15.5. 1975
Specialia
Table II. Content of intercellular dermal components in 6-day cultured skin explants supplemented whith CS or E nutritional medium CS
E
NANA Uronic acids Hexosamines
0.019 9 0.021 0.033
0.015 0.025 0.015
NANA/Ur ac NANA/Hexos Ur ac/Hexos
0.90 0.54 0.63
0.60 0.62 1.04
amg/mg of proteins; each walue is the mean of 2 independent experiments each in duplicate.
587
O u r r e s u l t s clearly d e m o n s t r a t e a c o r r e l a t i o n b o t h in v i v o a n d in v i t r o b e t w e e n t h e h i s t o c h e m i c a l a n d bioc h e m i c a l f e a t u r e s of d e r m a l s k i n a n d t h e o n s e t of epiderm a l d i f f e r e n t i a t i o n a n d t h e d e p e n d e n c e in v i t r o of m e s e n c h y m e m o d i f i c a t i o n o n e n v i r o n m e n t a l factor(s) p r e s e n t in t h e c h i c k e n serum. T h e y t h e r e f o r e s u p p o r t t h e s u g g e s t e d m e c h a n i s m t h a t e n v i r o n m e n t a l factor(s) c a n a c t i v a t e m e s e n c h y m a l cells t o p r o d u c e s u b s t a n c e ( s ) able t o a c t specifically u p o n e p i d e r m a l d i f f e r e n t i a t i o n ~.
Riassunto. E s t a t a e v i d e n z i a t a u n a c o r r e l a z i o n e I r a c a r a t t e r i s t i c h e i s t o c h i m i c h e e c o m p o s i z i o n e b i o c h i m i c a del m e s e n c h ~ m a e l ' i n s t a u r a r s i del d i f f e r e n z i a m e n t o dell'epitelio nella c u t e di 17ollo sia in vitro, i n e s p i a n t i p r e l e v a t i al 6 ~ giorno di i n c u b a z i o n e e m a n t e n u t i i n t e r r e n o n u t r i t i z i o c o n t e n t e siero, che in vivo. P. CARINCI, G. PANE, g . SIMONELLI a n d G. ZANIBON115
a n d a m i n o r a n a o u n t of u r o n i c acids is p r e s e n t in CS c u l t u r e d e x p l a n t s i n c o m p a r i s o n w i t h E c u l t u r e d ones (Table II). A s s u m i n g t h e a m o u n t of sialic acid as a n i n d e x of G P c o n t e n t , o f u r o n i c acids of GAG, a n d t h e a m o u n t of h e x o s a m i n e s as a n i n d e x of b o t h G P a n d G A G c o n t e n t , we c a n d e d u c e t h a t in CS e x p l a n t G P a c c u m u late to a g r e a t e r e x t e n t t h a n GAG. A s i m i l a r s i t u a t i o n h a s b e e n p r e v i o u s l y d e m o n s t r a t e d in v i v o 4. T h e c o n c e n t r a t i o n r a t i o H A / C S A in v i v o (6-day 1 ; 1 0 - d a y 1.52; 14-day 1.62) c o n f i r m s t h e p r e v a l e n t a c c u m u l a t i o n of H A .
Istituto di Istologia ed Embriologia dell' Universitg, Via Fossato di Mortars 66, 1-44100 Ferrara (Italy); and Istituto di Istologia ed Embriologia generale dell' Universit~ di Bologna (Italy), 23 December 797d.
la These studies were supported in part by Italian CNR grant No 74,00128, 04.
I n t e r r e l a t i o n s h i p B e t w e e n M e a n A r t e r i a l B l o o d P r e s s u r e , B l o o d F l o w , and V a s c u l a r R e s i s t a n c e in S o l i d T u m o r T i s s u e of D S - C a r c i n o s a r c o m a C a p i l l a r y m o r p h o m e t r i c s a n d blood flow m e a s u r e m e n t s in i m p l a n t a t i o n t u m o r s of D S - c a r c i n o s a r c o m a in r a t k i d n e y s show t h e following r e s u l t s : W i t h i n c r e a s i n g t u m o r w e t weight, t h e v o l u m e of t h e v a s c u l a r s p a c e decreases e x p o n e n t i a l l y , t h e r e d u c t i o n of t h e v a s c u l a r b e d b e i n g especially d i s t i n c t d u r i n g t h e first p e r i o d of t h e t u m o r g r o w t h 1. D u r i n g t u m o r g r o w t h in t h e k i d n e y , t h e r e r e s u l t s a n e x p o n e n t i a l e n l a r g e m e n t of t h e m e a n i n t e r c a p i l l a r y d i s t a n c e s a b o u t t h e f a c t o r 2.5-3.0, as well as a l e n g t h e n i n g a n d a n increase in d i a m e t e r of t h e capillaries. T h e r a r e f a c t i o n of t h e t e r m i n a l v a s c u l a r b e d w i t h inc r e a s i n g t u m o r w e i g h t or age is a t t e n d e d b y a n expon e n t i a l decrease of t h e t o t a l t u m o r b l o o d flow, t h e r e g i o n a l m i c r o c i r c u l a t i o n of t h e o u t e r areas of t h e t u m o r s h o w i n g h i g h e r blood flow v a l u e s t h a n t h e t u n e r p a r t s ~. T h e pres e n t i n v e s t i g a t i o n a t t e m p t s to a n a l y z e t h e i n t e r r e l a t i o n ship b e t w e e n m e a n a r t e r i a l b l o o d pressure, t u m o r b l o o d flow, a n d v a s c u l a r r e s i s t a n c e i n t u m o r t i s s u e of DSc a r c i n o s a r c o m a u n d e r s t a n d a r d i z e d in v i v o c o n d i t i o n s d u r i n g d i f f e r e n t stages of g r o w t h . Materials and methods. Ascites cells of D S - c a r c i n o s a r c o m a are i m p l a n t e d i n t o r a t k i d n e y s w h e r e t h e y are m a i n t a i n e d 'tissue-isolated'~, 4, A f t e r a n a v e r a g e of 7-12 days, t h e i n f i l t r a t i n g a n d d e s t r u c t i v e l y g r o w i n g t u m o r cells c o m p l e t e l y replace t h e k i d n e y tissue, a n d t h e t u m o r m a s s is c o n n e c t e d t o t h e h o s t b y o n l y a single a r t e r y a n d vein. T o t a l t u m o r b l o o d flow (TBF) is det e r m i n e d b y c a n n u l a t i n g t h e t u m o r v e i n b y m e a n s of a b u r e t t e , u s i n g a roller p u m p a n d a c o n s t a n t p r e s s u r e buffering chamber. The operative procedure, the blood flow d e t e r m i n a t i o n a n d m o n i t o r i n g of t h e m e a n a r t e r i a l b l o o d p r e s s u r e ( M A B P ) are d e s c r i b e d elsewhereS. To s t u d y t h e i n f l u e n c e of t h e m e a n a r t e r i a l b l o o d p r e s s u r e o n t o t a l t u m o r b l o o d flow u n d e r i n v i v o condi-
tions, blood flow m e a s u r e m e n t s in 10 a n i m a l s ( a n e s t h e t i z e d w i t h p e n t o b a r b i t o n e sodium, 25-30 m g / k g i.p.) are p e r f o r m e d u s i n g a b u b b l e flow meter. M u l t i p l e m e a s u r e m e n t s of t h e b u b b l e p a s s a g e in t h e b u r e t t e are m a d e n o t o n l y w h e n b l o o d p r e s s u r e v a l u e s s p o n t a n e o u s l y occur in t h e t h o r a c i c a o r t a b u t also a f t e r c h a n g i n g t h e p e r f u s i o n pressure. A f t e r d e c r e a s i n g t h e m e a n a r t e r i a l b l o o d pressure b y c o n t r o l l e d bleeding, or a f t e r i n c r e a s i n g i t b y blood t r a n s f u s i o n f r o m o t h e r t u m o r b e a r i n g a n i m a l s , we a l w a y s w a i t for s t e a d y s t a t e c o n d i t i o n s before b l o o d flow is m e a s u r e d a t a n e w p r e s s u r e level. Results. U n d e r s t e a d y s t a t e c o n d i t i o n s t h e d e p e n d e n c e of t u m o r b l o o d flow on m e a n a r t e r i a l b l o o d p r e s s u r e is e x a m i n e d a t n o r m a l r e s p i r a t o r y gas p a r a m e t e r s in t h e a r t e r i a l blood. As c a n b e seen f r o m t h e r e p r e s e n t a t i v e
1 p. VAIJPEL, W. BRAUNBECK, V. SGHULZ, H. GfJNTHER and G. T~EWS, Biblphia anat. 72, 527 (1973). P. VAIJPEL, in Funktionsanalyse biologischer Systerad (Ed. G. THEWS; Steiner, Wiesbaden 1974), vol. 1 3 p. 1~{. GULLINO
and F. H. GRANTHAM,
J. natn. Cancer
Inst. 27,
679 (1961). W. SCH~ArARZ,V. SCHULZ, M. KERSTEN, R. WORZ and P. VAUPEL, Z. Krebsforsch. 75, 161 (1971). 5 p. VAUPEL, H. Gf3NTHER, J. GROTE and G. AUMOLLER, Z. ges. exp. Med. 756, 283 (1971).
588
Specialia
e x p e r i m e n t s in Figures 1 a n d 2 (upper parts), t h e blood flow t h r o u g h t u m o r s w i t h d i f f e r e n t w e i g h t or age shows linear d e p e n d e n c e on t h e m e a n a r t e r i a l blood p r e s s u r e w i t h i n t h e r a n g e of 40 t o 135 m m Hg. A c c o r d i n g to t h e t u m o r weight, t h e b l o o d flow values of b o t h t h e t u m o r s differ a t t h e s a m e m e a n a r t e r i a l blood pressure 9 A u t o r e g u l a t i o n of blood flow, as is t y p i c a l of t h e h o s t organ, can no longer be observed. The ratio of p e r f u s i o n p r e s s u r e t o t u m o r blood flow indicates t h e v a s c u l a r resistance of t h e t u m o r s . W i t h increasing m e a n arterial blood pressure, v a s c u l a r r e s i s t a n c e decreases f r o m 160 t o 141 PIRU 6 for t h e l i g h t t u m o r (Figure 1, lower part) a n d f r o m 574 t o 219 PIRU for t h e older, h e a v i e r one (Figure 2, lower part). Correspondingly, t h e t u m o r blood flow increases w i t h rising perfusion pressure, t h e v a s c u l a r r e s i s t a n c e d e c r e a s i n g a t t h e s a m e time. Discussion. The blood flow values of D S - c a r c i n o s a r c o m a in r a t k i d n e y s c o r r e s p o n d w i t h o t h e r d a t a t h a t were o b t a i n e d n o t only in several ' t i s s u e - i s o l a t e d ' i m p l a n t a t i o n t u m o r s b u t also in t u m o r s growing s p o n taneousiyT-X3. C o m p a r e d t o p a r e n c h y m a t o u s organs, t h e b e h a v i o u r of t h e v a s c u l a r s y s t e m d u r i n g t u m o r g r o w t h c a n e x p l a i n t h e low t u m o r b l o o d flow a n d its d e p e n d e n c e on t h e weight. The t u m o r m a i n l y holds t h e large vessels of t h e h o s t o r g a n only. I n case of progressive g r o w t h of tile t u m o r cells, especially in quickly growing t u m o r s , v a s c u l a r i z a t i o n can no longer keep up w i t h t h e r a p i d p r o l i f e r a t i o n of t h e cells. I n c o n s e q u e n c e of t h e i r e x p a n s i v e g r o w t h , t h e t u m o r ceils w i d e n t h e e x i s t i n g capillary n e t w o r k , in tile sense of a general v a s c u l a r r e d u c t i o n , t o such a n e x t e n t t h a t t h e t u m o r blood flow, r e l a t e d t o tile weight, m u s t decrease t o t h e s a m e degree. Moreover, t h i s increasing r a r e f a c t i o n of t h e t u m o r vessels d u r i n g g r o w t h is p r o v e d b y t h e increase of v a s c u l a r r e s i s t a n c e
O.t,O TBF
EXPERIENTIA 31/5
f r o m 145.5 t o 234.5 P R U ( M A B P = 100 m m Hg) a t an increase in w e i g h t f r o m 2.5 t o 8.2 g. A c o m p a r i s o n w i t h t h e v a s c u l a r r e s i s t a n c e s of i m p o r t a n t r a t organs c a l c u l a t e d f r o m a l r e a d y k n o w n blood flow d a t a a t c o r r e s p o n d i n g m e a n arterial blood pressures (liver ~4: 8.2 PIRU, k i d n e y s 15 : 11.7 P R U , brainlS: 51.8 P R U , a n d h e a r t ~ 7 : 5 2 . 6 P R U ) illustrates t h e influence of t h e general r e d u c t i o n of t h e v a s c u l a r space a n d r a r e f a c t i o n of t h e t e r m i n a l v a s c u l a r b e d on h e m o d y n a m i c s a n d ' o n t h e tissue perfusion r a t e of solid t u m o r s .
Zusammen/assung. I n v i v o - U n t e r s u c h u n g e n d e r T u m o r d u r c h b l u t u n g all I m p l a n t a t i o n s t u m o r e n eines DSC a r c i n o s a r k o m s in der R a t t e n n i e r e zeigen eine d e u t l i c h e
s 1 PRU = 8x10 adynXsecxclI1-5. P. l~I. GULLINO, Acta Un. int. Cancer 20, 1645 (1964). s p. M. GULLINO and F. H. GRANTHAM,J. natn. Cancer Inst. 27, 1465 (1961). e p. M. GULLINOand F. H. GRAnT,AM,J, natn. Cancer Inst. 28, 211 (1962), 1O S. CAT.A.LAND, C. COHEN and L. A. SAPIRSTEIN, J. natn. Cancer
Inst. 29, 389 (1962). ix F. E. GUMPand R. L. WHITE, Cancer 21, 871 (1968). 18 W. ROGERS, R. F. EOLICH, D. V. LEWIS and J. B. AusT, Surg. Clins N. Am. d7, 1473 (1967). 18 S. H. WOLLMANand F. E. REED, J. natn. Cancer Inst. 31, 1479 (1963). 14 W. SCHWARZand V. SCHULZ, Z. ges. exp. Med. 155, 225 (1971). 15 H. GONTHER, G. AUMOLLER, S. KUNKE, P. VAUPEL and G. T~Ews, Res. exp. Med. 763, 251 (1974). 16 K. NORBERGand B. K. SIESJ6, Aeta physiol, scan& 91,154 (1974). 17 W. BRIZULL, D. REDEL, H. DAHNERS, J. SCHOTTE and H. FLOHR,
Biblphia anat. 71, 174 (1973).
0.07- TBF
0.35
0.050.30
Tumor: 2.5g
~
Tumor: 8.2g . J " ~ . .
'
/:
0.25
y= 0.003x- 0,025 r = 0.947 2=< 0.001
0,20
, ~
y=0.0007x-0.0203 r= 0.989 2~< 0.001
"
0.01 0
~o
60
80
loo
60
80
100
~2o
1~o
700- VR [PRU)
0.15 9
0.10:
~..
0.03-
9
MABP (mmHg)
/,0
60
80
100
120
140
600
170" VR (PRU)
500
160-
400
X.
X. 150
300
I/,0
MABP (mmHg) "'4'0
"
I~0
'
80
"
100 "
120
"
11.0
Fig. 1. Effect of mean arterial blood pressure (MABP) on blood flow (TBF) through a young tumor of 2.5 g (upper part). Dependence of vascular resistance (VR) in tumor tissue of DS- careinosareoma on mean arterial blood pressure (MABP) in the thoracic aorta (lower part).
200 0
40
MABP(mmHg) 120 140
Fig. 2. Effect of mean arterial blood pressure (MABP) on blood flow (TBF) through an old tumor of 8.2 g (upper part). Dependence of vascular resistance (VR) in this tumor on mean arterial blood pressure (MABP).
15.5. 1975
Specialia
Abhgngigkeit der Durchblutungsgr6sse vom Tumorgewicht. B e i V a r i a t i o n des a r t e r i e l l e n M i t t e l d r u c k s zwischen 40 u n d 135 m m H g s t e i g t die D u r c h b l u t u n g d e r einzelnen Tumoren linear mit zunehmendem Perfusionsd r u c k an. E i n e z u n e h m e n d e R a r e f i z i e r u n g d e r t e r m i n a l e n Strombahn mit ansteigendem Tumorgewicht wird belegt
zs Supported by the Landesversicherungsanstalt Rheinland-Pfalz, Speyer/Rh.
589
d u r c h eine s t a r k e Z u n a h m e des S t r 6 m u n g s w i d e r s t a n d e s , der s c h o n bei sehr j u n g e n T u m o r e n w e s e n t l i c h h 6 h e r e W e r t e als b e i v e r s c h i e d e n e n O r g a n e n aufweist. P . V A U P E L ls
Physiologisches Institut der Universitiit, Saarstrasse 2 7, D-65 M a i m (German Federal Republic, BRD), 15 January 7975.
A S t u d y of the P r i m o r d i a l G e r m Cells D u r i n g their M i g r a t o r y P h a s e in Steel M u t a n t Mice T h e p r i m o r d i a l germ cells (PGCs) of t h e m o u s e e m b r y o c a n first be i d e n t i f i e d in t h e c a u d a l e n d of tile p r i m i t i v e streak, a l l a n t o i c b u d a n d y o l k sac s p l a n c h n o p l e u r e a r o u n d d a y 8 of g e s t a t i o n ~-5. W h e n first d e t e c t e d , t h e P G C s n u m b e r a b o u t 100. O n d a y 9 t h e y r e a c h t h e h i n d g u t s p l a n c h n o p l e u r e a n d n u m b e r a b o u t 500. On d a y 10 t h e y r e a c h t h e dorsal m e s e n t e r y , m e s e n t e r i c r o o t a n d coelomic angles a n d n u m b e r a b o u t 1,000. B y d a y 11 t h e g o n a d a l ridges are h e a v i l y p o p u l a t e d w i t h a b o u t 2,000 g e r m cells ~-~. T h e m i g r a t o r y p h a s e o f g e r m cell d e v e l o p m e n t a p p e a r s to e n d d u r i n g d a y 12 a t w h i c h t i m e t h e y n u m b e r a b o u t 5,0002. T h e g e r m cells are k n o w n to d i v i d e a l o n g
50~ day 9 n=18 40 30
1 W. OZDZENSKI,Zoolica Pol. 17, 367 (1967). 2 B. MINTZ and E. RUSSELL,J. exp. Zool. 134, 207 (1957). 3 A. CHIQUOINE, Anat. Rec. 118, 135 (1954).
20
4 E. SPIEGELMAN and D. BENNETT, J. Embryol. exp. Morph. 30, 97 (1973). L. ZAMBONI and H. MERCHANT,Am. J. Anat. 737, 299 (1973). P. SAEVELLAand L. RUSSELL, J. Heredity 47, 123 (1956). E. V. YOUNGLAIand D. H. K. CHuI, Biol. Reprod. 9, 317 (1973). 8 M. C. GREEN,in Biology of the Laboratory Mouse, 2nd edn. (Ed. E. L. GREEN;McGraw-HiI1, New York 1966), p. 115. 9 D. BENNETT, J. Morph. 98, 199 (1956).
~...:.:..+:
40
t h e m i g r a t i o n p a t h before i n f i l t r a t i n g t h e g o n a d a l ridges 5. Mice c a r r y i n g 2 m u t a n t genes a t t h e Steel (S 1) locus are sterile, a n e m i c a n d lack h a i r p i g m e n t 6. T h e sterility, w h i c h is d u e to t h e a b s e n c e oI g e r m cells in t h e m a t u r e g o n a d 7,s, m a y b e d u e t o a failure in t h e p r o l i f e r a t i v e a n d / o r m i g r a t o r y c a p a c i t y of tile PGCs% T h e p r e s e n t w o r k is a s t u d y of m u t a n t Steel mice a n d t h e i r n o r m a l l i t t e r m a t e s t o f u r t h e r e l u c i d a t e t h e process of p r i m o r d i a l g e r m cell d e v e l o p m e n t . W C / R e - S 1 / + females a n d C 5 7 B L / 6 J - S l a / + m a l e s were m a t e d o v e r n i g h t . T h e p r e s e n c e of a v a g i n a l p l u g a t 08.00 h i n d i c a t e d d a y 0 of g e s t a t i o n . P r e g n a n t females were killed b y cervical d i s l o c a t i o n e x a c t l y 9, 10 a n d 11 d a y s l a t e r (08.00 h). T h e e m b r y o s a n d s u r r o u n d i n g m e m b r a n e s f r o m t w o 9 d a y litters, a n d 4 e a c h of 10 a n d 11 d a y litters, were fixed i n 9 5 % e t h a n o l , e m b e d d e d in p a r a p l a s s (M.P. 50-52 ~ a n d s e c t i o n e d a t 10 Fro. PGCs are recognized c y t o l o g i c a l l y f r o m t h e i r tligh c o n t e n t of a l k a l i n e p h o s p h a t a s e ~. Sections were s t a i n e d for t h i s e n z y m e in F a s t R e d T R s a l t coupled t o ~ - N a p t h y l
day 10
n=33
3o
~ 20 1o
6OO
A
LL
[ ] Total [ ] Normal []Mutant
5OO
o
4OO 30
/
day 11
n=33
~ 3O0
2O g 10
0
co
.....
1
3
5
9 " ' 3 3 65 1051 Total
128
1025
PGCcounts
Fig. 1. The percent frequency of total PGC counts arranged on a logarithmic scale, showing a bimodal distribution of PGCs on days 10 and 11.
200 100
on 9
10
11 Day
Fig. 2. An increase in the number of normal germ cells is seen on day 10 and 11 whereas the mutant PGCs remain about the same.