peripheral nerves of
respiration. Victor CASTLEBERRY*, T. H. BARNETT* and H. A.
HOLTMAN*, Department of Anatomy, The University of Texas Medical
Branch, Galveston. (Introduced by Donald Duncan)
A film illustrating tolerance of the dog to bilateral phrenectomy and section of
8 pairs of intercostal nerves, and a series of cats t o show effects of various
substitutions for the phrenic nerves. One cat illustrates the effects of vagophrenic anastomosis ; namely, a well preserved and powerful diaphragm but
inadequate due to lack of effective natural impulses. Other cats display the
adequacy of one phrenic nerve alone, the effectiveness of the levatores costarum,
and bilateral substitutions of the phrenic nerves by the nerves to the infrahyoid
muscles.
ABSTRACTS O F DEMONSTRATIONS
477
M P 11. An experimental study 01%the passage of air through the nose. Seymour
FRIEDBERG*, Department of Anatomy, Duke University. (Introduced
by Kenneth Duke).
No abstract submitted.
M P 8. Neurosonic surgery.' William J. FRY*, Frank J. FRY* and John W.
BARNARD, Bioacoustics Laboratory, University of Illinois.
The multiple, focussed beam, ultrasonic method for producing selective accurately localized reproducible changes in the central nervous system is illustrated
in this motion picture. Surgical preparation of the subject (monkey), the technique
of irradiation and histologically pipepared tissue sections are shown. The operation
of the instrumentation is demonstrated by producing a lesion in the fiber tracts
of the internal capsule.
This technique is in current use in studies of brain structure and function.
A lesion of practically any desired size (as small a s 1 mm in diameter) and shape
can be produced a t almost any specified position in the central nervous system
without disturbance of intervening tissue and without disruption of blood vessels
even within the site of the lesion. I n addition, it is possible by proper choice of
dosage to irreversibly affect fiber tracts without destroying surrounding or neighboring nuclei which receive the same dosage of acoustic radiation. The
stained tissue sections shown in the motion picture illustrate the production
of ultrasonic lesions of a variety of sizes and shapes in both white and gray
matter a t various depths in the brain.
'The support in this research field of the Physiology Branch of the Office of Naval Research is acknowledged.
M P 3 . Alkaline tolerance of the isolated embryonic rat heart.l E. K. HALL,
Department of Anatomy, University of Louisville School of Medicine.
The effects of alkalosis were studied on isolated hearts of ll$-day r a t embryos
in Ringer made alkaline with NaOH. I n the pH range 8.5-9.49 (30 cases), the
hearts accelerated during the first 4 minutes (from 158 to 177/minute), then
decelerated slowly during the rest of the 60-minute experimental period (to 132),
then accelerated slightly in the terminal control solution. I n the p H range
9.5-10.5 (30 cases), the same sequence of events occurred, except that changes
were more marked. The rate in the terminal control solution attained the initial
control rate. I n the p H range 110.6-11.5, there was immediate acceleration in
27 of 34 cases, then rapid deceleration and arrest within several minutes; upon
immediate return to normal Ringer, 25 hearts began to beat again, some within
a few minutes.
Results with NH,OH were similar (80 cases), except that deceleration and arrest,
with subsequent recovery in control solutions, occurred a t lower p H levels
(9.2-10.7). The alkaline tolerance of the embryonic r a t heart is evidently greater
than is generally stated for adult mammalian myocardium.
1 Supported by Grants-in-Aid from the -4mericau Cancer Society, upon recommendation of
the Committee on Growth of the National Research Counci!, from the Kentucky Siate, Medical
Research Commission, and from the National Heart Institute of the National Institutes of
Health. Public Health Service.
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AMERICAN ASSOCIATION O F ANATOMISTS
M P 4 . The effect of poliomyelitis virw on luman brain cells grown in tissue
culture.' Mary J. HOGUE, Robert McALLISTER", Arthur E. GREENEX
and Lewis L. CORIELL', Departments of Anatomy and Pediatrics, School
of Medicine, University of Pennsylvania.
A large neuron, a Purkinje cell, in a 94-day-old culture of cerebellum from a
127mm crown rump human fetus has been photographed for 4 hours and 20
minutes. The tips of the cell processes are active and there is streaming of cytoplasmic granules in the cell body, but little locomotion is seen. This feature is
followed by pictures of a group of three cells from a 108-day-old culture of the
cerebellum of a 127mm human fetus. The culture has been inoculated with
Photographing began 28
poliomyelitis virus, type I, Mahoney strain, titer
minutes after the cuIture was inoculated with the virus and continued for 5 hours
and 40 minutes. The pictures show the contraction of the cell processes and the
withdrawal of these processes into the cell body. During this activity, the cells
are pulled about by the powerful contraction of their processes. They become
masses of granules. There is no recovery from this reaction to the polio virus.
Supported by a grant from the Foundation of Infantile Paralysis.
MP8. The ingu.ina1 region and scrotal contents.' Joseph E. MARKEE, Department of Anatomy, Duke University School of Medicine.
This 16 mm color motion picture with optical sound illustrates the anatomy of
the inguinal region and canal and the scrotal contents. Emphysis is placed on the
relationships which are pertinent to femoral and inguinal herniation. The morphological details and the concepts are illustrated partly by models, drawings and
animation and partly by the demonstration of dissections.
P a r t of the film deals with inguinal ligament and the relationship of the structures which pass through the lacuna1 musculorum and vasorum. By means of
animation, another part of the film illustrates the development of the inguinal
canal and the continuity of the coverings of the testicle with the layers which
form the abdominal wall. Another animation sequence deals with the additional
layers which are formed during the various types of inguinal herniation. A dissection of the inguinal canal in the female is demonstrated. The final sequence
demonstrates a dissection of the scrotum and its contents.
Aided by a grant from the National Foundation for Infantile Paralysis.
MPl. Effects of narcosis and hypotherrnia upon resistance to asphyxia in newborn guinea pigs.' A Kodachrome moving picture. James A. MILLER,
Jr. and Faith S. MILLER*, Department of Anatomy, Emory University.
Earlier experiments have demonstrated that hypothermia is effective in protecting newborn guinea pigs from asphyxia (Miller and Miller, '54). Since narcosis reduces motor activity, blocks shivering, reduces muscle tone, particularly
at low temperatures, and depresses basal metabolism, experiments were performed
to test the effects of narcosis upon asphyxia1 resistance a t body temperatures
between 40°C. and 14°C. Two mg/cc/100 gm nembutal (sodium pentobarbital)
ABSTRACTS O F DEMONSTRATIONS
479
in water was injected intraperitoneally into day-old or younger guinea pigs.
Littermate controls received water only. Temperatures were adjusted by radiant
heat or immersion to the neck in ice water. Asphyxia was produced by exposure
in a bell jar to 95% N,
5% C02.
Results: Pentobarbital narcosis prolongs asphyxia1 survival a t all temperatures
tested, but is most effective a t temperatures below 20°C. Narcotized animals can
recover completely from exposures which are lethal to non-narcotized littermates
a t the same temperature. With cooling superimposed upon narcosis most animals
recover from two times the lethal exposure. As employed here nembutal narcosis
has the protective action of a 10°C. depression of body temperature. An analysis
of the data suggests that the benefits of narcosis in asphyxia result chiefly from
its effect upon cellular metabolisim.
The results are demonstrated in a Kodachrome moving picture.
+
Aided by grants from the National :Institutes of Health and the Emory Research Committee.
M P 7. T h e H e L a cell -its activities as seen w i t h phase microscopy.' C. M.
POMERAT and C. G. LEFEBER", Tissue Culture Laboratory, Department
of Anatomy, University of Texas Medical Branch, Galveston.
This is the first of a series of films based on living human cells being prepared
as medical school teaching aids.
The phenomena of cellular outgrowth, nuclear rotation, normal and abnormal
mitoses, pinocytosis, intercellular filopodial activity, and mitochondria1 movements
are shown with the use of phase contrast, time-lapse cinematography.
Aided by grant from the Association of American Medical Colleges.
M P 9. Peripheral and central faoial nerve paralysis. Othmar C. SOLNITZKY,
Department of Anatomy, Georgetown University.
This 16mm silent kodachrome teaching film shows the anatomy of the facial
nerve by drawings and diagrams in relation to facial paralysis. Patients with
various lesions and deficits are shown.
I n peripheral facial paralysis, both the temporofacial and cervicofacial divisions are affected with homolateral flaccid paralysis of one side of the face. Unilateral peripheral facial paralysis is shown in a patient following a n attack of
bulbar polio. A case of bilateral peripheral facial paralysis, resulting from CNS
syphilis is also shown.
Central facial paralysis is of two types. The first type of central facial paralysis
is characterized by loss of emotional facial expression (amimia) i n the lower half
of the face on the contralateral side with retention of voluntary facial expression.
This type is associated with lesions of the thalamus, basal ganglia or tegmental
region of the upper brainstem. Iit will be demonstrated by diagrams.
I n the second type, there is loss of voluntary facial expression in the lower half
of the face on the contralateral slide, with retention of emotional facial expression. This type results from involvement of the voluntary motor pathway and
is demonstrated by two patients with hemiplegia.
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AMERICAN ASSOCIATION O F ANATOMISTS
MP 19. The termination f o r t h e bile ducts. Juliaii A. STERLING*, Department
of Surgery, Graduate School of Medicine, University of Pennsylvania.
(Introduced by Oscar V. Batson)
This motion picture film demonstrates untraumatized specimens of the termination for the bile ducts. Photography of cleared specimens, the ducts having
been injected with pigmented gelatin-iodide, indicates the relationships of the
ducts, the papillary sphincter and the ‘common channel. ” The characteristics
of the duct terminations are examined in the normal, in choledocholithiasis and
in pancreatitis. Forty per cent of specimens were found to have separate orifices
for bile and pancreatic ducts. The other sixty percent were evaluated further
with regard to the significance of the common channel. An ampulla was found
present in only 5 % . I n these interductal reflux is possible. I n other instances
the presence of a common channel does not signify the existence of interductal
reflux. The author indicates that the terminations for the bile and pancreatic
duct is not through an ‘ ampulla, ” but through a ‘papilla. ”
ABSTRACTS O F DEMONSTRATIONS
481
TISSUE CULTURE ASSOCIATION
ABSTRACTS
TC 9. Yucopolysaccharide production b y normal human tendon cells in mass
tissue culture.' C. Andrew L. BASSETT and Karl MEYER ", Departments of Orthopedic Surgery and Medicine, and Histochemistry Research Laboratory, Columbia University College of Physicians and Surgeons.
Cells arising from plasma iinbedded explants of normal human toe extensor tendon, obtained at operation, grew readily through the clot periphery
onto glass. From D 3.5 Carrel flasks, in which the central explant and plasma
clot had been excised, T-60 cultures were established, with cells directly on a
glass substrate. The nutrient consisted of 20% chick EE-50, 40% human
placental cord serum, and 40% Earle's saline with antibiotics. Medium removed
a t bi-weekly fluid changes was subjected to m u c h clot test, pooled, frozen, and
mucopolysaccharide content determined.
The mucin clot test was positive during the first 3 months of cell propagation.
However, it rapidly became negative during the 4th month and remained negative f o r the next 6 months that the strain was maintained. Certain growth and
morphologic alterations occurred concomitant with the change in the mucin clot
test. Medium collected during the first 3 months showed a total mucopolysaccharide content about 5 times the amount accounted for by chick embryo extract in the medium. After 5 months in vitro, the cells produced less than 3 the
total mucopolysaccharide of earlier 3 month samples. Acetone powder residues,
containing chiefly protein and nualeic acid, remained unchanged in amount during the entire observation period. Hyaluronic acid was isolated only from the
first 3 month collections. The study indicates that these tendon cells in vitro
are capable of producing mucopolysaccharides, but in decreasing amounts with
the passage of time.
1 Supported in part by U. S. Public Health Service Grant A 817 and a grant from the
Masonic Foundation for Medical Research and Human Welfare.
TC40. Gonads of W mice cultured in vitro. E. BORGHESE, Department of
Anatomy, University of Pavia, Italy.
The relationship between anemia and gonad sterility has been studied in
mouse embryos of the W strain, by means of tissue culture. The gonads used
were from embryos a t the 12th day of gestation. These embryos were obtained
from crossing W+ X W+. At this age neither anemia nor gonad sterility are
recognizable. After cultivation for 4 days, four different structures were found:
fertile testes, sterile testes, fertile ovaries, sterile ovaries.
Of the total number of cultures, the ratio of fertile gonads to the sterile
ones appears to be 33:s. This number is not significantly different from the
3 : l ratio of fertile-normal embryos to sterile-anemic ones on the 16th day of
gestation. Sixteen days (12
4) is the total age of the cultured gonads. The
+
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AMERICAN ASSOCIATION OF ANATOMISTS
-
Xa is 0.6585, with P 0. 45. I n each of seven embryos, the right and left
gonad behaved the same. The probability of getting this result by chance was
calculated to 0.4970. Thus, the development of a gonad in culture as fertile or
sterile depends upon its genotype and not upon environmental conditions.
It can be concluded that gonads taken from embryos before the appearance
of anemia, continue to develop in culture into sterile or fertile gonads aecording to their genotype. The gene acts directly on gonads and not through the
anemia.
TC 39. The behavior of nuclear and nucleolar sizes in tissne cultures of different growth intensity. Otto M. BUCHER, Department of Histology
and Embryology, University of Lausanne, Switzerland.
Frequency curves of nuclear and nucleolar sizes, obtained from hanging-drop
cultures of fibroblasts and osteoblasts, correspond to normal distribution (for
the technique see Internat. Review of Cytology 111, 69-111, '54). Moreover, a
linear correlation exists between the nuclear and nucleolar volume. I n order
to characterize mathematically the quantitative relationship between the nuclei
and the nucleoli we calculate a quotient E / N by dividing the nuclear size (K)
by the nucleolar size (N). I f the intensity of growth of tissue cultures is influenced experimentally, the nucleoli react to a greater extent than the nuclei,
which can be explained by the participation of the former in protein formation. As the nucleolar size represents the denominator i n the nucleo-nucleolar
quotient, the latter decreases, if the tissue growth is augmented and vice-versa.
Therefore, the variation curves of the quotients become displaced respectively to
the left and t o the right. This conception, can be easily proven since it is not
difficult t o regulate the growth rate of the cultures, for instance, by changing
the conditions (temperature or composition of the medium, see Zschr. Anat.,
118: 150-164 and 531-542, '54-'55) as well as by administrating appropriate
chemical substances (see Aeta. anat., 25: 45-52, '55 and Zschr. mikrosk.-anat.
Forsch., '56 in press).
TC 68.
Some factors affecting the growth promoting properties of serum in
tissue culture.' Relda CAILLEAU*, Cancer Research Institute University of California Medical Center, San Francisco. (Introduced by
Morgan Harris)
Cord, human, monkey, horse, ox, sheep, hog, rabbit, chick and r a t sera were
tested as growth media for HeLa, strain L, and mouse liver epithelial cells. The
medium consisted of 40% serum, 40% medium 199 and 20% Tyrode's solution.
Three or more subcultures were carried out before a medium was considered to
be growth promoting.
The effects of sex, age and heating (30 minutes at 56°C.) were studied with
certain sera.
Sera which showed good growth promoting qualities when unheated were usually
adversely affected by heat. Sera which proved toxic when unheated were generally
483
ABSTRACTS OF DEMONSTRATIONS
less toxic after exposure to heat. Toxic sera often caused the cells t o clump or
become extremely granular.
No universal relationship could be established between sex, age or f a t content
of the serum and it growth promoting activity.
~~~
~-
Supported by a grant from the U. 8. Public Health Service, National Institutes of Health,
to Dr. P. L. Kirk, Department of Bioclhemistry, University of California, Berkeley.
I
T C 3 3 . Growth of carrot root tissue i n the presenca of coconut milk.’ Samuel
M. CAPLINI, Department of Biology, University of Rochester, and Eastman Kodak Company, Rochester, N. Y . (Introduced by M. S. PARSHLEY)
A cylindrical explant of secondary phloem, 1.8 mm in diameter, 1mm high and
weighing 2.5mg, was placed on 100 ml of White’s medium containing 0.8%
agar and 10% coconut milk. I t was photographed in color during a period of
38 days a t the rate of one frame every 64 minutes, the culture exposcd t o a few
seconds of light for each picture. Growth began as a uniform, progressive swelling
over the visible surface, like rising dough, which soon became somewhat nodular;
growth proceeded, nevertheless, in a steady manner. To prevent condensation of
moisture on the vertical viewing window a piece of flexible heating tape was placed
above it against the container. After 50 days the cultiw weighed 2.63 gm, making
a relative increase of 1,050 times (as compared with 2-4 times in the absence of
coconut milk). According to Blackman’s (Ann. Bot., 93: 353. 1919) compound
interest formula for growth, this represents an average relative growth rate of
15.9% per day for the 50-day period, comparable in relative rate t o the normal
vegetative growth of an intact plant. Other (unpublished) work has shown
that explants from 46 different carrots in the presence of indole3-acetic acid
averaged in 14 days less than 25q.b of the final weight attained in medium containing coconut milk.
Isupported by n research grant, No. C-755 (CS), from the National Cancer Institute,
Public Health Service.
P C l l . Studies on the growth of mammalian cells in agitated fluid media. William R. CHERRY and Robert N. HULL, Lilly Research Laboratories,
Indianapolis, Indiana.
The growth of cell suspensions in flasks incubated on a rotary Brunswick shaker
has been described by Earle et al. This accomplishment has been successfully
duplicated in our laboratory using cell strains other than those employed by Earle
and with some modification in the procedure. I n addition to the duplication of
the shaker flask cultures a second technic employing a suspended magnetic stirrer
bar f o r agitation has been developed. Results obtained by this method compared
favorably with those achieved with the shaker cultures grown in our laboratory.
The technics of both of these methods will be described as well as the variations
in medium, speed of rotation and size of cell inoculum required for each cell type
studied. The relative efficiency of the fluid culture to conventional flasks in terms
of cell populations obtainable will be discussed.
484
AMERICAN ASSOCIATION O F ANATOMISTS
TC 34. Functional and histological observations on islet tissue grown ‘ i n vitro’.
Robert E. COALSON”, Department of Anatomy and The Tissue Culture
Laboratory, University of Oklahoma School of Medicine. (Introduced
by Kenneth M. Richter)
Explants of histologically undifferentiated 9-10 day chick embryo pancreata
and of fetal C3H and C57 brown mice pancreata were grown in culture by roller
tube and hanging-drop methods for periods ranging up to 58 days.
Histologic studies of chick pancreatic cultures revealed (1) little or no evidence
of acinar tissue development or differentiation, but ( 2 ) a differentiation of tinctorially identifiable alpha and beta islet cells.
Limited histologic preparations of cultivated mouse pancreas showed no differentiation of either acinar tissue or tinctorially identifiable islet cells.
Assays for ‘insulin activity’ (Randle’s rat diaphragm technique, ’54) were
made a t specific intervals on the supernates of mouse pancreatic and liver (control)
cultures during 58 days of cultivation. The assays demonstrated clearly that the
supernates from the pancreatic cultures from the 17th day of cultivation on
possessed a high degree of insulin activity. ‘Insulin activity’ assays and expressed as milligram of glucose utilized by 100mg of rat diaphragm per unit of
time showed that pancreatic supernates effected an average glucose utilization
of 2.987 mg whereas control liver supernates effected an average glucose utilization of 1.960 mg. This data comprises the first positive demonstration of the
production of insulin or an insulin-like substance by pancreatic tissue grown in
culture.
1 Part of study submitted to the Graduate Faculty of the University of Oklalioma in partial
fulfillment of the requirements for the degree of Doctor of Philosophy.
TC20. A comparison of the Nissl substance of living and fixed spinal ganglion
cells. Arline D. DEITCH” and Margaret R. MURRAY, Departments of
Surgery and Anatomy, College of Physicians and Surgeons, Columbia
University.
Tissue culture preparations of chick embryo spinal ganglia were studied by
phase-contrast microscopy and ultraviolet photography. I n a medium of plasma,
chick embryo extract and Parker’s 858, cultures were maintained i n good health
up to 5 weeks; within a week neurones less than 10 p thick could be found without
cellular over- or under-lay. Such cells, in various stages of chromatolysis and
regenerative maturation, were photographed living, fixed with osmium tetroxide
followed by formalin, and rephotographed under phase-contrast, then stained
with azure A or cresyl violet and again rephotographed. The phase-contrast image
was thus related to the stained Nissl picture. I n living neurones there are mediumdark relatively homogeneous cytoplasmic areas in the form of larger flattened
plates or smaller flakes. These occupy the same positions as the fixed, stained
Nissl substance. The granular and fibrillar elements of the cytoplasm are sharply
delimited from the Nissl masses. The latter appear practically unchanged after
fixation; dehydration, however, produces some shrinkage and distortion. It is
nevertheless possible to homologize at least the larger masses with the same Nissl
areas after staining. Ultraviolet photographs taken by Dr. Montrose J. Moses
ABSTRACTS O F DEMONSTRATIONS
485
at 262 mp show Nissl bodies in living cells to be discrete darkly absorbing cytoplasmic bodies remaining unchanged after formalin fixation. These observations
support the concept that the cla.ssica1 Nissl bodies represent essentially similar
structures pre-existing in the living state.
1 Postdoctoral Fellow of the National Institute of Neurological Diseases and Blindness,
National Institutes of Health, U. 9. Public Health Service.
T C 3 . Recent results from the growth of cells of long-term strains in shaken fluid
suspension cultures. Wiltcin R. EARLE, J a y C . BRYANT and Edward
L. SCHILLIKG, National Cancer Institute, Bethesda, Maryland.
No abstract was submitted.
TC 16. T h e growth of cells on a transparent gel of reconstituted rat-tail collagan.
Robert L. EHRMANN* and George 0. GEY, Division of Cellular Pathology, Department of Surgery, Johns Hopkins University, Baltimore,
Maryland.
With the knowledge that collagen is a universal component of connective tissues,
serving as a kind of solid substratum and support for cells in the organism, an
effort has been made to reduplicate some aspects of this natural system i n tissue
cultures. By dializing an acetic acid solution of rat-tail collagen against distilled
water, a clear gel is formed. Slices of this gel were placed in roller tubes,
equilibrated with culture medium, and tested as a substratum for the growth of
28 cell strains, of which 23 were of human origin. Besides these, a strain of human
kidney fibroblasts was grown on collagen which had been jelled by exposure of
the acetic acid solution to ammonia vapor. Collagen gel proved superior to the
glass wall of roller tubes as a surface on which t o cultivate these strains. On
collagen gel, colony outgrowth wa,3 frequently more extensive, and almost always
occurred without the retractions so commonly seen on glass. Even after continual
exposure, the gel showed no eivdence of lysis by the cells.
TC 2. Nutritional studies on certain long-term cell strains. Virginia J. EVANS,
Benton B. WESTFALL, Ka,therine K. SANFORD, Naomi M. HAWKINS"
and William T. McQUILKIN, National Cancer Institute, Bethesda, Maryland.
No abstract was submitted.
TC.99. Toxicity of blood serum of schizophrenics in tissue culture. II.I Sergery
FEDOROFF* and Anthony F. MESZAROS* ', Department of Anatomy,
University of Saskatchewan and Saskatchewan Hospital, North Battleford,
Canada. (Introduced by Rudolf Altschul)
Continuing the work reported last year on the tosicity of blood serum of schizophrenics in tissue culture (Anat. Rec., 1 8 2 : 394, 1955), it was found that although
there was no correlation between the degree of serum toxicity and the clinical
condition of the schizophrenic patients, the toxicity was higher in female than in
male patients, and generally less in patients of older age groups. The 8erums of
a few normal individuals who were under conditions of stress a t the time blood
was obtained from them, were also highly toxic in tissue cultures.
486
AMERICAN ASSOCIATION O F ANATOMISTS
Toxic serum can be made non-toxic by heating it a t 56°C. for 35 minutes. Blood
serum which was toxic to strain L cells was never toxic to strain HeLa cells, despite
the fact that both cell strains had been grown i n the same medium for 6 months
prior to the tests.
The cerebrospinal fluid of schizophrenics is also toxic to strain L cells. Preliminary results indicate an even greater difference in the toxicity of cerebrospinal
fluid of schizophrenics and of non-schizophrenics than in the toxicity of the
blood serums of the two groups.
1 Supported by a National Health grant. Saskatchewan Committee on Schizophrenia Research.
TC
r.
Exfoliative cytology as n tool in tissue culture. Robert H. FENNELL, Jr.”,
Department of Pathology, University of Pittsburgh School of Medicine.
(Introduced by Joseph Leighton)
Contact smears from gelfoam particle tissue cultures were fixed and stained
using Papanicolaou ’s technique. The examiner had no previous experience
with tissue culture preparations and applied criteria used i n clinical exfoliative
cytology. By these criteria, smears were classified as ‘‘malignant’ ’ or “benign. ”
The cells examined included 4 derived from normal tissues (Chang’s conjunctiva, Earle’s skin, Henle ’s intestinal epithelium and Leighton’s D-189)
and three derived from cancers (Gey’s HeLa, Eagle’s K B and Osgood’s J-96).
The cytological criteria ordinarily used t o classify a smear as malignant were
found in smears from several of the “normal” cell lines as well as from the
“tumor” cells’ lines.
The results of this exploratory study indicate that the techniques of exfoliative
cytology can contribute to the evaluations of tissue cultures. The alcohol-ether
mixture is a good fixative and the stain, a modified hematoxylin-eosin method
is excellent, especially for nuclear details. Identification of cells of a certain
striin and detection of even minor alterations in cells should be possible, but to
answer the question on morphology alone of whether a given cell or group of cells
is “malignant” may not be possible in some cases where cells from many
different sources are considered.
TC $0. Tissze culture studies in rheumatic fever.’
Lloyd FLORIO*, Gertrud
WEISS* and Gladys Kinsman LEWIS* ’, Department of Preventive Medicine and Public Health, University of Colorado School of Medicine. (Introduced by C . M. Pomerat)
Among the more commonly accepted theories of causation of rheumatic fever,
is one that it is due t o a delayed hypersensitivity of the tuberculin type. Since
measurement of this type of sensitivity has been reported in tissue culture,
rheumatic and non-rheumatic tissues have been compared in their responses t o crude
streptococcal filtrates and t o disintegrated organisms derived from strains presumed
to have caused the disease, both in the presence of pooled serum from normal
individuals as well as in serum from rheumatic fever cases. Migration was
observed after one day and spindle shaped transformation of certain white
blood cells was measured about one week after explantation in both rheumatic
and non-rheumatic bloods. The effect on migration after phagocytosis of dead
ABSTRACTS OF DEMONSTRATIONS
487
streptococci by rheumatic andl non-rheumatic white blood cells was likewise
observed. Fibroblasts were compared from skin and heart auricular tips of
rheumatic and non-rheumatic individuals and from an occasional rheumatic
nodule grown in various concentrations of those same materials. Using similar
tissue culture techniques, we also re-examined the concepts involved in the
delayed type of tuberculin hypersensitivity using tissues from tuberculous and
non-tuberculous humans and guinea pigs. Our results in these experiments were
not nearly as definitive as many of the reports in the literature. We were
not seeking a statistical answer, but, a n “all or none phenomenon” that would
clearly distinguish a rheumatic from a non-rheumatic individual. We failed
to find it.
1
Supported by
a.
b.
c.
d.
grants from
National Heart Council United States Public Health Service.
The Delta Delta Delta Alliance of Denver, Colorado.
The Mary Ruby Murphy Fund.
The Barth Foundation.
TC 14. Use of manometric method in nutritional studies of strain HeLa.’ George
E. GIFFORD” ”, Hugh N. ROBERTSON* ’ and Jerome T. SYVERTON,
Department of Bacteriology and Immunology, University of Minnesota,
Minneapolis. (Introduccd by Thurlo B. Thomas)
The respiration of growing HeLa cells was measured with a standard Warburg
apparatus. The oxygen consumption rate was proportional to cell number,
while the rate of change of oxygen consumption rate was directly proportional
to rate of cellular multiplication and inversely proportional to inoculum size.
Maximum populations for growth were determined. Addition of human serum
to growth medium in amounts greater than 40% did not increase HeLa cell
respiration. Yeast extract, 0.1 %, added to media containing 40% serum stimulated
respiration significantly. Cells in 10% human serum with 0.1% yeast extract or
in 40% serum without yeast extract respired @t equal rates. The amount of
glucose commonly added to media, 100 mg%, limited respiration after 49
hours when approximately 1 million cells per milliliter were employed. For
short term maintenance of a coiistant rate of cellular respiration 10% chicken
serum in Hanks ’ balance salt solution was adequate. Respiration accelerated
slightly with 20% chicken serum, and was increasingly depressed in the absence
of serum. With 10% serum, semi-synthetic and synthetic media were not found
superior to Hanks’ solution for cell maintenance. Phosphite 0.02 M, phosphate
0.02 M and tris (hydroxymethyl) aminomethane 0.01 M buffers were not toxic f o r
HeLa cells.
1
Supported by a grant from the National Foundation for Infantile Paralysis, Inc.
T C Q 6 . T h e mitotic activity in vitro of rat kidney tubular epithelium and preliminary @dings on the effects of milateral nephrectomy. And& D.
GLINOS’ and Allan M. CAMPBELL* ”, Growth Physiology Laboratory,
Division of Surgery, Walter Reed Army Institute of Research, Washington
12, D. C. (Introduced by Duncan C. Hetherington)
The tube slip method was used in developing a short-term culture test for
this study. Cortex, outer and inner medulla were cultured separately. Inner
488
AMERICAN ASSOCIATION OF ANATOMISTS
medulla yielded pure epithelial outgrowth and was therefore used in all
subsequent work. Mitotic and total cell counts were performed on fixed and
stained slips a t the end of desired culture periods. It was found that following
the establishment of the epithelial membranes, the mitotic index decreases rapidly
between the fifth and seventh day.
Five adult male rats were subjected to a unilateral nephrectomy, and cultures
were prepared from the removed kidney. The animals were sacrificed at 1, 2,
and 7 days after the operation, and cultures were prepared from the remaining
kidney. All cultures were fixed on the fifth day of incubation. The ratios of the
mitotic indices of the cultures from the remaining kidney t o those of the cultures
from the removed kidney were as follows: First day 1.6, second day 2.3,
seventh day 1.7.
The significance of these preliminary results with regard to the study of the
mechanism controlling kidney tubular epithelium regeneration will be discussed.
TC 3.2. Morphological and cytochemical observations of normal and leukemic
blood cells in tisszse culture. Milton N. GOLDSTEIN* and Terence McCORDepartment of Biology, Roswell Park Memorial Institute,
MICK*
Buffalo, N. P. (Introduced by S. Culver WILLIAMS)
O,
Peripheral blood from normal and leukemic individuals and bone marrow
samples from leukemic patients were collected in citrated vials. After centrifugation, fragments of the buffy coat and bone marrow were explanted i n D-35
Carrel flasks on the surface of perforated cellophane and in plasma clots formed
on cellophane. The cultures were maintained at 36°C. in a medium consisting of
two parts Gey’s solution and one part pooled human sera.
The cells i n buffy coat and bone marrow cultures from patients with
myeloblastic, lymphoblastic and monocytic leukemias developed giant cells which
resembled those seen in normal blood cultures. The formation of giant cells on
cellophane from leukemic leucocytes took longer to develop, but resembled those
giant cells seen in preparations of normal blood cultures. There was an increase
in polysaccharide and acid phosphatase, which was also seen i n normal blood
cultures. Many cells in cultures of normal as well as leukemic Ieucocytes developed
a fibroblast-like appearance i n plasma clots, and showed the same cytochemical
staining reactions as the giant cells.
The potentiality of leukemic cells t o differentiate like the normal monocyte in
vitro suggests the possibility of a maturation defect in some leukemias,
TC 41. Tissue1 culture studies of fish melanomas. Sylvia S. GREENBERG
M. J. KOPAC and Myron GORDON Department of Biology, Graduate
O,
School of Arts and Science, New York University.
The 4 expressions of spontaneous pigment cell tumors of hybrid xiphophorin
fishes (typical melanomas, melanomas with pigmentless zones, amelanotic melanomas of albino hybrids, and amelanotic melanomas with melanotic zones) were
grown in tissue culture. The medium contained human ascitic fluid, chick embryo
extract, fowl plasma, and Holtfreter’s solution. Growth was faster and more
luxurious with ascitic fluid than with carp serum, the usual component of fish
culture media.
ABSTRACTS O F DEMONSTRATIONS
489
The pigmentless zones of melanomas are of two diverse types. Growth of cells
from fibrous, white zones is slow, with only a sparse proliferation of small spindle
cells. Disintegrating pigment cells are present in the esplants. Such zones
within the melanoma, indicate a cessation of pigment cell growth. I n contrast,
the pigmentless, soft, edematous, and translucent zones of- some melanomas grow
rapidly, with nielanocytes as the preponderant cell type. The cells are not well
differentiated, as manifested by a sparsity or complete lack of pigmented granules,
and inability to maintain the typical dendritic shape. Rapid growth, or a n
increase in number of immature pigment cells can therefor account for pigmentless
zones in some melanomas.
I n cultures of amelanotic melanomas, the inelanocyte with colorless granules
is the preponderant cell type. The pigmented zones contain mature, non-dividing
cells with brown granules.
Cytochemical studies on the cultures indicate that variations in pigment patterns
of xiphophorin pigment cell tumors are expressions of a single tumor, and
cannot be considered as 4 distinct tumor typeg.
1 Damon
Runyon Cancer Research Fellow.
TC 2.9. Acid ?nzicopolysaccharide’s produced in tissue culture.’ Henry GROSSFELD*, Karl MEYER’ ’, and Gabriel C. GODMAN*, Histochemistry
Research Laboratory and the Department of Medicine, Columbia University, College of Physicians and Surgeons, New York. (Introduced by
Wm. R. Duryee)
Mass cultures from explants of human foetal skin, and bone, of embryonic
bovine bone and rat subcutaneous tissue were grown in Petri or Petroff dishes.
They were fed biweekly with a Holution of 20-250/0 EE, 10% serum and 60-650/0
beef amniotic fluid. The pooled “used” medium, and the harvested cell mass were
submitted to analysis for mucopolysaccharides.
From human skin cultures a mixed polysaccharide was obtained, approximately
3 of which was hyaluronic acid and about & chondroitin sulfate C. The fraction
containing the latter was “undersulfated,” possibly owing t o the production in
vitro of insufficiently sulfated chondroitin.
Cells from human bone produced almost equal amounts of hyaluronic acid
(26.0 mg/100 mg acetone dry material) and chondroitin sulfate (31.9 mg/100 mg
acetone dried material). Bovine bone cells also produced appreciable quantities
of mucopolysaccharide, including a sulfated fraction.
705 ml of supernatant from cultures of r a t subcutaneous fibroblasts yielded
101 mg of a mixed polysaccharide (14.1 mg/100 ml) containing equal amounts of
chondrosamine and glucosamine. From its sulfate content and from its optical
rotation, this material is believed t o be a mixture of hyaluronic acid and
chondroitin sulfate C, which fractions are in process of being isolated.
The content of mucopolysaccharide in EE could in no case account for more
than 140 of that recovered and was considered negligible.
1
Supported in part by grants A-21 end A-817 of the U. S. Public Health Service.
490
AMERICAN ASSOCIATION O F ANATOMISTS
T C W . The virus spectra of several new cell strains. Janet (Lavelie) HERBERG" and Robert N. EIULL", Lilly Research Laboratories, Indianapolis,
Indiana. (Introduced by Duncan C. Hetherington)
The ability of established cell strains of mouse, rat, calf, monkey and human
origin t o support the growth of a group of viruses has been studied. The group
included polio-virus, mumps, measles, herpes simplex, A. P. C. vaccinia, influenza,
lymphocytic choriomeningitis, lymphogranuloma venereum, MM virus of mice and
others. The problems encountered in the adaptation of some of the viruses to
growth in several of the cell strains will be presented. Other problems occurred
in the preparation of certain cell strains for virus studies and will be discussed.
The latter difficulties primarily involved the elimination of virus inhibitors from
the complex medium used to sustain rapid proliferation of the cell strain.
The significant or characteristic cytopathogenic effects produced by some
viruses in a particular cell strain have been compared. Also differences in the
C. P. E. of a given virus has been noted in several cell strains. The relative
sensitivity of various cell strains to a given virus has been observed as well
as the capacity of the cell strains to produce the virus. Preliminary observations
on the growth of viruses in suspended cell cultures have been made.
TC 12. T h e adaptation and maintenance of mammalian cells to continuous
growth in tissue culture. Robert N. HULL, William R. CHERRY and
Irving S. JOHNSON, Lilly Research Laboratories, Indianapolis, Indiana.
During the past 5 years a number of cell straiirs have been isolated and
maintained in our laboratories. Several variations of the plasma clot technic
have been used t o initiate growth of cells on glass as well as the direct preparation
of cell suspension from animal tissues by the method of trypsin digestion. These
technics as well as procedures used for maintenance and subculture of various
cell strains will be discussed. Cell strains started in our laboratory include two
normal, and one malignant strains of mouse origin, one malignant rat cell, four
normal monkey cells of embryonic torso, adult kidney and testicular origin, one
of normal human embryonic origin, and one from calf embryonic tissue.
Our experience in,the isolation and handling of other cell strains not included
in the list above will be presented. These include cell strains which we developed
but which were lost by accident as well as strains from other laboratories.
T C 1 8 . Depletion of some of t k e serum proteins in tissue culture mediunc. H.
Naim KENT* and George 0. GEY, Division for Cellular Pathology,
Department of Surgery, The Johns Hopkins University School of
Medicine.
O
Experiments have shown that significant variations occur in the protein
composition of the media user1 to cultivate cells. The cells tested were from
the rat normal strain 14p; the rat tumorous strains T-72 and T-333; and the
human tumorous strain A. Fi. from Gey and Gey stock. Microchemical analyses
have demonstrated a considerable decrease of the protein concentration levels
during in vitro growth. Electrophoretic studies show that only the alpha and
beta globulins reveal a significant decrease in concentration. The strains listed
ABSTRACTS O F DEMONSTRATIONS
491
above produce varying degrees of depletion. The analytical results confirm the
depletion seen in the electrophoretic patterns obtained by zone and conventional
electrophoresis. The question of the mode of incorporation of these two
protein fractions by the cells will be discussed. I n direct tests of the utilization
of euglobulins and pseudoglobulins isolated from human placental cord serum
there was evidence of a depletion of these added globulins. This was also
confirmed by direct chemical analysis. The question of whether this depletion is
due to an intracellular funcion rather than extracellular protein degradation will
be discussed.
T C 13. Growth characteristics of two mouse fibroblast strains in stationary and
in shaken cultures.'
Robert J. KUCIILER and Donald J. MERCHANT,
Department of Bacteriology, University of Michigan, Ann Arbor, Michigan.
Two stable strains of mouse fibroblasts (Strain L and Strain LLCMI) were
grown both in stationary and in shaken cultures and growth curves were plotted
from the results of quantitative measurements of the cell populations. The growth
curves for each strain were similar in both stationary and in shaken cultures.
When either stationary or shaken cultures were started with cells taken from
the stationary growth phase the lag phase lasted for 48 to 96 hours. Starting
cultures with cells from the log phase of growth shortened the lag phase to
24 hours or less. Taking advantage of this fact it was possible to develop
a growth and feeding cycle for Elhaken cultures which approached a steady state.
Under these conditions no appreciable p H shift occurred during the 4-5 day
growth cycle and no aeration was required. A continuous large yield of cells was
obtained.
Supported by U.S.P.H.S. grant no. (32539 ( c ) M & I.
T C B 7 . A method of cultivating the normal adult mammary gland of the mouse.'
E. Y. LASFARGTJES*, Departments of Microbiology and Surgery,
Columbia University, Go llege of Physicians and Surgeons, New York.
(Introduced by M. ChBvremont)
A collagenase which can hydrolyze 25mg of collagen in 18 to 24 hours at the
concentration of 0.02mg/ml has been used to separate ducts and acini of the
mammary gland from the adipose tissue surrounding them. The glands from
C. 57 mice in an early stage of pregnancy are minced in a collagenase solution
10 times as concentrated (0.2 mg/ml), then agitated for two hours a t 37" C.
After 10 minutes centrifugation a t 1200 rpm most of the f a t can be discarded. The
sedimented cells are washed twice in plain Simrns' solution and resuspended in
the culture medium. One drop of this tissue suspension is spread on clotted plasma
on a Maximow cover slip and 1 m l is introduced into each roller tube prepared.
The slide preparations are washed and refed every 3 to 4 days; the medium in
the roller tubes is renewed twice a week. The epithelial sheets, many of them in
pure form, have spread widely by the 4th day of cultivation.
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AMERICAN ASSOCIATION O F ANATOMISTS
Human placental serum seems t o be a fundamental element. A 50% dilution
of this serum in Simm’s solution supports the growth of the mammary
epithelium for periods over one month. I n roller tubes, a nutritive fluid composed
of Parker’s 858 supplemented with 25% of human placental serum still supports
the growth of the epithelial cells after 5 weeks.
Isupported by research grant C-2520 from the National Cancer Institute, U. S . Public
Health Service.
TC 6. Some histologic featurt-s of human “normal” and “cancer” cell strains
in sponge matrix tissue culture. Joseph LEIGHTON, I r a KLINE, Morris
BELKIN O , Frances LEGALLAIS and Henry C. ORR, Laboratory of
Chemical Pharmacology, National Cancer Institute, Bethesda, Maryland.
Sponge matrix cultures of three cell strains that arose from human tumor
tissue - Gey ’s HeLa, Eagle’s KB and Osgood ’s J-96 -were examined in histologic
sections and found to be morphologically indistinguishable from each other:
Sections of D-189, a strain that arose in vitro as a morphologic “malignant
transformation” from normal connective tissue, had the same appearance a6 the
three strains originally from cancer tissue. All four had malignant features, i.e.
absence of architectural patterns indicating tissue of origin, hyperchromatism,
high nuclear, cytoplasmic ratio, prominent and often multiple nucleoli, and
many mitoses including numerous tripolar and other abnormal forms.
Two strains of “normal” human cells - Henle’s Intestine 407 and Chang’s
conjunctiva - were indistinguishable on histologic examination from the four
strains mentioned above.
The morphologic transformation in vitro of normal human cell into cells
resembling those from cancer does not necessarily imply that they are equivalent
in any other quality. The similarity may have resulted from the selection of
variants capable of rapid growth. Cell strains arising from many kinds of
tissues, fed on the same kinds of media and selected through many subculture
generations for rapid growth may eventually possess many biologic properties
in common, including microscopic structure.
Variations in the ability of these strains to invade and replace histologically
normal tissues are being studied.
T C 2 4 . Some problems involved in the culture of cells in a perfusion chamber.’
P. J. LEINFELDER” and B. Shannon DANES”, Department of
Ophthalmology and Physiology, State University of Iowa, College of
Medicine, Iowa City. (Introduced by Robert Chambers)
I n the development of a perfusion chamber for the study of the metabolic
and morphological activities of cells grown in culture, it was necessary to set
up a control environment in which the physiologic factors that effect growth and
maintenance of cells can be experimentally established.
The stainless steel chamber that permits continuous perfusion with nutrient
fluid is a modification of the Pomerat (’51), Christiansen (’53)’ and Buchsbaum
(’54) perfusion chambers. The unit is composed of twin chambers so that
simultaneously the activities of sister cultures under control and experimental
ABSTRACTS O F DEMONSTRATIONS
493
conditions can be determined. The perfusion fluid (4 parts horse serum, 4 parts
Txrode's and two parts embryonic chick extract), is gassed during delivery
to the chamber with 5% carbon dioxide and 95% air.
Cultures used were primary 7-day embryonic chick heart explants set up in
hanging-drop preparations. After 12 hours sister cultures were transferred to the
twin perfusion chambers.
I n order to simulate flow of tissue fluid continuous perfusion was used.
Perfusion rates from 7 p 1 per hour to 1600pl per hour were used. A definite
curve of activity based on migration and mitoses was obtained. The experimental
data indicates that under these controlled conditions flow of approximately
1 5 0 ~ 1per hour is optimum for the maintenance and growth of the chick heart
fibroblast.
1
Supported by a grant from the Iowa Division of the American Cancer Society.
TC31. Effects of antibodies on cells i n tissue culture. Charles E. LUMSDEN",
Maida Vale Hospital, London, W. 9, England. (Introduced by William
F. Windle)
A study has been made on the eff ects, upon cells in tissue culture, of heterologous
non-immune and immune sera, and of homologous agglutinating sera. This communication deals chiefly with the morphological changes, the phenomenon of
resistance, and - in the case of human tissue elements- the presence in the
cells of haemagglutinogens. The mechanism of these effects has been compared
in mammalian erythrocytes, nucleated erythrocytes (fowl), and in mammalian
fibroblasts, mesenchyme and nerve fibres. Though the various stages of the
antibody effect succeed each other extremely rapidly, this begins primarily in
the ground substance of the cytoplasm, and is followed by mitochondria1 fragmentation, and then by nuclear shrinkage associated with - or due to -passage
of nuclear substances into the cytoplasm. Complement is essential to the process:
in its absence, fibroblasts can fix antibody and grow unhampered but, even after
washing out of the serum, the antibody effect occurs a t once if complement is
added. Compared with red cells, living tissue cells have a relatively enormous
carrying poxer for antibody before succumbing to its effects.
TC 38. Radiosulphur intake b y mucopolysaccharides of embryonic and oultured
connective tissues. R. E. MANCINI ",E. 8. LUSTIG and C. NfifiEZ
O,
Instituto de Oncologia and A. H. ROFFO, Comisidn Naeional de la
Energia Atbmica, Buenos Aires, Argentina.
Using autoradiographic techniques employing stripping film" of high resolving power, the following studies were undertaken: (1) Intake pathway of
S3Gin adult, embryo and cultured connective tissue cells; (2) Nature of the
substance responsible for the intake by submitting sections to specific chemical
extractions or incubation with connective tissues enzymes.
Pele film was applied directly t o hanging-drop cultures in a liquid medium.
Six microcuries of radioactive substance proved to be a minimum useful dose
to obtain satisfactory images without disturbing culture growth. First evidences
of sulphur intake were given by cells of the central zone from the third hour
494
A M E R I C A N ASSOCIATION O F A N A T O M I S T S
of culturing with progressive increase up t o 24 hours. I n the migration zone,
the 5% appeared to be concentrated selectively in the perinuclear cytoplasm of the
cells. Intake increased until it reached a peak at 48 hours at which time the
sulphur diffused into all the cytoplasm even into the prolongations of the
fibroblasts.
From the third hour on, as intracrllular intake increased, an extracellular
arrangement of sulphur can be observed in the neighborhoood of the cells and
later on in the intercellular spaces, as observed in chick embryo mesenchyme.
Images are more evident and intense near the explant where the cellular population
is denser. Autoradiographs were negative in the controls using a non-cellular
medium. A partial removal of S35from fibroblasts can be obtained by previous
treatment with normal hydrochloric acid or with saturated barium hydroxide
or by hyaluronidase. Collagenase and elastase, which are specific enzymes for
collagen and elastic fibers respectively did not act upon the uptake of the
radioisotope.
T C 4 . Tissue culture) as a biochemical tool. Joseph F. MORGAN and Helen
J. MORTON, Laboratory of Hygiene, Department of National Health
and Welfare, Ottawa, Canada.
Freshly explanted chick embryonic heart tissues have been used t o study
cell nutrition and metabolism. The effect of variations in the synthetic media
and culture survival has been correlated with changes in these media during the
cultivation period. A specific requirement for L-cystine and a supplementary
requirement for either L-or D-methionine were found. Paper chromatography was
used to follow changes in the amino acid content of the synthetic medial during
tissue cultivation. The observed changes were related to the effect of each
individual amino acid on culture survival, employing a nutritional depletion
technique. Total protein determinations showed a progressive decrease during
cultivation i n synthetic media but the addition of certain natural extracts caused
a net protein increase. Synthetic media published by various workers were
compared, using one standardized type of culture, and the efficacy of these media
was related t o specific components. The advantages and disadvantages of fresh
explants and established cells ' strains are discussed in relation to the above
experiments.
!CC 5. Altered animal cell strains f o r quantitative cultlire work.' Raymond C.
PARKER, LaRoy N. CASTOR ' * and Ernest A. McCULLOCH
ConO,
naught Medical Research Laboratories and Department of Therapeutics,
Faculty of Medicine, University of Toronto.
Over the past several years, there have been numerous instances i n our
laboratory of the development of strains of altered cells that cannot readily
be identified with any cell type existing i n the animal body. These altered cells
are characterized mainly by their ability to multiply with great rapidity to
yield uniform populations of free-living cells that can be centrifuged, washed
and subcultured by quantitative procedures. Sometimes, these altered cells appear
during sudden bursts of activity i n old cultures undergoing marked cellular
495
ABSTRACTS O F DEMONSTRATIONS
degeneration. At other times, healthy cultures become altered either i n part
or in their entirety, Sometimes, the alterations take place in tissues only recently
explanted from the body; a t other times, the ce1l:s affected have been cultivated
for months or years. The cells of some of these altered strains live and multiply
while attached to the glass walls of the culture containers. Cells of other
strains, smaller in size, have a tendency to live and multiply in suspension, even
in stationary cultures. Strains of altered cells of one type or the other have been
obtained from bone marrow, kidney, liver and peripheral blood, from several
species, and from various long-established strains of cells from other sources.
A description will be given of the development of the altered strains and of
efforts that are being made t o determine their nature.
Supported in part by grants from the National Cancer Institute of Canada.
a Bellow of the American Cancer Society. Present addreams
: Johnson Foundation for Medical
Physics, University of Pennsylvania.
TPC 37. Netabolic changes in chick embryonic heart and liver during adaptation
H.E.R.T.
t o in vitro conditions. John PAUL and Enid S. PEARSON
O,
Tissue Culture Unit, Biochemistry Department,
Glasgow, Scotland.
Glasgow University,
Immediately after explantation 15-day chick embryonic liver had a very high
oxygen uptake, associated with almost compleie utilization of lactic acid
in the medium and production of hexose in addition to that originally present.
On the other hand heart tissue had vitrtually no oxygen uptake but rapidly
utilized glucose with production of pyruvate and lactate. One day later both
tissues had a low oxygen consumption of approximately the same order and
both utilized glucose with formation of pyruvate and lactate. By €he second
day these changes were exaggerated and liver often had a lower oxygen consumption than heart. Immediately after feeding, at this stage, liver oxygen
consumption increased while heart oxygen consumption tended t o decrease. Both
used glucose and produced pyruvate and lactate. By the third day the picture
had reverted to one similar to that a t the second day.
I n both tissues there was an initial decrease in DNA followed in a day or
two by an increase, the decrease being more prolonged i n the case of liver.
incorporation took place into lipid phosphorus, acid-soluble phosphorus,
Active P32
RNA and DNA a t all stages in both tissues. The explants were also analysed
for total lipid, carbohydrate and protein and these results along with the histochemical picture will be reported in full elsewhere.
T C d l . T h e behavior of chick spinal ganglia explanted in a chemically defined
medium.’ Edith R. PETERSON*, Department of Surgery, College of
Physicians and Surgeons, Columbia University, New York. (Introduced
by Margaret R. Murray)
While Parker’s synthetic medium 858 will maintain growth in Earle’s L-stain
indefinitely, it will not support organized growth of chick spinal ganglia. It is
496
AMERICAN ASSOCIATION OF ANATOMISTS
inadequate for this tissue even though supplemented with chicken plasma, which
is necessary t o provide a proper physical substrate. I n addition to the plasma,
6 biological components have beeen tested as supplements to the synthetic medium.
Only with the addition of embryo extract has complete differentiation, including
myelinization, occurred. This and the action of serum and plasma will be discussed
in detail in relation to supporting cells m d to neuronal Nissl pattern, mitochondria,
fibrous sheath and myelin. I n inadequate media a reduced percentage of cells
survive but with abnormal form and behavior.
I n these ganglion explants one is dealing with a mixed population of cells
whose regeneration, reconstitution, and progressive differentiation must be
maintained in delicate balance for several weeks, and in which survival and growth
alone are not sufficient for orderly development.
1 Supported in part by research grant B-858 from the National Institute of Neurological
Diseases and Blindness, U. S. Public Health Service.
l'C42. Further studies on connective tissue mechanics in vitro. Hans H. P F E I F
FER, Laborat. f. Polarization Microscopy, Bremen I, Germany.
The experiments tended to bring out a standardization of the culture medium
to elude deviations of the results. Objects studied were reticular connective tissue
of axolotl and perichondrial fibroblasts of chicken embryos. A mixture of plasma
and chicken embryo extract was suspended on a stocking woof of colourless
Dralon stained with HARRIS' hematoxylin.' Just before clotting a fragment of
tissue was transferred into the medium, and the whole preparation was placed
in a moist chamber, and incubated. A quick emigration of fibroblasts began
around the explant, and repeated passages were possible. The movement of the
fibroblasts is determined by tactile excitement starting from the Dralon net.
The cells do not grow out as isolated elements, but in a reticular formation.
Sporadic elements one finds, a t best, near the margin of the preparation. Step
by step, the cultures stop as a veil, the gaps of the Dralon net. Using Y.
GERENDAS' apparatus the Dralon woof, in this state, was fixed in a stretched
position. Measurements of double refraction, by means of a micra compensator,
prove that the cells stretch themselves while growing and utilizing the most
suitable fibers of their Dralon medium. Always the main directions of growth
correspond to midpoints of the distances of the connections of Dralon net. I f
now the fibrillar stroma of the cells was separated by digestion with alkaline
pancreatine, unbranched fibrils of leptonic thickness came across each other
and formed fibers and bundles undigestible by trypsin, swelling in acetic acid,
and showing double refraction like collagen. Some fibroblasts stretched between
Dralon fibers are, no doubt, in a state of increased tension and show dark parallel
striations indicating intracellular fibrils. They give evidence of the fact that their
arrangement corresponds (just like hematocytes repopulating a trypsin digested
omentum membrane [PFEIFFER, Anat. Rec., 121, n. 2, 19553) to the tensile
trajectories within the culture medium. Other cells, verisimilar, not exposed t o
any tractions, find their way, during the growth, down into the medium.
'1 have to thank Doctor Harms (Farbenfabriken Bayer, Leverkusen) for yielding up this
and other stains for histochemical purposes.
ABSTRACTS OF DEMONSTRATIONS
497
TC36. A sample holding vessel permitting the p H measurement of onn/fourth
cc samples w i t h ordinary-sized electrodas.‘
Kenneth M. RICHTER,
Department of Anatomy and The Tissue Culture Laboratory, University
of Oklahoma School of Medicine.
It would seem that one of the principles followed in the design and fabrication
of electrodes has been ‘to make the electrodes fit the size of the sample.’ It is
possible, by utilizing a different approach, namely one of “making the sample
fit the electrode,” to extend downward the present useful range of conimercially
available electrodes. This approach literally is a matter only o f fabricating a
sample holding vessel in which the sample chamber proper redeets closely the size
and shape of the tips of the electrodes available.
For electrodes measuring 7/16” by 54” the following type of vessel su&m for
samples down to ace. It consists simply of a block of plexiglass (1” x 13’’ J( 2 ” 1
in which two parallel but closely spaced 3” diameter wells are drilled afii3 interconnected a t their bottoms in U-tube fashion by a 1/16” o r Q” channel. The
electrodes when inserted into the arms of the U-shaped chamber displace the
sample upwards and effectively increase sample/electrode contact.
Hundreds o f check measurements attest to the reliability of determinations
made with this sort of vessel. It has been used routinely in this laboratory
for several years and has been especially useful in measuring the p H of roller tube
supernates ( 2 cc and less).
1
Supported by grantein-aid from the Helen Hay Whitney Foundation.
TC35. Studies on the individual and joint e r e c t s of ATP, glutathione and
histamine-HC1 on the fibroblast growth of chick heart in culture.’ Kenneth
M. RICHTER, B. R. RITCHESON” and .J. MURRAY”, Department of
Anatomy and the Tissue Culture Laboratory, University of Oklahoma
School of Medicine.
Experiments involving planimetric measurements and microscopic study of
1845 cultures bearing on the individual and joint effects of ATP, glutathione
and histamine-HC1 at concentrations of 100 pg/cc of nutrient on the growth and
cytomorphology of chick heart tissue in roller tube cultivation were made.
ATP and glutathione, when used singly or t,ogether, had no statistically
significant effect on growth during 48 hours of experimental treatment. Histamine,
given alone, had a statistically significant and marked (46%) growth inhibitory
effect. The growth inhibiting action of histamine was completely nullified when
histamine was administered in combination with either glutathione or ATP.
The combined administration of ATP, glutathione and histamine produced a
marked (223%) and statistically significant growth stimulating effect.
No specific correlations were evident between the p H changes observed in the
nutrient media and the effects of the above compounds on growth.
Phase contrast studies and study of the cultures after staining by a modified
Giemsa revealed no significant cytomorphic alterations from the normal controls
relative to the mitochondrial, golgi, nucleolar or nuclear components.
1
Supported by grants-in-aid from the Helen Hay Whitney Foundation.
498
A M E R I C A N ASSOCIATION O F A N A T O M I S T S
T C 1. Immunologic and growth properties of low and high sarcoma-producing lines
of cells derived in vitro f r o m a single adult mouse cell. Katherine K.
SANFORD, Gwendolyn L. HOBBS and Mary C. FIORAMONTI, National
Cancer Institute, Bethesda, Maryland.
No abstract submitted.
TC 19. Pineal tumor giant cell.' Machteld E. SANO, Department of Pathology,
Newton and Morristown Memorial Hospitals and Department of Tissue
Culture, Temple University Medical School.
The giant cell of a human pineal tumor was studied in tissue culture by cinematography. Rhythmic phases of nuclear and cytoplasmic activity were observed.
Interpretation of these phenomena from a cytophysiologic standpoint have been
made and will be discussed.
1
Supported in part by the Chase Research Foundation and the F. E. M. Sano Fund.
TC 15. T h e influence of elevated temperatures on the growth and morphology of
H e L a cultures.' Oleg S . SELAWRY"
and Terence McCORMICK*
Department of Biology, Roswell Park Memorial Institute, Buffalo, N. Y .
(Introduced by Machteld E. Sano)
O
O,
HeLa cells were cultured in D/35 Carrel1 flasks in a fluid medium composed s f
one-third calf serum and two-thirds medium 199 a t 36" C. The cells were subjected
to heat treatment (waterbath) in order to determine the upper level of their
biokinetic temperature and their morphological changes under such treatment.
100% of a cell population show irreversible damage after exposure to 42 f 0.1"C.
for 10-12 hours, to 43°C. for three hours, to 44°C. for two hours and to
45°C. for 55 minutes as determined by further observation of the culture for 25
to 30 days. Surviving cells after shorter exposure time are less heat susceptible,
especially in the range o f 42°C. After intermittent treatments i n the course
of 8 months they became adapted to 42°C. for more than 100 hours, t o 45°C.
for up to 75 minutes. The influence of elevated temperatures on the mitotic cycle
and on cytoplasm and nucleus of the interpliases will be described.
1
Supported by the L-S-W Qrant.
T C 8 . T h e Detroit lines of human epithelium-like cells: observations on morphology and biological behavior.' Cyril S. STULBERG, Lawrence BERMAN ' and Frank H. RUDDLE, The Child Research Center of Miehigan
and the Department of Pathology, Wayne University College of Medicine,
Detroit.
Previous communications have described a stable strain of human epitheliumlike cells (Detroit-6) derive2 from a tissue culture of bone marrow. Further
studies have resulted in the development of seven additional lines with similar
niorphologic and growth characteristics. Three of the strains (Detroit-6, 32,
-34) came from cultures of sternal marrow of patients with carcinomatosis.
Two strains (Detroit-30a, -56a) were obtained from the carcinomatous ascites
of two patients, and one strain (Detroit-116a) from the pleural effusion of a
ABSTRACTS OF DEMONSTRATIONS
499
patient with lymphosareoma. Two strains (Detroit-52, -98) developed from
the sternal marrow of individuals with no known malignancies.
I n the bone marrow cultures the epithelium-like cells first appeared a s
isolated plaques of polygonal cells on the 50th to 65th day of continuous culture.
Each marrow culture passed through myeloid, monocytoid, and sometimes fibroblastic phases before epithelium-like cells were observed. Epithelium-like cells
occurring in the original ascitic and pleural fluids gradually increased in number
during one to six weeks. Once epithelium-like cells appeared, they were
mechanically separated from the parent culture or primary cultures were trypsinized, and after subculture, other morphologic cell types disappeared.
Comparative studies on morphology, growth behavior, transplantation t o
heterologus hosts, sponge culture, and virus susceptibilities, illustrating similarities and differences in these cell lines, will be presented.
Supported by a grant (E859)from the National Microbiological Institute, and by 8 grant
(C-2149) from the National Institutes of Health, Public Health Service.
TC 17. The amino acid requirements of Auman uterine fibroblasts, strain U 12.'
H. E. SWIM* and R. 1. PARKER*
Department of Microbiology,
Western Reserve University. (Introduced by Ivor Cornman)
O,
The results of pre,vious studies indicate t h a t chronic toxicity of conventional cell
culture media is partially responsible for the failure of normal (unaltered) human
fibroblasts to proliferate after 4 to 8 months of serial cultivation. I n an attempt
to circumvent some of these difficulties thc amino acid requirements of uterine
fibroblasts have been investigated. Strain U12 in contrast to other human fibroblasts studied in this laboratory, became altered in vitro, as indicated by changes
in morphology of the cells and in their loss of susceptibility to infection with
poliomyelitis virus. Strain U12 was selected for study because it probably represents the closest practical parallel available at the present time to a n unaltered
human fibroblast. The method employed was based on studies of Fischer employing a defined medium supplemented with dialysed horse serum and dialysed
embryo extract. The cells degenerated rapidly when each of the following was
omitted from the medium: arginine, cystine, glutamine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine and
valine. The concentration of each amino acid and glutamine required for maximal
growth under the experimental conditions has. been established. The quantitative
aspects of these studies will be presented and discussed in relation to the continuous serial propagation of unaltered human fibroblasts.
1
Supported by a grant from The National Foundation for Infantile Paralysis.
T C I O . Some quantitative studies on the growth o f Strain L (Earle) mouse cells
in serum-free nutrient solutions. Charity WAYMOUTH, Roscoe B. Jackson
Memorial Laboratory, Bar Harbor, Maine.
The growth of strain L cells in media containing no serum or tissue extracts
has been studied with a simple quantitative method using a microhematocrit
(Waymouth, '56, in press). The effects on cell proliferation of omitting or varying
the concentration of certain components of the medium have been examined and
new information on nutrients essential f o r cell proliferation will be presented.
500
AMERICAN ASSOCIATION O F ANATOMISTS
TC25. Air-ion concentration and the growth of cells in vitro? John L. WORDEN*, Department of Biology, St. Bonaventure University and James R.
THOMPSON* ', Inst. f o r Cellular Research, University of Nebraska.
(Introduced by Tobie Mullerj
Recent investigations have demonstrated the effects of air-ion concentration
and polarity on various physiological values i n intact mammals. Statistically significant changes have been noted in carbon dioxide capacity of plasma, p H of
whole blood, nerve regeneration time, adrenal secretory activity and others.
Apparatus for the generation and dissemination of ions of determined polarity
has been adapted to tissue culture procedures so that cells may be propagated in
liquid medium with an overlay of 5% CO, in air with (1) normal ion content,
( 2 ) increased negative ion content, ( 3 ) increased positive ion content. Experimentation has shown that an excess of ions of one polarity is essential for physiological effects. I n conditions 2, 3, above, ions of opposite polarity are a t near
zero level.
Cultures of pure strain L cells (Earle's) were employed. Replicate planting8
and cell enumerations mere made according t o techniques of Earle, Evans, Sanford et al.
Determinations were run in sets of 36 replicates, 9 used to determine initial
counts and uniformity of planting, and 9 placed in each of three incubators at
37.5"C. for 72 hours. Atmospheric conditions were normal in one incubator, elevated
negative ion concentration i n the second, elevated positive ion concentration in
the third.
Statistically significant differences in rate of proliferation occurred -reproduction was accelerated under conditions of negative ionization and decelerated
under conditions of positive ionization. The nature of the effects is compatible
with that observed in intact animals.
1
Supported by a grant from the Wesix Research Foundation, San Francisco, California.
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