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Adrenergic innervation of tumour blood vessels
Cancer Letters, 3 (1977} 347--351 © Elsevier/North-Holland Scientific Publishers Ltd.
347
ADRENERGIC INNERVATION OF TUMOUR BLOOD VESSELS
J. MATTSSON, L. APPELGREN, B. HAMBERGER* and H.-I. PETERSON
Department of Surgery I, University of G6teborg, GSteborg, *Department of Surgery, Danderyds Hospital, Danderyd and *Department of Histology, Karolinska Institute, Stockholm (Sweden) (Received 20 June 1977) (Accepted 18 July 1977)
SUMMARY
The adrenergic innervation of two intramuscularly transplanted rat tumours was studied by a histochemical technique for visualization of catecholamines by fluorescence microscopy. While catecholamines were f o u n d in normal muscle vessels, no adrenergic innervation of t u m o u r vessels was found.
INTRODUCTION
Morphological studies on experimental tumours have mostly shown a very immature micro-vasculature. In a recent microangiographic study of two intramuscularly transplanted rat tumours, one turnout, a sarcoma, was f o u n d topographically to have good connections with vessels in the transplantation area (muscle), while a second t u m o u r , a hepatoma, showed a more isolated vascular bed [5]. In one earlier study [6] no muscles or nerves could be identified in experimental t u m o u r vessels. On the other hand, recent studies have suggested t h a t t u m o u r vessels might be influenced by vasoactive drugs [1,8]. The aim of the present study was to investigate the adrenergic vessel innervation of the two t u m o u r s described above, based on their different vascular supply. A histochemical technique for visualization of catecholamines in t u m o u r biopsies was used. MATERIAL AND METHODS
Animals and tumours Inbred Lister rats with a mean body weight of 200 g were transplanted intramuscularly into one hindleg with a 20-methylcholanthrene sarcoma in its 109th Address correspondence to: Dr. H.-I. Peterson, Department of Surgery I, Sahlgrenska University Hospital, S-413 45 GSteborg, Sweden.
348
transfer generation or with a hepatoma in its 206th transfer generation. Both tumours were recently described in detail [5]. The tumours were studied 2 weeks after transplantation having grown to a mean t u m o u r diameter of 15 mm.
Histochemical technique The animals were sacrificed under ether anaesthesia and the tumours were immediately dissected out. Multiple specimens from various parts of the rumours were taken and rapidly frozen in propane chilled with liquid nitrogen. Tissue specimens were then freeze-dried, treated with formaldehyde gas, embedded and sectioned for visualization of catecholamines by fluorescence microscopy [2,3]. In some animals the hindleg with the transplanted t u m o u r was perfused with a suspension of fluorescent plastic particles (methylmetacrylate spheres labelled with fluorescein -- kindly supplied by AB Bofors Nobel Kemi, Sweden) for a b o u t 15 sec and the animals were subsequently sacrificed [~/].
Fig. 1. F l u o r e s c e n c e m i c r o p h o t o g r a p h o f a 2 0 - m e t h y l c h o l a n t h r e n e i n d u c e d sarcoma. N o f l u o r e s c e n c e specific f o r n o r a d r e n a l i n e can b e seen. T h e n u m e r o u s small dark areas are b l o o d vessels, x 160.
349
To increase the fluorescence intensity of the adrenergic nerves, thin slices were prepared from some tumours and incubated for 15 min at 37~C in a Krebs-Ringer bicarbonate buffer containing alpha-methyl-noradrenaline in a concentration of 1 ug/ml [ 4 ] . RESULTS
In central turnout specimens a large n u m b e r o f small blood vessels could be seen b u t no adrenergic nerves (Fig. 1). The large a m o u n t of blood vessels was more obvious after injection o f fluorescein-labelled methylmetacrylate spheres (Fig. 2). In the borderline between muscle and infiltrating tumour, muscle vessels, mainly arteriolae, with an adrenergic innervation were found (Fig. 3). However, no b l o o d vessels with adrenergic innervation could be followed into turnout tissue. No principle differences were found b e t w e e n the sarcoma and the hepatoma.
Fig. 2. Fluorescence m i c r o p h o t o g r a p h o f a h e p a t o m a . The hindleg into which the t u m o u r was transplanted was perfused with a suspension o f fluorescent plastic particles. A large n u m b e r of small vessels are clearly d e m o n s t r a t e d , cf. Fig. 1. X 160.
350
Fig. 3. F l u o r e s c e n c e m i c r o p h o t o g r a p h o f a h e p a t o m a . The t u m o u r is seen t o infiltrate the s u r r o u n d i n g muscle b u n d l e s in w h i c h b l o o d vessels with adrenergie nerves are seen. x 160.
351 DISCUSSION T h e a d r e n e r g i c i n n e r v a t i o n o f t h e b l o o d vessels o f t w o t r a n s p l a n t a b l e r a t t u r n o u t s was s t u d i e d b y a h i s t o c h e m i c a l t e c h n i q u e . A d r e n e r g i c nerves w e r e f o u n d in n o r m a l m u s c l e b l o o d vessels at t h e p e r i p h e r y o f t h e t u r n o u t s , b u t c o u l d n o t b e f o l l o w e d i n t o t u m o u r tissue. In a p r e v i o u s m i c r o a n g i o g r a p h i c s t u d y o f t h e s e t w o t u r n o u t s a n a p p a r e n t d i f f e r e n c e in v a s c u l a r m o r p h o l o g y b e t w e e n t h e t u m o u r s was f o u n d . A 20methylcholanthrene induced sarcoma had good connections with surrounding n o r m a l m u s c l e vessels, while a h e p a t o m a h a d a m o r e i s o l a t e d vascular b e d . In spite o f t h i s d i f f e r e n c e n o a d r e n e r g i c nerves w e r e f o u n d in a n y o f t h e t u m o u r s . A larger a m o u n t o f vessels w e r e visualized in c e n t r a l s p e c i m e n s o f t u m o u r b y i n j e c t i o n o f f l u o r e s c e i n - l a b e U e d m e t h y l m e t a c r y l a t e spheres, c o m p a r e d w i t h a p r e v i o u s m i c r o a n g i o g r a p h i c s t u d y in w h i c h a m o d i f i e d S p a l t e h o l z t e c h n i q u e was used. T h i s m i g h t b e e x p l a i n e d b y d i f f e r e n t m i c r o a n g i o g r a p h i c t e c h n i q u e s , b u t m i g h t also b e d u e t o smaller t u m o u r s in t h e p r e s e n t s t u d y , in w h i c h a progressive d e v e l o p m e n t o f t u m o u r n e c r o s e s d u r i n g g r o w t h h a d o n l y s t a r t e d [ 5 ] . T h e a b s e n c e o f a d r e n e r g i c i n n e r v a t i o n o f t u m o u r vessels in t h e 2 0 - m e t h y l c h o l a n t h r e n e i n d u c e d s a r c o m a w i t h its r a t h e r ' n o r m a l ' vascular b e d d o e s n o t e x c l u d e t h a t t h e t u m o u r vessels m i g h t still h a v e s - r e c e p t o r s a n d m i g h t b e inf l u e n c e d b y v a s o a c t i v e drugs. T h i s will b e s t u d i e d f u r t h e r . REFERENCES 1 Ackerman, N.B. and Hechmer, P.A. (1977) Effects of pharmacological agents on the microcirculation of tumours implanted in liver. IX World Conference Europ. Soc. Microcirc., 1976. Bibl. Anat., in press. 2 Corrodi, H. and Jonsson, G. (1967) The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. A review on the methodology. J. Histochem. Cytochem., 15, 65--78. 3 Falck, B., Hillarp, N.-A., Thieme, G. and Torp, A. (1962) Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem., 10, 348--354.
4 Hamberger, B. (1967) Reserpine-resistant uptake of catecholamines in isolated tissues of the rat. A histochemical study. Acta Physiol. Scand. Suppl. 295. 5 Kjartansson, I. (1976) Turnout circulation. An experimental study in the rat with a comparison of different methods for estimation of tumour blood flow. Acta Chit. Scand. Suppl. 471. 6 Krylova, N.V. (1968) Characteristics of microcirculation in experimental turnouts. Bibl. Anat., 10,301--305. 7 Svendgaard, N.A., Bj6rklund, A. and Stenevi, U. (1975) Regenerative properties of central monoamine neurons. Adv. Anat., Embryol., Cell Biol., 51, Fase. 4. 8 Wickersham, J.C., Barrett, W.P., Furukawa, B., Puffer, H.W. and Warner, N.E. (1977) An evaluation of the response of the microvasculature in tumours in C3H mice to vasoactive drugs. IX World Conf. Europ. Soc. Microcirc., 1976. Bibl. Anat., in press.
347
ADRENERGIC INNERVATION OF TUMOUR BLOOD VESSELS
J. MATTSSON, L. APPELGREN, B. HAMBERGER* and H.-I. PETERSON
Department of Surgery I, University of G6teborg, GSteborg, *Department of Surgery, Danderyds Hospital, Danderyd and *Department of Histology, Karolinska Institute, Stockholm (Sweden) (Received 20 June 1977) (Accepted 18 July 1977)
SUMMARY
The adrenergic innervation of two intramuscularly transplanted rat tumours was studied by a histochemical technique for visualization of catecholamines by fluorescence microscopy. While catecholamines were f o u n d in normal muscle vessels, no adrenergic innervation of t u m o u r vessels was found.
INTRODUCTION
Morphological studies on experimental tumours have mostly shown a very immature micro-vasculature. In a recent microangiographic study of two intramuscularly transplanted rat tumours, one turnout, a sarcoma, was f o u n d topographically to have good connections with vessels in the transplantation area (muscle), while a second t u m o u r , a hepatoma, showed a more isolated vascular bed [5]. In one earlier study [6] no muscles or nerves could be identified in experimental t u m o u r vessels. On the other hand, recent studies have suggested t h a t t u m o u r vessels might be influenced by vasoactive drugs [1,8]. The aim of the present study was to investigate the adrenergic vessel innervation of the two t u m o u r s described above, based on their different vascular supply. A histochemical technique for visualization of catecholamines in t u m o u r biopsies was used. MATERIAL AND METHODS
Animals and tumours Inbred Lister rats with a mean body weight of 200 g were transplanted intramuscularly into one hindleg with a 20-methylcholanthrene sarcoma in its 109th Address correspondence to: Dr. H.-I. Peterson, Department of Surgery I, Sahlgrenska University Hospital, S-413 45 GSteborg, Sweden.
348
transfer generation or with a hepatoma in its 206th transfer generation. Both tumours were recently described in detail [5]. The tumours were studied 2 weeks after transplantation having grown to a mean t u m o u r diameter of 15 mm.
Histochemical technique The animals were sacrificed under ether anaesthesia and the tumours were immediately dissected out. Multiple specimens from various parts of the rumours were taken and rapidly frozen in propane chilled with liquid nitrogen. Tissue specimens were then freeze-dried, treated with formaldehyde gas, embedded and sectioned for visualization of catecholamines by fluorescence microscopy [2,3]. In some animals the hindleg with the transplanted t u m o u r was perfused with a suspension of fluorescent plastic particles (methylmetacrylate spheres labelled with fluorescein -- kindly supplied by AB Bofors Nobel Kemi, Sweden) for a b o u t 15 sec and the animals were subsequently sacrificed [~/].
Fig. 1. F l u o r e s c e n c e m i c r o p h o t o g r a p h o f a 2 0 - m e t h y l c h o l a n t h r e n e i n d u c e d sarcoma. N o f l u o r e s c e n c e specific f o r n o r a d r e n a l i n e can b e seen. T h e n u m e r o u s small dark areas are b l o o d vessels, x 160.
349
To increase the fluorescence intensity of the adrenergic nerves, thin slices were prepared from some tumours and incubated for 15 min at 37~C in a Krebs-Ringer bicarbonate buffer containing alpha-methyl-noradrenaline in a concentration of 1 ug/ml [ 4 ] . RESULTS
In central turnout specimens a large n u m b e r o f small blood vessels could be seen b u t no adrenergic nerves (Fig. 1). The large a m o u n t of blood vessels was more obvious after injection o f fluorescein-labelled methylmetacrylate spheres (Fig. 2). In the borderline between muscle and infiltrating tumour, muscle vessels, mainly arteriolae, with an adrenergic innervation were found (Fig. 3). However, no b l o o d vessels with adrenergic innervation could be followed into turnout tissue. No principle differences were found b e t w e e n the sarcoma and the hepatoma.
Fig. 2. Fluorescence m i c r o p h o t o g r a p h o f a h e p a t o m a . The hindleg into which the t u m o u r was transplanted was perfused with a suspension o f fluorescent plastic particles. A large n u m b e r of small vessels are clearly d e m o n s t r a t e d , cf. Fig. 1. X 160.
350
Fig. 3. F l u o r e s c e n c e m i c r o p h o t o g r a p h o f a h e p a t o m a . The t u m o u r is seen t o infiltrate the s u r r o u n d i n g muscle b u n d l e s in w h i c h b l o o d vessels with adrenergie nerves are seen. x 160.
351 DISCUSSION T h e a d r e n e r g i c i n n e r v a t i o n o f t h e b l o o d vessels o f t w o t r a n s p l a n t a b l e r a t t u r n o u t s was s t u d i e d b y a h i s t o c h e m i c a l t e c h n i q u e . A d r e n e r g i c nerves w e r e f o u n d in n o r m a l m u s c l e b l o o d vessels at t h e p e r i p h e r y o f t h e t u r n o u t s , b u t c o u l d n o t b e f o l l o w e d i n t o t u m o u r tissue. In a p r e v i o u s m i c r o a n g i o g r a p h i c s t u d y o f t h e s e t w o t u r n o u t s a n a p p a r e n t d i f f e r e n c e in v a s c u l a r m o r p h o l o g y b e t w e e n t h e t u m o u r s was f o u n d . A 20methylcholanthrene induced sarcoma had good connections with surrounding n o r m a l m u s c l e vessels, while a h e p a t o m a h a d a m o r e i s o l a t e d vascular b e d . In spite o f t h i s d i f f e r e n c e n o a d r e n e r g i c nerves w e r e f o u n d in a n y o f t h e t u m o u r s . A larger a m o u n t o f vessels w e r e visualized in c e n t r a l s p e c i m e n s o f t u m o u r b y i n j e c t i o n o f f l u o r e s c e i n - l a b e U e d m e t h y l m e t a c r y l a t e spheres, c o m p a r e d w i t h a p r e v i o u s m i c r o a n g i o g r a p h i c s t u d y in w h i c h a m o d i f i e d S p a l t e h o l z t e c h n i q u e was used. T h i s m i g h t b e e x p l a i n e d b y d i f f e r e n t m i c r o a n g i o g r a p h i c t e c h n i q u e s , b u t m i g h t also b e d u e t o smaller t u m o u r s in t h e p r e s e n t s t u d y , in w h i c h a progressive d e v e l o p m e n t o f t u m o u r n e c r o s e s d u r i n g g r o w t h h a d o n l y s t a r t e d [ 5 ] . T h e a b s e n c e o f a d r e n e r g i c i n n e r v a t i o n o f t u m o u r vessels in t h e 2 0 - m e t h y l c h o l a n t h r e n e i n d u c e d s a r c o m a w i t h its r a t h e r ' n o r m a l ' vascular b e d d o e s n o t e x c l u d e t h a t t h e t u m o u r vessels m i g h t still h a v e s - r e c e p t o r s a n d m i g h t b e inf l u e n c e d b y v a s o a c t i v e drugs. T h i s will b e s t u d i e d f u r t h e r . REFERENCES 1 Ackerman, N.B. and Hechmer, P.A. (1977) Effects of pharmacological agents on the microcirculation of tumours implanted in liver. IX World Conference Europ. Soc. Microcirc., 1976. Bibl. Anat., in press. 2 Corrodi, H. and Jonsson, G. (1967) The formaldehyde fluorescence method for the histochemical demonstration of biogenic monoamines. A review on the methodology. J. Histochem. Cytochem., 15, 65--78. 3 Falck, B., Hillarp, N.-A., Thieme, G. and Torp, A. (1962) Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. Cytochem., 10, 348--354.
4 Hamberger, B. (1967) Reserpine-resistant uptake of catecholamines in isolated tissues of the rat. A histochemical study. Acta Physiol. Scand. Suppl. 295. 5 Kjartansson, I. (1976) Turnout circulation. An experimental study in the rat with a comparison of different methods for estimation of tumour blood flow. Acta Chit. Scand. Suppl. 471. 6 Krylova, N.V. (1968) Characteristics of microcirculation in experimental turnouts. Bibl. Anat., 10,301--305. 7 Svendgaard, N.A., Bj6rklund, A. and Stenevi, U. (1975) Regenerative properties of central monoamine neurons. Adv. Anat., Embryol., Cell Biol., 51, Fase. 4. 8 Wickersham, J.C., Barrett, W.P., Furukawa, B., Puffer, H.W. and Warner, N.E. (1977) An evaluation of the response of the microvasculature in tumours in C3H mice to vasoactive drugs. IX World Conf. Europ. Soc. Microcirc., 1976. Bibl. Anat., in press.