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Program and abstracts of papers presented at the thirty-fifth annual meeting of the midwest association of anatomists October 7 У9 1977 Omaha Nebraska (pp. 534 У553)

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ABSTRACTS
ADAMS, H.G., E.D. PRENTICE. E.H. JELINEK,
W.W. STINSON, R.H. JENSEN, AND W.K. METCALF,
Department of Anatomy, University of Nebraska
M e d k a l Center, Omaha, Nebraska. Anatomy in
cross section: c o r r e l a t i o n with a x i a l body
scans.
With t h e advent of computerized a x i a l tomography (CAT) new emphasis is being placed on
i d e n t i f i c a t i o n of a n a t m i c a l s t r u c t u r e s i n the
a x i a l plane by anatomists and physicians a l i k e .
CAT scanning o f f e r s new incentive t o reemphas i z e a forgotten dimension of gross anatomy.
This p r o j e c t was, therefore, i n i t i a t e d t o produce a series of photographs displaying c r o s s
sections of anatany and corresponding a x i a l
scans from an EM1 CT5005 Body Scanner. A cadaver was frozen, scanned a t lcm i n t e r v a l s , and
then sectioned a t corresponding i n t e r v a l s in
t h e same plane a s t h e scan. Each anatomical
cross-section and scan w a s then printed photographically. I n a d d i t i o n a s e r i e s of scans of
t h e head and trunk of hospitalized p a t i e n t s
(done f o r diagnostic purposes but read as normal) was selected and printed photographically.
Unlike the head and trunk where scans of t h e
fixed cadaver produced l i t t l e information, t h e
scans of the extremities showed excellent tissue density d i f f e r e n t i a t i o n and c o r r e l a t i o n
with t h e anatcmical cross-sections.
Very reasonable c o r r e l a t i o n , however, was found between cross sections of t h e head and trunk and
the selected "live" scans. The "live" scans
produced additional information and added
c l i n i c a l realism through t h e use of a contrast
medium infused, i n j e c t e d , o r swallowed. Res u l t s obtained thus f a r i n d i c a t e t h a t t h i s proj e c t w i l l r e s u l t in a valuable reference t o o l
f o r t h e scanning industry and an excellent
teaching a i d f o r students of a n a t m y i n cont r a s t t o more c l i n i c a l l y oriented CAT scan
atlases.
-
ALBERTINE, K.H. and C.C.C. O'MORCHOE, Department of Anatomy, Loyola University S t r i t c h
School of Medicine, Maywood, I l l i n o i s . TransLymphatic Transport i n t h e Canine Kidney: &
U l t r a s t r u c t u r a l Study.
(Supported by a grant from t h e National I n s t i t u t e s of Health #AM 18473. )
Discrete openings through or between adjacent lymphatic endothelial c e l l s a r e considered,
according t o one p r i n c i p a l theory, t o be t h e
major route whereby substances e n t e r t h e lumen
of lymphatic c a p i l l a r i e s (Casley-Smith, Microc i r c u l a t i o n , Vol. I , 1977). These openings
have been more commonly described i n association
with t i s s u e trauma o r edematous s t a t e s than i n
control t i s s u e . The purpose of t h e present
study was t o examine t h e f i n e s t r u c t u r e of r e n d
lymphatic c a p i l l a r i e s with s p e c i a l reference t o
possible routes of trans-lymphatic t r a n s p o r t
under control and experimental conditions. For
t h i s purpose dog kidneys were subjected t o unil a t e r a l u r e t e r i c obstruction f o r 3 days followed
by lymphatic obstruction f o r 4 hours. Control
kidneys were subjected t o lymphatic occlusion
only. Two p o t e n t i a l pathways f o r trans-endot h e l i a l movement were v i s i b l e u l t r a s t r u c t u r a l l y :
( a ) i n t e r c e l l u l a r and ( b ) v e s i c u l a r t r a n s p o r t .
Lymphatic endothelial c e l l s showed l a r g e numbers
of both smooth and coated v e s i c l e s of various
s i z e s , e s p e c i a l l y where t h e cytoplasm was attenuated. Many v e s i c l e s were seen t o comunicate
with t h e e x t e r i o r o f t h e c e l l on i t s luminal and
abluminal surface suggesting t r a n s - c e l l u l a r
movement. I n t e r c e l l u l a r t r a n s p o r t depends upon
a v a r i e t y of which were
j u n c t i o n a l regions
seen i n t h i s study: ( a ) end-to-end, ( b ) overlapping and ( c ) elaborate i n t e r d i g i t a t i o n of
c e l l u l a r extensions. Junctional complexes
associated with these contacts were ( a ) zonula
occludens, ( b ) zonula o r f a s c i a adhaerens and
( c ) macda adhaerens. The zonula occludens w a s
most frequently located close t o t h e lumen. Although a v a r i e t y of c e l l contacts were o b s e y e d ,
as w e l l as junctional complexes, i n both control
and experimental t i s s u e , d i s c r e t e openings 'between adjacent c e l l s were not observed. The
absence of d i s c r e t e openings, p a r t i c u l a r l y i n
t h e experimental t i s s u e , together with t h e prominence of cytoplasmic v e s i c l e s seen i n t h e present study, c d l s i n t o question t h e hypothesis
t h a t open junctions a r e t h e major mechanism of
trans-lymphatic t r a n s p o r t i n t h e dog kidney.
-
J . G . SHARP, and W.W. STINSCIN,
Department of Anatomy, University of Nebraska
Medical Center, Omaha, Nebraska. Characterist i c s of cultured bone marrow stromal c e l l s .
The microenvironment of hematopoietic stem
c e l l s has been implicated as playing a r o l e in
the control of stem c e l l d i f f e r e n t i a t i o n . In
the present i n v e s t i g a t i o n bone marrow stromal
c e l l s have been grown i n c u l t u r e t o f a c i l i t a t e
i n v e s t i g a t i o n of the mechanism through which the
influence of the microenvironment i s expressed.
The cultured c e l l s have been characterized morphologically and a preliminary assay of t h e i r
a b i l i t y t o induce granulocyte-monocyte colony
(CFU-c) formation has been performed. Femoral
bone frsgments w e r e suspended i n agar and supplemented McCoy's 5A medium employing a method
s i m i l a r t o t h a t of Daniels (Anat. Rec. =.:388,
1976). The f i r s t cells t o appear within a dayof
i n i t i a t i o n of the c u l t u r e s were small round
c e l l s of uniform s i z e . These were probablyblood
mononuclear cells which had migrated out of t h e
bone fragments. By day 5 l a r g e ovoid c e l l s cont a i n i n g an oval nucleus with a prominent nucleol u s and numerous basophilic cytoplasmic granules
had appeared. S t e l l a t e f i b r o b l a s t o i d c e l l s ,
ANDERSON, R.W.,
534
first seen in a few cultures by day 9, appeared
in 66X of the cultures on day 15. This celltype
became the prominent constituent of all thecultures and grew to confluence by day 40. Cellsin
some cultures grew diffusely but most of the
cells grew compactly. Acellular matrix accumulated between cell clusters. Histochemical procedures carried out on compact cell clusters
demonstrated the stellate cel.ls had oval nuclei
containing one or more nucleoli. Their cytoplasm contained basophilic granules, vacuoles,
ribosomes and methanamine silver positive material around their periphery. Electron microscopy
verified the presence of numerous ribosomes and
demonstrated aggregates of immature fiber components at the cell periphery. Such cells appeared equipped to produce acellular matrix
material. However, when either cultured stromal
cells or concentrated culture supernate were
co-cultured with normal bone marrow cells, with
or without the presence of human urinary or
mouse endotoxin colony activation factor, in the
agar colony forming assay system there were no
striking increases in granulocyte-monocyte
colony formation attributable to the cultured
stromal cells or their supernate. (Supported by
NIH Grants CA18548, 1T32HD07097 and NSF RIASSER77-06922).
ASHIRU, O.A., Department of Anatomy, University
of Nebraska Medical Center. Omaha. Nebraska.
The role of medical education on the development of Nigeria.
Many areas of disparity between a developed
and a developing nation were projected with an
analysis of medical education. America has a
doctor-to-population ratio of about 1:EOO and
a per capita income of about $4,240. In contrast, Nigeria has a doctor-to-population ratio
of 1:20,000 and a per capita income of $90.
Although health problems such as malaria, pneumonia, gastroenteritis, helminthiasis, malnutrition and tuberculosis are relatiiely uncommon in America, Nigerian doctors are well acquainted with such diseases as they find themselves treating them daily. Such diseases can
be prevented by proper health programs, adequate water supplies, proper environmental
sanitary health services, dependable power
service and many of such basic services which
are taken for granted in a developed country.
The Federal Government of Nigeria is aware that
a sufficient number of well-trained physicians
could alleviate most of the health problems
through their treatment of patients and their
assistance in planning proper environmental
sanitary conditions. The government plans to
increase the number of medical schools from
the present 6 to 9 and the number of students
per class from the present 100-150 to 200-250
in each school. It is anticipated that over
1,500 physicians will graduate yearly by the
mid-1980's. In order to assure dissemination
of the doctors throughout the country, a one
year National Youth Service Posting has been
established. It is viewed that adequate
government programming can go a long way in
improving medical education and subsequently,
the health problems. However, there should
not be an urge to maneuver for an ideal situation overnight it may never work.
(Supported in part by the University of Lagos,
College of Medicine, Lagos, Nigeria).
-
BAUER, T.W., G. PEREIRA and J.G. SHARP, Department of Anatomy, University of Nebraska Medical
Center, Omaha, Nebraska. Morphological observations of cell types present in monolayer cultures of thymic non-lymphoid cells.
A potential problem in theapplication of cultured monolayers of thymic non-lymphoid cells to
the study of the thymic microenvironment &
is the possibility that the cultured cells
may differ morphologically and functionally from
the cells from which they originated in vivo.
We have undertaken a preliminary morphological
comparison of the cell types observed in confluent monolayer cultures of rat thymic nonlymphoid cells with the cells of the thymus &
vivo. Two cell types predominate in the cultures. The most common is an elongated cell
with dendritic processes which appears to be
phagocytic when co-cultured with syngeneic lymphocytes. Rather less co-n
is a large rounded
cell whose nucleus often has two prominent nucleoli. This cell is sometimes seen with several large lymphocytes in association with its
cytoplasmic processes when the monolayer is cocultured with lymphocytes. These cells are
similar to those described previously (Owen
aJ., Cold Spr. Harb. Symp. Quant. B i o . XLI:129,
1977 and WaksalsaJ., Ann. N.Y. Acad. Sci.
249:492, 1975). Also, a large multinucleated
cell is seen in the cultures. Such cells were
first described in the inner connective tissue
medulla invivoby Pereira (Ph.D. Thesis, McGill
University, 1968). Similar cells have recently
been described by Scheiff (Cell Tiss. Res. 170:
350, 1976; Acta Anatomica 2:311, 1977) who believes, however, that these cells are epithelial
i n nature. Additionally, several types of granule-bearing cells are observed in vitro. The
_in vivo equivalents of these cell types are not
known, however, one cell type, which in culture
has granules appearing less densecentrally than
peripherally may be seen in micrographsofchick
thymus (Hakauson eta.,Histochem. 2~25,1974)
and has recently been observed by us in rat
thymus in vivo. It is possible that the accumulation of granules in some of these cells is
an artifact of the culture system. Alternatively,
our current experience suggests that a more detailed investigation of the thymus in vivo,
with particular emphasis on spatial morphometry
is needed and may reveal more morphological correlates of the cells of the monolayers. (Supported by NIH Grants CAl8548 and 1T32 HD07097).
BECKMAN, W . C . , JR., D e p a r t m e n t o f
Anatomy, R e p r o d u c t i v e E n d o c r i n o l o g y P r o aram. U n i v e r s i t v o f M i c h i a a n , Ann A r b o r ,
f i i c h i g a n . A method f o r t h e p r e p a r a t i o n
o f thin sections suitable f o r electron
microscopy from t h i c k sections previousl y stained w i t h methylene blue.
635
The p u r p o s e of t h i s p r o c e d u r e i s t o
p e r m i t an i n v e s t i g a t o r t o examine t i s s u e
s e c t i o n s embedded i n e p o x y r e s i n s w i t h
l i g h t m i c r o s c o p y and t h e n t o r e e x a m i n e
selected sections with the electron
microscope.
S e c t i o n s as t h i n as 5 p a r e
p l a c e d i n sequence on albumen-coated
glass slides.
The s l i d e s a r e t h e n
stained l i g h t l y with methylene blue t o
f a c i l i t a t e l i g h t microscopic examination
and/or photography.
A p l a s t i c capsule
containing unpolymerized r e s i n i s then
i n v e r t e d o v e r t h e s e c t i o n s chosen f o r
electron microscopic examination. A f t e r
polymerization, the capsule i s separated
from t h e s l i d e by immersion f i r s t i n a
d r y - i c e / a l c o h o l b a t h and t h e n i n b o i l i n g
w a t e r . The f l a t f a c e o f t h e c a p s u l e i s
t r i m m e d away t o l e a v e a p y r a m i d w i t h t h e
thick section a t i t s t i p .
This i s then
o r i e n t e d c a r e f u l l y on t h e u l t r a m i c r o t o m e
t o be p e r f e c t l y p a r a l l e l t o t h e k n i f e .
About f o r t y u s a b l e t h i n s e c t i o n s can b e
o b t a i n e d f r o m such a specimen,
Electron
m i c r o g r a p h s o f t h e s e s e c t l o n s show a
s l i g h t i n c r e a s e i n g r a n u l a r i t y and
e l e c t r o n o p a c i t y o f t h e n u c l e u s and
ribosomes, b u t o t h e r o r g a n e l l e s appear
I f such
t o be u n a f f e c t e d b y t h e s t a i n .
e f f e c t s are undesirable, t h i c k e r
s e c t i o n s g e n e r a l l y p r o v i d e enough
c o n t r a s t f o r adequate s e l e c t i o n o f t h e
a p p r o p r i a t e r e g i o n and s t a i n i n g w i t h
methylene b l u e i s n o t necessary.
This
method p e r m i t s t h e s a m p l i n g o f an e n t i r e
b l o c k o f t i s s u e t o o b t a i n t h e most des i r a b l e sections f o r electron microscopy.
I n addition, i t i s a great aid
i n t h e s t u d y o f m o r p h o l o g i c a l l y complex
regions, since i t permits the development o f a t h r e e - d i m e n s i o n a l a p p r e c i a t i o n
f o r the area surrounding the region
which i s being studied w i t h e l e c t r o n
microscopy.
BLAKE, C . A . , Department of Anatomy, University
of Nebraska Medical Center, Omaha, Nebraska.
Central nervous system a c t i n g drugs: A biphasi c e f f e c t on p i t u i t a r y LH release.
There a r e many drugs which a c t on t h e cent r a l nervous system t o block the spontaneous,
pre-ovulatory surge of LH i n rat plasma when
administered a t proper dosages j u s t p r i o r t o
the s t a r t of the LH surge on proestrus. In
t h i s study, several of these drugs were t e s t e d
for t h e i r a b i l i t y t o i n h i b i t LH r e l e a s e a f t e r
the start of the LH surge. Four-day c y c l i c
r a t s were each f i t t e d with an indwelling r i g h t
a t r i a l cannula during t h e morning of proestrus.
S e r i a l blood samples were collected through t h e
cannula during the afternoon of proestrus
s t a r t i n g a t a time when plasma LH was expected
t o be elevated. Drugs were then administered
a t surge-blocking dosages. Additional blood
samples were c o l l e c t e d and subsequently, plasma
LH was measured by radioimmunoassay.
In r a t s
with elevated LH, i p i n j e c t i o n of phenobarbital
o r pentobarbital, s c i n j e c t i o n of atropine, or
i v i n j e c t i o n of ethanol caused plasma LA t o
f a l l rapidly s t a r t i n g within 20 min a f t e r inj e c t i o n . On the contrary, s c i n j e c t i o n of
n i c o t i n e , i p i n j e c t i o n of urethane, o r continuous exposure t o e t h e r fumes r e s u l t e d in a biphasic e f f e c t on plasma LH. Plasma LH rose
rapidly, peaked within 5-20 Din, and declined
rapidly t h e r e a f t e r . Nicotine, urethane and
e t h e r caused a small, t r a n s i e n t increase in
plasma LH when administered p r i o r t o t h e start
of the spontaneous LH surge, and these drugs
have been found t o have no apparent e f f e c t on
LHRH-induced LH r e l e a s e . These d a t a and the
f a c t t h a t LHRH is f a r more e f f e c t i v e in elevating plasma LH when given a f t e r the s t a r t of the
LH surge than j u s t p r i o r to i t lead the author
t o suggest t h a t nicotine. urethane, and e t h e r
a l t e r LH r e l e a s e in proestrous r a t s by f i r s t
releasing endogenous LHRH and then by blocking
LHRH r e l e a s e above b a s a l r a t e s . Caution is
warranted i n the use of these drugs a s blockers
of the pre-ovulatory LH surge when they are
f i r s t administered a f t e r t h e start of the LB
surge. (Supported by N I H Grant No. H D l l O l l and
t h e University of Nebraska Medical Center
Foundation).
BROWNSCHEIDLE, C.M., B.L. DRESSER AND P.T.
RUSSELL, Department of Anatomy and
Obstetrics and Gynecology, University of
Cincinnati College of Medicine, Cincinnati,
Ohio, Department of Animal Science, The Ohio
S t a t e Universitv. Columbus. Ohio and The
Cincinnati Zoo. ObseNati& on t h e
remoductive cycle of t h e African bonm,
Boocercus euwcerus.
The c e l l u l a r c h a r a c t e r i s t i c s of t h e
contents of t h e vaginal vestibule were studied
through two e s t r u s cycles i n t h e bongo (iamily
Bovidae) and c o r r e l a t e d with observations of
i t s breeding behavior. FraD these s t u d i e s ,
t h e duration o f t h e estrus cycle, t h e length
of heat and t h e morphology of both vaginal
c e l l s and sperm a r e established. Samples were
obtained d a i l y by lavage of t h e vaglnal
v e s t i b u l e with a dropper containing
approximately 3 m l . of physiologic saline.
Smears of t h e samples were stained with Harris
hematuxylin and 0.1% a l c o h o l i c eosin. Cell
s i z e was measured with a c a l i b r a t e d micraneter
and c e l l numbers were estimated by a standard
"battlement" technique and expressed a s percent
of total cell8 counted. These s t u d i e s
demonstrate t h a t t h e s t a g e s of e s t r u s a r e
characterized a s f o l l o w : 1) Proestrus: 2 days:
recognized by t h e presence of numerous
neutrophila (9%) and round or ovoid basophilic
c e l l s about 20 JIUI diameter, 2) Estrus: 2 days:
large, p a r t i a l l y cornified and a n t e d c e l l s
are present; neutrophils decreased (8%); 3)
Metestrus: 3 days: c o r n i f i e d cells a r e most
prevalent during t h i s period (2@) w h e r e a s
neutrophils occur much less frequently (4%).
Two types of basophilic c e l l s a r e also found,
4) M e s t r u s : 15 days: very large (25-90 JIUI),
round pale-staining c e l l s a r e c h a r a c t e r i s t i c ;
" I
536
neutrophils increase (SO$). Spermatozoa were
studied In several samples obtained a t estrus
immediately after breeding. The sperm are
appMximately 50 m long. The head is pearshaped with a blunt base and i s about 7 urn long
and 4
wide.
The bongo does not exhibit any external
signs of estrus. However, this investigation
demonstrates that the vaginal cytology may
ofler a means of cantrolled breeding
management in this spcies.
CAMPBELL, C. and J . G . SHARP, Department of
Anatomy, University of Nebraska Medical Center,
Omaha, Nebraska. Effects of thymectomy on the
femoral growth plate of the rat.
The thymus is not only the primary site of
differentiation of a major lymphocyte subpopulation; thymus processed, T lymphocytes, but
also it is an endocrine gland whose function is
still incompletely described. There have been
unconfirmed suggestions that thymectomy has an
effect on bone growth in mice as indicated by
changes in histochemical staining of acid mucopolysaccharides at the light microscopic level
in the femur (Berek gal. Experientia %:721,
1968; Experientia 5:982, 1969). Therefore,the
present investigation was undertaken to determine if any changes were evident in the femoral
growth plate of neonatally and infant thymectomized rats. Sprague-Dawley rats were thymectomized or sham-thymectomized at 1 or 5 days of
age and subsequently maintained under aseptic
conditions to minimize the risk of infection.
At autopsy, the body, femur and thymus weights
were recorded and measurements made of the femoral diaphyseal length, total femoral length
and the area at the epiphysis. Femora were
paraffin embedded and sections cut at 711. These
sections were stained with alcian blue and periodic-acid Schiff reagent. The width of the
growth plate was determined using an eye-piece
micrometer. An additional group of rats was injected intraperitoneally with 3wCi/g bodyweight
each of radioactive sulphur. The right femora
were removed for determination of radioactive
sulphur content and the left femora were removed for macroautoradiography. The results revealed differences that were most pronounced in
female animals that were thymectomized on day 5
and sacrificed at 3 months. In the thymectomized
animals, the width of the growth plate, particularly the columnar zone, and the radioactive
sulphur uptake, were significantly lower than
in the sham-thymectomized rats. In addition, the
intensity of alcian blue staining was decreased
in the growth plate of thymectomized rats.
These differences support the hypothesis that
the thymus is required for the normal development and growth of bone. Whether the influence
of the thymus is the result of a direct or indirect hormonal action, or a direct or indirect
cellular interaction remains to be determined.
(Supported in part by NIH Grants CA18548, 1T32HD07097 and NSF RIAS-SER77-06922.)
Castro, A.J., Department of Anatomy, Stritch
School of Medicine, Maywood, Illinois.
Corticospinal and rubrospinal pro.1ections after
yramidotorny in newborn rats.
?Supported by NIH Grant NS 13230)
In previous studies, unilateral frontal
cortical lesions in newborn rats caused the
development of aberrant ipsilateral corticospinal projections but caused no observable
alterations in rubrospinal projections. The
present study was conducted to determine
whether aberrant corticospinal or rubrospinal
projections would develop in response to neonatal pyramidotomy.
The corticospinal tract was uni- o r bilaterally sectioned at medullary levels in 1-3
day old Long-Evans black-hooded male rats. At
2-6 months after surgery, animals received a
second (adult) lesion. One group of rats that
underwent unilateral pyramidotomy sustained
adult cortical lesions on the side corresponding to the intact corticospinal tract. In a
second group that had sustained unilateral
pyramidotomy, the rubrospinal tracts were bilaterally ablated by stereotaxically transecting the ventral tegmental decussation. The
rubrospinal tracts were also sectioned in a
third group of animals that had sustained bilateral pyramidotomy. Animals were sacrificed
3-12 days after the second lesion, and cervica
spinal cord sections were stained using the
Fink-Heimer degeneration technique in order t o
determine cortico- and rubrospinal projections
Previous studies found no stainable degeneration two months after neonatal lesions.
Similar to observations after neonatal cortical lesions, an aberrant ipsilateral corticospinal tract was observed in animals that sustained neonatal unilateral pyramidotomy. A l though much smaller quantitatively than the
normal contralateral projection, the ipsilateral projection pattern was judged to be
qualitatively equivalent. There were no observable changes in the distribution of rubrospinal fibers after uni- o r bilateral neonatal
pyramidotomy in comparison to controls.
CATON, C.B. and P.J. GARDNER, Department of Anatomy, University of Nebraska Medical Center,
Omaha, Nebraska. Localization of hormones in
the male reproductive system by the unlabeled
antfbody peroxidase-antiperoxidase complex technique: Luteinizing hormone (LH).
Stimulation of the adult male testis by luteinizing hormone (LH) results in androgen synthesis and secretion. Although evidence suggests
that Leydig cells are the major source of androgenic steroids, the exact sites of LH action in
the testis have not been clearly demonstrated.
Previous studies (biochemical andmorphological)
using labeled hormones and/or labeled antisera
indicate that LH binds primarilytoLeydigcells,
although some binding occurs in the peritubular
and tubular elements. In the present investigation the cellular and subcellular binding sites
for anti-LH in the testis were studied by the
~
537
~~
unlabeled antibody, peroxidase-antiperoxidase
(PAP) complex technique. P i t u i t a r y g l a n d s and
t e s t e s of normal, a d u l t male rats were prepared
by r o u t i n e procedures f o r l i g h t and e l e c t r o n
microscopy. S e c t i o n s were incubated s e q u e n t i a l l y
i n anti-LH (NIAMDD S-3), g o a t a n t i - r a b b i t IgG.
and PAP, w i t h a p p r o p r i a t e i n t e r v e n i n g b u f f e r
washes. They were then t r e a t e d w i t h diaminobenzidine-Hz02.
U l t r a t h i n s e c t i o n s were a l s o
t r e a t e d w i t h Os04 t o r e n d e r t h e r e a c t i o n product
e l e c t r o n dense. Method c o n t r o l s c o n s i s t e d of
o m i t t i n g one of t h e s t e p s i n t h e s t a i n i n g sequence, w h i l e s p e c i f i c i t y was t e s t e d by u s i n g a
series of d i l u t i o n s of anti-LH o r anti-LH prev i o u s l y absorbed w i t h LH (NIAMDD 1-4). S t a i n i n g
i n t e n s i t i e s were measured a t t h e l i g h t micros c o p i c level w i t h a L e i t z MPV microscope photometer. Reaction product w a s l o c a l i z e d i n p i t u i t a r y gonadotrophs and t e s t i c u l a r Leydig cells.
Also, s t a i n i n g w a s observed i n c o n s i s t e n t l y i n
t h e p e r i t u b u l a r boundary t i s s u e . Q u a n t i t a t i v e
e v a l u a t i o n confirmed t h e s p e c i f i c i t y of t h e
s t a i n i n g r e a c t i o n . No s p e c i f i c s t a i n i n g w a s detected i n t h e testis a t the u l t r a s t r u c t u r a l
l e v e l , although s t a i n i n g w a s heavy i n p i t u i t a r y
gonadotrophs. The r e s u l t s of t h i s p r e l i m i n a r y
s t u d y confirm t h e f i n d i n g s of previous i n v e s t i g a t o r s t h a t LH b i n d s t o Leydig c e l l s and p e r i t u b u l a r elements and s u g g e s t t h a t i t may be
p o s s i b l e t o l o c a l i z e LH i n t h e t e s t i s a t t h e
u l t r a s t r u c t u r a l l e v e l by t h e PAP technique. Furt h e r e l e c t r o n microscopic s t u d i e s are in progress.
(Supported i n p a r t by N I H Grant No.
HD 09881 and NSF RIAS-SER-77-06922).
CLAYTON, F.C., G.L. TODD and G.M. PIEPER,
Cardiovascular Center and Department of Anatomy,
U n i v e r s i t y of Nebraska Medical Center, Omaha,
NE.
E f f e c t s of v a r y i n g doses o f i s o p r o t e r e n o l
i n f u s i o n s on c a r d i a c hemodvnamics and morphol-
Catecholamine s t i m u l a t i o n of t h e h e a r t produces both a p o s i t i v e c h r o n o t r o p i c and i n o t r o p i c response. It h a s a l s o been shown t o produce t h e p a t h o l o g i c a l c o n t r a c t i o n band l e s i o n s
of c o a g u l a t i v e myocytolysis. An i n v e s t i g a t i o n
was made t o determine t h e hemodynamic and morp h o l o g i c a l responses t o i n c r e a s i n g d o s e s of t h e
s y n t h e t i c , 8-adrenergic catecholamine, isoprot e r e n o l . Dogs were a n e s t h e t i z e d and t h e h e a r t s
exposed v i a a m i d l i n e sternotomy. S a l i n e or
i n c r e a s i n g doses o f i s o p r o t e r e n o l (0.1, 1.0 or
2.5 pg/kg/min) were continuously i n f u s e d f o r
one hour. Subsequently, t h e h e a r t s were excised, fixed, s l i c e d i n t o transverse s l i c e s ,
s e c t i o n e d and s t a i n e d w i t h H & E or phosphot u n g s t i c acid-hematoxylin.
Both t h e chronot r o p i c and i n o t r o p i c responses i n c r e a s e d w i t h
i n c r e a s i n g doses of i s o p r o t e r e n o l . The peak
occurred more r a p i d l y i n h e a r t rate than i n
I n g e n e r a l , both parameters t h e n dedP/dt.
c l i n e d d u r i n g t h e remainder of t h e e x p e r i m e n t a l
p e r i o d . Morphologically, t h e t o t a l number of
c o n t r a c t i o n bands i n c r e a s e d w i t h i n c r e a s i n g
doses. A d d i t i o n a l l y , t h e r e was a t r a n s m u r a l
d i s t r i b u t i o n of t h e c o n t r a c t i o n band l e s i o n s
i n d i c a t i n g a subendocardial v u l n e r a b i l i t y t o
t h i s sympathetic s t i m u l a t i o n . There is, t h e r e f o r e , a dose r e l a t e d response both f o r t h e
hemdynamic parameters and t h e production of
myocardial l e s i o n s . The p h y s i o l o g i c a l responses
concomitant with t h e production of hyperfunct i o n a l l e s i o n s s u p p o r t s t h e concept of mechani c a l o v e r d r i v e as one of t h e f a c t o r s i n t h e
u n d e r l y i n g p a t h o p h y s i o l o g i c a l mechanisms of
catecholamine-induced n e c r o s i s .
(Supported by U n i v e r s i t y of Nebraska Medical
Center, Cardiovascular C e n t e r Development
Award and Nebraska Heart A s s o c i a t i o n )
COOPER, M.H., Department of Anatomy, Wayne State
U n i v e r s i t y School of Medicine, D e t r o i t , Michigan
-J=ahums:
measurements of f i b e r s .
The b a s i l a r p o n t i n e gray
- . r e c e i v e s its l a r g
-e s t
i n p u t from t h e c e r e b r a l c o r t e x v i a corticopont i n e f i b e r s ; i t s neurons p r o j e c t t o t h e c e r e h l lum a s p o n t o c e r e b e l l a r f i b e r s w i t h i n t h e middle
c e r e b e l l a r peduncle. From t i s s u e removed a t t h e
l e v e l of t h e b a s i l a r pons of t h e s q u i r r e l monkey
( S a i m i r i s c i u r e u s ) v a r i o u s areas of t h e c o r t i c o F a n t i n e , c o r t i c o s p i n a l , and p o n t o c e r e b e l l a r f i b e r s were s t u d i e d . Three micra p l a s t i c s e c t i o n s
were c u t i n both t h e t r a n s v e r s e and s a g i t t a l
p l a n e , s t a i n e d w i t h t o l u i d i n e b l u e and photographed. The d i a m e t e r s of c r o s s s e c t i o n s of
a x i s c y l i n d e r s of each group were measured and
counted. Two d i f f e r e n t a r e a s of t h e c o r t i c o p o n t i n e / c o r t i c o s p i n a l bundles were s t u d i e d on
t r a n s v e r s e s e c t i o n s of t h e b a s i l a r pons: i n i t i a l l y a small group of 625 f i b e r s and a second
l a r g e r group of 4484 f i b e r s .
In both samples
over 70% of t h e a x i s c y l i n d e r s had a diameter of
2 micra o r l e s s w i t h t h e m a j o r i t y between .8 t o
1 . 3 micra. The range of a l l f i b e r s w a s - 4 t o
15.4 micra. 7% of t h e f i b e r s measured over 4.0
micra. The a x i s c y l i n d e r s of t h e pontocerebell a r f i b e r s of v a r i o u s p o r t i o n s of t h e middle
c e r e b e l l a r peduncle were measured on s a g i t t a l
s e c t i o n s of t h e b a s i l a r pons. A t o t a l of 805
a x i s c y l i n d e r s from t h e v e n t r a l edge of t h e midd l e of t h e b a s i l a r pons were observed. The d i ameter of t h e a x i s c y l i n d e r s range between . 4 t o
3.6 micra w i t h over 90% measuring under 2 micra.
A second area from t h e d o r s a l a s p e c t of t h e midd l e c e r e b e l l a r peduncle and e q u a l i n s i z e t o t h e
f i r s t r e v e a l e d a d i f f e r e n c e i n range and s i z e of
t h e diameter of 613 a x i s c y l i n d e r s . These a x i s
c y l i n d e r s w e r e somewhat l a r g e r ranging from .44 . 8 micra. 73% of t h e s e w e r e 2 micra o r less.
The m a j o r i t y measured between .9-1.6 micra
whereas t h e m a j o r i t y of t h o s e placed v e n t r a l l y
were between .7-1.2 m. I n an a r e a s e l e c t e d
from t h e more l a t e r a l a s p e c t of t h e middle cere b e l l a r peduncle 1214 a x i s c y l i n d e r s were meas u r e d and counted. Over 70X of t h e s e had a d i ameter under 2 micra w i t h t h e m a j o r i t y between
1.0-1.5 micra. They ranged i n s i z e from .4-7.2
micra. Observations of e l e c t r o n micrographs
t a k e n from t h e two t y p e s of bundles r e v e a l s i m ilar results.
(Supported by N I H Grant NS 06925-11 from t h e
NINCDS).
638
COX, V. S., WALUCE, L. J. and JESSEN, C. R.,
College of V e t e r i n a r y Medicine, U n i v e r s i t y
of Minnesota, S t . Paul, Irtlnnesota. Anatomic
and g e n e t i c s t u d i e s of cryptorchidism i n t h e
dog w i t h comparison t o t h e h o r s e .
Eleven cases of cryptorchidism were found
in a colony of Miniature Schnauser dogs.
T e s t i c u l a r maldescent w a s b i l a t e r a l i n 6
c a s e s and u n i l a t e r a l i n 5 cases. With only
one exception, t h e e c t o p i c testicles w e r e in
t h e abdominal p o s i t i o n . The morphological
appearance of t h e abdominal gonads was very
primitive i n the b i l a t e r a l cases but nearly
normal developmentally i n t h e u n i l a t e r a l
c a s e s . The degree of inbreeding was g r e a t e s t
f o r t h e b i l a t e r a l cases. The high incidence
of cryptorchidism i n t h i s colony provides
i n d i s p u t a b l e evidence for t h e h e r e d i t a r y
n a t u r e of t h e c o n d i t i o n in t h e dog. I n
c o n t r a s t , an endocrine b a s i s f o r equine
cryptorchidism is proposed based on study
of f e t u s e s and s u r g i c a l c a s e s .
CRALLEY, J.C. and E.J. WINGFIELD 111,
Department of B i o l o g i c a l Sciences, I l l i n o i s
S t a t e University, Normal, I l l i n o i s .
florphometric s t u d i e s of the dorsum of t h e human
foot.
Morphometric measurements were made of t h e
muscles and tendons of 50 human cadaver f e e t .
These measurements included t h e determination
of t h e s i z e of e x t e n s o r digitorum b r e v i s and
extensor h a l l u c i s b r e v i s a s w e l l as determinat i o n of t h e p o s i t i o n of t h e extensor tendons of
t h e dorsum. These observations were used t o
c o r r e l a t e muscle v a r i a t i o n w i t h bunion deformity
i n t h e cadaver f e e t . The deformity was d e t e r mined from X-rays taken p r i o r t o t h e d i s s e c t i n n .
The r e s u l t s of t h i s s t u d y showed no c o r r e l a t i o n
between tendon p o s i t i o n of t h e e x t e n s o r tendons
and bunion deformity; however, t h e g r o s s s i z e
of t h e e x t e n s o r b r e v i s muscle b e l l y (dry weight)
was smaller i n bunion deformed than i n normal
f e e t . Several tendon v a r i a t i o n s including,
s p l i t tendons, e x t r a s l i p s , and double tendons
were found.
CROUSE, D.A., E.J. AINSWORTH, and J.S. HULESCH,
Department of Anatomy, U n i v e r s i t y of Nebraska
Medical Center, Omaha, Nebraska, and Division
of B i o l o g i c a l and Medical Research, Argonne Nat i o n a l Laboratory, Argonne, I l l i n o i s . Thymid i n e s u i c i d e s t u d i e s on hematopoietic s t e m c e l l s
in aging i r r a d i a t e d mice.
Treatment of p r o l i f e r a t i n g c e l l populations
with high s p e c i f i c a c t i v i t y t r i t i a t e d thymidine
(3HTdR) has f r e q u e n t l y been used t o d e s t r o y
cells i n t h e DNA-synthetic (S) phase of t h e
c e l l cycle. U t i l i z i n g an i n v i v o 3HTdR s u i c i d e
technique and t h e s t a n d a r d T i l l and McCullough
s p l e e n colony a s s a y , we i n v e s t i g a t e d t h e p r o l i f e r a t i v e s t a t u s of hematopoietic s t e m c e l l s
(CFU-S) from young, aged, and aged-irradiated
B6CF1 male mice. T r i t i a t e d thymidine (53.7 Ci/
24 hours b e f o r e CFU-S assay. The CFU-S level i n t h e t r e a t e d mice was
t h e n compared t o t h e c o n t r o l CFU-S l e v e l s i n unt r e a t e d young, aged, and a g e d - i r r a d i a t e d mice.
The s u r v i v i n g CFU-S p e r femur f o r each c o n t r o l
and t r e a t e d group was determined i n t h e followi n g : e n t i r e femur, ground and f i l t e r e d ; marrow
plug only a f t e r a s p i r a t i o n , suspension, and f i l t r a t i o n ; hollow femur s h a f t , ground and f i l t e r e d
after marrow p l u g a s p i r a t i o n . P r e p a r a t i o n s were
a p p r o p r i a t e l y d i l u t e d and i n j e c t e d i n t o i r r a d i a t e d female B6CF1 mice (1100 R 24 h r s e a r l i e r ) .
Spleen c o l o n i e s were counted 8 days post-inject i o n . For t h e e n t i r e femur p r e p a r a t i o n , with
young mice (110 days o l d ) t h e CFU-S s u r v i v a l was
b e t t e r than 90%, compared t o a s u r v i v a l of 66%
i n the aged mice (518 days o l d ) and 48% i n t h e
a g e d - i r r a d i a t e d mice (518 days o l d , 408 days
a f t e r a 300-rad neutron exposure). In o t h e r
animals an a n a l y s i s of t h e a s p i r a t e d CFU-S popul a t i o n showed s i m i l a r l e v e l s of s u r v i v a l (%50%)
i n a l l t h r e e groups; however, i n t h e r e s i d u a l
CFU-S compartment t h e s u r v i v a l was reduced t o
72% in t h e aged group and was s t i l l lower in
t h e a g e d - i r r a d i a t e d group (31%). These observ a t i o n s suggest that t h e p r o p o r t i o n of CFU-S i n
S phase is s l i g h t l y g r e a t e r i n aged t h a n i n
young mice, and s i g n i f i c a n t l y higher i n agedi r r a d i a t e d mice. Furthermore, t h e c e l l s most
a f f e c t e d by t h i s treatment and thus a c t i v e l y
p r o l i f e r a t i n g , e s p e c i a l l y in t h e aged-irradia t e d group, appear t o be c l o s e l y a s s o c i a t e d
w i t h t h e e n d o s t e a l s u r f a c e . (Work supported by
t h e U. S. Energy Research and Development Administration.)
mM) was i n j e c t e d i . p .
CULLAN, G.E.
and G.L. TODD, Department of Anatomy and Cardiovascular Center, U n i v e r s i t y of
Nebraska Medical Center, Omaha, NE. Scanning
and t r a n s m i s s i o n e l e c t r o n microscopy o f lympha t i c endothelial lining.
Scanning e l e c t r o n microscopy (SEM) has been
used e x t e n s i v e l y t o study t h e e n d o t h e l i a l l i n i n g s of much of t h e c a r d i o v a s c u l a r system, b u t
t h e v a s t majority o f t h e s t u d i e s have been
l i m i t e d t o t h e blood v a s c u l a r system. This
study w a s undertaken t o examine t h e c e r v i c a l
and c a r d i a c lymphatic channels and t o compare
t h e i r s u r f a c e f e a t u r e s w i t h SEM and transmiss i o n e l e c t r o n microscopy (TEM). Adult mongrel
dogs were a n e s t h e t i z e d with p e n t o b a r b i t a l and
t h e lymphatic c o l l e c t i n g v e s s e l s v i s u a l i z e d
with Evans blue dye. Dye was introduced i n t o
t h e nose f o r t h e c e r v i c a l v e s s e l s and i n j e c t e d
i n t o t h e e p i c a r d i a l connective t i s s u e l a y e r s
f o r t h e c a r d i a c v e s s e l s . The vessels were
f i x e d i n 2 . 5 % glutaraldehyde s o l u t i o n , washed
with phosphate b u f f e r , p o s t f i x e d i n 1%osmium
t e t r o x i d e , washed and dehydrated. The t i s s u e
was then c r i t i c a l p o i n t d r i e d (Cog) and sputtercoated with gold f o r SEM o r embedded i n a r a l d i t e f o r TEM. T i s s u e examination revealed a
r e g u l a r p a t t e r n of s t r e t c h e d rhomboid t o d i a mond shaped c e l l s , w i t h t h e long a x i s i n t h e
general d i r e c t i o n of lymph flow along with o v a l
shaped n u c l e i p r o t r u d i n g i n t o t h e lumen in
g e n e r a l l y t h e same d i r e c t i o n , although some
angular v a r i a b i l i t y was noted. In a d d i t i o n t o
n u c l e a r p r o t r u s i o n s , t h e r e were s e v e r a l o t h e r
c a t e g o r i e s of i r r e g u l a r i t i e s of t h e e n d o t h e l i a l
l i n i n g . Course r i d g e s i n v u l v i n g a number of
cells g e n e r a l l y p a r a l l e l e d t h e d i r e c t i o n of
flaw, while f i n e r irregular f i l a m e n t s and small
cytoplasmic p r o c e s s e s had a more random o r i e n t a t i o n and d i s t r i b u t i o n . With t r a n s m i s s i o n
e l e c t r o n microscopy, t h e e n d o t h e l i a l cells
e x h i b i t e d only cytoplasmic p r o c e s s e s , most of
which were d i s t r i b u t e d a l o n g t h e e n d o t h e l i a l
j u n c t i o n s . C o r r e l a t i o n of SEM and TEM of t h e
o f t e n n e g l e c t e d lymphatic v e s s e l s p r o v i d e s a
more complete p i c t u r e o f t h e o r i e n t a t i o n and
luminal p r o t r u s i o n s of t h e i r e n d o t h e l i a l
lining.
(Supported by Nebraska Heart A s s o c i a t i o n )
CULLAN, G.M. and G.L. TODD, Department of Anatomy and Cardiovascular Center, U n i v e r s i t y of
Nebraska Medical Center, Omaha, NE. Different i a t i o n between myocardial l e s i o n s due t o i s o p r o t e r e n o l and a r t i f a c t s produced by biopsy.
A v a r i e t y of f a c t o r s such as temporary OCc l u s i o n of a coronary a r t e r y and a d m i n i s t r a t i o n
of v a r i o u s catecholamines produce c o n t r a c t i o n
band l e s i o n s i n t h e myocardium. Morphologicall y similar p a t h o l o g i c a l l e s i o n s , termed "zonal
l e s i o n s " , a r e c h a r a c t e r i s t i c of hemorrhagic
shock. This s t u d y w a s undertaken t o determine
whether t h e morphological l e s i o n s were d i r e c t l y
due t o catecholamine i n f u s i o n s o r due t o "instantaneous" hemorrhagic shock produced by
biopsy. Mongrel dogs were a n e s t h e t i z e d and t h e
h e a r t exposed v i a m i d l i n e sternotomy. They
were continuously i n f u s e d w i t h e i t h e r s a l i n e or
2 . 5 pglkglmin o f i s o p r o t e r e n o l (ISP) f o r one
hour. I n t h e c o n t r o l group, without ISP o r
biopsy, t h e h e a r t c o n t r a c t u r e s were stopped
with hypothermia. The biopsy group involved
removal of a t r a n s m u r a l biopsy 2 cm i n diameter
from t h e p o s t e r o l a t e r a l w a l l o f t h e b e a t i n g
h e a r t following t h e ISP i n f u s i o n . The f i n a l
group which r e c e i v e d ISP, b u t without b i o p s y ,
was terminated by s e v e r i n g t h e systemic v e s s e l s
The h e a r t s were r a p i d l y e x c i s e d , f i x e d w i t h 10%
formalin, and t r a n s v e r s e l y s l i c e d . The 2 m
t h i c k s l i c e s were r o u t i n e l y processed f o r h i s t o l o g y and s t a i n e d w i t h H & E, PAS and PTAH.
Results r e v e a l e d t h e p r e s e n c e o f l a r g e numbers
o f c o n t r a c t i o n bands only i n t h e animals rec e i v i n g ISP i n f u s i o n s . Two t y p e s of contract i o n bands were d i s c e r n e d depending on t h e
number of involved sarcomeres. The small cont r a c t i o n bands were more numerous, f r e q u e n t l y
o c c u r r e d a t t h e end of t h e c e l l s and superThere
f i c i a l l y resembled the "zonal lesions".
w e r e v e r y few c o n t r a c t i o n bands of e i t h e r s i z e
i n c o n t r o l s (both w i t h and w i t h o u t biopsy).
This was i n c o n t r a s t t o s e v e r a l hundred formed
with ISP i n f u s i o n s . High numbers of l a r g e
c o n t r a c t i o n bands were a l s o c o n s i s t e n t l y seen
along t h e edge of t h e biopsy. The p r e s e n c e of
c o n t r a c t i o n bands i n s i g n i f i c a n t numbers only
w i t h ISP and u n r e l a t e d t o t h e removal o f a
biopsy s t r o n g l y s u p p o r t s t h e conclusion t h a t
t h e l e s i o n s a r e due s o l e l y t o ISP and a r e not
a r t i f a c t s due t o t h e biopsy procedure.
(Supported by U n i v e r s i t y o f Nebraska Medical
Center, Cardiovascular Center Development
Award and Nebraska Heart Association)
DELLMA", H.-D.,
Department of V e t e r i n a r y
Anatomy, Pharmacology and Physiology, Iowa
S t a t e U n i v e r s i t y , Ames, Iowa. Scanning and
t r a n s m i s s i o n e l e c t r o n microscopy of t h e subf o r n i c a l organ of t h e g r a s s f r o g (Rana pipiens).
The s u b f o r n i c a l organ o f t h e f r o g is a n
ovoid s t r u c t u r e that p r o t r u d e s i n t o t h e lumen
of t h e t h i r d v e n t r i c l e r o s t r a l t o t h e r o s t r a l
c o r n i s s u r e and between t h e i n t e r v e n t r i c u l a r
foramina. Numerous a p i c a l m i c r o v i l l i a r e pres e n t on t h e covering ependyml c e l l 8 as w e l l as
o c c a s i o n a l cytoplasmic p r o t r u s i o n s and many
v a c u o l e s of v a r y i n g s i z e which f r e q u e n t l y prot r u d e as l a r g e b l e b s i n t o t h e v e n t r i c u l a r
lumen. Tmycytes p r o j e c t deep i n t o t h e organ.
Between t h e s e cells d e n d r i t e s of cerebrospimt
fluid-contacting neurons r e a c h t h e v e n t r i c l e
and t e r m i n a t e i n bulbous enlargements. In
a d d i t i o n , flask-shaped encephato-cbomffin
cells c o n t a i n i n g g r a n u l a t e d v e s i c l e s and aggreg a t e s of f i l a m e n t s i n t h e i r cytoplasm, p r o j e c t
i n t o t h e c e r e b r o s p i n a l f l u i d . A network of
c e n t r a l l y l o c a t e d c a p i l l a r i e s i s surrounded by
enlarged dendrites and axons of heterogeneous
morphology, some of which appear t o o r i g i n a t e
w i t h i n t h e s u b f o r n i c a l organ, intermingled
w i t h d e n d r i t e s and axons of normal s t r u c t u r e .
The g l h l c e l l s i n t h i s r e g i o n , e s p e c i a l l y t h e
m i c r o g l i a l cells, o f t e n c o n t a i n l a r g e l i p o f u s cin i n c l u s i o n s , s u g g e s t i v e o f d e g e n e r a t i o n and
subsequent phagocytosis of some of t h e enlarged
d e n d r i t e s and axons. The normally s c a r c e
neurosecretory peptidergic axons become more
e v i d e n t and form t y p i c a l Herring bodies i n
s t a l k - t r a n s e c t e d animals. Neuronal perikarya
some o f which c o n t a i n g r a n u l a t e d vesicles
(80-160 w diameter) and v a c u o l e s of v a r y i n g
s i z e s and amounts, are predominantly l o c a t e d
p e r i p h e r a l t o t h e r e g i o n of enlarged d e n d r i t e s
and axons. SupmependymaZ macrophages a r e
p a r t i c u l a r l y numerous on t h e s u b f o r n i c a l organ.
(Supported, i n p a r t , by N I H g r a n t NB 07492)
DINSMORE,C.E., Department o f Anatomy, Rush
Medical College, Chicago, I l l i n o i s . P r o t e a s e
i n h i b i t o r a c t i v i t y i n t h e r e g e n e r a t i n g and
non-regenerating humerus of t h e a d u l t newt,
Notophthalmus v i r i d e e c e n s .
S e v e r a l r e c e n t i n v e s t i g a t i o n s have demns t r a t e d t h e p r e s e n c e i n c a r t i l a g e of a l o w
molecular weight (less t h a n 13,000) c a t i o n i c
p r o t e i n which is a p r o t e a s e i n h i b i t o r (reviewed by K u e t t n e r , e t a l . , A r t h r i t . Rheumat., i n
p r e s s , 1977). Crude e x t r a c t s of t h i s material
have, i n a d d i t i o n , been shown t o i n h i b i t t h e
-i n v i t r o growth of e n d o t h e l i a l cells more than
they do f i b r o b l a s t s ( E i s e n s t e i n , et al., Am J.
Path. 81: 337-348, 1975).
S i n c e t h e humerus
540
of the! adult newt has a large amount of cartilage relative to bone, an assay for antiprotease activity was run as follows. Huraeri
from several anesthetized newts were removed,
cleaned of adhering muscle fibers and individually embedded in agar in small petri dishes.
When the agar had solidified, 1.0 mm. holes
were punched at intervals around the humerus
and innoculated with trypsin and chymotrypsin
alternately. The cultures were examined and
pictures taken at four and twenty-four hours.
Enzymatic digestion of the agar was distinctly
inhibited on the side nearest the humerus indicating the presence of a diffusible protease
inhibitor. Protease (cathepsin) activity has
been noted in regenerating newt limbs during
early stages (reviewed by Schmidt, 1968). The
anti-protease assay was, therefore, repeated
on limbs amputated through the humerus 10 days
prior to sacrifice. The presence of protease
inhibitor was indicated at both proximal and
regenerating distal ends of the humerus. The
significance of these findings for regenerating and non-regenerating limbs will be
discussed.
FATEMI, S.H. and G. PEREIRA, Department of Anatomy, University of Nebraska Medical Center, 0maha, Nebraska. Selective damage induced in the
gastric parietal cells of the adult mouse by
dietary deficiency.
In previous experiments designed to develop
an animal model for the study of pancreatic regeneration, ultrastructural observations showed
that maintenance of mice on a diet restricted
solely to sugar precipitates a condition that
bears a suggestive similarity to the syndrome
of diabetes mellitus by rapidly producing nearly complete destruction of the acinar and islet
beta cells of the pancreas and the appearance
inthe pancreatic interstitial tissue of various
stromal cells (macrophages, lymphocytes and
plasma cells) associated with the humoralimmune
response (Pereira, h a t . Rec. =:381,
1972).
Since it is well known that pancreatic disease
often coexists with "auto-immune" types of degenerative processes in various organs (including the stomach), the effects of the puresugar
diet on the gastric mucosa were investigated.
Male mice aged 3-4 months and weighing about 36
gm were used in this investigation. Some of
these were fed a stock diet of Purina Chow and
used as controls, while the others were given
only dextrose and water ad libitum for 10 days.
Ultrastructural observations of the gastric mucosa of animals on the pure sugar diet revealed
that, of the four cell types composing the gastric glands, only the parietal cells displayed
signs of degeneration. Nuclear degenerative
changes were evident in all parietal cells examined and consisted of a loss of nucleoli and
a clumping and peripheral margination of the
chromatin. The cytoplasm was highly vacuolated
and accumulated large membrane-bound structures
containing membranous or other debris including
amorphous osmiophilic material. Similar struc-
tures were also found in the gastric gland lumen. Other cytoplasmic alterations included membrane densification, augmentation of the number
of lysosome-like bodies, and heavy deposits of
flocculent and particulate densities in the cytoplasmic and mitochondria1 matrices and within
the microvilli lining the secretory canaliculi.
Thus, caloric intake restricted to sugar alone
rapidly causes selective degeneration of parietal cells in the mouse gastric mucosa; whether
or not this corresponds to an "auto-imune"
type of degenerative process remains to be determined.(Sup orted in part by NIH Grants GM15289 and RR-85391).
FATEMI, S.H. and W.W. STINSON, Department of
Anatomy, University of Nebraska Medical Center,
Omaha, Nebraska. Experimentally induced connective tissue complexes in the rat.
Human wound repair studies by Stinson et&.,
(1974) correlated with previous studies on dogs.
However his studies did not coincide with other
studies conducted on smaller animals (guinea
pigs and rats). The differences in observations
have been noted by other investigators (Hartwell).
Two possible factors are suggested to
account for these differences: (1) the site of
the induction devices which were adjusted for
smaller anbals could create repair differences
or (2) wound repair is species-dependent. It,
therefore, seemed necessary to examine the
effect of overall size of induction devices on
rats and compare these findings with previous
results on humans.
Fibrocollagenous tissue complex were induced
in 15, 300 gm rats using stainless steel wire
mesh cylinders. Each rat received one cylinder
1 cm in diameter by 3cm long and one cylinder
0.5cm in diameter by 3cm long. The two cylinders were implanted in the superficial fascia
of the lower abdominal quadrants. The tissues
were reclaimed at 8. '10 and 12 weeks of growth.
Seven of the 15 rats developed necrotic areas
over the implanted sites of the larger cylinders.
These animals were destroyed. In the remaining
rats, at all growth stages. the large cylinders
developed 2 times more tissue than the small
cylinders. Upon examination at both E.M. and
light microscopic levels, the fibrocollagenous
tissue complexes in both large and small cylinders demonstrated highly ordered lamellated
patterns consisting of alternating zones of
fibroblasts and collagenous fibers comparable to
those found in humans and dogs. Further, the
initial role of fibroblasts in fibrillogenesis
both in apocrine-like and holocrine-like processes were confirmed.
The observed differences between small and
large cylinders in rats, suggest that the actual
size of the induction device significantly
alters the development of the fibrocollagenous
tissue complexes. Further, the normal developmental process indicative of the action of
fibroblasts in fibrillogeneais did compare to
previous works on humans and dogs and did not
indicate a species-dependent phenomenon.
641
Feely, D.E. and S.L. Erlandsen. Department of
Anatomy, University of Minnesota, Minneapolis,
Minnesota. Isolation of the intestinal protozoan Giardia muris: viability and the effect
of diet.
A simple method of isolation of the intestinal protozoan Giardia muris from rat small
intestine has been developed in our laboratory.
Mucosal scrapings were made from isolated small
intestine and suspended in Hanks balanced salt
solution. Trophozoites of Giardia muris were
concentrated by centrifugation and G
o
w
d
e to
attach to the surface of a warmed petri dish.
Trophozoites were purified from intestinal
debris by detaching them at 4OC, followed by
reattachment to a clean surface at 37°C.
Viability of isolated trophozoites was tested
at 10, 30, 60, 90, 180, and 210 min. by trypan
blue exclusion; more than 80% were viable
through 180 minutes, but viability decreased
to 4oX after 210 minutes. The influence of
rat chow, beef, and low protein diets on the
yield of trophozoites was also investigated.
Rats fed commercial rat chow yielded 4.7 x
103 trophozoites, rats f e a beef diet for
3-5 days yielded 178 x 10 trophozoites/animal,
and those fed a low protein diet for 2 months
yielded 73.1 x 103 trophozoites/animal. This
technique for isolation of Giardia muris
provides an excellent model system to conduct
acute in vitro studies on this protozoan and
also has greatly facilitated their examination
by various light and electron microscopic
techniques. (Supported in part by USPHS
Grant No. AM18344).
1
GARBER, S.L., and O'MORCHOE. P.J., Departments
of Anatand Pathology, Loyola U n i v e r s i t y
S t r i t c h School of Medicine, Maywood, I l l i n o i s .
A Morphometric and Q u a n t i t a t i v e Study of t h e
Lymphocytes i n Canine P e r i p h e r a l Blood and
Renal H i l a r Lymph.
The c e l l s of p e r i p h e r a l (prenodal) r e n a l
lymph, obtained by cannulation of r e n a l h i l a r
lymphatic v e s s e l s i n a n e s t h e t i z e d dogs were
compared with t h o s e of p e r i p h e r a l blood i n t h e
same animal. The white c e l l s were counted and
t h e n suspended i n c u l t u r e medium and incubated
w i t h l a t e x p a r t i c l e s t o d i s t i n g u i s h t h e phagoc y t i c elements. Monolayer c e l l p r e p a r a t i o n s
were obtained by c y t o c e n t r i f u g a t i o n and were
s t a i n e d with Mac Neal's tetrachrome. Measurements were made of t h e non-phagocytic c e l l s
using a micrometer eyepiece. D i f f e r e n t i a l c e l l
counts were performed on both whole blood
smears and t h e c y t o c e n t r i f u g e p r e p a r a t i o n s .
Three major d i f f e r e n c e s were observed between t h e c e l l s i n t h e p e r i p h e r a l blood and
h i l a r lymph samples. F i r s t l y , t h e t o t a l white
c e l l counts i n t h e p e r i p h e r a blood ranged from
4 . 7 ~ 1 0t o~ 2.54~104 cells/mm', whereas i n h i l a r
lymph t h e c e l l counts v a r i e d between 20 and 375
cells/mm3. There was no c o r r e l a t i o n between
t h e t o t a l white c e l l counts i n t h e blood and
lymph. Secondly, t h e r e w a s a s t a t i s t i c a l l y
s i g n i f i c a n t d i f f e r e n c e between t h e mean ciae t e r s of t h e lymphocytes i n t h e two samples.
In t h e blood t h e mean diameter was 1 0 . 3 ~ 1
whereas i n t h e h i l a r lymph it was 1 1 . 5 ~ ~ The
.
l a r g e s t non-phagocytic c e l l s , which measured
over l5um i n d i a e t e r , w e r e c h a r a c t e r i s t i c a l l y
seen o n l y i n t h e lymph specimens. Thirdly,
t h e r e was no c o r r e l a t i o n between t h e different i a l c e l l counts i n t h e blood smears and h i l a r
lymph p r e p a r a t i o n s . In t h e lymph 50-75% of t h e
white c e l l population was lymphocytes, i n contrast t o p e r i p h e r a l blood which contained 15-30%
lymphocytes. Monocytes, as i d e n t i f i e d by l a t e x
p a r t i c l e i n g e s t i o n composed only a minor p a r t
o f t h e c e l l population i n both t h e lymph and
blood samples.
GARDNER, P. J. and C . A. HON, Department of
Anatomy, U n i v e r s i t y of Nebraska Medical Center,
Omaha, Nebraska. L o c a l i z a t i o n of hormones i n
t h e male reproductive system by t h e unlabeled
antibody peroxidase-antiperoxidase complex
technique: S t e r o i d hormones.
For some y e a r s a f t e r t h e e a r l y d e s c r i p t i o n
by Regaud (1901) of r e s i d u a l bodies i n t h e t e s tis, t h e s e s t r u c t u r e s received r e l a t i v e l y
l i t t l e a t t e n t i o n . Recently, Lacy (1967) proposed t h a t r e s i d u a l bodies a r e u t i l i z e d by
S e r t o l i c e l l s i n t h e s y n t h e s i s of one o r more
s t e r o i d s and t h a t t h e s e substances then a c t a s
l o c a l hormones. Evidence from a preliminary
i n v e s t i g a t i o n in t h i s l a b o r a t o r y u t i l i z i n g t h e
unlabeled antibody peroxidase-antiperoxidase
(PAP) complex technique suggests t h a t r e s i d u a l
bodies in t h e cytoplasm of S e r t o l i c e l l s may,
indeed, contain a s t e r o i d , p o s s i b l y t e s t o s t e r one. F i x a t i o n s t u d i e s i n d i c a t e d t h a t t h e maximum s t a i n i n g i n t e n s i t y of a n t i - s t e r o i d binding
sites was obtained i n t e s t i c u l a r t i s s u e perfused with 3% glutaraldehyde in 0.1 M Sorens e n ' s phosphate b u f f e r followed by postOs04, a l s o i n phosphate b u f f e r .
f i x a t i o n in 1%
T i s s u e blocks w e r e dehydrated i n a graded
series of a l c o h o l s and embedded i n a r a l d i t e
6005 by r o u t i n e methods. A f t e r etching and
blocking s t e p s , u l t r a - t h i n s e c t i o n s , mounted
on n i c k e l g r i d s , were incubated i n a n t i t e s t o s t e r o n e , then in goat a n t i - r a b b i t IgG, and PAP.
They were t h e n t r e a t e d w i t h diaminobenzidineH 2 0 2 , and f i n a l l y w i t h 0 ~ 0 4 . The i n t e n s e
s t a i n i n g t h a t occurs over l i p i d i n c l u s i o n s i n
t h e cytoplasm of S e r t o l i c e l l s , p a r t i c u l a r l y
a t t h e edges of t h e adluminal r e s i d u a l bodies
and, t o a lesser e x t e n t , over t h e s u r f a c e of
l a r g e r l i p i d bodies near t h e base of t h e epithelium, i n d i c a t e s t h a t t h e s e s t r u c t u r e s bind
a n t i t e s t o s t e r o n e , and suggests t h a t t h e c y c l i c
changes i n t h e l i p i d content of S e r t o l i c e l l s
a s proposed by Lacy (1967) deserve continued
i n v e s t i g a t i o n . However, t h e p o s s i b i l i t y t h a t
binding sites f o r a n t i b o d i e s t o c l o s e l y rel a t e d s t e r o i d s a r e being s t a i n e d must be cons i d e r e d . S t u d i e s of t h e s p e c i f i c i t y of t h i s
r e a c t i o n a r e i n p r o g r e s s . (Supported i n p a r t
by a UNMC Seed Grant and NSF RIAS-SER 7706922).
542
GOULD, R.N., N.F. METCALF and W.K. METCALF, Department of Anatomy, University of Nebraska
Medical. Center, Omaha, Nebraska. Density profiles of lymphocytes during the immune response
of pregnancy and malignant disease.
The cytoplasmic refractive index of the
cytoplasm of circulating lymphocytes has been
shown to increase during immunological activity.
This parameter is presumably a measure of the
lymphocyte protein concentration. If this is
true, changes in LCRI should be mirrored by
changes in lymphocyte specific gravity. As
only a proportion, albeit a significant proportion, of the circulating lymphocytes seem to be
involved in the change in LCRI, determination
of mean lymphocyte specific gravity would be
unlikely to demonstrate a correlation. Accordingly, a discontinuous ficoll-hypaque density
gradient has been used to determine the density
profile of circulating blood lymphocytes. Both
LCRI and density profiles have been determined
in lymphocytes obtained from pregnant women and
from patients with malignant disease. The density profiles and LCRI showed the expected good
correlation in both conditions. In the last
trimester of pregnancy, there was both a significant increase in the number of lymphocytes
with a high LCRI and a similar increase in the
number of heavy dense cells. A similar phenomenon was observed in patients with malignant
tumors. Our data, therefore, although admittedly preliminary, would support the hypothesis
that both LCRI and density profile are measuring the same phenomenon, probably changes in
the lymphocyte cytoplasmic protein concentration. (Supported by a UNMC seed grant and NIH
grant T32 HD07097).
HARPER, C.M., T.W. BAUER and J.G. SHARP, Department of Anatomy, University of Nebraska Medical
Center, Omaha, Nebraska. Evaluation of the role
of cultured thymic epithelium in the in vitro
induction of PHA-responsiveness of splenocytes
from neonatally thymectomized rats.
The thymus provides a unique environment for
the proliferation, differentiation and immunological maturation of T-lymphocytes. These processes involve an interaction of T-cell precursors
with thymic reticuloepithelial (TRE) cells. TRE
cells have been grown in culture using a modification of the methods of Wekerle
(Eur. J.
Immunol. 3:745, 1973) and Waksalete. (Ann. N.
Y. Acad. Sci., %:492,
1975). Adherent TRE-cells
were cultured at 37OC in 25cm2 Corning T-flasks
in MEM-Hanks medium and 20% fetal calf serum.
Suspensions of 2x106 spleen cells from neonatally
thymectomized rats were co-cultured in vitro
with confluent monolayers of TRE-cells or with
2 . 5 ~ .7 . 5 ~or 25x concentrated supernatant from
confluent TRE-cell cultures. Spleen cells cocultured with fibroblasts were used as controls.
Spleen cells were harvested from the monolayers
and cultured for 4 days with either PHA, Con-A,
LPS, or no mitogen. 1pCi of H3-thymidine was
added to each culture on the third day of incubation. The cultures were harvested and the H3thymidine uptake into the acid insoluble cell
fraction determined as a measure of the proli-
cs.
ferative response to the mitogen. Spleen cells
which had been co-cultured with fibroblasts
showed no significant response to mitogens. In
contrast, spleen cells which were co-cultured
with TRE-cells or with low concentrations of
supernatant showed a small but significantly increased proliferative response to PHA when compared to the control. The responsestocon-A and
LPS were only minimally increased. Splenocyte
DNA synthesis was inhibited by high concentrations of supernatant. Thus, co-culture of spleen
cells from neonatally thymectomized rats with
TRE cells or their supernatant induced an increased response to a T-cell specific mitogen
(PHA). In addition, there was some evidence for
a spontaneous increase in thymidine uptake with
time in spleen cells co-cultured with the monolayer in the absence of mitogen, suggesting
spontaneous blastogenesis of lymphocytes in contact with the monolayer. These results suggest
that the interaction of T-cell precursors with
thymic epithelial cells may be investigated
vitro employing this monolayer culture system.
(Supported by NIH Grants CA-18548, 1T32 HD07097).
HOLYOKE, E.A., Department of Anatomy, University
of Nebraska Medical Center. Omaha. Nebraska.
The University of Nebraska College of Medicine.
The University of Nebraska College of Medicine grew from two roots, both established on
paper in 1869. The original charter of the
University of Nebraska provided for a medical
school which, however, did not open until 1883
and then only survived four years - until 1887.
The second root was the Omaha Medical College,
which was also organized on paper in 1869. The
Omaha Medical College opened in 1880 and operated continuously until the founding of our
present school. The present University of
Nebraska College of Medicine was established
in 1902. It was promptly joined by the Omaha
Medical College which took over the teaching
of the final two or clinical years. The basic
science years were taught in Lincoln on the
University campus. Following a recommendation
in the Flexner Report it was decided to establish entire college on one campus and the location finally selected was in Omaha. Land was
purchased and in 1913 the college moved to its
present site in one building - the present
Poynter Hall. Other buildings the University
Hospital and South Laboratory Building were
added in 1917 and 1918 respectively. The college continued in essentially this physical
plant with a few additions through two world
wars and f o r a few years beyond. Then the
pressure to establish a medical center updated
according to present standards, from which the
school had slipped somewhat in the immediate
post war years, led to a spectacular building
program dating from the late 1950's and the
present plant of the Medical Center. The University of Nebraska has always been proud of
its Medical School and except for a few years
following World War 11, has succeeded in maintaining a high standard of medical education.
From the date of its founding a minimum of two
years of college education was always required,
543
-
a distinction that a number of our sister
schools can not claim. We hope the future
trends of medical education may find this institution in the fore-front among state medical
colleges as it was in the early half of the
century
.
HULTMAN, B.A. and P.J. GARDNER. Department of
Anatomy, University of Nebraska Medical Center,
Omaha, Nebraska. Localization of hormones in
the male reproductive system by the unlabeled
antibody peroxidase-antiperoxidase complex
technique: Prolactin.
It is generally believed that prolactin promotes the growthandmaintenance of the prostate
gland by acting in synergy with testosterone.
Prolactin may sensitize the prostatetoandrogen
by providing a stirnulatory effect upon testosterone uptake. Biochemical studies have demonstrated specific binding of prolactin to membrane preparations of the prostate gland and
also that the binding is androgen dependent.
However, these biochemical studies have not determined the site of action of prolactin in the
prostatic cell. This investigation was designed
to identify the site of prolactin binding in
the prostatic cell at the light microscopic
level. A modification of the unlabeled antibody, peroxidase-antiperoxidase (PAP) complex
technique was used in the study. Ventral prostates from adult male ratswerefixedinBouin's
solution and embedded in paraffin. Paraffin
sections, approximately 5 microns in thickness,
were first dewaxed in xylene and brought to
alcohol. They were then treated with a solution of H202 in methanol to block endogenous
peroxidaae activity and normal goat serum to
block non-specific background staining. The
sections were then sequentially incubated in
the primary antiserum, anti-prolactin (NIAMDD
5-4). goat anti-rabbit IgG, PAP, diaminobenzidine-Hz02 and Os04.
dilutions of the primary antiserum were used to
determine the sensitivity of the staining reaction. In other control studies, phosphatebuffered saline was substituted for theprimary
antiserum. The specificity of the reaction was
confirmed by the staining of cell types previously described as prolactin cells in the
pituitaries of male and lactating female rats.
Preliminary results of this investigation suggest that binding of anti-prolactin occurs in
the Golgi zone of rat prostatic eplthelialcells.
Electron microscopic studies to localize prolactin at the ultrastructural level are in progress. (Supported in part by NIH Grant No. HD
09881 and NSF RIAS-SEX-77-06922).
HUTSON, J.C. and P.J. GARDNER, Departments of
Anatomy, Texas Tech University School of Medicine, Lubbock, Texas, and the University of
Nebraska Medical. Center, Omaha, Nebraska.
Localization of an FSH-like molecule using
immunocytochemistry: Are immunologicalcontrols
enough?
An FSH-like molecule was localized in testes of normal adult rats and in testes of
testosterone-treated hypophysectomized rats by
use of the unlabeled antibody (anti-FSH),
peroxidase-antiperoxidase complex technique.
On ultrathin sections, anti-FSH bound specifically to acrosomes of spermatids, and intranuclear bodies of early spermatids. Quantitation of staining intensity demonstrated that
FSH, when used as an absorbing antigen, significantly reduced this binding. There was less
anti-FSH binding to the acrosomes of spermatozoa in the body and tail of the epididymis as
compared to the less mature germ cells located
in the testis and head of the epididymis. The
acrosomal and nuclear staining of spermatids
of hypophysectomized animals was similar tG
staining observed in sham-injected animals.
This investigation emphasizes the importance
of conducting physiological experiments t o
determine if imnocytochemical staining is of
true biological significance.
logical significance cannot be demonstrated by
altering the concentration of the specific
tissue antigen (e.g., reducing FSH by hypophysectomy), then it is possible that the localized molecule possesses immunoreactive sites
similar to those of the molecule used as the
absorbing antigen.
HD 09881).
H A N G , Y.C.,
and D.J. EDWARDS, Department of
Anatomy, School of Veterinary Medicine, Purdue
University, West Lafayette, Indiana. Changes
in the canine leukocyte differential counts
following treatments by electro-acupuncture.
To develop a model for studying mechanisms
of acupuncture and to test thepossibility of
treating animals' blood problems by acupuncture,
a pilot study on the effect of electro-acupunctwe (EA) on the leukocyte differential
counts was undertaken. Four adult female dogs
were used in the study. The experiment was
designed as a "before" and "after" study and a
paired-data two-tailed t-test (p< O.2)was
performed. m m each dog two blood samples
were collected just before and 30 minutes after
each treatment. The blood smears were then
made for the blindly conducted differential
counts. The experiment included three consecutive days of EA treatments followed by
three sham EA treatments. In cases of EA,
needles were placed in selected acupuncture
points; while in the sham treatments they were
put in non-specific loci.
the animal was stimulated by an electric current
of 3 mA for 10 minutes through needled points.
Comparing each "before" and "after" blood counts
(short-term effect) showed that average
neutrophil counts increased 6.6%, 7.5%, and 8.5
% and average lymphocyte counts decreased 5 . 5 % ,
8.9%, and 9.3% after the three EA treatments.
The sham treatments, however, showed only 3.8%,
0.48, and 0.4% increase of neutrophils, and
4.8%, 0.1%, and 2.0% decrease of lymphocytes.
Contrary to the short-term effect, the "long"term effect (comparing %efore%blood samples
of the first and the third treatmnts) showed
that in the EA treatment neutrophils decreased
11% and lymphocytes increased 12.5% in average,
544
however, in the sham treatment changes in
leukocyte differential counts were statistically insignificant. Results of this study
indicate adequate electric stimulation of
properly selected acupuncture points can
significantly effect the dog's peripheral white
blood cell picture.
(Supported in part by Purdue Agt.icultum
Experiment Station Project 72001-36-11725)
JELINEK, E.H. and G. PEREIRA, Department of
Anatomy, University of Nebraska Medical Center,
Omaha, Nebraska. Verification of the existence
of a blood-splenic white pulp barrier.
Recent studies of the spleen in experimental
argyria have demonstrated that there is a barrier to the penetration of circulating silver
particles into the parenchyma of the white pulp
and that this barrier is effective in theregion
of the reticular cell layer demarcating the
white pulp from the extravasated blood of the
surrounding marginal zone and investing its
capillaries (Pereira, 35th Proc. Electr. Micr.
SOC. A m . , Boston, Mass., pp. 504-505, 1977).
The present study was undertaken to verify the
existence of such a blood-splenic white pulp
barrier using Thorotrast, an electron-opaque
colloidal tracer consisting of thorium dioxide
stabilized in suspension by dextrins. Young
adult male rats, aged 10-12 wk, were slowly injected via the inferior vena cava under anesthesia with 1 ml of Thorotrast 15 min and 6 hr
before splenectomy. Electron microscopic observations revealed the dense particles of
thorium dioxide to be present in high concentration in the elastic membranes of the splenic
capsule and trabeculae and in various extraand intra-cellular locations throughout the red
pulp and marginal zone, but not in the parenchyma of the white pulp. Indeed, no tracer was
detected in the reticular cells and fibers,
fixed macrophages or intercellular clefts of
the white pulp parenchyma. Thorium dioxide was
limited, on the one hand, to the reticular fiber that delineates the white pulp and in
which the tracer was present in low concentration, and, on the other, to the lumen of the
white pulp capillaries which were observed to
have, like those of the thymic cortex, impermeable endothelial junctions. An interesting
observation was that the boundary layer between
white pulp and marginal zone was often seen to
be traversed by free macrophages displaying a
multitude of cytoplasmic vacuoles and surface
invaginations filled with tracer; whether or
not this represents the transport mechanism of
antigens into the white pulp remains to be determined. Therefore, a blood-splenic white
pulp barrier to certain circulating macromolecules does exist. (Supported in part by NIH
Grants GM-15289 and RR-05391).
JORDAN, R.K.. Department of Anatomy, University
of Newcastle upon Tyne, Newcastle upon Tyne,
Great Britain. In vitro modelsofthymic microenvironment.
While there is now accumulating evidence in
favor of a T-committed pre-thymic stem cell a
sojourn within the thymic microenvironment still
appears to be a prerequisite for its further
proliferation and differentiation. A simple
monolayer culture technique has been used to
obtain thymic stromal cells free of lymphoid
elements as a first step to investigating the
nature of their interaction with immigrant stem
cells. The monolayer cultures are initiated
by explanting fragments of embryonic or newborn
mouse thymus into plastic flasks in RPMI 1640
medium supplemented With 10% FCS. The explants
produce outgrowths which growtoconfluent mnolayers within 4 weeks. The cells of the confluent monolayer are heterogeneous in morphology.
Two major types can be clearly distinguished.
The first type is a cell, rounded in profile
with a central oval nucleus. The other type is
elongated with cytoplasmic processes giving it
a dendritic appearance. Ultrastructural examination of these monolayer cells show them to
contain numerous osmiophilic inclusion vesicles.
In order to test the functional capabilities of
the cultured stromal cells with respect to
their permitting the functional maturation of
immigrant stem cells, reaggregates of cultured
cells have been transplanted back to an in vivo
environment. On grafting beneath the kidney
capsule of intact mice such reaggregates become
lymphoid as assessed by optical and electron
microscopy. These results lead to the hope
that models involving thymic stromal cells grown
in this manner may prove valuable in investigating the intrathymic events of T-precursor
maturation.
LAHEY, R.D. and G. PEREIRA, Department of Anatomy, University of Nebraska Medical Center,
Omaha, Nebraska. Re-evaluation of the concept
of a blood-thymus barrier.
Previous studies have demonstrated that the
thymic lobule is divisible into (1) an epithelial compartment rich in epithelial reticular
cells and comprising the cortex and the outer
zone of the medulla and (2) a connective tissue
compartment consisting of the inner medulla and
the prolongation of the latter in the form of
perivascular spaces (Pereira and Clermont, Anat.
Rec., =:613-625,
1971). Whereas the epithelial compartment is supplied exclusively by
capillaries, the connective tissue compartment
contains arterioles, capillaries, post-capillary
venules and lymphatics. Since numerous contradictory reports concerning the effectiveness of
the blood-thymus barrier in postnatal life have
appeared in the literature, the present study
was undertaken tc assess the penetration of
Thorotrast, an electron-opaque tracer consisting
of thorium dioxide stabilized in suspension by
dextrins, into the two compartments of the thymic lobule. Young adult male rats, aged 10-12
wk, were slowly injected via the inferior vena
cava under anesthesia with 1 ml of Thorotrast
15 min and 6 hr before thymectomy. In the cortex, ultrastructural observations showed the
dense particles of thorium dioxide to be limited to the luminal plasma of the capillaries and
the digestive vacuoles of an occasional peri-
646
vascular macrophage. In the outer medulla, however, thorium dioxide was present both in the
capillary lumen and in variable numbers of endothelial vesicles and multivesicular bodies;
also, some tracer was detected in isolated vesicular invaginations of the investing epithelial reticular cells but not in the intercellular clefts of the outer medullary parenchyma.
In the inner medulla, which forms the bulk of
the connective tissue compartment., large quantities of thorium dioxide were present in the
endothelium and adventitia of post-capillary
venules as well as in the reticular fibers and
intercellular clefts of the surrounding parenchyma. These preliminary observations indicated that the blood-thymus barrier is effective
in preventing some cjrculating macromolecul.es
from reaching the parenchyma of the epithelial
compartment of the thymic lobule, i.e., the
cortex and outer medulla, where thymus-derived
(T) lymphocytes are produced. (Supported in
part by NIH Grants GM-15289 and RR-05391).
LAMAR, C.H., Y.C. HWANG, and B.G. MILLER,
Department of Anatomy, Purdue University, West
Lafayette, Indiana. Growth and characterization of canine skin explant cultures.
Recent reports of the diagnostic potentials
of human skin fibroblastic cultures have
stimulated the evaluation of canine skin
explant cultures. Explants. 1-3 mm square
biopsies, were aseptically obtained from the
skin covering the legs and the lumbar areas of
adult female dogs. Cultures were established
in 35 mm plastic petri dishes using glass
coverslips to submerge the explants. Two
commerciallyprepared media, MEM and Dulbecco's
MEM, supplemented with fetal calf serum and
antiobiotics were utilized. The cultures were
examined using phase and light microscopic
techniques and with the aid of scanning electron microscopy (SEM). Cells migrated from
the epidermis and dermis at approximately 3
and 3.5 days, respectively. In 68% of the
cultures established, epidermal-derived cells
were observed concurrently, or prior to the
appearance of fibroblasts. In only 4% of the
cultures, epidermal cells were not observed.
Epidermal cellular outgrowth morphologically
consisted of 2 types. Cuboidal and stellate
cellular configurations were observed with
phase optics. Cytoplasmic vacuoles, observed
in the cuboidal cells, increased with time.
Multinucleated cells were observed to increase
with culture age. The cytoplasmic granules
of the young cuboidal cells reacted positively
for acid phosphatase. Later, cytoplasmic
vacuoles developed from the granules which
eventually coalesced. As the cuboidal cells
grew larger, cytoplasmic filamentous structures were observed which were suggestive of
tonofibrils, using light microscopic techniques.
Cells derived from the dermis were fusiform in
shape. These fibroblastic cells did not
contain prominent cytoplasmic granules. SEM
observations support the phase optic findings
and revealed microvilli on the surface of the
cells.
LAMPERTI, A . and BALDWIN, D.M., Departments of
Anatomy and Physiology, University of Cincinnati
College of Medicine, Cincinnati, Ohio.
The effects of estradiol benzoate on gonadotropin secretion in hamsters with a lesion of the
arcuate nucleus of the hypothalamus.
The arcuate nucleus of the hypothalamus has
been suggested a s a site for the negative
feedback action of estrogen on gonadotropin
secretion. This nucleus is selectively destroyed after administration of monosodium glutamate
(MSG) to neonates. The purpose of this investigation was to study the negative feedback
actions of estradiol benzoate (EB; Expt. 1) and
to study EB-induced surges of gonadotropins
(Expt. 2) in hamsters with a lesion in the
arcuate nucleus.
Hamsters were injected with saline or
8 mg MSG/gm b.w. on day 8 of the neonatal period. They were ovariectomized on day 60. In
Expt. 1, groups of 5-8 animals received 0.1 ml
of sesame oil or 5 kg of EB/O.I ml oil s . 4
day17 days. They were sacrificed on the day
following the last injection and the levels of
LH & FSH were measured by radioimmunoassay
(RIA) i n the plasma and anterior pituitary
glands. The levels of LH in the plasma and pituitary glands of EB-treated control & experimental animals were lower than those values in
oil-treated animals. EB lowered the postcastration rise of PSH in control animals but had no
effect on FSH levels in MSG-treated animals.
These results suggest that the arcuate nucleus
is not the site for the negative feedback action
of EB on LH secretion but that it is a site for
estrogen modulation of FSH secretion. In Expt.
2 , groups of 5-16 animals were treated with a
single S.C. injection of 50 @g EB at least 3
weeks after ovariectomy. Blood was drawn from
the external jugular vein at 1000 h on the third
day after EB treatment. At 1600 h blood was
drawn from the inferior vena cava and the levels
of LH measured by R I A . It was found that the
levels of LH in the morning sample were quite
low ( < 0.6 ng/ml) in both control and experimental animals. The levels of LH in the afternoon
sample were approximately 6 0 ng/ml in both
groups of animals. The results of this experiment suggest that EB can induce a surge of LH
in animals which have a lesion of the arcuate
nucleus.
(Supported by Biomedical Research & Support
Grant # 57.)
LEUSCHEN, M.P., Departments of Anatomy, PhysioloRv and Biouhvsics. Universitv of Nebraska
Meiical Center; Omaha, NebrasL. Cyclic AMP
level and cell shape in cultures from normal
and SSPE virus infected human brain tissue.
The study showed that a relationship exists
between the level of cyclic AMP and the cell
shape in normal and SSPE virus infected human
brain tissue. Cells were cultured from two
biopsies of normal brain tissue and from a biopsy derived from a patient with subacute sclerosing panencephalitis ( S S P E ) . In each experiment replicate cultures were used as controls.
The intracellular level of cyclic AMP was moni-
546
tored by r a d i o i m n o a s s a y . The system w a s
manipulated by a d d i t i o n of exogenous d i b u t y r y l
c y c l i c AMP (0.5 mM) o r c y c l i c AMP (0.5 mM) and
t h e u s e of t h e phosphodiesterase i n h i b i t o r s :
t h e o p h y l l i n e (1.0 mM), papaverine (0.1 mM) and
96
R020-1724 f o r periods ranging from 24
hours. A l t e r a t i o n s i n c e l l shape were appare n t as e a r l y a s 24 hours i n both t h e normal
c u l t u r e s and i n t h e SSPE v i r u s i n f e c t e d cult u r e s . C e l l d i v i s i o n was s i g n i f i c a n t l y inh i b i t e d i n t r e a t e d c u l t u r e s and l a r g e r i n t e r c e l l u l a r d i s t a n c e s were seen. The t r e a t e d
c e l l s h a d - a n increased number of cytoplasmic
extensions. This a l t e r e d c e l l shape increased t h e i r adhesion t o t h e s u b s t r a t e as
measured by a delayed a b i l i t y t o d i s s o c i a t e t h e
c e l l s w i t h t r y p s i n . The e f f e c t s w e r e reversi b l e a f t e r 24 hours i f t h e chemical treatment
was suspended and t h e c e l l s were allowed a t
l e a s t 24 hours t o recover. Changes i n c e l l
shape have been a s s o c i a t e d w i t h t h e detachment of c e l l s from t h e i r s u b s t r a t e by t r y p s i n i z a t i o n and w i t h t h e sequence of e v e n t s involved i n m i t o s i s . I n both i n s t a n c e s t h e
c y t o s k e l e t a l changes i n c e l l shape appear t o
be r e l a t e d t o t h e s t a t e of t h e microtubules.
I n normal and SSPE v i r u s i n f e c t e d b r a i n c e l l
c u l t u r e s t h e r e l a t i o n s h i p between t h e c e l l
shape, t h e r a t e of c e l l d i v i s i o n , and c e l l
adhesion appears t o be l i n k e d t o t h e l e v e l of
c y c l i c AMP.
-
H . J . KUEHN and W.K. METCALF, Departments of P e d i a t r i c s and Anatomy, U n i v e r s i t y
of Nebraska Medical Center, Omaha, Nebraska.
Congenital coronary a r t e r y f i s t u l a e .
Congenital coronary a r t e r y f i s t u l a , although
r e l a t i v e l y r a r e , provides a n i n t e r e s t i n g challenge to the c l i n i c i a n i n terms of d i a g n o s t i c
c r i t e r i a due to a v a r i e t y of anatomical s t r u c ture. I n t h i s study, seven new c a s e s of coronary a r t e r y f i s t u l a e a r e presented w i t h a
v a r i e t y of t e r m i n a t i o n s , i n c l u d i n g t h e l e f t
atrium, the l e f t and r i g h t v e n t r i c l e and the
pulmonary a r t e r y . These angiographically proven f i s t u l a e produced a wide v a r i e t y of murmurs
leading to a conclusion t h a t the " c l a s s i c " continuous murmur need n o t be thought of a s a prer e q u i s i t e f o r the d i a g n o s i s of a coronary
a r t e r y f i s t u l a . The complexity of c l i n i c a l
p r e s e n t a t i o n of coronary a r t e r y f i s m l a e furt h e r supports the need f o r coronary a r t e r y
angiography i n e s t a b l i s h i n g a d e f i n i t i v e diagnosis.
LINTON, R.L.,
LIPSCOMB, H.L., S. BOHBRINK and J.G. SHARP.
Department of Anatomy, University of Nebraska
Medical Center, Omaha, Nebraska. A model f o r
studying autoimmune-type endocrine changes employing i n f a n t thyrnectomized r a t s .
The thymus i s e s s e n t i a l d u r i n g pre- and neon a t a l periods i n t h e rat f o r processing h e l p e r
(Th) and c y t o x i c k i l l e r lymphocytes (Tk), which
t h e n m i g r a t e from t h e thymus t o populate o t h e r
lymphoid t i s s u e s . Also during t h i s period and
subsequently, perhaps i n t o a d u l t l i f e , t h e thy-
mus processes suppressor lymphocytes (T,) which
perform an immunoregulatory f u n c t i o n . P i e r p a o l i
and Besedovsky (Clin. Exp. I m u n o l . g:303,1975)
propose t h a t t h e thymus i s a l s o involved i n programming of neuroendocrine f u n c t i o n , s i n c e athymic mice and r a t s thymectomized i n t h e p e r i n a t a l
period develop endocrine abnormalities. Nishizuku and Sakakuru (Endocrinol.B:886,1971) showed
that i n some s t r a i n s of m i c e , thymectomy between
2 and 5 days of age, b u t n o t b e f o r e o r a f t e r ,
l e a d s t o ovarian dysgenesis and t h y r o i d i t i s . The
p r e s e n t study was designed t o compare t h e e f f e c t s
of n e o n a t a l (within 1 day of b i r t h , TMX-1) and
i n f a n t ( 5 days a f t e r b i r t h , TMX-5) thymectomyon
t h e growth and morphological c h a r a c t e r i s t i c s of
endocrine organs of male and female Fischer 344
(F344), Holtzman, and Sprague Dawley rats. The
g r e a t e s t changes were observed i n F344 female
r a t s , TMX-5, and autopsied a t 90 days of age,
when o v a r i e s , a d r e n a l s , thyroid and kidneyswere
decreased i n weight. No s i g n i f i c a n t changes i n
body weight were observed f o r any of t h e groups,
and no changes i n endocrine glands w e r e f o u n d i n
male r a t s of any of t h e s t r a i n s . H i s t o l o g i c a l l y ,
t h e t h y r o i d gland of F344 female r a t s appeared
t o be t h e most a f f e c t e d , showing lymphocytic inf i l t r a t i o n and degenerating f o l l i c l e s . Theblood
l e v e l of thyroxine was a l s o s i g n i f i c a n t l y decreased. These r e s u l t s are c o n s i s t e n t with t h e
h y p o t h e s i s t h a t i n f a n t thymectomy (day 5) i n
c o n t r a s t t o n e o n a t a l thymectomy (day 1) r e s u 1 . t ~
i n a s e l e c t i v e removal of immunoregulatory supp r e s s o r (T,) c e l l s . This may l e a d t o a l o s s of
r e g u l a t o r y f u n c t i o n toward a u t o a n t i g e n s , thus
p e r m i t t i n g t h e development of autoimmunedisease.
Since no changes were found by 90 days of a g e i n
male rats, t h i s s t r o n g l y s u g g e s t s t h a t sexr e l a t e d hormones, perhaps most importantly test o s t e r o n e o r i t s m e t a b o l i t e s , play a r o l e i n
modulation of immunoregulatory suppressor (Ts)
c e l l f u n c t i o n . (Supported i n p a r t by N I H g r a n t s
CA18548, lT32-HD07097 andNSF RIAS-SER77-06922).
LOVE, L.A., A.F. PELFRENE, AND H.G. GARCIA,
Eppley Cancer Research I n s t i t u t e , U n i v e r s i t y of
Nebraska Medical Center, Omaha, Nebraska.
Carcinogenicity of d i a z o a c e t i c e s t e r (DAAE)
The o b j e c t i v e of t h i s i n v e s t i g a t i o n was t o
study t h e - c a r c i n o g e n i c i t y and s p e c i e s s p e c i f ic i t y of DAAE, as w e l l a s t h e organotropic e f f e c t s and t h e i r dependence on a d m i n i s t r a t i o n
routes.
MRC-Wistar r a t s , Syrian hamsters and S w i s s
mice were maintained under standard experiment a l c o n d i t i o n s , and except f o r the newborn mice,
were 8 wks o l d a t t h e begining of treatment.
Experimental groups from a l l 3 s p e c i e s w e r e
given s o l u t i o n s of lOmg DAAE/kg BW i n d i s t . H20
continuously per 0s 5 days/wk f o r l i f e . Tap
H 0 was given on weekends and holidays. In t h e
m8use percutaneous experiment, DAAE was applied
t o i n t r a s c a p u l a r area. Groups 162 received
108mg DAAE once, followed 1 wk later by acetone
o r 0.1% Croton o i l (CO) i n acetone. 1 drop
twice weekly. Group 3 received 1 drop DAAE
twice weekly and Group 4, c o n t r o l s . Survivors
were k i l l e d a t t h e 50th experimental wk. I n
.
547
the mouse newborn experiment, 0.05ml of a 0.45%
aqueous fresh or light decomposed aAAE solution
was injected sc in the dorsal cervical region.
Orally ingested DAAE increased the total
tumor incidence a minimum of 2-fold in all 3
species tested, as compared to controls. DAAE
animals mainly showed epithelial digestive
tract tumors; mice also had an increased lung
adenoma incidence. Controls mainly showed endocrine or connective tissue tunors. DAAE applied
once on mouse skin followed by CO or acetone increased the number of lung adenomas, but no skin
tumors developed. Repeated DAAE applications
produced a 4-fold increase in the number of
lung adenomas; 172 of the animals also had skin
tumors. Both fresh and light-decomposed D M E
in newborn mice increased the tumor incidence.
This was more pronounced in the former group
which showed a predisposition towards lung tumors, which was not seen in the latter group.
From these results, we can conclude that
DAAJZ is potent carcinogen for all 3 species
tested. DAAE is mainly locally effective as
most tumors were of the digestive tract or skin,
although a systemic effect does occur as shown
by the increased number of lung adenomas in mice
following both oral and percutaneous administration.
(Supported by PHS contract 43-68-959 to
Eppley Institute and NIH Grant 1 T32 €DO7097 to
the Anatomy Department).
LUCKETT, W.P., Department of Anatomy, Creighton
University, Omaha, Nebraska, and Hubrecht
Laboratory, Utrecht, the Netherlands. Early
yolk sac development in the dwarf galago,
Galagoides demidovii: A model for early human
development.
The blastocyst of the dwarf galago undergoes
a type of 'interstitial' implantation and
subsequently develops an extensive meshwork of
extraembryonic tissue which fills the blastocyst cavity. These relationships are a striking example of convergent evolution to the condition which exists in early human embryos;
thus the dwarf galago provides a useful model
for studying the possible causal relationships
which have contributed to the specialized
pattern of early human development.
The earliest available blastocyst measures
0.35 x 0.29m and is implanted within an implantation chamber on the lateral wall of one
uterine horn. This condition may be designated
as 'pseudointerstitial', since the narrow opening of the implantation chamber is continuous
with the main uterine lumen. The blastocyst is
partially filled with an extensive epithelial
meshwork which is continuous with the lateral
margins of the embryonic hypoblast. This tissue exhibits numerous mitotic figures and is
considered to be a modification of the extraerabryonic hypoblast. A small, discrete portion
of the yolk sac cavity underlies the embryonic
hypoblast, and it is continuous in places with
the interstices of the hypoblastic meshwork.
In an expanded, 1.5m blastocyst, the yolk
sac cavity has enlarged, and the 'reticulated'
hypoblast is reduced and incorporated into the
endodermal lining of the yolk sac. There is
continued reduction of the reticulated hypoblast in a 2.6m blastocyst, and in many places
the yolk sac endoderm now forms a simple squamous epithelium. This attenuation and expansion
is complete in a 7 . h blastocyst.
The differentiation and subsequent disruption of the hypoblastic meshwork are strikingly
similar to the conditions in previllous human
embryos, and it is suggested that this developmental convergence is the result of the transitory retardation of blastocyst expansion during interstitial implantation in both species.
Absence of a hypoblastic meshwork in related
genera which exhibit superficial implantation
provides corroboration for this hypothesis.
MAGILTON, J.H. and C.S. SWIFT, Biomedical Engin e e r i n g Program, Iowa S t a t e U n i v e r s i t y , Ames,
Iowa. -Evidence o f temperature d i f f e r e n t i a l s
between s u r f a c e c a r r y i n g a r t e r i e s and metaboli c f i e l d s i n the brain.
By changing t h e temperature o f t h e blood i n
t h e s u r f a c e c a r r y i n g vessels o f t h e b r a i n , i t
was demonstrated t h a t temperature d i f f e r e n t i a l s e x i s t between these vessels and s i t e s
w i t h i n b r a i n parenchyma. Evidence i n d i c a t e s
t h a t 1 ) these temperature d i f f e r e n t i a l s a r e
due t o t h e a c t i o n o f s p h i n c t e r s l o c a t e d a t t h e
o r i g i n o f t h e vessels which supply i n t r a c e r e b r a 1 m e t a b o l i c f i e l d s and 2 ) t h e l o n g e r t h e
time i n t e r v a l t h a t t h e temperature o f t h e
blood i n t h e s u r f a c e c a r r y i n g vessels i s i n
near e q u i l i b r i u m w i t h t h e temperature i n t h e
m e t a b o l i c f i e l d s , t h e g r e a t e r i s t h e change i n
t h e temperature o f t h e m e t a b o l i c f i e l d s .
These o b s e r v a t i o n s o f f e r a p o s s i b l e explanat i o n f o r t h e f o c a l areas o f n e c r o s i s which a r e
o f t e n seen i n deep c o o l i n g o f t h e b r a i n .
McAuliffe, W.G. Department of Anatow,
University of Cincinnati, Cincinnati, Ohio.
rat has hereditary hygothalemic diabetes due
to an inability to produce Antidiuretic
Hormone (ADH). Animals homeygous for this
autosomal recessive trait (DI) lack ADH
completely, axe polydipsic, and excrete daily
a volume of hypotonic urine (176 Q&
H$,
S.E. 10) approximately equal to 70% of their
body weight. Heterozygotes (HZ)have a
partial deficiency of ADH and excrete urine
less concentrated than normal rats but
hypertonic to plasma (870 mOsm/kg €I20 S.E. 93).
Light microscopic observations on formalin
fixed kidneys reveal striking differences
between DI and M a n W s . In DI rats the
collecting tubule lumina axe wider and the
epithelial cells reduced i n height when
compared to either Hz or normal animale. The
dark or intercalated cells of the collecting
tubules of HZ rats resemble those seen in
normal rats but dark cells in DI animals are
very large end axe occasionally binucleate.
548
Their cytoplasm bulges into the tubule lumen
and stains intensely with a variety of dyes.
DI rats also seem to have an increased number
of dark cells. In both no&
and HZ rats
the medullary interstitium is extensive and
stains intensely with Hale's Colloidal Iron
(CI) or Alcian Blue at pH 2.5 or 1.0 (AB),
indicating high concentrations of acid
mucoplysaccharides (MPS). In DI animals the
amount of connective tissue is greatly
reduced and stains weakly with these methods.
I n normal and HZ a n W s CI or AB stain the
collecting tubule epithelial cell coat
intensely, but this reaction is weak or
absent in DI rats. The hypothesis is
advanced that the production of interstitial
Mps is influenced by ADH and that these Mps
Function in the process of urinary
concentration.
MCMASTER, B.A., Department of Biological
Sciences, Illinois State University, Normal,
Illinois. a h i s t o l o g y of the mental barbel of
certain aauatic turtles,
The function of papilla-like projections
(mental barbels) located on the skin of the jaw
immediately posterior to the mandibular symphysis of aquatic turtles in the families Chelydridae and Kinosternidae, is unknown. Histological
preparations of mental barbels from these
turtles contained no unusual glandular or neural
tissue. The core of the barbel, composed of
loose connective tissue, is covered by keratinized stratified squamous epithelium. This core
is highly vascular. In view of the known copulatory behavior of the turtle, the vasculatory
nature of the barbel suggests that it may be an
erectile tissue used to stimulate the copulatory
pair.
METCALF, N.F., E.D. PRENTICE, W.W. STINSON and
W.K. METCALF, Department of Anatomy, University
of Nebraska Medical Center, Omaha, Nebraska.
Functional anatomy: the logical basis for
patient examination.
Successful physical diagnosis depends largely on a thorough understanding of the underlying functional anatomy. Accordingly, in this
department, we offer, in conjunction with our
Gross Anatomy course, a "Living Anatomy" program which serves an an introduction to physical
examination. In this, the most clinically relevant section of our Gross Anatomy course, the
students learn to conduct a systematic anatomical evaluation of the normal individual and to
recognize basic anatomic pathology.
In order to enable students to efficiently
accomplish the objectives of Living Anatomy. we
designed a curriculum consisting of 24 x 1%
hour sessions which are coordinated with the
subject matter presented in the Gross Anatomy
course. To achieve greater uniformity of instruction, all sessions are conducted via
closed circuit television. With guidance provided by a live television production and the
assistance of instructors, groups of 16-24 stu-
dents work in pairs alternating as physician
and patient. Student examination of sensitive
areas in the opposite sex such as the breast.
vagina, male rectum, and inguinal region is
accomplished by the use of student organized
volunteer subjects, who were examined in private by fellow classmates under the direct supervision of either a medically qualified anatomist or a physician from the family practice
clinic.
Student learning in Living Anatomy is assessed by both multiple choice examinations and by
subjective examinations. In the subjective examinations, students, on an individual basis
randomly select question cards from a large pool
written in accordance with the educational
objectives. Each question requires the student
to describe orally and/or perform selected
physical examination procedures. Student performance is judged in three areas: knowledge,
psychomotor skill, and patient approach.
Student acceptance data from both preclinical
and clinical students suggest that Living Anatomy is a valuable, perhaps the most valuable,
component of our Gross Anatmy program.
MONTEMURRO, D.G., and J . E . BRUNI, Department of
Anatomy, University o f Western Ontario. London.
Ontario, Canada. Morphology of the ependymal .
lining o f the rabbit t h i r d ventricle following
intraventricular administration of synthetic
LH-RH: A scanning electron microscopic investioat
~- i on.
..
The morphology of the ependymal surface o f
the rabbit third ventricle was studied follwing intraventricular injection of an ovulatory
dose of synthetic LH-RH (200 ng/kg b wt). Six
identically treated female rabbits that received
injections of 0.15M NaCl only, served as
controls. Both groups of rabbits were sacrificed 5 and I5 minutes after injections. The
ependyma of the dorsolateral wall o f the ventricle and the transitional zone maintained a constant appearance from animal to animal regardless of treatment or time of sacrifice. The
ependyma of the ventrolateral wall and floor
(infundibular and mamniliary recesses) of the
ventricle, however, not only exhibited an
appearance quite distinct from either the dorsolateral wall or transitional zone b u t also
exhibited a transient responsiveness to LH-RH
treatment. in control rabbits, the ependymal
surface of this region o f the ventricle was nonciliated and irregularly contoured. Most
ependymal cells bulged conspicuously into the
ventricle. Their apical membranes were characterized by numerous pleomorphic bleb-like protrusions and occasional microvilli. In contrast
marked changes, suggestive of increased surface
activity were seen in the character of the
ependymal surface of this region in rabbits
sacrificed 5 minutes following LH-RH administration. These changes which principally took
the form o f numerous irregular microvillous-like
eruptions of the apical surface pervaded the
rostrocaudal extent of the recesses. Equivalent
regions of the ventricle in rabbits sacrificed
15 minutes after LH-RH injection, however, were
549
simila'r t o t h o s e of c o n t r o l s . These t r a n s i e n t
morphological changes noted i n t h e ependymal
s u r f a c e 5 minutes a f t e r LH-fUi injection may
r e f l e c t structural modifications associated with
a b s o r p t i o n o f t h e hormone a c r o s s t h e ependymalCSF i n t e r f a c e .
(Supported by a g r a n t from t h e Medical Research
Council of Canada).
PIENKOWSKI, MAREK M., Department of Anatomy,
Michigan S t a t e U n i v e r s i t y , E a s t Lansing,
Involvement of Biogenic Amines i n
Michigan.
Control of Development o f E a r l y Mouse Embryos.
The l i m i t e d developmental c a p a c i t y i n v i t r o ,
of one-celled embrvos,
. i n c o n t r a s t t o twoc e l l e d embryos, i m p l i c a t e s involvement of ext e r n a l regulating factor(s) i n controlling
e a r l y embryonic development. I n partenog e n e t i c development, an e l e c t r i c shock and/or
hyaluronidase-hypotonic t r e a t m e n t a r e known t o
promote growth. This s u g g e s t s t h a t a change of
c e l l u l a r charge and/or p o t e n t i a l i s prer e q u i s i t e f o r f u r t h e r development. I n v i v o ,
biogenic amines may be a c a n d i d a t e t o perform
t h i s f u n c t i o n . To test t h i s h y p o t h e s i s I
examined: 1 ) t h e e f f e c t of Noradrenalin on
t h e development of one-celled m u s e embryos,
i n v i t r o , and o b t a i n e d r e s u l t s i n d i c a t i n g a
growth promoting e f f e c t of t h i s b i o g e n i c amine;
2) t h e e f f e c t of r e s e r p i n e ( a b i o g e n i c amine
d e p l e t o r ) i n j e c t i o n s on development of f ertil i z e d ova i n vivo and r e s u l t s i n d i c a t e t h a t
n o r a d r e n a l i n e treatment r e t a r d e d growth of embryos a t t h e two t o f o u r - c e l l e d s t a g e ; 3) d i s t r i b u t i o n of b i o g e n i c amines i n t h e ovary and
oviduct using a h i s t o c h e m i c a l technique of
formaldehyde-induced f l u o r e s c e n c e i n f r e e z e d r i e d p r e p a r a t i o n s . I observed age-dependent
accumulation of b i o g e n i c a m i n e s i n b o t h the
w a l l of t h e o v i d u c t and i n s p e c i f i c s t r u c t u r e s
i n t h e ovary. These r e s u l t s p r o v i d e evidence
which s u p p o r t s t h e h y p o t h e s i s of a n involvement of b i o g e n i c amines i n t h e c o n t r o l of
development of e a r l y mouse embryos.
(Supported by: Biomedical Research Support
Grant f o r College of Human Medicine, from
N I H , RRO5656-09.)
PIEPER, G.M., G.L. TODD and F.C. CLAYTON,
Cardiovascular Center and Department of Anatomy,
U n i v e r s i t y o f Nebraska Medical C e n t e r , Omaha,
NE.
Dose-related changes i n t r a n s m u r a l mvocard i a l metabolism and morphology w i t h i s o p r o t e r enol.
Myocardial s t r e s s during i n t e n s e c a t e c h o l a mine stimulat'ion h a s been shown t o reduce highenergy phosphates and produce p a t h o l o g i c a l les i o n s . However, t h e t r a n s m u r a l myocardial met a b o l i s m and t h e c o e x i s t e n t morphology followi n g continuous i n f u s i o n s of i s o p r o t e r e n o l have
n o t been i n v e s t i g a t e d . Anesthetized, openc h e s t e d dogs were i n f u s e d w i t h e i t h e r saline or
0.1, 1.0 or 2.5 ug/kg/min i s o p r o t e r e n o l f o r one
hour. A transmural biopsy w a s removed from t h e
posterolateral l e f t ventricular w a l l , frozen i n
l i q u i d n i t r o g e n and d i v i d e d i n t o o u t e r , middle
and i n n e r t h i r d s . The remainder of t h e h e a r t
w a s f i x e d , s l i c e d , s e c t i o n e d , and s t a i n e d w i t h
H & E or phosphotungstic acid-hematoxylin.
With i n c r e a s i n g doses of i s o p r o t e r e n o l , highenergy phosphates and glycogen decreased w h i l e
m e t a b o l i c breakdown p r o d u c t s increased.
In
g e n e r a l , t r a n s m u r a l g r a d i e n t s w e r e observed
only a t t h e h i g h e s t dose. A t t h e 2.5 uglkglmin
dose, t r a n s m u r a l d e p l e t i o n s i n ATP and glycogen
occurred p r i m a r i l y i n t h e i n n e r one-third.
Phosphocreatine, however, d e c l i n e d t o approximately t h e same l e v e l i n a l l l a y e r s . Concomit a n t w i t h t h i s were t r a a s m u r a l accumulations
( i n c r e a s i n g from o u t e r co i n n e r ) of ADP, AMP,
glucose-6-phosphate and l a c t a t e . Histopathol o g i c a l l y , s i g n i f i c a n t dumbers of l e s i o n s w e r e
observed a t t h e h i g h e r doses of i s o p r o t e r e n o l .
The v u l n e r a b i l i t y of t h e i n n e r l a y e r was confirmed by t h e d i s t r i b u t i o n of myocardial lei o n s . G r e a t e r numbers of c o n t r a c t i o n bands
w e r e found i n t h e i n n e r l a y e r t h a n i n e i t h e r
t h e middle o r o u t e r l a y e r s . A p r e f e r e n t i a l
v u l n e r a b i l i t y of t h e subendocardial r e g i o n t o
i s o p r o t e r e n o l a d m i n i s t r a t i o n and t h e dose-rel a t e d r e s p o n s e s were, t h e r e f o r e , s u b s t a n t i a t e d
both biochemically and h i s t o l o g i c a l l y .
(Supported by U n i v e r s i t y of Nebraska Medical
Center, C a r d i o v a s c u l a r Center Development
Award and Nebraska Heart A s s o c i a t i o n )
RENDER, L.L. and C.A. BLAKE, Department of
Anatomy, U n i v e r s i t y of Nebraska Medical Center,
Omaha, Nebraska. E f f e c t s of long-term LHRH
i n f u s i o n on LH s e c r e t i n g c e l l s i n t h e p a r s
distalis.
Previous s t u d i e s i n o u r l a b o r s t o r y haveshown
t h a t one f a c t o r r e s p o n s i b l e f o r t e r m i n a t i n g t h e
spontaneous pre-ovulatory s u r g e of LH i n r a t
plasma d u r i n g p r o e s t r u s is a d e c r e a s e d p i t u i t a r y
r e s p o n s i v e n e s s t o LHRH. The p r e s e r t s t u d y w a s
undertaken t o i n v e s t i g a t e u l t r a s t r u c t u r a l
char,ges t h a t might occur i n LH s e c r e t i n g c e l l s
d u r i n g t h i s " r e f r a c t o r y period." Four-day cyc l i c r a t s r e c e i v e d two indwelling r i g h t a t r i a l
cannulas d u r i n g t h e morning of p r o e s t r u s . They
were i n j e c t e d w i t h p h e n o b a r b i t a l (75mg/kgBW) a t
1400 h t o b l o c k t h e spontaneous LH s u r g e d u r i n g
t h e a f t e r n o o n of p r o e s t r u s . A t 1500 h, s a l i n e
or s y n t h e t i c LHRH w a s i n f u s e d through one o f t h e
c a n n u l a s a t a c o n s t a n t rate (50ng/h) u n t i l dec a p i t a t i o n a t 2000 h. Blood samples w e r e withdrawn through t h e o t h e r cannula a t h o u r l y i n t e r v a l s during infusion. A t s a c r i f i c e , p i t u i t a r i e s
were removed and b i s e c t e d ; h a l f f o r determinat i o n of LH c o n c e n t r a t i o n and h a l f f o r e l e c t r o n
microscopy. Radioimmunoassay of plasma LH rev e a l e d t h a t LHRH i n f u s i o n simulated t h e r i s i n g
and p l a t e a u phases of t h e spontaneous LH s u r g e
and t h a t plasma LH decreased s t e a d i l y d u r i n g
t h e l a s t 2 h of i n f u s i o n . After 5 h of LHRHinf u s i o n , p i t u i t a r y LH c o n c e n t r a t i o n was reduced
t o about 1 / 3 t h a t of c o n t r o l s . Due t o c u r r e n t
c o n t r o v e r s y as t o whether or n o t t h e same c e l l
secretes b o t h gonadotropic hormones; t h e unl a b e l l e d a n t i b o d y , peroxidase-antiperoxidase
complex t e c h n i q u e was used t o s t a i n LH c o n t a i n i n g c e l l s . An a n t i - r a t LHB (NIAMDD) was v a l i -
550
dated for use and used at a dilution of 1:
100,000. One basic cell type stained in all
the control animals. It was ovoid to polygonal
but never stellate. Granules were, for the
most part, evenly distributed throughout the
cell or concentrated at one pole. The LHRHinfused animals exhibited a cell type with a
similar shape and granule size. The presenceof
many cells with very few granules along with
well granulated cells was characteristic of the
experimental group. The results suggest that
the mechanism of pituitary refractiveness to
LHRH may be the result of a reduced ability of
the LH cells to synthesize LH. (Supported by
NIH Grant H D l l O l l and the University of Nebraska Medical Center Foundation).
RUBEN, R.L. and K.A. RAFTERTY, JR.,Department of
Anatow, University of Illinois Medical Center,
Chicago, Illinois. Anchorage independence and
predictable crisis in SV40-transformed cells.
-In vitro transformation of human cells by
oncogenic viruses results in a spectrum of inheritable phenotypic alterations which vary not
only across but within cell populations. Such
properties as increased rates of glycolysis
and acid production in culture, morphologic
changes, aneuploidy, altered growth patterns,
and tumorigenicity in immunosuppressed animals
are often but not consistently observed. A
fairly general observation, however, is the
loss of such normal constraints on celldivision
as contact inhibition, lowered serum concentration, and absence of a solid substratum for anchorage. Of these, loss of anchorage dependence
is the single characteristic most consistently
associated with cellular transformation. In the
present study anchorage independence was confirmed for a line of SBkO-transformed human
parapharyngeal cells recently developed in o u r
laboratory. Designated SV-TGo, this epithelioid
cell line is characterized by increased acid
production in culture, increased growth rate
and doubling potential over untransformed controls, altered epithelioid morphology, and release of infectious virus. Original growth patterns and extreme sensitivity to contact inhibition were unaffected by transformation.
The ability to proliferate independent of a
substratum was determined by cloning cells
in s o f t agar. A f t e r 2-3 weeks and 14-16 population doublings, colonies ranging in diameter
from 0.3-2.0 mm were isolated and tested for
continued virus production. Following 1-3 subcultures (36-48 population doublings), all
clones senesced as evidenced by the accmulation of cellular debris, multinucleated giant
cells, and lysis. The importance of these findings lies in the facts that 1) transformation
does not confer unlimited division potential,
as is commonly assumed, 2) this limit falls
within the range predicted by Hayflick for fibroblasts, a population of considerably less
division potential in vivo than epithelial
cells, and 3) that from 77 2,500 lbs of cells
can still be produced within the confines of
these limits provided that every cell divides.
(Supported by NIH research grant CA 13494)
-
SINGER, R. and K. KIMURA, Department of Anatomy,
University of Chicago and Department of Anatomy,
National Defense Medical College, Tokorozawa,
Japan. Brachymesophalangia of the fifth finger.
The term “Brachymesophalangia V” was
introduced more than 50 years ago to describe a
shortening of the middle phalanx of the fifth
digit of the hand. A number of investigators
have recorded varying incidences of the trait
in different populations, for example, that it
is considerably higher in Mongoloid populations
than in Caucasoid or Negroid ones, and that sex
differences may be present. However, it is
noted that differing criteria for the trait
have resulted in differing results. A high
incidence of the trait has been observed in
Down’s syndrome where the dysplasia is
different from normal individuals and usually
associated with clinodactyly. Investigators
have also attempted to explain the trait as a
consequence of nutritional deficiencies. Our
study involves samples of Hottentot and Cape
Colored populations from Namibia and South
Africa. For determining the trait,
inspectional and metric methods were used. For
the latter, the following criteria were
adopted: the ratio of the lengths of the fifth
to the fourth middle phalanges is less than
0.59; the ratio of the lengths of the middle
to the proximal phalanges (fifth digit) is less
than 0.50; and the ratio of the breadth to the
length of the fifth middle phalanx is more
than 0.60. Our data suggest that nutrition
per se is not accountable for the trait.
STINSON, W.W., J.G. SHARP and J.W. OSBORNE. Department of Anatomy, University of Nebraska
Medical Center, Omaha, Nebraska and Radiation
Research Laboratory, University of Iowa, Iowa
City, Iowa. Histogenesis of radiation-induced
intestinal tumors.
Exposure of the hypoxic, temporarily exteriorized ileum and jejunum of male Holtzman rats
to 2000 R X-irradiation leads to the induction
of intestinal adenocarcinoma in approximately
60% of the irradiated rats. Similar tumors occur in the irradiated large bowel of rats (Kirchner ct. Radiat. Res. z : 6 0 6 , 1977). Various
attempted diagnostic procedures; weight loss
(Coop $t&.
Cancer Res. 3:1487, 1974); scintiscanning of injected radiolabeled anti-tumor antibodies (Cheng gt. Radiat. Res. 51:439,1972)
and assay of tumor-associated oncofetal protein
(Stevens eta. JNCI x:1011, 1975) did not consistently identify small tumors. Consequently,
intestine-irradiated rats were subjected to regular monthly laparotomies in an attempt to detect early lesions. At laparotomy the intestine
was inspected visually for abnormalities and
palpated gently in an attempt to detect any
areas of hardness or thickening of the mucosa.
Rats with suspected lesions were killed by decapitation and the intestine removed, slit longitudinally along its mesenteric edge and placed
immediately in cold buffered 2.5% glutaraldehyde
fixative. The mucosal surface was scanned carefully using a dissecting microscope and abnormal
areas removed for light and electron microscopic
551
study. The e a r l i e s t l e s i o n s observed involved
very small ulcer " p i t s " accompanied by a granul o c y t i c , eosinophilic, and mononuclear c e l l inf i l t r a t i o n . Abnormally located epithelium was
observed a t the edges of these u l c e r s . This epithelium may have been "trapped" by the connect i v e t i s s u e response to the ulcerated mucosa.
Structures whose c e l l constituents closely resembled adenomatous polyps were observed i n more
advanced lesions although these did not necess a r i l y project i n t o t h e i n t e s t i n a l lumen i n the
t y p i c a l fashion of polyps. These observations
would be consistent with a hypothesis t h a t rad i a t i o n impairs the a b i l i t y of r e s t i n g i n t e s t i n a l stem c e l l s t o maintain the i n t e g r i t y of the
mucosa, so t h a t subsequently, small a r e a s of ulceration occur. Whether these u l c e r s progress t o
polyp-like l e s i o n s and adenocarcinoma o r whether
the l a t t e r have an a l t e r n a t i v e o r i g i n i s curr e n t l y unclear. (Supported i n p a r t by USPHS
Grant EC-00066).
STRAWSER, R.T., F.F. BARTONE, and P.J. GARDNW.
Departments of Anatomy and Urology, University
of Nebraska Medical Center. %aha. Nebraska
Histometric observations i n the descended testis
of u n i l a t e r a l l y cryptorchid r a t s .
It is well known that degenerative changes
occur i n the undescended t e s t i s of u n i l a t e r a l l y
cryptorchid individuals. These changes a r e mani f e s t e d by a decrease in t h e diameter of t h e
seminiferous tubules and a l o s s of germinal
cells. Such seminiferous tubules are composed
primarily of S e r t o l i c e l l s . It i s o f t e n assumed t h a t , a f t e r the undescended t e s t i s is surgic a l l y relocated in t h e scrotum, f e r t i l i t y w i l l
r e t u r n to normal. However, recent r e p o r t s ind i c a t e t h a t f e r t i l i t y is reduced in 27-46% of
these individuals. In t h e present investigat i o n , histometric techniques were employed t o
determine the e f f e c t of u n i l a t e r a l cryptorchidism on the descended ( s c r o t a l ) t e s t i s of r a t s .
Spragueaawley r a t s were rendered u n i l a t e r a l l y
cryptorchid a t 2 1 days of age. A t that age,
the t e s t i s is located a t t h e entrance t o t h e
deep inguinal ring. Thus, the r i n g i s r e a d i l y
closed and t h e t e s t i s anchored t o the abdomina l w a l l through t h e epididymal f a t pad. Three
months a f t e r operation, both t h e cryptorchid
and descended t e s t i s were excised and t i s s u e
blocks processed f o r l i g h t and e l e c t r o n microscopy. Seminiferous tubule diameters were measured i n both t h e experimental and control
groups by t h e use of an eyepiece micrometer.
Measurements were performed on 2pm t o l u i d i n e
blue-stained araldite-embedded sections. The
mean diameter of t h e seminiferous tubules of
descended t e s t e s from u n i l a t e r a l l y cryptorchid
animals was 22.22 2 l . l l u m , which showed a
s i g n i f i c a n t decrease (21%. p = 0.005) from that
of t h e control animals (27.98 k 0.83umm). The
tubular diameters in cryptorchid testes (mean
of 13.34 t 0.87urn) manifested a 52% decrease
(p < 0.001) from those of controls. These
preliminary r e s u l t s suggest t h a t h i s t o l o g i c a l
a l t e r a t i o n s occur i n t h e descended testis as
well a s i n the undescended testis of u n i l a t e r a l l y cryptorchid r a t s . Additional u l t r a s t r u c ~
~~
t u r a l s t u d i e s are i n progress t o determine t h e
e f f e c t s of these changes on t h e f e r t i l i t y of
t h e animals.
(Supported in p a r t by a U.N.M.C.
Seed Grant,
NIH Grant 1 T32 HD07097, and NSF RIAS-SER 7706922).
SWEENEY, L.J. and G.C. ROSENQUIST, Departments
of Anatomy and P e d i a t r i c s , University of Nebraska Medical Center, Omaha, Nebraska. The relationship of t h e g r e a t v e s s e l s t o underlying
cardiac s t r u c t u r e s i n t r a n s p o s i t i o n of the g r e a t
vessels.
In Transposition of the Great Vessels (TGV)
abnormal d i v i s i o n of t h e truncus a r t e r i o s u s reverses the positions of t h e Aorta (Ao) and Pulmonary Artery (PA). Present knowledge of t h e
junction of t h e g r e a t v e s s e l s (GV) with the
h e a r t i s inadequate fornewer techniques of noninvasive diagnosis of t h i s anomaly, such a s
cross-sectional echocardiography. While t h e
a n t e r i o r GV in TGV ( t h e Ao) is known t o be more
a n t e r o l a t e r a l than innormal h e a r t s , t h e changes
i n r e l a t i o n t o i n t r a c a r d i a c s t r u c t u r e s t h i s produces a r e unknown, a s well as the degree of
displacement from normal position. We, theref o r e , studied t h i s junction i n 80 human h e a r t s
with TGV with i n t a c t v e n t r i c u l a r s e p t a and compared these findings with those f o r 90 normal
h e a r t s . F i r s t , we found t h a t t h e t h r e e cusps
of t h e PA ( p o s t e r i o r GV) were not aligned normally with e i t h e r t h e Aortic cusps o r the underlying Membranous Ventricular Septum (MeVS).
While t h e p o s t e r i o r G V ' s point of contact with
the a n t e r i o r GV is normally separated by one
f u l l cusp (or one-third of the GV circumference)
from i t s point of contact with the MeVS, i n TGV
t h e two points of contact a r e always c l o s e r
than normal: t h e separation averages only ones i x t h of t h e GV circumference (56%). Since t h e
MeVS remains fixed i n the fibrous base of the
h e a r t , t h i s separation can be used a s a measure
of t h e displacement of the a n t e r i o r GV in TGV.
The a m u l u s of t h e PA i n TGV is a l s o lower than
normal in t h e LV outflow t r a c t . The PA annulus
is continuous with t h e Mitral annulus i n 46% of
the h e a r t s , and i t s average separation is only
.9mm, whereas i n only 23% of normal h e a r t s a r e
t h e A 0 and m i t r a l annuli continuous, and t h e i r
average sepraation is 1 . k . Further, the tricuspid annulus s e p t a 1 commissure is on o r
superior t o the PA annulus i n TGV. whereas i t
i s s e v e r a l mm below the Ao i n normal h e a r t s .
These changed r e l a t i o n s h i p s i n TGV should provide useful information f o r developing noninvasive diagnostic s k i l l s . (Supported by N I H
Grant No. 86-208-172-02).
F.C. CLAYTON and G.M. PIEPER,
Department of Anatomy and Cardiovascular Center,
University of Nebraska Medical Center, Omaha,
NE. Transmural d i s t r i b u t i o n of glycogen f o l lowing l e f t circumflex l i g a t i o n and isoproteren01 infusion.
Glycogen provides a source of myocardial
high-energy phosphate production during stress-
TODD, G.L.,
552
ful conditions such as hypoxia, ischemia or
catecholamine overdrive. A preferential vulnerability of the subendocardial region has
been reported for a variety of parameters. The
presence of a transmural gradient in glycogen
stores following continuous infusions of varying doses of isoproterenol (ISP) was investigated both biochemically and histochemically.
Anesthetized, open-chested mongrel dogs were
continuously infused with saline, or 0.1, 1.0
or 2.5 pglkgfmin ISP for one hour. Left circumflex ligation was used as the standard
model of ischemic necrosis f o r comparison. A
transmural left ventricular biopsy was assayed
for glycogen concentrations. The heart was
then fixed in 10% neutral buffered formalin
and transversely sliced. Alternate 2 mm slices
were sectioned and stained with H & E or PAS
for microscopic examination. There was an
inverse relation between the presence of PASstainable material and increasing doses of ISP.
The most pronounced decrease occurred in the
inner one-third. There was, however, a maintenance of glycogen in the subendocardial conduction system. The transmural depletion of
glycogen was verified biochemically at all
doses of I S P . Permanent ligation produced
large decreases in glycogen stores, again predominantly in the inner layer. The values for
ligation were not statistically different from
those at the highest dose of ISP. The presence
of transmural gradients with both ligation and
ISP adds further support to the concept of
increased vulnerability of subendocardium to
stress.
(Supported by University of Nebraska Medical
Center, Cardiovascular Center Development
Award and Nebraska Heart Association)
TURBES, C.C., G.T. SCHNZIDER and J.M. SIMARD,
Department of Anatomy, Creighton University,
Omaha, Nebraska 68178. Studies on the
influence of septal and caudate nuclei
stimulation on electrical activity of the
cerebral cortex and limbic system.
Multiunit and slow wave were recorded from
extracellular microelectrodes in the dorsal
hippocampus and the amygdala in twenty-eight
cats. The electroencephalogram was recorded
from the right and left frontal, parietal,
temporal and occipital cerebral cortex. Three
channels were recorded on an F.M. tape
recorder for further data analysis. The data
were processed with a Varian V-72 minicomputer.
Channels were sampled alternately at 2.5 ms
and 120 data segments for each channel of 2.56
seconds duration each. These data were
transformed using a Fast Fourier Transform
algorithm to give a power spectral estimate.
Stimulating electrodes were located in the
left medial and lateral septal nuclei, nucleus
accumbens and caudate nucleus. Septa1 stimulation enhanced evoked potentials at the
cerebral cortex, amygdala and dorsal hippocampus. Stronger septal stimulation resulted
in afterdischarge at the cerebral cortex and
the limbic nuclei. Focal application of
penicillin to the cerebral cortex resulted in
focal cortical seizures. Generalized seizures
were induced with pentylenetetrazol.
Caudate stimulation resulted in general
cortical desynchronization of synchronized
activity in normal cerebral cortex and focal
synchronized seizure activity induced by
local application of penicillin to the
cerebral cortex. Stimulation of the caudate
using high voltages (25 volts) resulted in
inhibition of generalized seizures induced
with pentylenetetrazol.
WOOD, STEVEN R
.
'
,
JERRY D. HUNTER' and NANCY
SEMKOz, lDepartment of Anatomy, University of
Nebraska Medical Center, Omaha, Nebraska, 'Department of Biology, University of Texas at El
Paso. The action of hepatic growth factor on
normal mouse liver.
Hepatic Growth Factor (BGF) was obtained
from Charles River CD-1 albino out-bred rats
and injected into CD-1 albino mice inorder to
study hepatic regeneration responses. Mice of
both sexes were employed lnorder to initially
find the optimal time for secretion of HGF
following a partial hepatectomy (Hep-X) , This
was found to occur 30 hours after the operation.
In turn, the maximum response to the HGF, as
indicated by tritiated Thymidine ('H-TdR)
uptake into hepatocyte nuclei, was found to occur
36 hours after injection into the mice. The
experimental auto-radiographs re resented an
average 20-fold increase in the $-TclR Percent
Label Index (XLI) when compared to control
animals. This further represented a 4-fold
increase over previous XLI's reported using
different pairings of HGF secretion and injection response times.
553
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