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Southern society of anatomists twentieth annual meeting october 16 У18 1980.

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THE ANATOMICAL RECORD 199:287-308 (1981)
SOUTHERN SOCIETY OF ANATOMISTS
lbentieth Annual Meeting
October 16-18, 1980
Augusta, Georgia
Host
MEDICAL COLLEGE OF GEORGIA
287
SOUTHERN SOCIETY OF ANATOMISTS
OFFICERS 1979-80
President
DALE E. BOCKMAN
(Medical College of Georgia)
President-Elect
RICHARD J. WEYMOUTH
(South Carolina-Columbia)
Secretary-Peasurer
HERBERT SCHAPIRO
(Eastern Virginia)
Councilors
W.J. DOUGHERTY
(South Carolina)
M.L. ZIMNY
(LSU)
R.P. STURTEVANT
(Loyola)
M. SHARAWY
(Medical Collge of Georgia)
This meeting was dedicated to the memory of
DR. B . RAJ BHUSSRY
President of the Southern Society of Anatomists, 1978-79
288
CORPORATE SPONSORS
The following firms have generously supported this, the 20th Annual
Meeting of the Southern Society of Anatomists, by their participation in
the commercial exhibits and/or sponsoring of social events:
AMERICAN
OPTICAL
CORPORATION
AMMHAY, INC.
BECKMAN
INSTRUMENTS,
INC.
CAROLINA
BIOLOGICAL
SUPPLY,
CO.
DE NEMOURS
& COMPANY,
INC.
E.I. Du PONT
SCIENCES
ELECTRON
MICROSCOPY
GEORGIA
INSTIlUMENTS, INC.
HAIXOINSTRUMENTS,
INC.
H l T A C H l S C I E N T I F I C INSTRUMENTS
ISI, INC.
JEOL
USA, INC.
L.K.B. INSTRUMENTS, INC.
MEDICAL
INTERNATIONAL S O U T H E A S T E R N , INC.
NEWCOMB
ASSOCIATES
PGT
PHI L I tJsELECTRON
IC I N STRU M E NTS, I Nc .
PHY SI M E T HI c s, I Nc .
POK1’ER INSTRUMENTS S E R V I C E , INC.
SOUTIIE
MICRO
I ~ N INSTRUMENTS,
INC.
SCIENTIFIC,
INC.
VASHAW
289
OPENING SESSION
Dr. Dale E . Bockman, Professor and Chairman
Department of Anatomy, Medical College of Georgia
President, Southern Society of Anatomists
Dr. William H. Moretz
President, Medical College of Georgia
A total of 54 free papers were presented in 7 sessions as follows
SESSION I
“Neurobiology”
(Chairpersons;E.G. Hamel, Jr. and M.L. Kirby)
SESSION I1
“Neuromuscular”
(Chairpersons: D.W. Sickles and G.S. Sohal)
SESSION I11
“Special Senses, Salivary Glands”
(Chairpersons:W.K. O’Steen and M. Sharawy)
SESSION IV
“Methods in Anatomy”
(Chairpersons;R.R. Cowden and J.B. Longley)
SESSION V
“Cell Biology, Immunology, Tissue Culture”
(Chairpersons: M.L. Zimny and D.B. Lause)
SESSION VI
“Reproduction, Endocrinology”
(Chairpersons: W.P. Jollie and W.J. Dougherty)
SESSION VII
“Pathology, Environmental Pathology”
(Chairpersons:D.E. Hinton and J.M. Barrett)
290
AUGUSTINE, J a m e s R . , D e p a r t m e n t o f Anatomy,
U n i v e r s i t y o f S o u t h C a r o l i n a , S c h o o l o f Medic i n e , Columbia, South C a r o l i n a . A L u c i t e
p l a t e method f o r t h r e e - d i m e n s i o n a l r e c o n s t r u c t i o n of neuronal populations.
The combined u t i l i z a t i o n o f HRP h i s t o c h e m i s t r y coupled w i t h a ].“cite p l a t e reconstruct i o n method p r o v i d e s a n e x c e l l e n t means o f
s t u d y i n g t h e f u n c t i o n a l o r g a n i z a t i o n of neuronal populations.
T h i s i s a n a d a p t a t i o n of a
method p r e v i o u s l y e m p l o y e d by B a e t e n s _
c t a_l .
( 1 9 7 9 ) t o d e m o n s t r a t e t h e two t y p e s o f i s l e t
c e l l s i n t h e e n d o c r i n e p a n c r e a s of t h e r a t .
C o l o r p h o t o m i c r o g r a p h s w e r e made o f e v e r y s i x t h
s e c t i o n ( 4 0 m i c r o n s e c t i o n s w i t h 200 m i c r o n s
between s e c t i o n s ) through t h e baboon oculom o t o r n u c l e u s . Each o f t h e r e s u l t i n g 18 s l i d e s
was t h e n p r o j e c t e d w i t h t h e a i d o f a p h o t i i g r a p h i c e n l a r g e r o n t o a 9” X 1 2 ” L u c i t e p l a t e
a n d t h e c o n f i g u r a t i o n of t h e o c u l o m o t o r n u c l e u s
a t e a c h r e p r e s e n t a t i v e l e v e l drawn t o s c a l e
on t h e p l a t e s .
The L u c i t e p l a t e s w e r e t h e n
s t a c k e d one i n f r o n t of t h e o c h e r y i e l d i n g a
s e e - t h r o u g h , 3-D r e c o n s t r u c t i o n (it t h e e n t i r e
nucleus.
Color t r a n s p a r e n c i e s of e v e r y s i x t h
HRP-processed s r c t i o n were made and t h e image
of each section t h e n projected onto the i.ucite
p l a t e s on w h i c h t h e c o n f i g u r a t i o n o f t h e o c u l o motor n u c l e u s w a s p r e v i o u s l y o u t l i n e d .
Using
d i f f e r e n t colored s t a r s tu represent the d i f f e r e n t o c u l o m o t o r m u s c l e s , t h e number a n d l o c a t i o n o f H R P - p o s i t i v e n e u r o n s was t h e n p l o t t e d .
A c o m p o s i t e 3-D r e c o n s t r u c t i o n n f t h e e n t i r e
l a b e l e d p o p u l a t i o n of t h e oculomotor n u c l e u s
on L u c i t e p l a t e s was t h e e n d r e s u l t .
This
method p r o v i d e s a n u n p a r a i l e d a p p r e c i a t i o n o f
t h e e n t i r e n u c l e a r complex t h a t is anatomic a l l y a c c u r a t e and t h a t c a n h e viewed f r o m a n y
s i d e o r a t any a n g l e .
T h i s method c a n be
a p p l i e d t o any neuronal p o p u l a t i o n i n any animal
form. R e c o n s t r u c t i o n s from s e r i a l e l e c t r o n
micrijgraphs might be p o s s i b l e w i t h t h i s method.
Finally, w i t h adequate reference points availa b l e on t h e L u c i t e p l a t e s t o a l i g n s e c t i o n s and
maintain s p a t i a l r e l a t i o n s , t h i s d a t a can be
e n t e r e d v i a A two-dimensional d i g i t i z e r , s t o r e d
i n a c o m p u t e r d a t a b a s e and l a t f , r v i e w e d a n d
m a n i p u l a t e d i n 3-7) f a s h i o n u s i n g a n a p p r o p r i a t r
computer g r a p h i c s d e v i c e .
( S u p p o r t e d by n
U n i v e r s i t y o f S o u t h C , i r o l i n a , S c h o o l o f Medicine Intramural Crant).
BELTZ*, A l v i n J . a n d J a m e s BUGGY”, D e p a r t m e n t s
o f Anatomy and P h y s i o l o g y , U n i v e r s i t y r i f S o u t h
C a r o l i n a , School of Medicine, Columbia, South
Carolina.
( I n t r o d u c e d by J a m e s A . H i g h t o w e r )
Mapping o f c h a n g e s i n b r a i n f u n c t i o n a l a c t i v i t y
f o l l o w i n g midbrain periaqueductal gray stimul a t i o n : A 3H-2-deoxyglucose s t u d y .
The m i d b r a i n p e r i a q u e d u c t a l g r a y (PAG) h a s
b e e n i m p l i c a t e d as a c e n t r a l s i t e o f morphines
a n t i n o c i c e p t i v e a c t i o n and a c e n t r a l l o c u s f o r
s t i m u l a t i o n produced a n a l g e s i a .
The p r e s e n t
i n v e s t i g a t i o n was undertaken t o d e t e r m i n e which
CNS s i t e s show c h a n g e i n t h e i r f u n c t i o n a l a c t i v i t y f o l l o w i n g PAG s t i m u l a t i o n . T h i s w a s accomp l i s h e d by p r o d u c i n g f u n c t i o n a l maps o f CNS
a c t i v i t y u t i l i z i n g a m o d i f i c a t i o n o f t h e 2d e o x y g l u c o s e t e c h n i q u e d e v e l o p e d by S o k o l o f f
and c o l l a b o r a t o r s ( 1 9 7 7 ) .
Chronic e l e c t r o d e s
w e r e i m p l a n t e d s t e r e o t a x i c a l l y i n t o t h e I’AG o f
20 m a l e S p r a g u e Dawley r a t s (200-250 gm).
E a c h a n i m a l was t e s t e d 4 8 h o u r s a f t e r s u r g e r y
t o d e t e r m i n e i f PAC, s t i r n u l a t i o n r e s u l t e d i n
a n a l g e s i a t o i n c r e a s i n g i n t e n s i t i e s of e l e c t r i c a l s h o c k . Animals which d i s p l a y e d s t i m u l a t i o n
produced a n a l g e s i a were s u b s e q u e n t l y i m p l a n t e d
w i t h a lemoral venous c a t h e t e r . Twenty-four
h o u r s a f t e r c a t h e t e r i z a t i o n t h e a n i m a l s were
i n j e c t e d i n t r a v e n o u s l y w i t h 3H-Z-deoxygl u c o s e
( 1 6 7 p c i / I O O g m body w e i g h t ) and t h e PAG w a s
subsequently stimulated f o r a period of 45
min.
The a n i m a l s were i m m e d i a t e l y s a c r i c i c e d
by i n t r a c a r d i a c p e r f u s i o n w i t h c o l d f i x a t i v e ;
t h e i r b r a i n s were removed a n d f r o z e n i n l i q u i d
n i t r o g e n ; and 1 5 p t h i c k s e c t i o n s o f b r a i n
were c u t on a c r y o s t a t . The s e c t i o n s w e r e
e x p o s e d t o LKB u l t r o f i l m f o r a p e r i o d o f 3
weeks and t h e r e s u l t i n g a u t o r a d i o g r a p h i c images
were a n a l y z e d u s i n g a c o m p u t e r i z e d image p r o c e s s i n g system.
‘The m o s t s t r i k i n g c h a n g e s i n
i u n c t i o n a l a c t i v i t y f o l l o w i n g PAC s t i m u l a t i o n
o c c u r r e d i n t h e v e n t r a l hippocampus. n u c l e u s
paragigantocellularis, nucleus cuneiformis,
zona i n c e r t a , nucleus c e n t r a l i s s u p e r i o r ,
n u c l e u s a m b i g i n i s , n u c l e u s s o l i t a r i u s and t l l c
d o r s a l h u r n o f tiif s p i n a l c o r d .
The d a t a obt a i n e d f r o m t h i s s t u d y s u g g e s t t h a t many o f
t h e above n u c l e a r groups a r e involved i n t h e
p r o d u c t i o n o f a n a l g c s i n b r o u g h t a b o u t by PAC
stimulation.
( S u p p o r t e d by NSF (:rant BNS
7906486 and USC C r a n t 11040 E - 1 3 3 . )
BICKNELL, H. R.* a n d J. A . B e a l , D e p a r t m e n t
o f Anatomy, L o u i s i a n a S t a t e U n i v e r s i t y M e d i c a l
Center, Shreveport, Louisiana.
Atypical
neuronal cell types i n the substantia
g e l a t i n o s a ( l a m i n a 11) o f t h e a d u l t c a t
lumbosacral s p i n a l cord.
The n e u r o n s o f t h e s u b s t a n t i a g e l a t i n o s a
(SG) w e r e e x a m i n e d i n G o l g i p r e p a r a t i o n s t a k e n
from t h e l u m b o s a c r a l s p i n a l c o r d o f t h e a d u l t
cat. I n a d d i t i o n t o s e v e r a l t y p e s of charact e r i s t i c SG n e u r o n s , a f e w n e u r o n s were f o u n d
w i t h a t y p i c a l c h a r a c t e r i s t i c s which have n o t
been previously reported i n the adult s p i n a l
c o r d . T h e a t y p i c a l c e l l s f a l l i n t o two
c a t e g o r i e s : 1) n e u r o n s w i t h a s t a r s h a p e d ,
r a d i a t i n g d e n d r i t i c p a t t e r n and 2 ) neurons
w i t h d u a l axons.
The s t a r s h a p e d n e u r o n s
were observed i n Rexed‘s (‘52) i n n e r zone
o f l a m i n a 11. T h e s e n e u r o n s h a v e a s m a l l ,
p o l y g o n a l s h a p e d c e l l b o d y ( 1 4 x 18 p)
p o s s e s s i n g numerous, s h o r t primary and
secondary d e n d r i t e s which extend r a d i a l l y
f r o m t h e soma i n a s t a r - l i k e f a s h i o n .
Both
t h e c e l l body a n d d e n d r i t e s o f t h e s t a r
s h a p e d n e u r o n h a v e an i r r e g u l a r c r a g g y
contour.
A strong structural similarity
e x i s t s between t h e s t a r shaped neuron and
i m m a t u r e , u n d i f f e r e n t i a t e d SG n e u r o n s
previously described i n the k i t t e n (Falls
and Gobel, ’ 79).
The s i m i l a r i t y s u g g e s t s
t h a t t h e star shaped c e l l has n o t completed
its d i f f e r e n t i a t i o n i n t o a mature neuronal
f o r m . N e u r o n s p o s s e s s i n g d u a l a x o n s were
found i n b o t h t h e i n n e r and o u t e r zones of
R e x e d ’ s ( ‘ 5 2 ) lamina 11. The two a x o n s
o r i g i n a t e from d e n d r i t e s a t v a r i o u s
d i s t a n c e s f r o m t h e c e l l body a n d g e n e r a t e
an extensive plexus that extends f o r several
hundred micra.
The two a x o n a l a r b o r s d i s p l a y
c o n s i d e r a b l e o v e r l a p . S i n c e d u a l axon c e l l s
a r e few i n number and s t r u c t u r a l l y s i m i l a r
t o o t h e r SG n e u r o n s w i t h s i n g l e a x o n s i t i s
most p r o b a b l e t h a t t h e y r e p r e s e n t a
developmental aberrancy r a t h e r than a
f u n c t i o n a l l y d i s t i n c t c l a s s of n e u r o n s .
(Supported i n p a r t by N I N C D S , NS 16642-01).
BOSHELL, J . L . , Department of O r a l B i o l o g y /
Anatomy, M e d i c a l C o l l e g e o f G e o r g i a S c h o o l of
D e n t i s t r y , Augusta, G e o r g i a . A Comparison of
t h e E f f e c t s of I s o p r o t e r e n o l and T e s t o s t e r o n e
P r o p i o n a t e on A c i n a r C e l l R e g e n e r a t i o n i n
Suomandibular Glands o f R a t .
S t u d i e s on f a c t o r s c o n t r o l l i n g p a r e n chymal r e g e n e r a t i o n of s u b m a n d i b u l a r g l a n d s of
r a t s a r e c u r r e n t l y ongoing i n o u r l a b o r a t o r i e s .
The p u r p o s e of t h i s r e p o r t is t o compare t h e
e f f e c t s of i s o p r o t e r e n o l ( I S O ) and t e s t o s t e r o n e
p r o p i o n a t e (TP) on a c i n a r c e l l m i t o s e s f o l l o w i n g s u r g i c a l removal of a p o r t i o n of t h e l e f t
s u b m a n d i b u l a r g l a n d . Male Sprague-Dawley r a t s ,
325-350 grams i n w e i g h t , were a n e s t h e t i z e d and
a wedge was removed from t h e m e d i a l s u r f a c e of
B e g i n n i n g on
t h e l e f t submandibular gland.
t h e t h i r d day a f t e r t h e o p e r a t i o n , t h e a n i m a l s
were i n j e c t e d I P w i t h I S 0 (16 mglkg) i n s a l i n e
Control
o r TP (100 u g l a n i m a l ) i n sesame o i l .
g r o u p s were g i v e n i n j e c t i o n s of e i t h e r s a l i n e
o r sesame o i l .
A p p r o p r i a t e e x p e r i m e n t a l and
c o n t r o l a n i m a l s were k i l l e d on 5 , 1 0 , 1 5 , 20,
2 5 and 30 d a y s p o s t o p e r a t i o n . S u b m a n d i b u l a r
g l a n d s were removed, f i x e d i n f o n a l i n and proc e s s e d f o r l i g h t m i c r o s c o p i c e x a m i n a t i o n . One
thousand a c i n a r c e l l s were c o u n t e d i n two
a r e a s o f t h e o p e r a t e d g l a n d , and t h e number of
m i t o t i c f i g u r e s was r e c o r d e d . A t t h e wound
s u r f a c e , m i t o s e s of a c i n a r c e l l s i n s a l i n e i n j e c t e d c o n t r o l s were h i g h t h r o u g h 1 0 d a y s .
A f t e r I S 0 o r sesame o i l , m i t o t i c a c t i v i t y on
t h e wound s u r f a c e d e c l i n e d s h a r p l y and remained n e a r z e r o from day 10 t h r o u g h day 30. I n
c o n t r a s t a f t e r TP, t h e r e is d drop i n m i t o t i c
a c t i v i t y a t day 5 i n comparison t o s a l i n e cont r o l s b u t t h e n a s h a r p r i s e a t day 1 0 f o l l o w e d
by a d e c l i n e t o n e a r z e r o t h r o u g h day 30.
Only a f t e r I S 0 was a b u r s t o f m i t o s e s of a c i n a r
c e l l s observed i n t h e remainder of t h e gland.
I t a p p e a r s t h a t sesame o i l and I S 0 s u p p r e s s
t h e induced m i t o s e s on t h e c u t s u r f a c e , wherea s , a f t e r TP a marked d e p r e s s i o n on day 5 i s
f o l l o w e d by an i n c r e a s e nn day 1 0 . T h i s sugg e s t s t h a t t h e s e exogenous a g e n t s s u p p r e s s t h e
normal r e g e n e r a t i v e p r o c e s s i n r a t suhmandibul a r glands.
S u p p o r t e d by N I D R G r a n t ii DE 05072 -02.
BRINN*, Jack E.; Carol SMITH* and
Carl R.MORGAN, Department o f Anatomy,
East C a r o l i n a U n i v e r s i t y School o f Medicine,
Greenville, North Carolina. F i x a t i o n o f
p a n c r e a t i c i s l e t s f o r morphometric a n a l y s i s .
Sampling o f t h e p a n c r e a t i c i s l e t s f o r
morphometric a n a l y s i s i s o f t e n a problem
because o f t h e i r uneven d i s t r i b u t i o n i n t h e
pancreas and because o f t h e d i f f e r e n t c e l l
types. For those i n t e r e s t e d i n i s l e t
morphometry a t b o t h t h e EM and LM l e v e l s , t h e
problem i s f u r t h e r complicated by t h e
necessity o f using d i f f e r e n t f i x a t i v e s f o r the
two types o f microscopy. Being convinced o f
t h e e f f i c a c y o f p e r f u s i o n f i x a t i o n we have
devised a procedure t h a t p r o v i d e s an i n i t i a l
f i x a t i o n , sampling each r e g i o n o f t h e
pancreas, and p r o c e s s i n g o f those samples f o r
b o t h LM and EM. The p e r f u s i o n c o n s i s t s o f a
v e r y b r i e f washout w i t h a balanced s a l t
s o l u t i o n f o l l o w e d by a m i x t u r e o f 4-5%
paraformaldehyde, 0.05% p i c r i c a c i d and 0.01%
t o 0.05% g l u t a r a l d e h y d e i n 0.05 M phosphate
b u f f e r f o r 3-4 minutes. Y e l l o w i n g o f t h e
pancreas by t h e p i c r i c a c i d i s i n d i c a t i v e o f
adequate p e r f u s i o n . Samples o f t i s s u e a r e
p l a c e d i n B o u i n ' s f l u i d f o r 2-3 hours f o r LM
o r i n 2.5% g l u t a r a l d e h y d e f o r EM. Subsequent
processing i s routine.
I n t h e LM p r e p a r a t i o n s
we have s u b s t i t u t e d a l c i a n b l u e f o r aldehyde
f u c h s i n t o o b t a i n a more d e l i c a t e s t a i n i n g o f
t h e beta c e l l s . A d d i t i o n a l experiments a r e
b e i n g done t o determine t h e a p p l i c a b i l i t y o f
the technique t o other tissues, p a r t i c u l a r l y
t h e hypophysis. A m a j o r advantage o f t h i s
procedure i s t h e r e d u c t i o n o f animal c o s t s by
e l i m i n a t i n g t h e n e c e s s i t y o f u s i n g separate
animals f o r LM and EM morphometry w h i l e
p r o v i d i n g good f i x a t i o n f o r b o t h l e v e l s .
BURDEN, H. W., M. L. CAPPS*, and
I . E. LAWRENCE, JR., Department o f Anatomy,
School o f Medicine, East C a r o l i n a U n i v e r s i t y .
p e r i f o l l i c u l a r t i s s u e has been demonstrated i n
numerous p h y s i o l o g i c a l and pharmacological
experiments. It has been suggested t h a t
c o n t r a c t i o n o f p e r i f o l l i c u l a r t i s s u e may a i d
i n t h e o v u l a t o r y process. The f o r m a t i o n o f
s p e c i a l i z e d j u n c t i o n s between c o n t r a c t i l e
c e l l s may p l a y a r o l e i n t h e spread o f
e l e c t r i c a l i n f o r m a t i o n between c e l l s and
f a c i 1 it a t e s y n c h r o n i z a t i o n o f c o n t r a c t i 1 it y o f
p e r i f o l l i c u l a r tissue.
I n t h e present study,
o v a r i a n p e r i f o l l i c u l a r t i s s u e was examined a t
s p e c i f i e d i n t e r v a l s p r i o r t o t h e expected t i m e
o f ovulation.
Emphasis was p l a c e d on t h e t y p e
and r e l a t i v e number o f c e l l - t o - c e l l c o n t a c t s .
Rat o v a r i a n t i s s u e was taken f o r e l e c t r o n
m i c r o s c o p i c s t u d i e s a t d i e s t r u s , a t 1500 hours
on t h e a f t e r n o o n o f p r o e s t r u s ( 0 hour group),
a t 2000 hours ( 5 h o u r group) and 0100 hours
(10 hours group). Three t y p e s o f j u n c t i o n s
were observed between c e l l u l a r components i n
t h e theca e x t e r n a : c l o s e c o n t a c t s ,
i n t e r m e d i a t e j u n c t i o n s , and gap j u n c t i o n s .
A l l groups s t u d i e d e x h i b i t e d c l o s e c o n t a c t s
and i n t e r m e d i a t e j u n c t i o n s .
The number and
l e n g t h o f i n t e r m e d i a t e j u n c t i o n s was increased
i n t h e p r e o v u l a t o r y p e r i o d as compared t o t h e
d i e s t r u s p e r i o d . The average l e n g t h o f c l o s e
c o n t a c t s was g r e a t e r a t 5 and 10 hours than a t
d i e s t r u s o r 0 hours. There was an abrupt
i n c r e a s e i n t h e number o f gap j u n c t i o n s a t 10
hours. These o b s e r v a t i o n s p r o v i d e
m o r p h o l o g i c a l evidence f o r a dynamic r o l e o f
p e r i f o l l i c u l a r tissue i n the ovulatory
process.
"Supported i n p a r t by USPHS Grant
No. HO 06899.
CHIBUZO, G . A. AND J . F. CUMMINGS
Department of Anatomy, S c h o o l o f V e t e r i n a r y
M e d i c i n e , Tuskegee I n s t i t u t e , Alabama 36088
L i n g u a l P r o p r i o c e p t i o n : a n enzyme t r a c e r s t u d y .
I t h a s b e e n assumed t h a t t h e t o n g u e , a n o r g a n
of d i v e r s e and r e f i n e d movements would b e endowed w i t h a n e x t e n s i v e p r o p r i o c e p t i v e i n n e r v a t i o n . However, t h e r e e x i s t s no c o n c l u s i v e
e x p e r i m e n t a l e v i d e n c e f o r t h e s o u r c e of s e n s o r y
( p r o p r i o c e p t i v e ) i n n e r v a t i o n of t h e t o n g u e musc u l a t u r e . Although some a u t h o r s r e p o r t e d t h e
p r e s e n c e of l i n g u a l m u s c l e s p i n d l e s ( p r o p r i o r e c e p t o r s ) i n some a n i m a l s , o t h e r i n v e s t i g a t o r s
a r g u e d t h a t a t l e a s t i n some a n i m a l s l i n g u a l
p r o p r i o c e p t i o n was s u b s e r v e d by r e c e p t o r s
o t h e r t h a n m u s c l e s p i n d l e s . U s i n g t h e method
of induced c h r o m a t o l y s i s a c c o m p l i s h e d by select i v e c r a n i a l nerve neurectomies, d i f f e r e n t inv e s t i g a t o r s suggested t h e mesencephalic n u c l e u s
of t r i g e m i n a l n e r v e , i n t r a h y p o g l o s s a l n e r v e
g a n g l i a , d i s t a l v a g a l ( n o d o s a l ) g a n g l i a and
c e r v i c a l s p i n a l g a n g l i a as p o s s i b l e s o u r c e s f o r
lingual proprioceptive innervation.
The r e s u l t b e i n g p r e s e n t e d h e r e r e l i e d on h o r s e r a d i s h p e r o x i d a s e r e t r o g r a d e l a b e l i n g of s e n s o r y
n e u r o n s t o t h e e x t r i n s i c l i n g u a l m u s c l e s . The
enzyme (30% s o l u t i o n ) was i n j e c t e d i n microl i t e r amounts i n t o t h e g e n i o g l o s s u s , s t y l o g l o s s u s and h y o g l o s s u s i n v e r y young d o g s . Twentyfour t o forty-eight hours post injection, the
d o g s were e u t h a n a t i z e d and p e r f u s e d t r a n s c a r d i a l l y w i t h a Karnovsky-type f i x a t i v e . S e v e r a l
c r a n i a l nerve g a n g l i a , c e r v i c a l s p i n a l g a n g l i a ,
t h e b r a i n stems and h y p o g l o s s a l n e r v e s were
collected, s e r i a l l y sectioned at 60Nm with
f r e e z i n g s l i d i n g microtome and p r o c e s s e d a c c o r d i n g t o t h e method of Nauta e t a l . ( B r a i n Res.
67 ( 1 9 7 4 ) : 219). H o r s e r a d i s h p e r o x i d a s e rea c t i o n p r o d u c t s were demonstrated histochemic a l l y i n t h e i p s i l a t e r a l upper c e r v i c a l s p i n a l ,
p r o x i m a l v a g a l ( j u g u l a r ) and t r i g e m i n a l g a n g l i a .
The r e s u l t t h e r e f o r e c o n t r a d i c t e d t h e suggest i o n t h a t t h e mesencephalic n u c l e u s of trigemin a l nerve, t h e intrahypoglossal nerve ganglia
and t h e d i s t a - l v a g a l ( n o d o s a l ) g a n g l i a c o n t a i n ed t h e l i n g u a l p r o p r i o c e p t i v e n e u r o n a l somata.
T h i s r e s e a r c h was s u p p o r t e d by N I H G r a n t 15F34GMO6013-02.
CLARK, George, V e t e r a n s A d m i n i s t r a t i o n M e d i c a l
C e n t e r ; and Department o f Anatomy, M e d i c a l
U n i v e r s i t y of S o u t h C a r o l i n a , C h a r l e s t o n ,
South C a r o l i n a .
Biological
Commis s i on.
The
Stain
P r i o r t o World War I t h e Gi-iibler l a b e l was
a c c e p t e d as q u a l i t y a s s u r a n c e f o r b i o l o g i c a l
d y e s . The w a r and i t s a f t e r m a t h e l i m i n a t e d
t h i s s o u r c e . I n t h i s c o u n t r y a domestic dye
i n d u s t r y d e v e l o p e d . T h e i r p r o d u c t s were o f t e n
q u i t e i n f e r i o r and sometimes m i s l a b e l e d . A
committee h e a d e d b y H . J . Conn found t h a t some
s a m p l e s w e r e f u l l y e q u a l t o t h e pre-war d y e s s o
t h e problem c o u l d h e s o l v e d b y s e l e c t i o n . Out
o f t h i s t h e B i o l o g i c a l S t a i n Commission
f i n a l l y developed.
P r o c e d u r e s were d e v i s e d
f o r c h a r a c t e r i z a t i o n o f many of t h e commonly
used dyes. A system of l a b e l s , i d e n t i f y i n g
good h a t c h e s w a s d e v e l o p e d .
I would l i k e t o
summarize t h e c u r r e n t work o f t h e B i o l o g i c a l
S t a i n Commission. F o r some dyes c h e m i c a l
t e s t s a r e a d e q u a t e h u t many a r e a l s o t e s t e d
as s t a i n s . No c h e m i c a l t e s t s h a v e b e e n d e v i s e d
f o r h e m a t o x y l i n , t h e most c o m n l y u s e d d y e , s o
i t i s t e s t e d s o l e l y as a s t a i n .
Fifty-six
O t h e r s t a i n s are now a v a i l a b l e a s c e r t i f i e d
s t a i n s . The Comnission c h a r g e s a s i n g l e f e e
f o r e a c h b a t c h o f dye t e s t e d and a n o t h e r f o r
t h e c e r t i f i c a t i o n l a b e l s which are a f f i x e d t o
each b o t t l e of c e r t i f i e d dyes s o l d b y t h e
v a r i o u s s u p p l i e r s . I n t h e p a s t few y e a r s l a b e l
sales h a v e d e c r e a s e d . T h i s , i f c o n t i n u e d ,
w i l l s e r i o u s l y l i m i t t h e work of t h e
Commission. T h i s l o s s i n l a b e l sales is
p r o b a b l y d u e t o t h e i n c r e a s e d sale of comm e r c i a l l y prepared s t a i n i n g s o l u t i o n s . I n
a d d i t i o n t o t h e r e g u l a r membership t h e r e are
representatives of various societies; Arthur
L a V e l l e i s t h e r e p r e s e n t a t i v e from t h e
American A s s o c i a t i o n of A n a t o m i s t s .
Trustees
a r e e l e c t e d f o r terms o f t h r e e y e a r s .
“ P a r t i a l l y supported by t h e Veterans
A d m i n i s t r a t i o n and by t h e S t a t e Appropriation
f o r Biomedical Research t o t h e Medical
Un i v e rs i t y of S o u t h C a ro 1 i n a
.
C O L E , T . B . , J R . and SCHWEISTHAL, M . H . ,
Department o f Andtomy, O r a l R o b e r t s Univ e r s i t y , T u l s a , Oklahoma.
A c h a l a s i a : H i s t o l o g i c a l f i n d i n g s of a
case study.
Gross a n a t o m i c a l f i n d i n g s on n e c r o p s y
o f an 8 5 - y e a r - o l d male w i t h s e v e r e d r h a l a s i a have heen r e p o r t e d i n a n acrompariying
abstract.
H i s t o l o g i c a l examination of t h e
d i l a t e d e s o p h a g u s was c o n d u c t e d i n o r d r r t o
i n v e s t i g a t e t h i s c a s e more t h o r o u g h l y .
Approximately twenty t i s s u e samples t a k e n
from t h e w a l l o f t h e e s o p h a g e a l d i l a t i o i i
and a d j a c e n t r e g i o n s were c o l l e c t e d f o r
examination.
The t i s s u e s p e c i m e n s were
p r o c e s s e d and o b s e r v a t i o n s made employing
r o u t i n e l a b o r a t o r y procedures. Examination
of the t i s s u e s e c t i o n s r l e a r l y demonstrated
t h e f o u r g e n e r a l l a y e r s of t h e a l i m e n t a r y
tract.
The
innermost
mucosa
appeared
normal
and
displayed
non-keratinizing
s t r a t i f i e d squamous e p i t h e l i u m which o c c a s i o n a l l y appeared thickened.
Lymphocyte
i n f i l t r a t i o n of t h e mucosal and submucosal
l a y e r s was ronunoii. S o l i t a r y and a g g r e g a t e d
l y m p h a t i c n o d u l e s were o b s e r v e d i n t h e
submucosa. Specimens t a k e n from t h e openi n g t o t h e d i l a t e d esophagus and t h e t r a n s v e r s e r i d g e p r o j e c t i n g i n t o t h e esophageal
lumen
demonstrated
a
thick
muscularis
externa
composed
of
numerous
skeletal
muscle f a s c i c u l i .
T h i s t h i c k e n e d muscularis
externa
represents
the
cricopharyngeus,
t h u s confirming g r o s s anatomical
observations.
Relative t o
the
c r a n i a l end o f t h e d i l a t e d e s o p h a g u s , t h e
m u s c u l a r i s e x t e r n a appeared t h i n i n t h e
r e m a i n i n g a r e a s examined. T h i s t h i n n i n g o f
t h e m u s c u l a r l a y e r mdy b r i n r e s p o n s e t o
d i l a t i o n of
t h e esophagus.
The o u t e r
advaritit i a l l a y e r of t h e esophagus appeared
n o r m a l . The h i s t o l o g i c d l f i n d i n g s s u p p o r t
the c o n r l u s i o n t h a t t h e tipper e s o p h a g e a l
s p h i n c t e r p l a y e d a n i m p o r t a n l r o l e 111 t h i s
r a s e of a t h a l d s i a .
~
293
COWDEN, RONALD R. AND S.K. CURTIS, DEPARTMENT
OF BIOPHYSICS, COLLEGE OF MEDICINE, EAST
TENNESSEE STATE UNIVERSITY, JOHNSON CITY,
TENNESSEE 37614
Microfluorometric Comparison of DNA Specific
Fluorochromes
A series of six dyes, putatively selective f o r
DNA under certain conditions, were compared
using isolated mouse thymic lymphocyte and
hepatocyte nuclei. The list included:
mithramycin (M) (G-C specific), Hoechst 33258
(H), D.A.P.I., proflavine (PR), quinacrine
mustard (QM) (all A-T specific), and propidium iodide (PI) (double-stranded nucleic
acid specific). Since PI fluorochromes
double-stranded RNA and DNA, these preparations were pretreated with RNase.
All the methods used, except QM, produced
values for thymus lymphocyte nuclei and 2c
hepatocytes that were within the C.V. f o r
the measurement. In general, QM values for
thymocyte nuclei were greater than those for
2c hepatocytes, and values for 4c and 8c
hepatocyte nuclei were lower than the anticipated values. While pretreatment with RNase
reduced PI values by ca. lo%, the reduction
with M and particularly with PR and QM were
substantially greater than would be expected
from known proportions of DNA:RNA. RNase
appears to interfere with either binding or
fluorescence emissions when used with these
dyes.
CURRY*, T. E., JR., C. A. HODSON*,
D. E. D. JONES*, P. D. MOZLEY*, M. L. LAPPS*,
and I . E. LAWRENCE, JR. ( I n t r o d u c e d by
H. W. Burden) Departments o f Anatomy and
O b s t e t r i c s and Gynecology, School o f
Medicine, East C a r o l i n a U n i v e r s i t y ,
G r e e n v i l l e , North C a r o l i n a . A m o r p h o l o g i c a l
s t u d y o f t h e human o v a r i a n ligament.
I n t h e human female, t h e o v a r y i s
attached t o t h e l a t e r a l angle o f t h e u t e r u s ,
p o s t e r o - i n f e r i o r t o t h e u t e r i n e tube, b y a
round c o r d termed t h e o v a r i a n l i g a m e n t . The
o v a r i a n ligament has been s t u d i e d p r e v i o u s l y
w i t h h i s t o l o g i c a l techniques and has been
r e p o r t e d t o be a h i g h l y v a s c u l a r f i b r o muscular s t r u c t u r e , r i c h l y s u p p l i e d w i t h
nerve elements (Mahran e t a l . , 1971,
Obstet. Gynec., 3 7 : 7 1 1 ) 7 5 t h e p r e s e n t
study, t h e f i n e s t r u c t u r e o f t h e human o v a r i a n
ligament was i n v e s t i g a t e d w i t h t r a n s m i s s i o n
e l e c t r o n microscopy. Emphasis was placed on
c h a r a c t e r i z a t i o n o f t h e muscular and n e u r a l
components i n t h e ligament. Specimens were
o b t a i n e d by hysterosalpingectomy from women o f
c h i l d b e a r i n g age. T i s s u e was f i x e d i n
phosphate b u f f e r e d g l u t a r a l d e h y d e paraformaldehyde, s t a i n e d en b l o c w i t h u r a n y l
a c e t a t e , sectioned, and s t a i n e d w i t h lead
c i t r a t e b e f o r e examination. The o v a r i a n
ligament c o n s i s t s o f smooth muscle c e l l s and
f i b r o b l a s t s d i s p e r s e d i n a collagenous m a t r i x .
Close c o n t a c t s and i n t e r m e d i a t e j u n c t i o n s were
observed between a d j a c e n t smooth muscle c e l l s .
Large nerves embedded i n t h e collagenous
m a t r i x c o n t a i n e d b o t h m y e l i n a t e d and
nonmyelinated axons. These nerves may p l a y a
r o l e i n f u n c t i o n a l r e l a t i o n s h i p s between t h e
o v a r y and u t e r u s . Furthermore, j u n c t i o n a l
complexes between smooth muscle c e l l s i n t h e
o v a r i a n l i g a m e n t may c o o r d i n a t e muscular
contractions i n c e r t a i n reproductive
processes.
DA\’ENPORT, W i l l i a m D., J r . and C a r r o l l R. B a l l ,
Department o f Anatomy, U n i v e r s i t y o f M i s s i s s i p p i
Medical Center, Jackson, M i s s i s s i p p i .
E n d o t h e l i a l damage by h i g h f a t - l o w p r o t e i n d i e t
seen by scannin e l e c t r o n microscopy.
Mice f e d a’semipurified,
high f a t (lard)
low p r o t e i n ( c a s e i n ) d i e t show an e x t r e m e l y
h i g h i n c i d e n c e o f a t r i a l thrombosis. A f t e r
s i x weeks o f d i e t f e e d i n g , e x p e r i m e n t a l animals
were p a i r k i l l e d w i t h c o n t r o l s , a t weekly i n t e r v a l s . Mice which d i e d a t i r r e g u l a r i n t e r v a l s
were a l s o s t u d i e d .
The e n d o t h e l i a l s u r f a c e o f a t r i a was
examined by scanning e l e c t r o n microscopy (SEM).
The f i r s t s i g n s o f damage were prominent n u c l e a r
b u l g i n g and a l t e r a t i o n s i n t h e rhomboidal shape
o f t h e e n d o t h e l i a l c e l l s . More severe damage
i n c l u d e d c r a t e r - l i k e h o l e s i n t h e cytoplasm o f
e n d o t h e l i a l c e l l s w i t h exposure o f sub-endot h e l i a l t i s s u e t o which p l a t e l e t s w i t h pseudop o d i a were adhering. S i m i l a r i n j u r y t o endot h e l i a l c e l l s has been r e p o r t e d when l e t h a l
doses o f f a t t y a c i d s were i n j e c t e d i n t o r a b b i t
e a r veins. T h i s a l t e r e d e n d o t h e l i a l i n t e g r i t y
probably underlies the s u r c e p t i b i l i t y o f these
s i t e s t o formation o f diet-induced thrombotic
lesions.
-
Supported by NIH Biomedial Research Support
Grant #5-S07 RR05386)
D i a n i , A r t h u r R. and D. Davis*, Department o f
Anatomy, E a s t C a r o l i n a U n i v e r s i t y School o f
M e d i c i n e and Department o f B i o l o g y , East
Carolina University. A l t e r a t i o n o f Caliber
Spectra and Numerical D e n s i t y A s s E i a t e d t h
Nerve F i b e r s m y m p a t h e t i c Trunk o f The
K e t o n u r i c D i a b e t~
i c Chi nese Hamster
~
~.
_
M y e l i n a t e d and unmyelinated f i b e r s from
t h e sympathetic t r u n k (ST) o f 7 k e t o n u r i c d i a b matched none t i c Chinese hamsters and 7 age
d i a b e t i c s were q u a n t i t a t i v e l y analyzed t o det e r m i n e frequency d i s t r i b u t i o n and numerical
d e n s i t y . Animals were p e r f u s e d w i t h Karnovs k y ’ s f i x a t i v e and each ST was embedded i n
Durcupan. For m y e l i n a t e d f i b e r s , s e r i a l c r o s s
s e c t i o n s were c u t a t 1 pm and randomly s e l e c t ed t o e l i m i n a t e o b s e r v e r b i a s . F a s c i c l e s o f
ST were photographed and combined i n t o photomontages (875X). A p l a s t i c g r i d w i t h 16 u n i form s e c t o r s was p l a c e d o v e r t h e geometric cent e r o f each f a s c i c l e . E x t e r n a l diameter and
d e n s i t y o f m y e l i n a t e d axons w i t h i n 2 randomly
s e l e c t e d s e c t o r s were measured. F o r unmyelina t e d f i b e r s , s e r i a l c r o s s s e c t i o n s were c u t a t
60-90 um and randomly s e l e c t e d f o r a n a l y s i s .
Every t h i r d g r i d space ( w i t h nervous t i s s u e )
was photographed (1400X) i n an e l e c t r o n m i c r o scope u n t i l each f a s c i c l e was sampled. Diam e t e r and numerical d e n s i t y o f a l l unmyelina t e d f i b e r s i n each p r i n t (5DOOX) was recorded.
-___-
-
294
M y e l i n a t e d and unmyelinated f i b e r s i n d i a b e t i c hamsters d i s p l a y e d a s i g n i f i c a n t reduct i o n i n d i a m e t e r which was r e l a t e d t o p r o g r e d i e n t k e t o n u r i a . Mean numerical d e n s i t y
o f m y e l i n a t e d f i b e r s was s i g n i f i c a n t l y r e duced whereas t h a t o f unmyelinated f i b e r s was
increased. The a l t e r a t i o n s i n sympathetic
nerve p o p u l a t i o n s a r e b e l i e v e d t o be a manif e s t a t i o n o f p r e v i o u s l y observed d e m y e l i n a t i o n
and " d y i n g back" neuropathy. On t h e b a s i s o f
axon diameter, i t appeared t h a t b o t h v i s c e r a l
a f f e r e n t and e f f e r e n t f i b e r s were p a t h o l o g i c .
These d a t a s t r o n g l y i m p l y t h a t neuropathy i n
t h e sympathetic t r u n k o f t h e d i a b e t i c Chinese
hamster may be a c r i t i c a l f a c t o r u n d e r l y i n g
g a s t r o i n t e s t i n a l dysfunction.
Supported by Upjohn Company g r a n t and
N I H g r a n t AM-21933-02.
W . J . DDUGHERTY and S. I . COLEMAN, Department
o f Anatomy, Medical U n i v e r s i t y o f South
C a r o l i n a , Charleston, SC 29403. E f f e c t s o f
hypophysectomy and growth hormone o r t h y r o x i n e
replacement on t h e occurrence and c a l c i f i a b i l it.y o f m a t r i x v e s i c l e s i n r a t t i b i a l epiphyses.
Young female Long-Evans r a t s were hypophysectomized o r hypophysectomized p l u s t h y r o p a r a t h y r o i d e c t o m i z e d , h e l d f o r 14 days, and
then i n j e c t e d w i t h normal s a l i n e , growth
hormone, o r L - t h y r o x i n e t w i c e d a i l y f o r 2 o r 4
days. Proximal t i b i a e were prepared f o r
a n a l y s i s by l i g h t and e l e c t r o n microscopy a t
t h e t e r m i n a t i o n o f these t r e a t m e n t s . I n
a d d i t i o n t o measuring whole body w e i g h t s and
t h e w i d t h s o f t i b i a l e p i p h s e s , t h e number of
m a t r i x v e s i c l e s p e r 100 un o f e x t r a c e l l u l a r
m a t r i x , t h e percentage o f m a t r i x v e s i c l e s
c o n t a i n i n g m i n e r a l c r y s t a l l i t e s and t o t a l
serum Ca l e v e l s i n each o f t h e c o n t r o l and
e x p e r i m e n t a l groups o f animals were recorded.
Hypophysectomi zed animals l o s t w e i g h t and
showed a s i g n i f i c a n t decrease i n t h e w i d t h s o f
t h e epiphyseal p l a t e s . Serum Ca l e v e l s r e mained normal. While t h e r e a r e a p p r o x i m a t e l y 276
m a t r i x v e s i c l e s p e r 100 urn2 i n b o t h c o n t r o l
and hypophysectomized animals, hypophysectom i r e d animals e x h i b i t e d 50% fewer m a t r i x
v e s i c l e s c o n t a i n i n g m i n e r a l c r y s t a l l i t e s than
c o n t r o l animals. Replacement o f 0.68 I . U . r a t
growth hormone produced an i n c r e a s e i n t h e
number o f m a t r i x v e s i c l e s w i t h c r y s t a l l i t e s
t h a t was 124% g r e a t e r than i n hypophysectomized
animals and 24% g r e a t e r than i n c o n t r o l s . Thyr o x i n e replacement i n hypophysectomized animals
r e s t o r e d t h e number o f m a t r i x v e s i c l e s w i t h
c r y s t a l l i t e s approximately t o control levels.
Thus, w h i l e t h e numbers o f m a t r i x v e s i c l e s p e r
u n i t area a r e n o t a f f e c t e d by hypophysectomy o r
hormone replacement, t h e c a l c i f i a b i l i t y o f
m a t r i x v e s i c l e s i n v i v o i s a f f e c t e d by b o t h
growth hormone and t h y r o x i n e .
3
DYESS, * E r i c M.and W. D. Davenport, J r . ,
Department o f Anatomy, U n i v e r s i t y o f M i s s i s s i p p i
Medical Center, Jackson, M i s s i s s i D o i .
E f f e c t s o f d i f f e r e n t f u c h s i n s on t h e
a l d e h d e - f u c h s i n method f o r t h e d e m o n s t r a t i o n
o f e1;stic
siute.
--
_____
A m o d i f i c a t i o n o f t h e Cameron and S t e e l e
Method f o r t h e i d e n t i f i c a t i o n o f n e u r o s e c r e t o r y
substance (1959) was i n t r o d u c e d by Davenport
(1979) as a polychrome technique f o r t h e i d e n t i f i c a t i o n o f t i s s u e types. T h i s m o d i f i c a t i o n was
f u r t h e r discussed as a q u a l i t a t i v e technique by
Davenport and B a l l (1979).
The s p e c i f i c component o f t h i s technique i s
t h e p r i m a r y s t a i n , namely a l d e h y d e - f u c h s i n , which
i s used f o r t h e i d e n t i f i c a t i o n o f e l a s t i c t i s s u e .
As w i t h a l l o t h e r techniques employing aldehydef u c h s i n , a major concern has been which i s t h e
b e s t f u c h s i n t o use and a l s o , what i s t h e
p r o b a b l e mechanism o f attachment o f t h e aldehydef u c h s i n complex t o t h e e l a s t i c t i s s u e .
I n the
p r e s e n t study, t h r e e f u c h s i n s were used:
r o s a n i l i n , p a r a r o s a n i l i n , and magenta 111 (new
f u c h s i n 111). Q u a l i t a t i v e l y , t h e paraldehydep a r a r o s a n i l i n was observed t o be t h e most
s p e c i f i c f o r e l a s t i c t i s s u e as w e l l as g i v i n g
t h e most i n t e n s e s t a i n i n g r e a c t i o n .
These d a t a a l s o p r o v i d e d some s u p p o r t f o r
t h e [mechanism o f a c t i o n o f t h e paraldehydepararosanilin i n attaching t o the e l a s t i c tissue;
namely, t h e i n t e g r i t y o f t h e resonance s t r u c t u r e
r e s p o n s i b l e f o r t h e r e a c t i v e n e s s o f t h e azo(1979)
methines as proposed by Buehner
seems t o be p o s i t i v e l y a f f e c t e d by t h e s p e c i f i c
c o m b i n a t i o n o f p a r a r o s a n i l i n and paraldehyde.
The more s t r u c t u r a l l y complex f u c h s i n s ,
r o s a n i l i n and new f u c h s i n 111, p o s s i b l y cause a
d e s t a b i l i z a t i o n o f t h e carbonium i o n o r perhaps
i n t r o d u c e t h e problem o f s t e r i c hindrance i n t h e
i n t e r a c t i o n o f t h e azomethines w i t h t i s s u e
aldehydes.
st
T h i s work was supported by N I H Biomedical
Support Grant f5-SO7-RR05386.
Biomechanics of Two Joint Muscles in the
Human Lower Extremity. ROBERT M. FISK,
JAMES P. WELLS, West Virginia School of
Osteopathic Medicine
The biomechanical significance o f t w o - ~ o l n t
muscles has remained an unanswered question in
the literature. The two-joint muscles o f the
human thigh provide several interesting models
for such a mechanical system because o f their
close association with single-joint muscles
which have similar functional roles; furthermore, these two-joint muscles normally participate in both stereotyped, phasic movements
(during locomotion) and in special, non-phasic
movements. A series o f muscle function experiments with male and female subjects and representing a range of body types were initiated
to determine the mechanical role o f these
muscles in a variety of different movements.
The experiments ernplayed radiotelemetered EMG,
in conjunction with indwelling (fine wire)
electrodes to determine muscle activity.
Correlative kinematic data were recorded with
standard video techniques. The subjects performed normal locomotor and postural activities and, in addition, worked against various
resistances to elicit differential activity
and/or phasic patterning between one- and twojoint muscles. It has been proposed that
two-joint muscles have evolved to meet require
ments necessary for moving three limb segments
s i m u l t a n e o u s l y d u r i n g p h a s i c locomotor a c t i v i t i e s ; however, t h e t w o - j o i n t muscles o f t h e
t h i g h are r a r e l y c a l l e d upon t o do t h i s
d u r i n g locomotion. I n f a c t , t h e unique b i p e d a l s t a n c e o f man and t h e important r o l e o f
i n e r t i a l f o r c e s i n human locomotion b r i n g
about a r e l a t i v e l y low l e v e l of a c t i v i t y i n
t h e s e muscles d u r i n g such p h a s i c movements.
Our s t u d y demonstrates t h a t t h e f u n c t i o n a l
d i f f e r e n c e s between one- and t w o - j o i n t
muscles appear d u r i n g non-locomotor of t h e
lower l i m b . We conclude from t h e s e EMG d a t a
and from comparative morphological s t u d i e s
t h a t t h e p r e s e n c e o f t w o - j o i n t muscles i n t h e
human t h i g h i s due p r i m a r i l y t o mechanical
needs i n t h e a n c e s t r a l t y p e s .
S.D. Gaede and S.L. Q u a t t r o p a n i , Departments of
Anatomy, U n i v e r s i t y of V i r g i n i a , C h a r l o t t e s v i l l e ,
V i r g i n i a and Medical C o l l e g e of V i r g i n i a ,
Richmond, V i r g i n i a . E n d o t h e l i a l m i t o s i s i n t h e
immature gonadotrophin-stimul a t e d r a t o v a r y .
The m i t o t i c a c t i v i t y o f vasclrlar endothelium
i n t h e ovary was examined i n normal and gonadot r o p h i n - s t i m u l a t e d immature r a t s u s i n g 1 i g h t
microscopy and a u t o r a d i o g r a p h y . The l a b e l i n g of
e n d o t h e l i a l n u c l e i with t r i t i a t e d thymidine was
u t i l i z e d a s an index o f v a s c u l a r growth. A n i mals r e c e i v e d an i n t r a p e r i t o n e a l i n j e c t i o n of
thymidine 20 m i n u t e s p r i o r t o r e t r o g r a d e a l d e hyde p e r f u s i o n v i a t h e a o r t a . Twenty-one day o l d
r a t s were a d m i n i s t e r e d a s i n g l e i n j e c t i o n o f PMS.
An i n j e c t i o n of HCG 56 hours l a t e r induced sup e r o v u l a t i o n by 17 hours a f t e r t r e a t m e n t (method of France and Pincus, ‘ 6 4 ) . Four groups were
s t u d i e d : 1 ) 28 hours a f t e r t h e PMS i n j e c t i o n ,
2 ) 4 h o u r s , 3 ) 17 hours, and 4 ) 24 hours a f t e r
HCG i n j e c t i o n . Twenty-one and twenty-four day
o l d animals which had n o t r e c e i v e d i n j e c t i o n s o f
hormones were g i v e n s i m i l a r dosages of thymid i n e and examined a s c o n t r o l s .
The p e r c e n t a g e s of l a b e l e d endothel i a l c e l l s
v a r i e d according t o t h e r e g i o n o r t i s s u e and t h e
s t a g e of d i f f e r e n t i a t i o n of t h a t r e g i o n o r t i s sue. Stromal v e s s e l s were g e n e r a l l y l e s s heavily
l a b e l e d than t h e c a l v e s s e l s . Thecal v e s s e l s s u r rounding l a r g e heal thy f o l l i c l e s were more heavi l y l a b e l e d t h a n surrounding s m a l l e r o r a t r e t i c
f o l l i c l e s . The h e a v i e s t l a b e l i n g was seen i n the
v e s s e l s surrounding r u p t u r e d f o l l i c l e s and w i t h i n developing c o r p o r a l u t e a found i n t h e PMSHCG t r e a t e d o v a r i e s .
Labeling i n d i c e s o f t h e endothelium in t h e
PMS-treated o v a r i e s were s i g n i f i c a n t l y e l e v a t e d
in a l l regions cmpared t o the control ovaries.
This r e s u l t was n o t a l t e r e d by time o r HCG
treatment.
These r e s u l t s i n d i c a t e t h a t endothel i a l mitos i s i s a s e l e c t i v e r e s p o n s e occuring i n s p e c i a lized regions of the ovary. I t i s suggested t h a t
t h e gonadotrophic s t i m u l i promote t h e production
o f f a c t o r s which e l i c i t such a r e s p o n s e .
GARRIS, David R., J. FAIR*, and D. S.
WHITEHEAD*, Department o f Anatomy, East
Carolina U n i v e r s i t y School o f Medicine,
27834. A l t e r a t i o n s i n
Greenville, N. C.
u t e r i n e blood f l o w d u r i n g deciduoma f o n n a t i o n
7 n t h e pseudopregnant r a t .
The a l t e r a t i o n s i n u t e r i n e blood f l o w
(UBF) occurring d u r i n g deciduoma (DT)
formation i n t h e r a t were studied t o d e l i n e a t e
t h e e x i s t i n g developmental r e l a t i o n s h i p s
between c e l l u l a r d i f f e r e n t i a t i o n and t i s s u e
vascularization.
DT-formation was induced i n
pseudopregnant (PSP: Day 0 = o v u l a t i o n ) r a t s
on Day 4 by a k n i f e scratch along t h e
antimesometrial border o f each u t e r i n e horn.
Sham-operated animals served as c o n t r o l s (C).
Between Days 4-15 o f PSP. r a t s were prepared
f o r UBF measurements by s u r g i c a l exposure o f
each u t e r i n e artery. Blood f l o w was measured
w i t h an e l e c t m a g n e t i c blood f l o w monitor and
analyzed f o r mean f l o w r a t e (ml/min).
The
u t e r i were subsequently removed, cleaned,
b l o t t e d and weighed t o t h e nearest mg.
U t e r i n e weight remained constant a t basal
l e v e l s (127-167 mg/horn) i n c o n t r o l r a t s
throughout PSP. I n contrast, r a t s subjected
t o u t e r i n e trauma e x h i b i t e d a s i g n i f i c a n t
increase (p < 0.001) i n u t e r i n e weight (Day 9:
1440 v s 138 mg/horn; DT vs C) as a r e s u l t o f
t h e DT fonnation between Days 5 and 9 o f PSP.
S i m i l a r l y , UBF remained at constant basal
l e v e l s (0.5-0.8 ml/min) i n c o n t r o l r a t s d u r i n g
PSP. DT-bearing r a t s e x h i b i t e d a dramatic
r i s e i n UBF between Days 4-7, followed by a
p l a t e a u i n UBF l e v e l s (3-4.5 m l / m i n ) on Days
7-9.
Between Days 9 and 15, both u t e r i n e
weight and UBF i n DT r a t s f e l l t o basal
l e v e l s . The r e s u l t s o f t h i s study i n d i c a t e
t h a t UBF increases i n response t o induced
d e c i d u a l i z a t i o n d u r i n g PSP and t h a t an
increase i n UBF preceeds t h e period o f maximal
s t romal d i f f e r e n t i a t i o n . These r e s u l t s
suggest t h a t an inTrease i n t i s s u e
v a s c u l a r i z a t i o n i s mandatory f o r t h e support
o f c e l l u l a r d i f f e r e n t i a t i o n i n t h e r a t uterus.
(Supported i n p a r t by a Grant fm t h e North
Carol ina United Way).
GASSLER”, John H . and Margaret L . KIRBY, D e partment o f Anatomy, Medical C o l l e g e o f
Georgia, A u g u s t a Georgia. E l e c t r i c a l recordi n g o f embryonic c h i c k m o t i l i t y .
T h e s t u d y o f a v i a n embryonic m o t i l i t y
t r a d i t i o n a l l y has u s e d t h e t e c h n i q u e of w i n d o w i n g eggs and d i r e c t l y o b s e r v i n g movement.
Shortcomings w h i c h may a f f e c t t h e r e l i a b i l i t y
of t h i s technique are:
i t involves a major
m o d i f i c a t i o n o f t h e embryo‘s environment, i t
i s i m p o s s i b l e t o o b s e r v e t h e e n t i r e embryo
a t a l l times, and i t i s d i f f i c u l t t o m a i n t a i n
a n o b j e c t i v e s t a n d a r d f o r movement among d i f f e r e n t observers. T h i s r e p o r t presents t h e
u s e o f a m o d i f i c a t i o n of a n e l e c t r i c a l recordi n g t e c h n i q u e ( J a c k s o n and R u b e l , J . Comp.
P h y s . P s y c h . , 1978) t o q u a n t i t a t i v e l y d e s c r i b e
m o t i l i t y during t h e last h a l f of incubation
i n t h e normal c h i c k embryo.
F e r t i l i z e d White Leghorn e g g s f r o m 11 t o
1 9 d a y s o f incubation i n c l u s i v e l y w e r e candled
t o d e t e r m i n e t h e p o s i t i o n o f t h e e m b r y o . Rec o r d i n g e l e c t r o d e s were i n s e r t e d and s e c u r e d
180° a p a r t on t h e e g g s . Eggs younger t h a n 14
d a y s were windowed, and t h e amnion c u t , t o
p r e v e n t amnionic c o n t r a c t i o n a r t i f a c t .
Eggs
were r e t u r n e d t o t h e i n c u b a t o r and c o n n e c t e d
t o a polygraph.
A f t e r 45 m i n u t e s o f a c c l i m a t i z a t i o n , 1 5 minute r e c o r d i n g s of m o t i l i t y
296
were made from f i v e e g g s on e a c h d a y . Recordi n g s w e r e a n a l y z e d and t h e t i m e s p e n t i n
m o t i l i t y was c a l c u l a t e d .
M o t i l i t y i n c r e a s e d to day 1 3 , p l a t e a u e d on
d a y s 1 4 and 1 5 , and s t e a d i l y d e c l i n e d from day
1 6 onward. Random (Type I ) and s t a r t l e (Type
1 1 ) movements w e r e n o t e d t h r o u g h day 1 5 . On
d a y 1 6 o c c a s i o n a l p r e h a t c h i n g (Type 111)
b u r s t s o f a c t i v i t y were o b s e r v e d between Types
I and I1 movements. From d a y 1 6 o n , Type 1
and I1 a c t i v i t y d e c r e a s e d a s t i m e s p e n t i n
Type 111 m o t i l i t y p a t t e r n s i n c r e a s e d .
These f i n d i n g s c o r r e l a t e w i t h d i r e c t obs e r v a t i o n s of embryonic c h i c k s made by o t h e r
i n v e s t i g a t o r s . This technique provides an
o b j e c t i v e , a c c u r a t e method o f q u a n t i t a t i n g
t o t a l s o m a t i c m o t i l i t y i n a v i a n embryos.
It
c a n b e used t o e v a l u a t e e f f e c t s of experiment a l p r o c e d u r e s o n embryonic a v i a n m o t i l i t y .
GULATI, A . K., L a b o r a t o r y of N e u r o c h e m i s t r y ,
NINCDS, N a t i o n a l I n s t i t u t e s o f H e a l t h , B e t h e s d a
Maryland and Department o f Anatomy, West V i r g i n i a U n i v e r s i t y , West V i r g i n i a . Macromolecular
c h a n g e s i n t h e n e u r a l r e t i n a d u r i n g r-egenerat i o n of a l e n s .
There i s e x t e n s i v e evidence t h a t n e u r a l
r e t i n a s t i m u l a t e s l e n s r e g e n e r a t i o n i n newts.
The p r e s e n t s t u d y was d e s i g n e d t o i n v e s t i g a t e
m a c r o m o l e c u l a r (RNA and p r o t e i n ) c h a n g e s i n
Incort h e n e u r a l r t i n a a f t e r l e n removal.
p o r a t i o n of 'H-uridine
and H - l e u c i n e was u s e d
as a n i n d e x t o e v a l u a t e RNA and p r o t e i n synthesis.
An i n i t i a l i n c r e a s e i n 3H-uridine u p t a k e
was s e e n o n e t o t h r e e d a y s a f t e r l e n t e c t o m y .
T h i s was f o l l o w e d by g r e a t e r i n c o r p o r a t i o n o f
H - l e u c i n e between day 5 t o 15. To f u r t h e r
c h a r a c t e r i z e p r o t e i n changes,
s o l u b l e prot e i n s of n e u r a l r e t i n a from n o r m a l eyes and
e y e s 1 2 d a y s a f t e r l e n t e c t o m y were a n a l y z e d by
SDS ( 0 . 2 % ) p o l y a c r y l a m i d e (7.5%) g e l e l e c t r o p h o r e s i s . Coomassie b l u e s t a i n i n g r e s o l v e d
a b o u t 30 b a n d s , among t h e s e t h e r e w e r e 1 6
prominent bands w i t h a p p a r e n t molecular
w e i g h t s r a n g i n g from 1 8 , 5 0 0 t o 1 8 5 , 0 0 0 . The
banding p a t t e r n of t h e n e u r a l r e t i n a w a s s i m i l a r i n normal e y e s and i n e y e s 1 2 d a y s a f t e r
lentectomy. Synthesis of s o l u b l e p r o t e i n s i n
n e u r a l r e t i n a were a l s o s t u d i e d by s l i c i n g
j i s c g e l s and by a u t o r a d i o g r a p h y on s l a b g e l s .
H - l e u c i n e and 5S-methionine were i n j e c t e d
i n t r a p e r i t o n e a l l y t h r e e h o u r s p r i o r to t i s s u e
p r e p a r a t i o n f o r g e l s l i c i n g and a u t o r a d i o g r a p h y
r e s p e c t i v e l y . Both d i s c and s l a b g e l s rev e a l e d a b o u t 11 r a d i o a c t i v e b a n d s . The
p a t t e r n o f s y n t h e s i s of t h e s o l u b l e p r o t e i n s
i n n e u r a l r e t i n a by b o t h e l e c t r o p h o r e t i c proc e d u r e s was q u i t e s i m i l a r . L i q u i d s c i n t i l l a t i o n c o u n t i n g on g e l s l i c e s showed some d i f f e r e n c e s i n t h e n e u r a l r e t i n a o b t a i n e d from normal
e y e s as compared t o l e n t e c t o m i z e d e y e s . Howe v e r , s u c h d i f f e r e n c e s were n o t s e e n w i t h
a u t o r a d i o g r a p h y . The p r e s e n t r e s u l t s demons t r a t e s i g n i f i c a n t c h a n g e s i n RNA and p r o t e i n
s y n t h e s i s i n n e u r a l r e t i n a d u r i n g l e n s regeneration.
These c h a n g e s may h e i n d i r e c t l y rel a t e d t o t h e production of n e u r a l r e t i n a l fact o r , although t h e p r e c i s e r e l a t i o n s h i p needs
f u r t h e r i n v e s t i g a t i o n . E l e c t r o p h o r e s i s of
soluble proteins indicates that neural
r e t i n a l f a c t o r ( i f a s o l u b l e protein) is
p r e s e n t i n normal, u n s t i m u l a t e d r e t i n a .
3
HIGHTOWER, James A . . Department of Anatomy,
S c h o o l of M e d i c i n e , U n i v e r s i t y of S o u t h Carol i n a , Columbia, S o u t h C a r o l i n a . The e f f e c t s
of s h a r k serum on DNA r e p l i c a t i o n i n murine
lymphocytes.
jlhole s h a r k serum d e m o n s t r a t e s a n t i n e o p l a s t i c p r o p e r t i e s , h u t t h e mechanism(s) by which
i t i n d u c e s tumor r e m i s s i o n is ( a r e ) unknown.
However, d u r i n g t h e p a s t s e v e r a l y e a r s a number
o f i n t e r e s t i n g r e p o r t s have encouraged f u r t h e r
s t u d y of t h i s q u e s t i o n . One i n v e s t i g a t i o n ,
f o r example, h a s d e m o n s t r a t e d t h a t n u r s e s h a r k
serum p o s s e s s e s a l e c t i n - l i k e m o l e c u l e c a l l e d
f r u c t o s a n - s p e c i f i c p r o t e i n (FSP). The f u n c t i o n
o f t h i s p r o t e i n i n v i v o and i n v i t r o i s unc e r t a i n , b u t workers have demonstrated t h a t
o t h e r ( p l a n t ) l e c t i n s can (1) be c y t o t o x i c f o r
c e r t a i n tumor c e l l s , ( 2 ) a c t i v a t e immunocompet e n t c e l l s and ( 3 ) s t i m u l a t e t h e r e t i c u l o e n d o t h e l i a l s y s t e m . Whether FSP p o s s e s s e s any of
t h e s e b i o l o g i c a l p r o p e r t i e s i s n o t known.
Hence, t h i s i n v i t r o s t u d y w a s u n d e r t a k e n t o
d e t e r m i n e t h e r o l e of whole n u r s e s h a r k serum
and FSP on a c t i v a t i o n o f irmnunocompetent c e l l s
a s a s s a y e d by DNA r e p l i c a t i o n i n m u r i n e thymic
and s p l e n i c lymphocytes.
Results indicate that
growth p r o m o t i n g a g e n t s , e v e n though l e s s e f f e c t i v e than t h o s e i n f e t a l c a l f s e r a , a r e pres e n t i n t h e s e r a of n u r s e s h a r k s . The a b i l i t y
of b o t h mammalian and c h o n d r i c h t h i a n s e r a t o
s u p p o r t m u r i n e s p l e n i c and thymic lymphocyte
p r o l i f e r a t i o n induced by c o n c a n a v a l i n A stimul a t i o n s u g g e s t s t h a t t h e mechanism by which
serum promotes growth of immunocompetent c e l l s
a r o s e e a r l y p h y l o g e n e t i c a l l y and h a s b e e n cons e r v e d . FSP was p r e c i p i t a t e d by t r e a t i n g n u r s e
s h a r k serum w i t h a n o p t i m a l amount of i c v a n P-6.
The p r e c i p i t a t e d complexes were s o l u b i l i z e d ,
t h e n chromatographed on Bio-gel P-150. P r o t e i n
c o n t e n t h e r e was done v i a t h e Lowry a s s a y u s i n g
b o v i n e serum a l b u m i n (BSA) a s a s t a n d a r d .
D i f f e r i n g l o t s of s h a r k serum y i e l d e d 90-18Opg
FSP/ml serum.
Preliminary d a t a suggest t h a t
FSP i s m i t o g e n i c f o r m u r i n e s p l e n i c l y m p h o c y t e s ,
b u t n o t thymic lymphocytes.
H i n t o n , David E . , E l i z a b e t h R . Walker, and John
H . S t r o h l . D e p a r t m e n t s of Anatomy and Chemistry,
West V i r g i n i a U n i v e r s i t y , Morgantown. West
V i r g i n i a . Manganese-induced a l t e r a t i o n i - n - g i l l
o f f a t h e a d minnow ( P i m e p h a l e s p r o m e l a s , R a f i n e s q u e ) : A l i g h t and e l e c t r o n m i c r o s c o p i c s t u d y .
U n t r e a t e d w a t e r from s t r i p mine s i t e s i n West
V i r g i n i a c o n t a i n e d h i g h l e v e l s of manganese ( M A ?
maximum 83 mg/L) which were n o t s i g n i f i c a n t l y
r e d u c e d by t r e a t m e n t w i t h a l k a l i n e s o l u t i o n s ( i .
e . , s o d a a s h . l i m e s t o n e , l i m e , or c a u s t i c s o d a ) .
have been recorded i n t h e
L e v e l s of 3 ppm M*+
Monongahela R i v e r and c o n c e n t r a t i o n s o f 10-20ipm
a r e n o t uncommon i n a m b i e n t waterways o f c o a l p r o d u c i n g r e g i o n s . S i n c e l i t t l e i n f o r m a t i o n was
a v a i l a b l e on t i s s u e e f f e c t s o f Mn++ i n t e l e o s t s ,
t h i s a c u t e s t u d y ( 9 6 h r s ) w a s p e r f o r m e d t o det e r m i n e s t r u c t u r a l e f f e c t s o f Mn++ O n g i l l s o f
t h e f a t h e a d minnow. Twenty randomly s e l e c t e d
f i s h ( 5 p e r e a c h of 4 g l a s s a q u a r i a ) were p l a c e d
i n w a t e r C o n t a i n i n g Mn++ as MnS04.H20 a t a f i n a l
c o n c e n t r a t i o n o f 0 , 10, 5 0 , o r 7 5 ppm. Mrt+conc e n t r a t i o n a t 96 h r s was w i t h i n 10% of t h e i n i t i a l c o n c e n t r a t i o n . Following f i x a t i o n (4% g l u t a r a l d e h y d e i n 200 mOSm p h o s p h a t e b u f f e r ) tiss u e s were p r o c e s s e d f o r s c a n n i n g (SEMI andtrans-
-
m i s s i o n e l e c t r o n microscopy a s w e l l as h i g h res o l u t i o n l i g h t microscopy (HRLM). SEM of g i l l s
from a l l f i s h exposed t o Mn++ showed a l t e r a t i o n
which i n c l u d e d : f o c a l a b s e n c e o f s e c o n d a r y l a m e l l a e ; d i s r u p t i o n o f m i c r o r i d g e p a t t e r n on p r i mary and s x o n d a r y l a m e l l a e ; and h i g h a m p l i t u d e
s w e l l i n g o f i n d i v i d u a l c e l l s on secondary l a mellae.
Changes on b u c c o p h a r y n g e a l s u r f a c e of
g i l l a r c h p s i n c l u d e d d i s r u p t i o n of m i c r o r i d g e
p a t t e r n and a p p a r e n t s w e l l i n g o f t a s t e b u d s .
HRLM r e v e a l e d r o u n d e d , p a l e c e l l s w i t h one or
more l a r g e v a c u o l e s which formed b u l g e s v i s i b l e
a t s u r f a c e s o f p r i m a r y and s e c o n d a r y l a m e l l a e .
I n a d d i t i o n , f o c i o f p r i m a r y l a m e l l a e showed
edema and c e l l d e g e n e r a t i o n i n s e c o n d a r y lame l l a r s i t e s which produced a smoother o u t e r cont o u r on p r i m a r y l a m e l l a e w i t h no s u r f a c e i n d i c a t i o n o f normal s e c o n d a r y l a m e l l a r p a t t e r n . T h i s
n o v e l l e s i o n , a p p a r e n t l y u n i q u e t o Mn++, may b e
u s e f u l o n f i e l d e v a l u a t i o n o f f e r a l f i s h e s exposed t? mine w a t e r s .
( S u p p o r t e d by O f f i c e of Water Research and Techn o l o g y , U.S. Department o f I n t , o r i o r . P r o j e c t
A-037-W.Va.).
H o l t , R . K . and G . S . S o h a l . Department o f
Anatomy, M e d i c a l C o l l e g e of C e o r e i a , A u g u s t a ,
t o r s have been p o s t u l a t e d t o d e s c r i b e t h e
manner i n which numerous d i f f e r e n t i a t e d
c h a r a c t e r i s t i c s of a d u l t s k e l e t a l m u s c l e a r e
m a i n t a i n e d . The r o l e o f i n n e r v a t i o n on t h e
embryonic development o f s k e l e t a l m u s c l e , howe v e r , i s p o o r l y documented.
The p r e s e n t s t u d y
was u n d e r t a k e n t o d e t e r m i n e t h e r e l a t i v e i m p o r t a n c e of m u s c l e a c t i v i t y and n e u r n t r o p h i c
f a c t o r s o n t h e d i f f e r e n t i a t i o n and m a i n t e n a n c e
of s k e l e t a l m u s c l e . Normal, u n i n n e r v a t e d and
p a r a l y z e d s u p e r i o r o b l i q u e m u s c l e s of w h i t e
P e k i n g duck embryos were u l t r a s t r u c t u r a l l y
e v a l u a t e d t h r o u g h o u t development w i t h t h e
a s s u m p t i o n t h a t normal m u s c l e d e v e l o p s u n d e r
che i n f l u e n c e o f b o t h nerve-evoked m u s c l e
a c t i v i t y and t r o p h i c f a c t o r s , t h e u n i n n e r v a t e d under n e i t h e r , and t h e p a r a l y z e d o n l y
under t h e t r o p h i c f a c t o r s r e l e a s e d by t h e
p r e s y n a p t i c n e r v e t e r m i n a l s . F o r embryos w i t h
u n i n n e r v a t e d m u s c l e s t h e t r o c h l e a r n u c l e u s was
d e s t r o y e d o n day 7 of i n c u b a t i o n , 3 d a y s p r i o r
t o normal i n n e r v a t i o n . P a r a l y s i s was produced
i n a n o t h e r g r o u p by d a i l y a p p l i c a t i o n of t h e
p o s t s y n a p t i c blocking a g e n t a-bungarotoxin t o
t h e v a s c u l a r i z e d c h o r i o a l l a n t o i c membrane from
d a y 10 onwards. Both t h e p a r a l y z e d and u n i n n e r v a t e d g r o u p s d i f f e r e d from normal i n t h a t
development was r e t a r d e d .
I n b o t h groups
f u s i o n o f m y o b l a s t s t o form myotubes was
d e l a y e d , m y o f i l a m e n t s and 2 l i n e s were o l t e n
d i s o r g a n i z e d , and l i p i d i n c l u s i o n s , f r e e
r i b o s o m e s , and c e l l d e a t h were f r e q u e n t l y
seen. C e l l s could n o t d i f f e r e n t i a t e p a s t t h e
myotube s t a g e i n u n i n n e r v a t e d m u s c l e w h e r e a s
many were a b l e t o p r o g r e s s t o t h e m y o f i b e r
s t a g e i n p a r a l y z e d muscle.
Reginning o n day
21 o r 2 2 a m a s s i v e wave o f c e l l d e a t h f o l l o w e d
by c o n n e c t i v e t i s s u e r e p l a c e m e n t o c c u r r e d i n
uninnervated muscles h u t not i n paralyzed
muscles.
These o b s e r v a t i o n s s u g g e s t t h a t b o t h
m u s c l e a c t i v i t y and t r o p h i c f a c t o r s a r e n e c e s -
s a r y f o r t h e normal development o f t h e m u s c l e .
T r o p h i c f a c t o r s a p p e a r t o p l a y a more c r u c i a l
r o l e than muscle a c t i v i t y .
( S u p p o r t e d by a
g r a n t from t h e ? l u s c u l a r D y s t r o p h y A s s o c i a t i o n
and by a g r a n t from NIH).
Kinney, F . C l e v e l a n d , Department of Anatomy
U n i v e r s i t y o f Alabama i n Birmingham. BirminPham, Alabama.
C i r c l i n g B e h a v i o r and Pathol o g i c a l Sleep i n the Cat.
P a t h o l o g i c a l s l e e p and c i r c l i n e" b e h a v i o r
have been observed i n a s e r i e s of experiments
f o l l o w i n g t h e a b l a t i o n o f o r i r r i t a t i o n of
d i f f e r e n t l e v e l s of t h e c e n t r a l n e r v o u s s y s t e m
i n the cat.
I n a s e r i e s of b i l a t e r a l s t e r e o t a x i c , e l e c t r o l y t i c l e s i o n s of t h e p o s t e r i o r
h y p o t h a l a m u s , i m m e d i a t e l y d o r s a l and l a t e r a l
t o t h e mamillary b o d i e s , p a t h o l o g i c a l s l e e p
( n a r c o l e p s y ) was o b s e r v e d . In o n e a n i m a l
a c a t a l e p t i c l i k e s t a t e coupled w i t h narcolepsy
was s t r i k i n g l y a p p a r e n t . I n a n o t h e r a n i m a l
d e c e r e b r a t e r i g i d i t y and coma r e s u l t e d from
a bilateral stereotaxic, electrolytic lesion
o f t h e p o s t e r i o r h y p o t h a l a m i c a r e a which i n c i d e n t a l l y involved t h e b a s i l a r a r t e r y . This
c a t was u n r e s p o n s i v e t o p a i n and l a c k e d a c o r n e a l response.
Following p e r i o d s of patholog i c a l s l e e p , c i r c l i n g b e h a v i o r was o b s e r v e d
from b i l a t e r a l l e s i o n s of t h e p o s t e r i o r hypot h a l a m u s which e x t e n d e d i n t o t h e o v e r l y i n g
v e n t r a l thalamus.
Using p e n i c i l l i n a s a n i r r i t a n t , c i r c l i n g
b e h a v i o r h a s been o b s e r v e d from i r r i t a t i v e
l e s i o n s o f t h e f r o n t a l and i n s u l a r c o r t i c a l
a r e a s i n t h e c a t , t h e d i r e c t i o n o f r o t a t i o n bei n g d e p e n d e n t upon t h e p a r t i c u l a r c o r t i c a l a r e a
involved. P e n i c i l l i n f i l l e d p e l l e t s placed over
t h e head o f t h e c a u d a t e n u c l e u s h a v e a l s o r e s u l t e d i n s e i z u r e s d u r i n g which t h e a n i m a l s
c i r c l e d toward t h e s i d e o f t h e i r r i t a t i o n . Oft e n t h e s e i z u r e s became so v i o l e n t t h a t t h e an i m a l s f e l l o v e r backward.
Immediately followi n g t h e s e s e i z u r e s s l e e p was o b s e r v e d which
l a s t e d f o r a s h o r t p e r i o d of two t o t h r e e minutes.
I n summary, c i r c l i n g b e h a v i o r h a s b e e n obs e r v e d from i r r i t a t i o n of v a r i o u s c o r t i c a l a r e a s , from i r r i t a t i v e l e s i o n s of t h e
h e a d of t h e c a u d a t e n u c l e u s and f o l l o w i n g des t r u c t i v e l e s i o n s which i n v o l v e d t h e p o s t e r i o r
h y p o t h a l a m u s and t h e v e n t r a l t h a l a m u s .
Sleep
h a s been o b s e r v e d f o l l o w i n g s t i m u l a t i o n ( i r r i t a t i o n ) o f t h e head o f t h e c a u d a t e n u c l e u s and
following d e s t r u c t i v e l e s i o n s of the p o s t e r i o r
hypo t h a l a m u s .
V
I
0
~~
L a n t z , R . C l a r k and David E . H i n t o n , Department
of Anatomy, West V i r g i n i a l l n i v e r s i t y , Morgantow
W.V.
A l t e r a t i o n o f l u n g s t . r u c t u r e by p a r t i c u l a t e e f f l u e n t s from f l u i d i z e d bed c o a l combus__
t i o n : A rnorphometric s t u d x .
F l u i d i z e d bed c o a l c o m b u s t i o n ( F B C ) , a r e c e n t l y d e v e l o p e d p r o c e s s f o r u t i l i z a t i o n of s u l f u r c o n t a i n i n g c o a l , i s b e i n g d e v e l o p e d f o r cumrnercia1 application. Particulate effluent (fly&)
from t h i s p r o c e s s had d i f f e r e n t p h y s i c a l and
chemical p r o p e r t i e s than conventional f l y a s h .
We a r e c o n d u c t i n g s t u d i e s on t h e pulmonary toxi c i t y o f FBC p a r t i c u l a t e s and p r e s e n t i n i t i a l
f i n d i n g s i n t h i s r e p o r t . Baghouse f i l t e r - c o l -
l e c t e d f l y a s h from t h e MERC 18" a t m o s p h e r i c
FBC b u r n i n g Montana Rosebud s u b - b i t u m i n o u s c o a l
was suspended i n s a l i n e ( 1 5 m g / m l ) and i n t r a t r a c h e a l l y i n s t i l l e d i n t o male S y r i a n g o l d e n
h a m s t e r s ( 3 mg f l y a s h i n 0 . 2 m l s u s p e n s i o n v o l ume). C o n t r o l s r e c e i v e d e q u a l volumes of s a l i n e
s o l u t i o n a l o n e . Exposures were g i v e n once/wk
for 4 wks f o l l o w e d by A w k s p o s t e x p o s u r e p r i o r
t o k i l l i n g . Airways and l u n g s were f i x e d &
s i t u by i n t r a t r a c h e a l p e r f u s i o n of 3% g l u t a r a l dehyde i n 0.1 m p h o s p h a t e b u f f e r a t 20 cm H z O
p r e s s u r e for 1 h r . T i s s u e s were embedded i n
Epon and s e m i t h i n s e c t i o n s ( 1 ~ )were s t a i n e d
w i t h t o l u i d i n e b l u e for h i g h r e s o l u t i o n l i g h t
m i c r o s c o p y . S t a n d a r d morphometric t e c h n i q u e s
were u s e d t o d e t e r m i n e volume d e n s i t i e s o f a l v e o l a r d u c t s and s a c s and parenchymal t i s s u e .
Two f i e l d s p e r s e c t i o n , one s e c t i o n p e r l o b e
were s e l e c t e d a t random from p r o c e s s e d t i s s u e s .
T o t a l f i e l d s a n a l y z e d were 32 for c o n t r o l s and
28 f o r experimental animals. N o s i g n i f i c a n t
d i f f e r e n c e s were found between volume d e n s i t y of
a l v e o l i o r a l v e o l a r d u c t s and s a c s i n c o n t r o l
and e x p e r i m e n t a l a n i m a l s . However, volume dens i t y o f parenchymal t i s s u e ( V v t ) w a s s i g n i f i c a n t l y i n c r e a s e d i n e x p e r i m e n t a l a n i m a l s ( V v cont r o l = 1 7 . 8 % + 6 . 8 ; e x p e r i m e n t a l = 2 2 . 4 + 8 . 9 P<O . 6 5 ) ,
T h i s may b e i n d i c a t i v e of f i b r o s i s , a l v e o l a r
pneumonocyte p r o l i f e r a t i o n o r b o t h . F u r t h e r
t e s t i n g i s [under way t o d e t e r m i n e t h e n a t u r e bf
t h e i n c r e a s e i n parenchymal t i s s u e volume densi t y .
S u p p o r t e d by Depart.ment o f Energy c o n t r a c t
number DE-AT21-79 MC 1 1 2 3 A .
-
L I E B , Richard J., Robert L . JORDAN and J .
S t u a r t OGLESBY, Department o f Anatomy, Medical
C o l l e g e o f V i r g i n i a , V i r g i n i a Commonwealth
U n i v e r s i t y , Richmond, V A 23298. E f f e c t s o f
p u l s e d 27.12 MHz e l e c t r o m a g n e t i c r a d i a t i o n on
p r e n a t a l development i n t h e r a t .
Recent r e p o r t s i n t h e l i t e r a t u r e have
d e s c r i b e d t h e t e r a t o l o g i c a l e f f e c t s o f noni o n i z i n g radiofrequency r a d i a t i o n i n the r a t
(Ann. N . Y . Acad. S c i . , 2 4 7 ~ 3 6 7 , 1975;
T e r a t o l o g y , E : 3 6 A , 1 9 7 r These e f f e c t s have
been a t t r i b u t e d t o thermal i n s u l t caused by
the i r r a d i a t i o n . The p r e s e n t i n v e s t i g a t i o n
u t i l i z e d a p u l s e d r a d i a t i o n source which d i d
n o t produce a p p r e c i a b l e temperature e l e v a t i o n
i n t h e e x p e r i m e n t a l animals. A t o t a l o f 110
pregnant Sprague-Dawley r a t s were d i v i d e d i n t o
two c o n t r o l ( n a i v e and sham i r r a d i a t e d ) and
n i n e experimental groups. The sham c o n t r o l and
t h e experimental animals were i r r a d i a t e d f o r
20 minlday on days 5 t o 15 o f g e s t a t i o n w i t h a
dose o f 0 (sham c o n t r o l ) t o 4.9 mW/gm o f
n o n i o n i z i n g i r r a d i a t i o n . C o n t r o l and e x p e r i mental animals were examined f o r b o t h s k e l e t a l
and v i s c e r a l d e f e c t s . A n a l y s i s o f t e r a t o l o g i c a l
data by means o f t h e Chi-square d i s t r i b u t i o n
w i t h Yates' c o r r e c t i o n f o r c o n t i n u i t y r e v e a l e d
no 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 c o n t r o l and e x p e r i m e n t a l animals. The
r e s u l t s o f t h i s s t u d y s u p p o r t t h e hypothesis
o f p r e v i o u s i n v e s t i g a t o r s t h a t ma1 f o r m a t i o n s
observed i n o f f s p r i n g o f animals t r e a t e d w i t h
27.12 MHz n o n i o n i z i n g i r r a d i a t i o n a r e t h e
r e s u l t o f thermal i n s u l t t o t h e d e v e l o p i n g
embryo and n o t a "frequency window" e f f e c t .
Supported, i n p a r t , by an A.D. W i l l i a m s
Summer Research F e l l o w s h i o .
MARK, Marvin R . , Cathy P e n n i n g t o n and Mohamed
Sharawy. S u r f a c e F e a t u r e s o f t h e Kat P a r o t i d
Gland. X e d i c a l C o l l e g e of G e o r g i a S c h o o l o f
D e n t i s t r y , Au$usta, G e o r g i a .
The s u r f a c e morphology of t h e r a t p a r o t i d
g l a n d was s t u d i e d i n t h e s c a n n i n g e l e c t r o n m i croscope following e i t h e r microdissection o r
e n z y m a t i c d i g e s t i o n w i t h c o l l a g e n s e and o t h e r
enzymes. Examination o f d i s s e c t e d f r e s h t i s s u e c o n f i r m e d t h e o r g a n i z a t i o n of t h e g l a n d
a s c o n c e p t u a l i z e d by c l a s s i c a l h i s t o l o g i c a l
methods, v i z . , a n e l a b o r a t e s y s t e m of branchi n g d u c t s w i t h g r a p e - l i k e t e r m i n a l a c i n i . The
number of c e l l s composing a n a c i n u s v a r i e d
w i d e l y from a s few a s 6 t o a s many a s a b o u t 20.
I n t i s s u e p r e p a r e d by d i s s e c t i o n a l o n e , a c i n a r
c e l l s were c o v e r e d by a f e l t w o r k of d e l i c a t e
c o l l a g e n f i b e r s . The f i b e r s t e n d e d t o f i l l
i n t h e s p a c e between t h e b a s a l s u r f a c e s o f adjacent cells.
Acinar c e l l s p r e s e n t e d s e v e r a l
d i s t i n c t i v e s u r f a c e f e a t u r e s . The c e l l s had
a t e x t u r e d s u r f a c e t h a t was o r g a n i z e d i n
p a r a l l e l KOWS g i v i n g a s c a l l o p e d a p p e a r a n c e .
P a r a l l e l t o t h e s e rows a c e l l was o f t e n s e e n
t o he d i v i d e d i n t o h e m i s p h e r e s by a s i n g l e
c l e f t . ?lost c e l l s were a l s o p u n c t u a t e d by
s e v e r a l c r a t e r s on t h e o r d e r o f 1 wn. A t highe r m a g n i f i c a t i o n a l a r g e number of v e r y s m a l l
p o r e l i k e s t r u c t u r e s was a l s o s e e n .
The i n t e r l o b u l a r d u c t s c o u l d n o t be
i d e n t i f i e d w i t h c e r t a i n t y by s u r f a c e morphology a l o n e .
I n g e n e r a l , however, d u c t s
had a much smoother s u r f a c e t h a n t h e a c i n a r
cells.
O c c a s i o n a l l y a d u c t was s e e n unassociated with acinar c e l l s . tecminatine in
a t u f t o f f i n g e r - l i k e p r o j e c t i o n s . Small
a r t e r i o l e s were o f t e n i d e n t i f i a b l e by t h e p r e sence of p e r i c y t e s d i s t r i b u t e d a t r e g u l a r
i n t e r v a l s along t h e s u r f a c e . Following
enzymatic treatment a c i n a r c e l l s appeared
a u i t e d i f f e r e n t from t h e i r u n t r e a t e d c o u n t e r parts.
B l e b s 0.5 t o 1 . 0 Um were p r e s e n t on
t h e m a j o r i t y of t h e t r e a t e d c e l l s .
The
c r a t e r s a l s o became more n o t i c a b l e , b u t were
l o c a l i z e d between :he b l e b s . The d i s t r i b u t i o n
of b l e b s a p p e a r e d q u i t e s i m i l a r t o t h a t s e e n
i n t r a n s m i s s i o n e l e c t r o n m i c r o g r a p h s of s i m i l a r l y t r e a t e d t i s s u e . These o b s e r v a t i o n s sugg e s t t h a t enzymatic d i g e s t i o n might c r e a t e c e l l
s u r face a r t i f a c t s .
ElcDONALD*, A l e x a n d e r J . , Department of Anatomy,
U n i v e r s i t y o f S o u t h C a r o l i n a S c h o o l of M e d i c i n e ,
Columbia, S o u t h C a r o l i n a . ( I n t r o d u c e d by W i l l i a m
L. P o t e a t ) C y t o a r c h i t e c t u r e of t h e b a s o l a t e r a l
amygdala: a G o l g i s t u d y i n t h e r a t .
The amygdala, a t e l e n c e p h a l i c n u c l e a r comp l e x i n v o l v e d i n b e h a v i o r and e m o t i o n , h a s been
t h e s u b j e c t of numerous a n a t o m i c a l , b i o c h e m i c a l ,
p h a r m a c o l o g i c a l , p h y s i o l o g i c a l , and b e h a v i o r a l
i n v e s t i g a t i o n s . S i n c e t h e r a t i s t h e a n i m a l most
o f t e n u t i l i z e d i n s u c h e x p e r i m e n t s , i t is of
i n t e r e s t t o d e t e r m i n e t h e c y t o a r c h i t e c t u r e of
t h e amygdala i n t h i s s p e c i e s . T h i s s t u d y i s
b a s e d on o b s e r v a t i o n s of t h e b a s o l a t e r a l p o r t i o n
of t h e amygdala i n t w e n t y rat b r a i n s s t a i n e d
w i t h t h e Golgi-Kopsch t e c h n i q u e and s i x b r a i n s
s t a i n e d w i t h t h e r a p i d Golgi technique. Three
major n e u r o n a l c l a s s e s a r e recognized i n t h e
b a s o l a t e r a l amygdhla. C l a s s I c e l l s , t h e main
p r o j e c t i o n n e u r o n s , a r e p y r a m i d a l OK s t e l l a t e
n e u r o n s whose d e n d r i t e s d i s p l a y a d e n s e c o v e r i n g
of s p i n e s . In many c a s e s t h e axon h i l l o c k a n d / o r
i n i t i a l segment may a l s o have s p i n e s . Axons g i v e
o f f a f e w beaded c o l l a t e r a l s and c o u r s e i n t h e
d i r e c t i o n of t h e main e f f e r e n t pathways of t h e
amygdala. C l a s s I1 neurons a r e c h a r a c t e r i z e d by
s p i n e - s p a r s e d e n d r i t e s and a moderate t o dense
l o c a l axonal a r b o r i z a t i o n . ’ These neurons v a r y i n
s i z e , d e n d r i t i c branching p a t t e r n , and axonal
morphology. T h e i r axons g i v e o f f numerous beaded
c o l l a t e r a l s which a r b o r i z e and c o n t a c t d e n d r i t e s
of c l a s s I neurons. The axonal c o l l a t e r a l s o f
one v a r i e t y of c l a s s I1 neuron, t h e “amygdaloid
chandelier c e l l “ , display clustered varicosit i e s t h a t appear t o c o n t a c t axon i n i t i a l segments of c l a s s I neurons. C l a s s I11 neurons a r e
n e u r o g l i f o r m c e l l s t h a t have 7-9 s h o r t , t o r t u o u s , s p i n e - s p a r s e d e n d r i t e s t h a t branch extens i v e l y . Axons of C l a s s I11 neurons g i v e r i s e t o
numerous c o l l a t e r a l s which branch p r o f u s e l y t o
form a dense l o c a l axonal a r b o r i z a t i o n . Both
d e n d r i t e s and a x o n a l c o l l a t e r a l s c o n t a c t dendr i t e s of c l a s s I neurons. The r e s u l t s of t h i s
s t u d y i n d i c a t e t h a t t h e c y t o a r c h i t e c t u r e of t h e
r a t b a s o l a t e r a l amygdala i s b a s i c a l l y similar t o
t h a t seen i n o t h e r mammals. F u t u r e s t u d i e s w i l l
i n v e s t i g a t e v a r i o u s u l t r a s t r u c t u r a l , hodologic a l , and neurochemical a s p e c t s o f t h e b a s i c c e l l
t y p e s found i n t h e b a s o l a t e r a l amygdala. (Supp o r t e d by t h e U n i v e r s i t y of South C a r o l i n a
Grant 11040-F411).
MCKINNEY, R. V., D. E . STEFLIK, J . L. BOSHELL,
R. BAKER and E. E. SINGH. Department o f O r a l
Pathology, Medical C o l l e g e o f Georgia, Augusta
and U n i v e r s i t y o f L o u i s v i l l e , Kentucky.
umns i n s k i n and t h e r e p o r t e d presence o f spec i f i c c e l l columns i n t h e f i l i f o m p a p i l l a e o f
mouse tongue (Hume and P o t t e n , J . C e l l S c i . 2 2 :
24, 1976) has p r o v i d e d an impetus t o i n v e s t i g a t e
the s t r u c t u r a l organization o f specialized epit h e l i a such as t h e p a p i l l a e o f t h e tongue. The
purpose o f t h i s study was t o examine t h e c e l l u l a r o r g a n i z a t i o n o f t h e tongue f i l i f o r m p a p i l l a e
o f cow, dog and monkey i n o r d e r t o c o r r e l a t e t h e
p a p i l l a e m o r p h o l o g i c a l development. Dorsal
tongue fran Rhesus monkey, cow and dog was f i x e d
i n 10% n e u t r a l b u f f e r e d f o r m a l i n , embedded i n
p a r a f f i n , c u t s e r i a l l y a t 5 pm and s t a i n e d w i t h
H & E. F o r scanning e l e c t r o n microscopy (SEM),
a d d i t i o n a l t i s s u e s were f i x e d i n Karnovsky’s
paraformaldehyde-glutaraldehyde and p o s t - f i x e d
i n 1% osmic a c i d . C o r r e l a t i o n o f SEM and t h e
s e r i a l s e c t i o n s o f monkey tongue showed t h e
presence o f two major d i f f e r e n t i a t i n g . c e l l l i n e s
comprising t h e a n t e r i o r ( s o f t k e r a t i n ) and post e r i o r (hard k e r a t i n ) portions o f the papillae.
A t h i r d c e l l l i n e culminated i n the i n t e r p a p i l l a r y area. Dog p a p i l l a e e x h i b i t e d a r a t h e r cornp l e x p a p i l l a r y s t r u c t u r e w i t h two major c e l l
l i n e s , b o t h w i t h r e c o g n i z a b l e boundaries, cons t i t u t i n g t h e p r i m a r y f i l i f o r m p r o j e c t i o n . Two
l a t e r a l c e l l l l n e s gave r i s e t o secondary p r o j e c t i o n s and 5-7 c e l l l i n e s c o n s t i t u t e d s m a l l e r
t e r t i a r y p r o j e c t i o n s i n dog tongue. I n b o t h
species t h e f i l i f o r m p a p i l l a c o n s i s t e d o f cent r a l l y p l a c e d v i a b l e e p i t h e l i a l c e l l s and
k e r a t i n i z a t i o n o c c u r r e d as these c e l l s m i g r a t e d
v e r t i c a l l y and l a t e r a l l y . The cow f i l i f o r m
p a p i l l a e was unique among species s t u d i e d i n o u r
laboratory i n t h a t the external p a p i l l a r y proj e c t i o n was f u l l y d i f f e r e n t i a t e d hard k e r a t i n
analogous t o t h e h a i r c o r t e x . The hard k e r a t i n
a p p a r e n t l y d i f f e r e n t i a t e s from a dominant post e r i o r c e l l l i n e and i t i s p a r t i a l l y ensheathed
by a t h i n l a y e r o f s o f t k e r a t i n . The obvious
species v a r i a t i o n i n tongue f i l i f o m p a p i l l a e
c e l l l i n e s and t h e r e s u l t i n g s i g n i f i c a n t l y d i f f e r e n t k e r a t i n i z a t i o n p a t t e r n s and p a p i l l a r y
morphology may suggest an e v o l u t i o n a r y p a t t e r n
o f development f o r s p e c i a l i z e d use.
MONG, F . S . F . , A.P. KAYS, and A . MIRANDA, Dept.
of Anatomy, Medical C o l l e g e of V i r g i n i a ,
Richmond, V a . , and Dept. o f P a t h o l o g y , Columbia
Medical School, N Y , NY. C r e a t i n e phosphok i n a s e (CPK) isozyme t r a n s i t i o n i n regener a t i n g f r e e muscle t r a n s p l a n t s o f mice.
CPK i n a d u l t s k e l e t a l muscle i s almost
e x c l u s i v e l y MM i s o z j m e , a dimer o f two ident i c a l subunits.
I n i t i a l l y , i n embryonic
muscle, o n l y t h e b r a i n form of CPK (BB) i s
present.
But a s muscle developed t h e r e i s a
r i s e of t o t a l CPK a c t i v i t y i n a g r a d u a l t r a n s i t i o n from BB t o MM. A h y b r i d form of CPK
(MB) i s a l s o d e t e c t e d d u r i n g t h i s p e r i o d . The
purpose of t h i s i n v e s t i g a t i o n w a s t o d e t e r m i n e
whether a s i m i l a r t r a n s i t i o n i n CPK isozymes
occurred i n regenerating transplants.
Free
muscle a u t o g r a f t i n a d u l t male mice(1CR s t r a i n )
w e r e performed w i t h or w i t h o u t i n j e c t i o n of
marcaine, a myotoxin t h a t l e a d t o n e c r o s i s and
e v e n t u a l r e g e n e r a t i o n of muscle f i b e r s . Transp l a n t s and s o l e u s muscle from sham o p e r a t e d
c o n t r o l were removed on day 1 , 3 , 5 , 7 , 9 , 1 5 & 25
a f t e r s u r g e r y and p r e p a r e d f o r h i s t o l o g y o r
e l e c t r o p h o r e s i s on c e l l u l o s e a c e t a t e membrane.
I n e a r l y t r a n s p l a n t s t r e a t e d w i t h marcaine most
f i b e r s were n e c r o t i c b u t i n u n t r e a t e d t r a n s p l a n t s , muscle f i b e r s a t t h e p e r i p h e r y o f t e n
remained i n t a c t . The CPK isozymes changed as
t h e r e g e n e r a t i o n of muscle p r o g r e s s morphologic a l l y . The f e t a l BB and MB isozymes appeared
t r a n s i e n t l y on day 3 5 7
By d a
t h e CPK
w a s almost e x c l u s i v e i y ’ a c i d t , an$ ?he regener a t i n g muscle f i b e r s reached an i n t e r m e d i a t e
s t a g e of morphological d i f f e r e n t i a t i o n . The
BB & MB isozymes seemed p a r t i c u l a r l y prominent
i n marcaine i n j e c t e d t r a n s p l a n t s .
Our r e s u l t s
resembled t h o s e r e p o r t e d p r e v i o u s l y for
r e g e n e r a t i n g a d u l t muscle grown i n t i s s u e
c u l t u r e s and s u g g e s t t h a t r e g e n e r a t i n g muscles
i n v i v o r e c a p i t u l a t e t h e ontogeny of muscle
b i o c h e m i c a l l y and m o r p h o l o g i c a l l y .
ODOR, D . L . ,
P. GADDUM-ROSSE, and R . J . BLANDAU.
Department o f Anatomy, U n i v e r s i t y of South
C a r o l i n a , Columbia, South C a r o l i n a and Department of B i o l o g i c a l S t r u c t u r e , U n i v e r s i t y of
Washington, S e a t t l e , Washington.
Secretory
c e l l s o f t h e o v i d u c t o f t h e p i g - t a i l e d monkey,
Macaca n e m e s t r i n a .
The l i g h t and e l e c t r o n microscopic s t r u c t u r e o f t h e s e c r e t o r y c e l l s of t h e o v i d u c t a l
e p i t h e l i u m of t h e p i g - t a i l e d monkey has been
s t u d i e d d u r i n g d i f f e r e n t s t a g e s o f t h e menstrua l c y c l e and a f t e r ovariectomy w i t h subsequent
estrogen treatment.
During t h e e a r l y f o l l i c u l a r phase t h e e p i t h e l i u m i s r e c o v e r i n g from
t h e d e c i l i a t i o n and s e c r e t o r y c e l l a t r o p h y
t h a t occurs during the l a t e l u t e a l stage.
O c c a s i o n a l f i m b r i a l and a f a i r number o f
ampullar and i s t h m i c c e l l s e x h i b i t a f e w
e l e c t r o n d e n s e , homogeneous s e c r e t o r y
g r a n u l e s a p i c a l l y . During t h e l a t e preovulat o r y and e a r l y p o s t o v u l a t o r y p e r i o d s t h e
secretory c e l l s t r u c t u r e v a r i e s appreciably
i n d i f f e r e n t animals and i n d i f f e r e n t samples
from t h e same animal. U s u a l l y f i m b r i a l c e l l s
have a p i c a l p r o t r u s i o n s c o n t a i n i n g no o r a
few s m a l l homogeneous s e c r e t o r y g r a n u l e s .
The cytoplasm i s crowded w i t h Golgi complex
e l e m e n t s , g r a n u l a r endoplasmic r e t i c u l u m
p r o f i l e s often intimately associated with
m i t o c h o n d r i a , and v a r i a b l e numbers o f polysomes
and glycogen g r a n u l e s .
Secretory granules
and l u c e n t v a c u o l e s are more abundant i n
ampullar and i s t h m i c t h a n i n f i m b r i a l c e l l s .
1) t h o s e
The g r a n u l e s a r e of two t y p e s :
having a n e l e c t r o n d e n s e , homogeneous m a t r i x
and 2 ) t h o s e p o s s e s s i n g l a m e l l a r s t r u c t u r e s
w i t h i n moderately dense m a t r i c e s .
The l a m e l l a e
c o u r s e i n p a r a l l e l a r r a y s and e x h i b i t a
periodicity.
Homogeneous g r a n u l e s predominate
i n t h e p r e o v u l a t o r y phase and l a m e l l a r g r a n u l e s
i n t h e p o s t o v u l a t o r y p e r i o d . Two t y p e s of
v a c u o l e s occur.
During t h e l a t e l u t e a l phase
and a f t e r ovariectomy i n a l l o v i d u c t a l segments
o n l y r a r e o r no s e c r e t o r y g r a n u l e s o c c u r and
o r g a n e l l e s involved i n s e c r e t i o n are u s u a l l y
poorly developed. I n ovariectomized-estradiol
t r e a t e d monkeys some a r e a s of a l l t h r e e segments
may be w e l l s t i m u l a t e d w h i l e o t h e r s have no
g r a n u l e s and minimally developed o r g a n e l l e s .
(Supported by s u b c o n t r a c t s from t h e U n i v e r s i t y
o f Washington's USPHS g r a n t HD 03752.)
OWERS, Noel 0.
Department of Anatomy, Xedical
College of V i r g i n i a , V i r g i n i a Commonwealth
U n i v e r s i t y , Richmond, V i r g i n i a 23298.
A p o s s i b l e c o r r e l a t i o n between o p a c i t y i n a
human c a t a r a c t o u s l e n s and t h e p r e s e n c e of
acid proteases.
Three human c a t a r a c t o u s l e n s were o b t a i n e d
from p a t i e n t s , who were o t h e r w i s e normal and
healthy.
Two were c h a r a c t e r i z e d a s b e i n g
n u c l e a r c a t a r a c t s and one as a p o s t s u b c a p s u l a r
c a t a r a c t based on s l i t - l a m p o b s e r v a t i o n .
A f t e r t h e i r removal, t h e l e n s e s were f r o z e n i n
T i s s u e Tek O . C . T . compound and 8-16 li s e c t i o n s
were c u t i n a c r y o s t a t maintained between -10
and -3OOC.
The s e c t i o n s were p l a c e d on a
g e l a t i n membrane s u b s t r a t e c o n t a i n i n g I n d i a i n k
and s u p p o r t e d on a g l a s s s l i d e , p r e v i o u s l y i n s o l u b i l i z e d by t r e a t m e n t w i t h g l u t a r a l d e h y d e
( O w e r s , 1970 J . C e l l B i o l . 47:152a, h e r s and
Blandau, 1971 i n The B i o l o
of t h e B l a s t o c st
Chicago U n i v e r s i t y P r e s s ,
2 0 7 - 2 2 d '
s e c t i o n was submerged under a t h i n l a y e r of
b u f f e r and t h e s l i d e s were i n c u b a t e d i n a humid
chamber a t 23, 37, 40 and 4a0c f o r about 20
h o u r s . A few drops of w a r m g l y c e r o l j e l l y
were a p p l i e d and a g l a s s c o v e r s l i p was p l a c e d
over t h e j e l l y .
C o n t r o l s l i d e s were p r e p a r e d
by h e a t i n g t h e t i s s u e s e c t i o n i n w a t e r a t 90°C
f o r 1 0 minutes, followed by b u f f e r t r e a t m e n t
t h e same a s f o r e x p e r i m e n t a l s l i d e s . The
b u f f e r s used w e r e 30-50 nS+ KC1-HC1 uH 1 and 2.
p h o s p h a t e - c i t r i c pH 3 , v e r o n a l acetate-HC1 pH
4 , 5 , 6 , and Tris-HCl pH 7 . 5 c o n t a i n i n p
e i t h e r 1 0 mM C a and 1 f i j Mg o r 1 0 d4 EDTA.
The a r e a under t h e t i s s u e where d i g e s t i o n o f
t h e membrane o c c u r r e d appeared as l i q h t s p o t s
a g a i n s t a d a r k background.
In t h e two n u c l e a r
c a t a r a c t o u s l e n s e s , d i g e s t i o n of t h e membrane
o c c u r r e d i n t h e n u c l e a r zone a t a c i d pHs. I n
g.
t h e post subcapsular cataractous l e n s , digest i o n o c c u r r e d mostly a s s o c i a t e d w i t h t h e post e r i o r s u b c a p s u l a r t i s s u e s a l s o a t a c i d pH's.
N e u t r a l p r o t e a s e s were u b i q u i t o u s .
Plaximw?
d i g e s t i o n o c c u r r e d a t 37 and bO°C, minimum o r
none a t 23 and 4 8 O C . F u r t h e r work needs t o be
done to determine whether t h e opaque t i s s u e
i t s e l f c a u s e s t h e d i g e s t i o n of t h e membrane.
P r e l i m i n a r y t r i a l s i n d i c a t e t h a t t h e a r e a s of
d i g e s t i o n could b e q u a n t i t a t e d u s i n g an
O p t r o n i c s drum scanning m i c r o d e n s i t o m e t e r
(Model C4100).
PEPPLER, R . , T . KWASIGROCH and M . HOUGLAND,
Departments of Anatomy and O b s t e t r i c s and Gynecology, Quillen-Dishner C o l l e g e of Medicine,
East Tennessee S t a t e U n i v e r s i t y , Johnson C i t y ,
Tennessee. C r o s s - s e c t i o n a l anatomy: An e a s y
and i n e x p e n s i v e %
prepare teaching specimens.
With t h e advent of computerized a x i a l tomography (CAT) s c a n s , g r o s s anatomy c o u r s e s must
now expose medical s t u d e n t s t o c r o s s - s e c t i o n a l
anatomy.
Commercial s o u r c e s f o r such t e a c h i n g
specimens a r e n o n - e x i s t e n t and academic s o u r c e s
a v a i l a b l e w i l l s u p p l y specimens f o r as l i t t l e a s
$ 1 0 0 / s e c t i o n up t o $9-10,00O/complete body.
These c o s t s a r e p r o h i b i t i v e t o many d e p a r t m e n t s .
R e c e n t l y , we prepared 40 c r o s s - s e c t i o n s which we
used f o r a CME seminar and u l t i m a t e l y , which
w e ' l l u s e i n our t e a c h i n g programs. A t h i n ,
male cadaver w a s s e l e c t e d and c i r c u m f e r e n t i a l
measurements i n t h e head, neck, t h o r a x , abdomen,
Subsep e l v i s and e x t r e m i t i e s were o b t a i n e d .
q u e n t l y , t h i s embalmed body was f r o z e n a t -2O'C.
f o r two weeks u n t i l s e c t i o n e d .
Bids were obt a i n e d from g l a s s companies f o r t h e c o n s t r u c t i o n
of 40 r e c t a n g u l a r boxes a c c o r d i n g t o o u r measurements. The boxes were made from ?I;'' p l a t e
g l a s s , had an i n s i d e d e p t h of 1" and a 4," h o l e
was d r i l l e d i n one s i d e . The i n s i d e d i a m e t e r s
of t h e boxes v a r i e d from 7%" x 1 2 " t o 1%'x 24".
F i v e s i d e s of t h e boxes were s e a l e d w i t h GE s i l icone c l e a r rubber c a u l k (GE 2567-012) by the
g l a s s company. The specimen was removed from
t h e f r e e z e r and s e c t i o n e d i n 1" increments usi n g a commercial band saw. R e p r e s e n t a t i v e sect i o n s were r i n s e d i n running w a t e r and placed
i n t o t h e a p p r o p r i a t e s i z e d g l a s s boxes which
A
c o n t a i n e d J o r e s I1 f l u i d a s a p r e s e r v a t i v e .
c o r k ( s i z e 0 ) covered w i t h p a r a f i l m was i n s e r t ed i n t o t h e ?' h o l e . S e c t i o n s were allowed t o
thaw o v e r n i g h t and t h e p r e s e r v a t i v e w a s changed
t h e f o l l o w i n g day i n o r d e r t o g e t r i d of e x c e s s
d e b r i s . A bead of t h e GE s i l i c o n e c a u l k was
placed around t h e top edges of t h e f d u r s i d e s
and t h e g l a s s t o p was p o s i t i o n e d .
After s u f f i c i e n t d r y i n g time, t h e boxes were edged w i t h y'
f i r c o r n e r molding which was then s e a l e d w i t h a
low g l o s s tung o i l v a r n i s h . T o t a l investment
f o r t h e 40 specimens was $1200 o r $ 3 0 / s e c t i o n
n o t c o u n t i n g o u r expenses i n o b t a i n i n g t h e cad a v e r . Self-assembly of t h e g l a s s boxes would
reduce t h i s c o s t c o n s i d e r a b l y .
POOLE, M. C., H. W. BURDEN, I . E. LAWRENCE,
J r . , and C. A. HODSON*, Department o f Anatomy
and Department o f O bs te tric s and Gynecology,
School o f Medicine, East Carolina University,
Greenville, N. C.
27834. Suppression o f
Gonadotropin Secretion by Hyperprol actinemia.
301
Hyperprol a c t i nemi a ( e x c e s s i v e pro1 a c t i n )
i s known t o suppress gonadotropin s e c r e t i o n ,
and i m p a i r r e p r o d u c t i v e f u n c t i o n i n women.
The f o l l o w i n g s t u d y i n v e s t i g a t e s t h e
suppression o f s t imu 1 a t e d gonadot ropes
f o l l o w i n g ovariectomy i n r a t s b e a r i n g a
p r o l a c t i n s e c r e t i n g tumor ( F u r t h MtTW15). A
group o f c y c l i n g female W i s t a r - F u r t h r a t s were
implanted subcutaneously w i t h t h e M t T W l S
tumor. A f t e r 35 days o f tumor growth, t h e
p r o l a c t i n l e v e l s were e l e v a t e d (26.7 t 3.8
ng/ml i n d i e s t r o u s tumor f r e e animals vs.
4162
297 ng/ml i n tumor b e a r i n g r a t s ) , and
t h e animals were a c y c l i c . These r a t s were
then o v a r i e c t o m i z e d and f o l l o w i n g 7 days t h e
p i t u i t a r i e s were removed f o r e l e c t r o n
microscopy and t h e serum assayed f o r LH
( l u t e i n i z i n g hormone) and FSH ( f o l l i c l e
s t i m u l a t i n g hormone). An a d d i t i o n a l group o f
tumor f r e e 7 day o v a r i e c t o m i z e d r a t s were
used as c o n t r o l s .
The c o n c e n t r a t i o n s o f
b o t h LH and FSH were suppreksed i n t h e tumor
b e a r i n g o v a r i e c t o m i z e d animals as compared t o
t h e 7 day o v a r i e c t o m i z e d tumor f r e e r a t s
(FSH, 868 i 109 ngiml i n tumor b e a r i n g vs.
2589 +_ 155 ng/ml i n tumor f r e e ;
LH, 40.1
10.7 ng/ml i n tumor b e a r i n g vs.
377 +_ 24 ng/ml i n tumor f r e e ) .
Electron
microscopy r e v e a l e d t h a t gonadotropes i n t h e
o v a r i e c t o m i z e d tumor f r e e animals were l a r g e
c e l l s , w i t h an e x t e n s i v e network o f d i l a t e d
R E R , and a l a r g e Golgi complex. The
gonadotropes i n t h e MtTW15 b e a r i n g animals
were s m a l l e r , w i t h decreased v e s i c u l a r R E R ,
and s m a l l e r G o l g i complexes. Furthermore,
t h e r e was an apparent decrease i n t h e volume
o f m i t o c h o n d r i a . These s t u d i e s suggest t h a t
h y p e r p r o l a c t i nemia suppresses b o t h t h e
s y n t h e t i c and s e c r e t o r y mechanisms o f t h e
gonadot ropes.
+_
+_
.!u:ittroyzni, S t e v e n >. DeDartment of Anatomy,
l e d i c a l Co1lei.e of V i r g i n i a , Richmond,
. i r g i n i a , 23298. ATPase i n t h e R e t e a n d
Cystadenomas o f t h e Guinea P i g Ovary.
S e r o u s cystadenomas form s p o n t a n e o u s l y i n
a l l g u i n e a p i g o v a r i e s as a c o r r e l a t e of a g e
and r e s u l t o f d i l a t i o n o f t h e r e t e o v a r i i .
CJ-sts r e a c h a d i a m e t e r of 5 cm and a r e l i n e d
w i t h a s i m p l e e p i t h e l i u m o f c i l i a t e d and
n o n c i l i a t e d b u t no s e c r e t o r y c e l l s .
I n an
e f f o r t to d e t e r n i n e t h e method of d i l a t i o n ,
c y s t i c and p r e - c y s t i c t i s s u e s h a v e b e e n
e x m i n e d f o r .ATPase a c t i v i t y . O v a r i e s from
12 and 26 day o l d and a d u l t a n i m a l s were
aldehyde-fixed.
Vibratome s e c t i o n s ( 1 0 0 u )
ng a n i m a l s c o n t a i n i n g n o n d i l a t e d and
d i l a t e d r e t e , and c y s t f r a g m e n t s
I t s were i n c u b a t e d a t 37' f o r 20 min.
i n a ne6iis c o n t a i n i n g 1 mM ATP, 1 0 0 mM N a C l ,
1 5 d 4 KC1, 10 mN I4gSO,,: 3.6 mM Pb ( N O ) and
3% s u c r o s e i n 0.1 I 4 T r i s b u f f e r ( p H 7353.
Control incubations included e l i m i n a t i n g t h e
s u b s t r a t e o r v a r i o u s c o f a c t o r s . Following
i n c u b a t i o n t i s s u e s were washed i n b u f f e r ,
f i x e d i n osmium and p r o c e s s e d f o r e l e c t r o n
microscopy.
Cyst o r r e t e e p i t h e l i u m of a l l a g e s
e x h i b i t e d r e a c t i o n Froduct w i t h i n t h e
i n t e r c e l l u l a r s p a c e . I n t h i S r e e i o n smooth
s u r f a c e d c a v e o l a e which were more common i n
a d u l t s t h a n immatures a l s o were p o s i t i v e f o r
p h o s p i i a t a s e . The r e a c t i o n p r o d u c t was
l i m i t e d t o t h e s e r e g i o n s , w a s d e p e n d e n t on
t h e p r e s e n c e o f ATP a n d w a s n o t formed when
f i - g l y c e r o p h o s p h a t e was u s e d i n p l a c e of ATP.
'%en 'la, K and Xg were e l i m i n a t e d , t h e r e w a s
no r e a c t i o n . When o n l y Na a n d K o r 14g was
e l i m i n a t e d , however, r e a c t i o n p r o d u c t ,
a l t h o u g h n o t as p r o m i n e n t , was s t i l l d e t e c t e d
i n some p r e p a r a t i o n s .
Previous s t u d i e s have i n d i c a t e d t h a t t h e
e p i t h e l i u n o f t h e c y s t i c and n o n c y s t i c r e t e
i s permeable t o t r a c e r s s u c h as l a n t h a n u m and
h o r o e r a d i s h peroxidase and e x h i b i t s no
e x o c r i n e s e c r e t o r y a c t i v i t y . The p r e s e n t
s t u d y i n d i c a t e s t h a t t h e l a t e r a l c e l l membranes
h a v e a n A'I'Pase a c t i v i t y which i s i o n d e p e n d e n t .
I t i s s u g g e s t e d t h a t t h i s may f a c i l i t a t e a
pum?ing s y s t e m f o r the d i l a t i o n o f t h e r e t e .
( S d p p o r t e d by N I l i G r a n t AG 0 0 6 5 2 ) .
ROSENQUIST, Thomas and Donald STEWART, D e p a r t ment of Anatomy, Medical C o l l e g e o f G e o r g i a ,
A u g u s t a , G e o r g i a . Measurement o f T i s s u e
D i s u l f i d e Groups by A l c i a n B l u e 8GX and
Atomic A b s o r p t i o n S p e c t r o p h o t o m e t r y .
R e c e n t l y BEck (1978) h a s r e p o r t e d a p r o c e d u r e
f o r histochemical demonstration of d i s u l f i d e
g r o u p s i n t i s s u e p r o t e i n s by c o n v e r s i o n o f
c y s t i n e t o S - s u l f o c y s t e i n e and s u b s e q u e n t
s t a i n i n g o f t h e r e s u l t a n t s u l f a t e s by t h e c r i t i c a l e l e c t r o l y t e c o n c e n t r a t i o n method f o r
A l c i a n b l u e 86X. I n t h i s s t u d y , k i d n e y s were
o b t a i n e d from mice and f i x e d i n a s o l u t i o n of
10% f o r m a l i n b u f f e r e d w i t h 0.5M p h o s p h a t e , pH
7 . 0 (NBF) o r i n a s o l u t i o n of 95 p a r t s e t h a n o l /
15 p a r t s formalin15 p a r t s g l a c i a l a c e t i c a c i d
(AFA). F o l l o w i n g p a r a f f i n embedding, s e c t i o n s
o f 1 0 pm were o b t a i n e d and were s t a i n e d i n a
0.52 s o l u t i o n o f A l c i a n b l u e 8GX ( P o l y s c i e n c e s ,
I n c . ) ; o r were s t a i n e d f o l l o w i n g t h i o s u l f a t i o n .
S t a i n e d and u n s t a i n e d c o n t r o l s e c t i o n s were
o b s e r v e d , t h e n were removed from t h e s l i d e s
and weighed t o t h e n e a r e s t
mg. Pooled
s e c t i o n s w e r e d i s s o l v e d i n 35% n i t r i c a c i d and
tile amount of A l c i a n i c c o p p e r was measured.
T i s s u e f i x e d i n 1 0 % NBF c o n t a i n e d 1 . 1 5 ug/Cu/mg
t i s s u e w i t h o u t t h i o s u l f a t i o n and 1 . 9 9 pgCu/mg
t i s s u e a f t e r t h i o s u l f a t i o n ; t h e r e f o r e 0.84
ugCu/mg were d u e t o t h i o s u l f a t i o n .
Tissue f i x ed i n AFA c o n t a i n e d 1 . 3 5 pgCu/mg t i s s u e w i t h o u t
t h i o s u l f a t i o n and 3 . 2 5 pgCuImg t i s s u e a f t e r
t h i o s u l f a t i o n ; t h e r e f o r e 1 . 9 0 iigCu/mg were
d u e t o t h i o s u l f a t i o n . No c o p p e r was d e t e c t e d
i n unstained samples. Yicroscopic o b s e r v a t i o n
showed t h a t most of t h e v i s u a l i z a b l e n a t i v e
s u l f a t e s w e r e i d t h e basement membrane o f
Bowman's c a p s u l e , whereas f o l l o w i n g s u l f a t i o n
A l c i a n b l u e was v i s i b l e i n g l o m e r u l a r , t u b u l a r
and x a s c u l a r b a s a l l a m i n a e a s w e l l : a s shown
by Bock. We c o n c l u d e d t h a t : 1 ) X a t i v e s u l f a t e s
a r e present i n quantities easily detectable
b y AAS b u t d e t e c t a b l e l e s s e a s i l y o p t i c a l l y
and t h e y a r e found p r i n c i p a l l y i n t h e b a s a l
l a m i n a o f Borjman's c a p s u l e ; 2 ) AFA p r e s e r v e s
more n a t i v e s u l f a t e s a s w e l l a s d i s u l f i d e s
( e . g . , i n Type I V c o l l a g e n ) t h a n d o e s 1 0 % NBF.
I t s h o u l d b e p o s s i b l e t o c a l c u l a t e t h e number
o f d i s u l f i d e s p e r u n i t w e i g h t o f t i s s u e by
m e a s u r i n g A l c i a n i c c o p p e r w i t h AAS a f t e r t h e
i f one u s e s a t y p i s t a i n i n g p r o c e d u r e of B:ck
c a l A l c i a n b l u e 8 G X which c o n t a i n s 1 c o p p e r
atom p e r m o l e c u l e .
Rudeen, P. Kevin, Department o f Anatomy, Bowman
Gray School o f Medicine, Winston-Salem, N o r t h
C a r o l i n a . Alcohol-induced i n c r e a s e o f serum
p r o l a c t i n i n male r a t s .
A l c o h o l has been shown t o induce a f e m i n i r a t i o n e f f e c t i n c h r o n i c a l c o h o l i c males. Associa t e d w i t h t h i s f e m i n i z a t i o n i s an i n c r e a s e i n
plasma p r o l a c t i n . T h i s s t u d y was made t o invest i g a t e t h e e f f e c t s o f an a c u t e a d m i n i s t r a t i o n
o f a l c o h o l on serum p r o l a c t i n i n male r a t s .
S i x t y - f o u r a d u l t male Sprague-Dawley r a t s were
used i n t h i s study. The animals were a l l o w e d
f o o d and water ad 1 ib i t u m f o r 2 weeks p r i o r t o
e x p e r i m e n t a t i o n T - O n t a y o f t h e experiment,
t h e animals were p l a c e d randomly i n t o groups
which r e c e i v e d e i t h e r an i n t r a p e r i t o n e a l i n j e c t i o n o f a l c o h o l (2.5 gm a l c o h o l / k g body w e i g h t )
o r an e q u i v a l e n t volume o f i s o t o n i c s ' a l i n e .
F o l l o w i n g t h e i n j e c t i o n , animals were k i l l e d
a t i n t e r v a l s o f up t o 120 minutes; b l o o d was
c o l l e c t e d and t h e serum was harvested f o r t h e
e v a l u a t i o n o f p r o l a c t i n , l u t e i n i z i n g hormone
(LH), and a l c o h o l . The r e s u l t s i n d i c a t e t h a t
serum p r o l a c t i n i n c r e a s e s above c o n t r o l l e v e l s
w i t h i n 10 minutes o f t h e i n j e c t i o n o f a l c o h o l .
T h i s i n c r e a s e i s t r a n s i e n t , s i n c e serum p r o l a c t i n l e v e l s r e t u r n t o c o n t r o l l e v e l s w i t h i n an
hour. Serum LH i s depressed below c o n t r o l
l e v e l s w i t h i n 10 minutes, b u t r e t u r n s t o n o r mal l e v e l s w i t h i n 2 h r s a f t e r t h e i n j e c t i o n o f
a l c o h o l . Serum a l c o h o l l e v e l s a r e increased t o
about 225 mg% w i t h i n 1 0 minutes a f t e r t h e
i n j e c t i o n and a r e s t i l l e l e v a t e d above c o n t r o l
l e v e l s 2 h r s a f t e r t h e i n j e c t i o n . These data
c o n f i r m o t h e r r e p o r t s showing reduced serum L H
a f t e r a l c o h o l a d m i n i s t r a t i o n and i n d i c a t e t h a t
an a c u t e i n j e c t i o n o f a l c o h o l w i l l induce a
concomitant r i s e i n serum p r o l a c t i n . T h i s sugg e s t s t h a t a l c o h o l o r an a l c o h o l m e t a b o l i t e
may a f f e c t p r o l a c t i n s e c r e t i o n by i n f l u e n c i n g
t h e p i t u i t a r y o r t h r o u g h i n h i b i t i o n o f hypothalamic p r o l a c t i n i n h i b i t i n g factor. Additiona l research i s continuing t o discount the
s t r e s s f u l e f f e c t s o f a l c o h o l i n j e c t i o n as a
p o s s i b i l i t y i n enhancing t h e serum p r o l a c t i n
l e v e l s seen under c o n d i t i o n s o f t h i s experiment.
T h i s r e p o r t i n d i c a t e s t h a t some o f t h e e f f e c t s
o f a l c o h o l on t h e hypothalamo-pituitary-gonadal
a x i s are involved w i t h the regulation o f prol a c t i n secretion.
(Supported by a g r a n t from t h e N o r t h C a r o l i n a
A l c o h o l i s m Research A u t h o r i t y . )
SCHIEBLER, T.H., ReTZEL, H., Department of
Anatomy, University of Wiirzburg, Wiirzburg/
FRG.
The prenatal and early postnatal
development of the glial cells of the rat
median eminence.
The development of the glial cells of the
median eminence ( M E ) , including the supraependymal cells, was investigated from embryonic day (ED)
14 through postnatal day
(PD) 7, and pituicyte development from ED
12 through ED 17. The anlage of the ME and
neurohypophysis shows a neuroepithelial-like
structure at ED 12. From ED 1 3 to 15, the
cells of both regions start to differentiate,
accompanied by the formation o f multiple cell
layers. At the ultrastructural level, only
one cell type occurs in the anlage of the ME
and neurohypophysis. A$ the start of ED 16,
gliohlasts of the cligodendrocyte and astrocyte series migrate laterally (from the
arcuate nucleus) into the ME. Also at this
time the first distinctive structural features appear in the neurohypophyseal anlage,
whose cells later develop into pituicytes.
Starting at ED 18, tanycytes and astrocytic
tanycytes form in the ME from local glial
cells, and somewhat later cligodendroblasts
and astrohlasts form from immigrant glioblasts of the arcuate nucleus. Owing to
their common features, the pituicytes, tanycytes and astrocytic tanycytes apparently
represent different forms of differentiation
of the same parent cell type. Microglial and
supraependymal cells are first seen at ED 12.
Both cell types are apparently of mesodermal origin.
Schneider", Peter J . and J . David Deck, Department of Anatomy, University of Virginia, Charlottesville, Virginia. Turnover measurements of
cardiovascular connective tissue components &
-~
in vitro labeling.
Connective tissues of the heart valve are regularly subjected to the stresses of cyclic valve
operation. We have previously shown by autoradiography that the tissues of normal rat valves
respond with a turnover o f protein and ground
substance, respectively higher in leaflet and
leaflet attachment, apparently associated in
each case with the regions of greatest tension
and flexion. We have now found that similar
evidences of tissue turnover can he obtained
from excised rat valves labeled in vitro. !hen
studied using liquid scintillation methods,
valve tissues exposed to a 1.5 hour pulse label
of tritiated-proline in vitro readily showed
evidence of marked-precursor uptake, demonstrating the continued viability of the tissues. In
addition, autoradiographic comparison of tissues labeled in vitro and in viva has produced
the same patterns of labeling in both cases,
namely higher labeling of proteins in leaflet
than elsewhere. These observations indicate
that tissue excision and short-term in vitro
culture do not alter the pattern of protein
synthesis in valvular tissues. We now expect
to apply this technique to valves of larger animals where experimental alteration of structure is more feasible. Changed structure reflected in how these valves function may result
in different turnover patterns and indicate the
regions most affected by the altered stresses.
The in vitro labeling method can thus strengthen our evidence for stress-related tissue turnover. Supported by NIH grant HL 16935.
SCHUSTER, G. S . , 6.6. SINGH, J . F . ERBLAND and
D. E . STEFLIK, School o f D e n t i s t r y , M e d i c a l
C o l l e q e o f Georgia, Augusta. Growth and
C h a r a c t e r i z a t i o n o f Hamster C h Z k T u c k
Epithelium I n Vitro.
V a r i o u s c m e t e c h n i q u e s have been
employed t o s t u d y o r a l e p i t h e l i a b u t most
r e s u l t e d i n l i m i t e d o r no o u t g r o w t h o f an e p i t h e l i a l c e l l sheet. The o b j e c t i v e o f t h i s i n v e s t i g a t i o n was t o d e v e l o p a method t o a c h i e v e
s u s t a i n e d outgrowths o f e p i t h e l i a l c e l l s from
hamster cheek pouch e x p l a n t s and d e s c r i b e t h e
c e l l s ' m a j o r c h a r a c t e r i s t i c s . E x p l a n t s o f hams t e r cheek pouches were s u r g i c a l l y e x c i s e d ,
washed i n T r i s b u f f e r e d s a l i n e and p l a c e d i n .1%
t r y p s i n f o r v a r y i n g t i m e p e r i o d s up t o 60 m i n u t e s , t h e n m e c h a n i c a l l y scraped t o remove l o o s e
c o n n e c t i v e t i s s u e . 1 x 2 mm p i e c e s o f t i s s u e
were p l a c e d on .22 Urn, .45 um, .8 um M i l l i p o r e
f i l t e r s o r on p o l y s t y r e n e cover s l i p s . A l l were
covered w i t h a p l a s t i c cover s l i p , f e d w i t h MEM
supplemented w i t h 102 ( v / v ) f e t a l b o v i n e serum
and i n c u b a t e d a t 37OC i n 95% A i r - 5% C02 f o r up
t o 15 days. Growth was m o n i t o r e d by f i x i n g
r e p r e s e n t a t i v e samples each day i n 10% n e u t r a l
b u f f e r e d f o r m a l i n and p r o c e s s i n g them by p a r a f f i n techniques f o l l o w e d by s t a i n i n g w i t h hemat o x y l i n and e o s i n . A d d i t i o n a l c u l t u r e s were
used t o c h a r a c t e r i z e t h e n a t u r e o f c e l l u l a r o u t growths employing scanning e l e c t r o n m i c r o s c o p i c
(SEM) and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p i c
(TEM) t e c h n i q u e s . P r e f e r e n t i a l e p i t h e l i a l c e l l
o u t g r o w t h was seen on .22 pm and .45 urn
M i l l i p o r e f i l t e r s w h i l e l i t t l e o r no o u t g r o w t h
f i l t e r s . F i b r o b l a s t s were
was e v i d e n t on .8
t h e predominant c e l l t y p e when t h e p l a s t i c c o v e r
s l i p was used as a g r o w t h s u r f a c e . I n g e n e r a l ,
immersion o f e x p l a n t s i n .l%
t r y p s i n f o r 10-15
minutes was deemed e s s e n t i a l f o r i n i t i a t i o n o f
c e l l u l a r o u t g r o w t h . 5EM s t u d i e s demonstrated
p o l y g o n a l c e l l s o r g a n i z e d i n s h e e t s . The cons t i t u e n t c e l l s were s i m i l a r i n shape and s i z e
and covered w i t h m i c r o v i l l i . The m i c r o v i l l i
were o f t e n seen c o n n e c t i n g a d j a c e n t e p i t h e l i a l
c e l l s w i t h occasional thickenings probably
r e p r e s e n t i n g i n t e r c e l l u l a r j u n c t i o n s . Observ a t i o n o f t h e c e l l u l a r o u t g r o w t h by TEM showed
t h e presence o f abundant t o n o f i l a m e n t s and
desmosomes, c o n f i r m i n g t h e e p i t h e l i a l n a t u r e o f
the constituent c e l l s .
Supported by DE 05449 frm N I D R .
SCHWEISTHAL, M.R. and COLE, T . B . , JR.,
Department o t Anatomy, Oral Roberts University, Tulsa, Oklahoma.
A~ case
~ _ of_ achalasia:
_ _ _ _ _gross
_ _ anatomical
_
-~
findix.
Achalasia is defined as a dilatation of
the esophagus due to the inability o f the
lower esophageal sphincter to relax arid to
the absence of peristalsis in the body of
the esophagus in response to swallowing.
This postmortem study was done on an 85year-old male who had a diagnosis of severe
achalasid. Examination o f the esophagus
revealed that it was greatly enlarged
between the upper and lower esophageal
sphincters. The lumen of the esophagus as
it passed through the diaphragm was so
small that i t would not accept an attempt
to pass the index finger into the stomach.
Three important anatomical features were
observed: one, the criropharyngeus muscles
were well defined; two, a distinct transverse ridge was found to project into the
esophageal lumen dlrectly helow the upper
esophageal opening; three, there was a
well-defined extension of the dilated
esophagus located posterior to the larynx.
Muscle fibers from the cricopharyngeus were
found to rontrihute to b o t h thr upper
esophageal sphincter and the transverse
ridge located helow the sphincter. Four
hundred and ten grams of solid food material was found in the esophagus, but none
was found 111 the respiratory tract. I t is
concluded that the upper esophageal sphincter wds functional a n d played an important
role o t longevity i n this rase. The anat o m i c a l findings from this study w ~ l l be
disriisscd wiLh regard t o somr of Lhe other
cases reported i n the literature.
SICKLES, Dale W. and Roger E. McLENDON, Department of Anatomy, Medical College of Georgia,
Augusta, Georgia. Metabolic Heterogeneity of
Rat Lumbosacral Motoneurons.
Skeletal muscle fibers are known to differ in
activity of enzymes involved in energy production and contraction. It has been suggested
that motoneurons also exhibit a heterogeneity
in activity of these metabolic enzymes. However, Campa and Engel (Neurology 20: 559, 1970)
found no difference in enzyme activities of
a-motoneurons. More recent cytophotometric
studies suggest that motoneurons are not identical (Penny et al., J Neurol Sci 26: 1 7 9 ,
1 9 7 5 ) . The current investigation examines
quantitatively the activities of certain
enzymes in the a-motoneuron pool of the rat
lumbosacral cord with the photographic densitometry and atomic absorption spectrophotometry technique of Troyer and Rosenquist (J
Histochem Cytochem 23: 941, 1 9 7 5 ) . Rats were
killed by decapitation; the spinal cords removed by rapid injections of saline into sacral vertebral column and quenched in liquid
nitrogen. Enzyme histochemical techniques for
the demonstration of succinic dehydrogenase
(SDH) NADH dehydrogenase, menadione-linked aglycero phosphate dehydrogenase, (MaGPDH) acid
phosphatase, phosphorylase and Mg+Z-activated ATPase activities were performed on 10
p frozen sections taken through the long axis
of anterior horn of lumbosacral cord. Photographs of the motoneurons were taken on Kodak
SO-115 film; the area corresponding to the
neuron cytoplasm was cut out, weighed and the
silver eluted o f f the negative with 2 ml of
35% nitric acid. The quantity of silver was
determined with a Perkin-Elmer atomic absorption spectrophotometer. The data indicates
that a spectrum of activities exist in the rat
~~~
304
~~
spinal cord motoneurons, similar to that previously reported in rat tibialis anterior
muscle fibers. The activities of Mg+2-activated ATPase, MaGPDH and SDH were uniformly
negative. The greatest differences were observed with the NADH-D, phosphorylase and acid
phosphatase techniques. The significance o f
the findings in reference to neuromuscular
organization and selectivity of motoneurons
to neuromuscular diseases will be discussed.
Supported by BRSG Grant from N L H #2507RR0536519.
SINGH, B. B., J . L. BOSHELL, D. E. STEFLIK,
S . L. ZUNT, R. V . MCKINNEY and D. L. SCHAFFNER.
Department o f O r a l Pathology, Medical C o l l e g e o f
Georgia, Augusta and I n d i a n a U n i v e r s i t y ,
I n d i a n a p o l i s . Observations on t h e K e r a t o h y a l i n
Granules o f Tongue F i l i f o r m P a p i l l a e fran
G e r b i l , Cow, S p e c i o s i s Monkey, P i g and R a b b i t .
P r e v i o u s s t u d i e s frm our l a b o r a t o r i e s have
shown t h a t t h e tongue f i l i f o r m p a p i l l a e o f t h e
hamster, r a t , mouse and guinea p i g c o n t a i n b o t h
b a s o p h i l i c k e r a t o h y a l i n g r a n u l e s (BKHG) and
e o s i n o p h i l i c k e r a t o h y a l in g r a n u l e s (EKHG) which
a r e i n v o l v e d i n tongue k e r a t i n p r o d u c t i o n (Anat.
Record 178:464, 1974). The dog tongue i n t e r e s t i n g l y e x h i b i t e d o n l y EKHG i n t h e f i l i f o r m
p a p i l l a e . Because o f these o b s e r v a t i o n s conc e r n i n g species v a r i a t i o n i n g r a n u l e morphology
we began a comparative s t u d y o f tongue k e r a t o h y a l i n g r a n u l e s . The tongues o f g e r b i l , cow,
s p e c i o s i s monkey, p i g and r a b b i t were f i x e d i n
10% n e u t r a l b u f f e r e d f o r m a l i n o r 80% methanol
f o r l i g h t microscopy and 3% g l u t a r a l d e h y d e and
1% Os04 f o r e l e c t r o n microscopy. For h i s t o chemical s t u d i e s p a r a f f i n s e c t i o n s were c u t a t
5pm and s t a i n e d w i t h t h e f o l l o w i n g :
H & E for
g e n e r a l morphology and KHG i d e n t i f i c a t i o n ;
H a r r i s ' hematoxylin t o i d e n t i f y t h e BKHG; e o s i n
o n l y t o i d e n t i f y EKHG; P a u l y ' s r e a g e n t t o ident i f y h i s t i d i n e and t y r o s i n e c o n t a i n i n g p r o t e i n s
i n t h e KHG; and, t h e Sakaguchi o x i n e r e a c t i o n
for arginine. L i g h t microscopic observations
r e v e a l e d t h a t t h e g e r b i l and cow tongue p a p i l l a
c o n t a i n e d EKHG as w e l l as a s m a l l e r number o f
BKHG; s p e c i o s i s monkey c o n t a i n e d p r i m a r i l y EKHG
and o c c a s i o n a l BKHG observed i n s e r i a l s e c t i o n s ;
and, t h e r a b b i t tongue g r a n u l e s s t a i n e d p r e f e r e n t i a l l y w i t h hematoxylin b u t also reacted
m i l d l y w i t h e o s i n . H i s t o c h e m i c a l l y t h e BKHG
r e a c t e d s t r o n g l y w i t h P a u l y ' s r e a g e n t and t h e
EKHG w i t h t h e Sakaguchi o x i n e r e a c t i o n suggesti n g t h e presence o f a s p e c i f i c p r o t e i n m o i e t y
for the respective granules. U l t r a s t r u c t u r a l l y
t h e BKHG and EKHG e x h i b i t e d s u b o r g a n e l l e v a r i ations i n t h e i r u l t r a s t r u c t u r e w i t h the l a t t e r
e x h i b i t i n g a l e s s e r e l e c t r o n d e n s i t y . Both
types o f g r a n u l e s were a s s o c i a t e d w i t h r i b o s a n e s
and/or t o n o f i l a m e n t s . These p r e l i m i n a r y h i s t o chemi c a l and m o r p h o l o g i c a l o b s e r v a t i o n s s t r o n g l y
suggest a species v a r i a b i l i t y i n t h e n a t u r e o f
t h e tongue f i l i f o r m p a p i l l a KHG which m i g h t i n
t u r n r e f l e c t p h y l o g e n e t i c development o f t h e
tongue p a p i l l a e .
STEFLIK, 0. E., 6 . B. SINGH, J. R. SMITH and
R. V. MCKINNEY. Department o f O r a l Pathology,
Medical C o l l e g e o f Georgia, Augusta and R i v e r -
banks Z o o l o q i c a l Park, Columbia, South C a r o l i n a .
A C o r r e l a t i G e TEM, SEM and LM Study o f Capybara
LHydrochoerus hydrochoerus) F i l i f o r m P a p i l l a e .
The comparative morphology and e p i t h e l i a l
d i f f e r e n t i a t i o n of the tongue f i l i f o n n p a p i l l a e
of v a r i o u s species has been o f r e c e n t i n t e r e s t
i n o u r l a b o r a t o r y . The o b j e c t i v e o f t h i s i n v e s t i g a t i o n was t h e examination o f t h e f i l i f o n n
p a p i l l a e o f t h e capybara w i t h p a r t i c u l a r m p h a s i s on i t s k e r a t o h y a l i n (KHG) g r a n u l e populat i o n . The capybara resembles a g i a n t guinea
p i g , i s t h e w o r l d ' s l a r g e s t l i v i n g r o d e n t and
weighs a p p r o x i m a t e l y 100 l b s . F o r t h i s study,
samples of v a r i o u s s i z e s were e x c i s e d frm an
a d u l t capybara post-mortem and r o u t i n e l y p r o cessed f o r scanning e l e c t r o n microscopy (SEM),
t r a n s m i s s i o n e l e c t r o n microscopy (TEM) 'and l i g h t
microscopy (LM). By SEM t h e d o r s a l s u r f a c e of
t h e capybara tongue showed a p p r o x i m a t e l y 16
p o s t e r i o r curved c o n i c a l p a p i l l a e p e r square
m i l l i m e t e r . The s u r f a c e o f t h e p a p i l l a e d i s p l a y e d desquamation o f f l a t t e n e d squamous c e l l s
which e x h i b i t e d r n i c r o r i d g e s . H i s t o l o g i c a l l y t h e
convex a n t e r i o r p o r t i o n o f t h e p a p i l l a appeared
t o a r i s e frm g r a n u l a r c e l l s w h i l e t h e concave
p o s t e r i o r p o r t i o n was c m p r i s e d p r e d m i n a n t l y o f
a g r a n u l a r c e l l s . The g r a n u l a r c e l l s d i s p l a y e d a
mixed p o p u l a t i o n o f b o t h e o s i n o p h i l i c and basop h i l i c g r a n u l e s w i t h t h e e o s i n o p h i l i c group
b e i n g dominant. B a s o p h i l i c g r a n u l e s appeared t o
e x i s t p r i m a r i l y a t t h e base o f t h e p a p i l l a .
U l t r a s t r u c t u r a l l y , t h e KHG's were a s s o c i a t e d
w i t h t o n o f i l a m e n t s and/or r i b o s m e s . These
g r a n u l e s were arranged r a n d a n l y t h r o u g h o u t t h e
c e l l ' s cytoplasm b u t o c c a s i o n a l l y appeared t o
form a necklace t y p e arrangement surrounding t h e
c e n t r a l l y l o c a t e d nucleus. KHG's o f t h e capyb a r a appeared t o c o n t a i n a s u b o r g a n e l l e s t r u c t u r e c o n s i s t i n g o f v a r i o u s amounts o f dense
homogeneous d e p o s i t s (DHD). These DHO were cont a i n e d w i t h i n t h e normal f i n e g r a n u l a r m a t r i x o f
t h e KHG and v a r i e d i n s i z e and shape. T h i s
s t u d y suggests a species s p e c i f i c e p i t h e l i a l
d i f f e r e n t i a t i o n p a t t e r n i n capybara tongue w i t h
d i s t i n c t r e g i o n a l o r i e n t a t i o n o f b a s o p h i l i c and
e o s i n o p h i l i c KHG's. F u r t h e r , t h e KHG's c o n t a i n
v a r i o u s degrees o f OHD and d i s p l a y d i s s i m i l a r
morphologies. The c o m p o s i t i o n o f these KHG's i s
c u r r e n t l y under study.
TERRACIO, L. and DOUGLAS, W.H.J., Department
of Anatomy, University o f South Carolina, Columbia, SC. and Tufts University, Boston, MA.
(Introduced by R . J . Weymouth). Densitometric
Evaluation of Growth in Primary Cultures of Rat
Ventral Prostate Epithelial Cells.
The use of a photovoltic cell densirometer
to assess clonal growth of cell lines is well
established. Densitometry has not been successfully applied to primary culture systems, especially those that are established from cellular aggregates. This study describes a method
for establishing primary cultures in 24-well
culture vessels and evaluating the effects of
serum, hormones, and other factors o n cell
growth using densirometry. Aggregates of rat
ventral prostate epithelial cells were prepared
by a collagenase digestion and selectSve attachment procedure (Terracio % &., Proc. EMSA
37:138-139, 1979). The epithelial aggregates
were uniformly distributed into 24-well culture
vessels containing F12K culture medium supple-
mented w i t h v a r i o u s c o n c e n t r a t i o n s o f t h e f o l lowing s u b s t a n c e s :
f e t a l b o v i n e serum, h o r s e
serum, t e s t o s t e r o n e , d i h y d r o t e s t o s t e r o n e , e s t r i d i o l , h y d r o c o r t i s o n e , z i n c , t r a n s f e r r i n , cys t e i n e , g l u t a m i n e , s e l e n i u m and a s c o r b i c a c i d .
The c u l t u r e s were m a i n t a i n e d a t 37% i n a n a t mosphere of 5% C O 2 i n a i r , and t h e media and
s u p p l e m e n t s r e p l e n i s h e d e v e r y o t h e r day. A f t e r
seven days _
in_
v i_
t r o t h e c u l t u r e s were washed
w i t h PBS, f i x e d i n 1 . 0 % g l u t a r a l d e h y d e and
s t a i n e d w i t h 0 . 5 % c r y s t a l v i o l e t f o r 2 min. The
e f f e c t o f e a c h supplement on c e l l growth was
evaluated using a photovoltic c e l l densitometer
d e s i g n e d t o r e a d t h e t o t a l c u l t u r e s u r f a c e of
a 16 nun w e l l . A d e c r e a s e i n t r a n s m i t t e d l i g h t
when compared t o a c o n t r o l c u l t u r e i n d i c a t e d
a n i n c r e a s e i n c e l l growth by a supplement. I n c r e a s i n g c o n c e n t r a t i o n s o f b o t h h o r s e and f e t a l
b o v i n e serum r e s u l t e d i n a n i n c r e a s e i n c e l l
growth. T e s t o s t e r o n e , e s t r i d i o l , z i n c , hydroc o r t i s o n e , t r a n s f e r r i n , and a s c o r b i c a c i d e a c h
had a s t i m u l a t o r y e f f e c t on c e l l growth.
Cys t e i n e , g l u t a m i n e , and d i h y d r o t e s t o s t e r o n e had
no e f f e c t on c e l l growth w h i l e s e l e n i u m was i n h i b i t o r y t o c e l l growth. T h i s d a t a compares
f a v o r a b l y w i t h t h a t produced by o t h e r methods
such a s , o r n i t h i n e decarboxylase production.
These r e s u l t s i n d i c a t e t h a t d e n s i t o m e t r y is a
u s e f u l method €or d e t e r m i n i n g t h e e f f e c t of
s u p p l e m e n t s on c e l l growth.
( S u p p o r t e d by G r a n t KA26063 f r o m N C I ) .
THOMAS*, D. A . , Department o f Anatcmy, west
V i r g i n i a U n i v e r s i t y , Morgantown, W e s t V i r g i n i a .
(Sponsored by J. L. C u l b e r s o n ) . Motor u n i t s i n
s e l e c t e d h i n d l i m b m u s c l e s o f t h e opossum,
Didelphis virginiana.
T h i s is a preliminary study of neuranuscular
innervation i n t w o a n t a g o n i s t i c hindlimb
muscles, t h e m e d i a l g a s t r o c n e m i u s (FIG), a n a n k l e
f l e x o r , and t h e t i b i a l i s a n t e r i o r (TA), a n a n k l e
extensor.
The i n n e r v a t i o n , motor u n i t s i z e a n d
f i b e r t y p e s of t h e s e m u s c l e s w e r e s t u d i e d .
D i s s e c t i o n of t h e lumbosacral p l e x u s r e v e a l e d
t h a t t h e s e m u s c l e s a r e s u p p l i e d by b r a n c h e s of
t h e s c i a t i c n e r v e , which arises from ( L ) L 4
One or t w o d e s c e n d i n g s c i a t i c b r a n c h e s 5
S2.
r e a c h t h e head o f MG a n d m u l t i p l e b r a n c h e s
e n t e r t h e head o f TA.
Nerves t o t h e s e muscles
w e r e osmium s t a i n e d , epoxy embedded, s e c t i o n e d
and f i b e r s were c o u n t e d a n d measured.
Mean
n e r v e f i b e r c o u n t was 227 f o r MG and 3 5 1 f o r TA.
D e a f f e r e n t a t i o n of t h e s e n e r v e s w a s a c c o m p l i s h e d
by removing d o r s a l root g a n g l i a L5-S2. E f f e r e n t
n e r v e f i b e r s were c o u n t e d i n p e r i p h e r a l n e r v e s
a f t e r a 45-day s u r v i v a l p e r i o d .
The MG and TA
have also been i n j e c t e d w i t h h o r s e r a d i s h
p e r o x i d a s e t o l o c a l i z e t h e i r motoneuron pools.
E a r l y r e s u l t s demonstrated motoneurons i n L
and L6 o f t h e v e n t r a l - l a t e r a l h o r n . Muscle?
from normal a n i m a l s w e r e embedded i n p a r a f f i n ,
s e c t i o n e d , s t a i n e d w i t h H & E and used for t o t a l
f i b e r c o u n t s . A d j a c e n t b l o c k s were removed,
f r o z e n s e c t i o n e d and r e a c t e d f o r a l k a l i n e
adenosine triphosphatase.
The MG h a s a 1:l
r a t i o o f f a s t t o s l o w f i b e r s ; a 211 r a t i o of
f a s t t o s l o w f i b e r s e x i s t s i n TA. Crosss e c t i o n a l areas o f m u s c l e f i b e r s w e r e measured
u s i n g a n o p t i c a l image a n a l y z i n g s y s t e m (MOP/AM
01). The mean c r o s s - s e c t i o n a l area o f a TA
s l o w f i b e r w a s 3 9 3 5 IJ‘
and t h e mean c r o s s -
s e c t i o n a l area of a TA f a s t f i b e r w a s 3 7 5 1 u z ;
t h e r e w a s no s i g n i f i c a n t d i f f e r e n c e i n s i z e
between t h e t w o f i b e r t y p e s . The mean crosss e c t i o n a l area of a MG s l o w f i b e r was 6 1 7 9 uz
and 6 4 9 8 1-1’ f o r a MG f a s t f i b e r ; there was n o
s i g n i f i c a n t d i f f e r e n c e i n s i z e between t h e t w o
f i b e r t y p e s . The f a s t f i b e r s o f t h e MG w e r e
s i g n i f i c a n t l y l a r g e r t h a n t h e f a s t f i b e r s of
t h e TA ( P < 0 . 0 2 ) ; t h e s l o w f i b e r s o f MG, however,
were n o t s i g n i f i c a n t l y l a r g e r t h a n s l o w f i b e r s
of TA (0.05<p<0.10).
( S u p p o r t e d i n p a r t by NSF g r a n t BNS-7924172).
TILLMAN, L a r r y J . , Bruce R. Parks, Ben H.
Douglas and John E. Rawson, Departments o f
Anatomy and P e d i a t r i c s , U n i v e r s i t y o f
M i s s i s s i p p i Medical C e n t e r , Jackson,
Mississippi.
v e r t i n g enzyme i n h i b i t o r C a p t o p r i l ( S q u i b b ) ,
(D-2-methyl-3-mercaptopropanoyl-L-pro1 in e ) was
a d m i n i s t e r e d t o t h r e e groups o f animals made
h y p e r t e n s i v e by uninephrectomy, d a i l y i n j e c t i o n s
of 5 ug/lOO g body d - a l d o s t e r o n e (Sigma) and
s u b s t i t u t i o n o f 0.9% NaCl f o r t h e d r i n k i n g
w a t e r . C a p t o p r i l was g i v e n t o p r e g n a n t and
nonpregnant animals d u r i n g t h e developmental
phase o f h y p e r t e n s i o n and t o nonpregnant animals
w i t h e s t a b l i s h e d h y p e r t e n s i o n ( f o u r weeks o f
t r e a t m e n t ) . Treatment w i t h C a p t o p r i l p r e v e n t e d
f u r t h e r increases i n blood pressure i n the
animals d u r i n g t h e developmental phase o f hypert e n s i o n , and reduced t h e b l o o d p r e s s u r e t o
normotensive l e v e l s i n t h e animals w i t h
e s t a b l i s h e d hypertension.
The pregnant and nonpregnant animals r e c e i v i n g C a p t o p r i l d u r i n g t h e developmental phase o f
h y p e r t e n s i o n demonstrated s i m i l a r u l t r a s t r u c t u r a l changes w i t h no s i g n i f i c a n t d i f f e r e n c e s .
G l o m e r u l i i n t h e s e animals showed l o c a l f o o t
process f u s i o n and areas of s l i g h t basement
membrane t h i c k e n i n g . E n d o t h e l i a l and e p i t h e l i a l
c e l l s o c c a s i o n a l l y appeared e n l a r g e d . I n cont r a s t , animals t r e a t e d w i t h C a p t o p r i l f o l l o w i n g
e s t a b l i s h e d h y p e r t e n s i o n demonstrated d r a s t i c
changes i n g l o m e r u l a r a r c h i t e c t u r e . There was
marked basement membrane t h i c k e n i n g w i t h redup l i c a t i o n and s p l i t t i n g . E p i t h e l i a l f o o t p r o cess f u s i o n i n c r e a s e d . E a r l y s u b e n d o t h e l i a l
e l e c t r o n d e n s i t i e s and s u b e p i t h e l i a l humps were
observed. U1t r a s t r u c t u r a l r e s u l t s suggest t h a t
Captopril i s e f f e c t i v e i n p r o t e c t i n g glomerular
a r c h i t e c t u r e w h i l e a t t e n u a t i n g developing hypertension. Although i t i s e f f e c t i v e i n reducing
e s t a b l i s h e d h y p e r t e n s i o n , C a p t o p r i l does n o t
appear t o r e v e r s e a l r e a d y e x i s t i n g h i s t o p a t h o l o g i c a l damage t o t h e k i d n e y .
T r i l l J.J.“, J.M. B a r r e t t , and J . A .
T i s c h f i e l d ” Department of Anatomy, X e d i c a l
C o l l e g e of G e o r g i a , Augusta, G e o r g i a
L l i m i n a t i o n o f Mvcoplasma C o n t a m i n a n t s
i n Mammalian C e l l C u l t u r e s .
Normal cells have adenine phosphroibosyltransferase (APRe) activity which converts
adenine to adenylate. One can select APRT
deficient mutants (APRT-) with toxic adenine
analogs such as 2,6-diaminopurine (DAP) and
2-fluoroadenine (FA). Several APRT- mouse (L)
and Chinese hamster ovary (CHO) cell clones
were selected for resistance to DAP (DAPR) and
FA (FAR). iiTe then selected for APRT'
revertants in alanosine-adenine medium (AA). Testing of two putative revertants revealed low
levels of APRT activity, yet there was a paradoxical growth increase with 0.5 to 5 uglml
FA, and cell death in AA. These latter two
observations are inconsistent with the APRT'
phenotype. Microbiological examination indicated contamination with an anaerobic mycoplasma.
Initial testing (APRT activity values 2
.05>:wild-type' growth in DAP & FA; death in
AA) of the DAPk CHO cells indicated that they
were APRT-. Subsequent analyses (11.4% of
wild-type adenine incorporation and 1.7 - 2.9%
of wild-type APRT activity) however, indicated
some APRT activity. ?licrobiological and biochemical testing revealed a mycoplasma of
bovine origin.
To eliminate these mycoplasma, we devised
a protocol involoving alternating passage of
cells between antibiotic-free medium and medium containing 25 u g l m l tetracycline, 100
uglml spectinomycin and 60 ug/ml tylocin. To
date, both microbiological and biochemical
examination reveal no recurrence of the mycoplasma in treated cells. These tests are
further substantiated by SEM examination of
treated vs. untreated cells. Adenine incorporation and APRT activity assays (5 1% and
c .05% of wild-type, respectively) also indicate that the treated cells are now within
the range of other APRT- mycoplasma-free cell
lines. Elvcoplasma may cause artifacts or
misinterpretations in studies of purine metabolism and prenatal diagnosis. SEM examination for this contaminant is extremely sensitive, rapid and definitive. (Supported by
NIH Crdnt GM-26917)
W a y , C h a r l e s F . , D e p a r t m e n t of A n a t o m y ,
M e d i c a l University o f S o u t h C a r o l i n a ,
C h a r l e s t o n , S.C. 2 9 4 0 3
Immunohistochemical comparison o f lysozyme activity in
t h e l i v e r s of g l u c a n - t r e a t e d and g l u c a n hydrocortisone-treated nice.
A d m i n i s t r a t i o n of t h e p o l y g l u c o s e
glucan t o rodents confers hyperfunctiona 1 status upon the reticuloendothelial
(RE) s y s t e m w i t h h y p e r t r o p h y o f t h e m a jor R E o r g a n s .
Livers in these animals
exhibit increased macrophage numbers
with prominent granuloma formation. Lys o z y m e (LZM), w h i c h i s s e c r e t e d by m a c r o p h a g e s , i s e l e v a t e d i n t h e s e r a of
t h e s e a n i m a l s . In this s t u d y , the l i v e r s of g l u c a n - t r e a t e d m i c e w e r e e x a m i n e d
for L Z M a c t i v i t y u s i n g a n i m m u n o g l o b u l i n
e n z y m e b r i d g e t e c h n i q u e . As h e p a t i c
m a c r o p h a g e s a r e t h o u g h t by many t o ' d e r i v e f r o m c i r c u l a t i n g m o n o c y t e s , we e x amined the a b i l i t y d glucan t o enhance
macrophage numbers in mice rendered mon-
o c y t o p e n i c by h y d r o c o r t i s o n e a c e t a t e
(HAc), a n d e x a m i n e d t h e s e t i s s u e s for
LZM a c t i v i t y . Male C 5 7 B l m i c e w e r e a d m i n i s t e r e d ~ l u c a n( . U 5 m g / m o u s e , i.p.)
f o r 5 days.
C o n t r o l s r e c e i v e d 5% d e x trose. Blood s a m p l e s w e r e t a k e n at 2'4 h r
i n t e r v a l s for W B C c o u n t s . Y o n o c y t e s
were detected by esterase cytochemistry.
Other mice were given a single subcutane o u s i n j e c t i o n of 15ng !!Ac. F o r t y - e i g h t
h r l a t e r , m o n o c y t e s w e r e no l o n g e r d e t e c t a b l e i n t h e blood a n d e l u c a n t r e a t m e n t (.45mg/mouse f o r 5 d a y s ) w a s begun.
Twenty-four hr after the final glucan
injection, tissues were excised and prep a r e d for LM. F o u r m i c r o m e t e r s e c t i o n s
w e r e i m m u n o s t a i n e d f o r LZM. L i v e r s i n
g l u c a n - t r e a t e d m i c e s h o w e d LZ?1 p o s i t i v e
c e l l s s c a t t e r e d i n s i n u s o i d s and i n
granulomas. Dextrose controls were L Z i l
n e g a t i v e . G l u c a n failed t o e n h a n c e m a c rophage numbers in monocytopenic m i c e ,
These hepatic tissues were also LZM negative and organ weights were normal.
These results indicate that glucan enh a n c e s h e p a t i c m a c r o p h a g e n u m b e r s by
by recruitment o f circulating nonocytes
into the sinusoids which differentiate
into resident macrophages.
WEAVER, Macon M . and Bruce R. Parks. Effect
recovery
of offspring of DCA/NaCl pregnant rats.
of Indomethacin 0" development
-
In prior reports we have shown that
prostaglandin synthesis inhibition by indomethacin (I) may affect the severity of desoxycorticosterone acetate (DCA)/salt induced
renal lesions in the pregnant rat and her
offspring. This study further Characterizes the
injury and recovery of the offspring of the
DCA-I rats. Sprague-Dawley rats with blood
pressure (BP) ranging from 110-123 mm Hg
and weighing from 200-22> gm were used.
Animals were uninephrectomized and following
recovery each received 4 mg/Kg/da (OK 28
mg/Kg/wk DCA and 0.9% saline for drinking
water. When BP stabilized (163 to 201 rmn Hg)
the animals were bred. Weights and BP were
determined weekly. All animals were killed
at intervals for gross and histological
evaluation. Thirty rats served as hypertensive
controls; 15 were given 3 mg/Kg/da indomethacin
seven to 10 days prior to delivery. Some rats
and their offspring from both groups were
killed at term or allowed to recover. Offspring
were killed at intervals up to weaning.
Offspring treated transplacentally with
indomethacin showed an increase in fetal
morbidity and mortallty and cannibalism occurred
more often. These findings support the
concept that prostaglandins may influence BP
of pregnant UCA/NaCl hypertensive rats and the
survival of offspring.
A Biomechanical Investigation of Selected
Locomotor Modes in the Green Monkey
(C. a. sabaeus). JAMES P. WELLS, ROBERT M.
FISK, West Virginia School of Osteopathic
Medicine.
This investigation focuses upon two locomotor modes habitually employed by
Cercopithecus in the natural environment;
horizontal and vertical leaping. In an
attempt to view holistically the positional
behavior of this species, these two modes
were studied in the context of both their
behavioral associations in the natural
substrate and the biomechanics o f the locomotor event. Techniques employed include the
use of high speed cinematography with
kinematic and kinetic analysis and the use of
telemetered electromyography.
The initial positional behavioral phase
of the investigation was carried out on 'the
island of St. Kits in the British West Indies
This study showed vertical leaping to be
highly correlated with entry into the trees
while horizontal leaping was associated with
branch diameter and orientation. The kinematic portion of the study provided segmental
displacements, velocities and accelerations.
These data formed the basis for the kinetic
analysis along with the information obtained
on segment mass distribution by dissection.
The inertial force versus muscle contribution
in leaping was found to be much larger than
expected. Finally, telemetered EMG of lower
extremity muscle groups provided a view of
the phasic patterns of muscle activity
during leaping.
WELSH,* Marcia G . , Department of Anatomy, Univ e r s i t y o f South C a r o l i n a , School o f Medicine,
Columbia, South C a r o l i n a . ( I n t r o d u c e d by D.L.
Odor). The b l o o d - b r a i n b a r r i e r , CSF-brain
b a r r i e r and p i n e a l c o n c r e t i o n s : T h e i r r e l a t i o n s h i p i n t h e p i n e a l g l a n d o f t h e Mongolian
g e r b i l (Meriones u n g u i c u l a t u s ) .
The p o s s i b l e e x i s t e n c e o f e i t h e r a b l o o d b r a i n b a r r i e r (BBB) and/or a CSF-brain b a r r i e r
i n the p i n e a l g l a n d o f t h e g e r b i l was examined
u l t r a s t r u c t u r a l l y u s i n g t h e t r a c e r s microperoxi d a s e (MP) and h o r s e r a d i s h peroxidase (HRP).
G e r b i l s were i n j e c t e d w i t h e i t h e r MP or HRP i n t r a v e n o u s l y ( I V ) o r they r e c e i v e d i n t r a v e n t r i c u l a r ( I V T ) i n j e c t i o n s o f HRP. Male g e r b i l s
which had been m a i n t a i n e d i n a c o n t r o l l e d l i g h t
environment (LO 14:lO) were a n e s t h e t i z e d w i t h
pentobarbital.
For t h e I V i n j e c t i o n s t h e r e n a l
a r t e r i e s were t i e d o f f t o p r e v e n t r a p i d c l e a r ance o f t h e t r a c e r s . MP and HRP ( b o t h 15mg/ml
s a l i n e ) were i n j e c t e d i n t o t h e i n f e r i o r vena
cava. For t h e I V T i n j e c t i o n s , HRP (3mg/0.02ml
s a l i n e ) was i n j e c t e d u s i n g an i n f l o w cannula
placed s t e r e o t a x i c a l l y i n t o the l a t e r a l v e n t r i c l e . The t r a c e r s were a l l o w e d t o c i r c u l a t e f o r
15 o r 60 minutes. F o l l o w i n g f i x a t i o n , t h e t i s sue was i n c u b a t e d f o r p e r o x i d a t i c a c t i v i t y as
r e p o r t e d b y Graham and Karnovsky ( J . Histochem.
291) f o k HRP and by SimonCytochem. 1966,
escu g a l . (J. C e l l B i o l . 1975, %, 586) f o r
MP. The t i s s u e was p o s t f i x e d i n 2% osmium
t e t r o x i d e f o l l o w e d by r o u t i n e p r e p a r a t i o n f o r
e l e c t r o n microscopy. The I V i n j e c t i o n s r e s u l t e d
i n b o t h MP and HRP t r a n s p o r t t h r o u g h the endo-
3,
t h e l i a l c e l l s o f t h e g l a n d by v e s i c u l a r t r a n s p o r t and through t h e i n t e r e n d o t h e l i a l j u n c t i o n s .
F o l l o w i n g t h e IVT i n j e c t i o n s , t h e t r a c e r was
demonstrated i n t h e subarachnoid space a s w e l l
as i n t h e g l i a l c e l l s a s s o c i a t e d w i t h t h e p e r i phery o f t h e gland. I n b o t h cases (IVT and I V ) ,
t h e r e a c t i o n p r o d u c t s were l o c a t e d w i t h i n t h e
e x t e n s i v e p e r i v a s c u l a r r e g i o n as w e l l as i n
b o t h g l i a l c e l l s and p i n e a l o c y t e s . There a l s o
appeared t o be s p e c i f i c l o c a l i z a t i o n i n t h e
r e g i o n o f t h e vacuolated p i n e a l o c y t e s and t h e
p i n e a l c o n c r e t i o n s . The r e s u l t s o f t h i s study
demonstrate t h a t n e i t h e r a BBB n o r a CSF-brain
b a r r i e r e x i s t s i n t h e p i n e a l g l a n d o f t h e gerb i l . I n a d d i t i o n , t h e techniques employed i n
t h i s i n v e s t i g a t i o n may be a v a l u a b l e means f o r
d e m o n s t r a t i n g s i t e s o f s e c r e t i o n and o f s t o r a g e
o f t h e p i n e a l s e c r e t o r y p r o d u c t s . (Supported by
USC Grant #11040 E128).
Zuchelkowski*, Edwin M., and I.antz,R. Clark,
DeparLment of Anatomy, West Virginia Universit~y,
Morgantown, West Vjrginia. (Sponsored by David
E. Hinton) Mucous cells in epidermis of control
and acid-stressed (pH 4 . 0 ) brown bullhead c a t fish (Tctalurus nebiilosus): 4 morphometric
-~~
study.
~
~
~
Mining and utilizdtion of coal affect quality
of surface waters, rendering them less suitable
for supporting aquatic l i f e . Previous quelitative studies in ot.her species of teleosts tmve
suggested an increase in epidermal mucosubstance s following exposure to severe acid stress.
This increase was attributed to hypertrophy of
mucous cells in epidermis of acid-exposed brook
trout (Daye and Garside, Can. J . Zool., 54:21402155, 1976). Previous study in o u r laboratory
demonstrated that hyperplasia but. not hypertrophy (of murous cells occurred in brown bullhead
epidermis at a sublethal pH level (5.7-6.15).
The purpose of the present study was to determine thr effect of a lethal pli level (3.8-4.7)
on epidermal mucous cells of the brown bullhead.
Randomly selccLed young, hatchery-reared fish
(n=9), obtained from a commercial supplier, were
housed for 5 days in a 10 gallon aquarium in
which water pH was adjusted via addition of sulfuric acid to t h e p H range 3.8-4.2. Control
fish (n=9) were maintained in a tank containing
no sulfuric acid (pH 6 . 8 - 7 . 0 ) .
Following exposure, epidermis from the anterior abdominal
wall was prucessed for histochemical analysis
using a combined AfI-PAS technique. A total of
144 microscopic fields were evaluated in each
group of fish, using standard morphometric p r o cedures. Average cell daLa indicated no change
in diameter or volume of individual mucous cells
jn acid stressed f i s h . Howevrr, mucous cell v o j .
ume density (MCVV) and number density (MCN,)
were significantly increased ( p C . 0 1 ) i n the acid
stressed group. These results drmonstrate that
even at lethal pH levels, hyperplasia b u t not
hypertrophy of mucous cells occurs in the epidermis of the brown bullhead. T t i l s flnding
suggests that the brown bullhead epidermis may
be a useful model for studies involving t h e
mechanism of riucous cell hyperplasia.
Supported by WaLer Research Institlite, WVU, as
part o f i'roject A-037-W.Va.
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