Z. Zellforsch. 120, 120-136 (1971) 4;) by Springer-Verlag 1971
Adrenergic Innervation of the Umbilical Vessels Light- and F l u o r e s c e n c e M icr o sco p i c S t u d i e s * LUTZ LACHENMAYER** Institute of Anatomy, University of Hamburg Received April 28, 1971
Summary. The intra- and extraietal portions of the umbilical vessels in the guinea-pig and the umbilical cord of man, mouse and rabbit have been investigated by means of the Falck-Hillarp method for the fluorescence microscopical demonstration of catecholamines. The umbilical cord was found to be devoid of nerves in all species investigated. Adrenergic nerves are present only in the immediate vicinity of the umbilicus. The intrafetal portions of the umbilical artery and umbilical vein receive adrenergic nerves, the distribution pattern of which is different for each vessel. In the guinea-pig the ductus venosus is an intrahepatic branch of the vena umbilicalis. No adrenergically innervated sphincter has been detected in the initial segment of the ductus venosus. Regional variations in the pattern of innervation of the intrafetal portion of the mnbilica] vein are paralleled by regional differences in the construction pattern of the vessel's wall. Regional differences in the noradrenalinc concentration (measured by iluorometry) which correspond to the fluorescence microscopical findings have been detected in umbilical vessels: low noradrenaline content of the umbilical cord, high concentrations in the intrafetal sections of the umbilical vessels. The noradrenaline concentration of the guinea-pig umbilical artery is three times that of the umbilical vein. Key-Words: Umbilical vessels - - Smooth muscle arrangement - - Adrenergic innervation Noradrenaline content.
Introduction Th e c o n t r o v e r s y concerning t h e i n n e r v a t i o n of the vessels of the umbilical cord has a long history. A l r e a d y in 1836 S c h o t t s u m m a r i z e d the earlier literature in a r e v i e w article. L a t e r publications h a v e been critically e v a l u a t e d by S p i v a c k (1943) who i n v e s t i g a t e d the extra- a n d i n t r a f c t a l sections of the umbilical vessels from m a n a n d guinea-pig with silver i m p r e g n a t i o n and m e t h y l e n e blue staining techniques. I n both species m e n t i o n e d she described t h a t the i n t r a f e t a l sections of t h e vessels are i n n e r v a t e d b u t t h a t the cord lacks nerves. I n recent years p h a r m a c o l o g i s t s considered t h e umbilical cord vessels as a model of a nerve-free effector organ a n d studied the effect of s y m p a t h o m i m e t i c amines and o t h e r p h a r m a c a (v. Euler, 1938; Somlyo, Woo an d Somtyo, 1965; Gokhale et al., 1966; Dyer, 1970). S c h m e r m u n d et al. (1959) studied the effect of serotonin on t h e umbilical cord a r t e r y of m a n using strip p r e p a r a t i o n s and a t t r i b u t e d to serot o n i n released f r o m t h r o m b o c y t e s i m m e d i a t e l y after birth a significant role in the p o s t n a t a l closure of t h e umbilical cord vessels. I n r e c e n t l y published papers some a u t h o r s claim t h a t nerves are present in the umbilical cord of different species (ten Berge, 1963; J a c o b s o n an d Chapler~ 1967; * Supported by the Joachim Jungius-Gesellschaft der Wissenschaften, Hamburg. ** For continuous advice and constructive criticism I am indebted to Prof. Dr. Dr. E. Horstmann.
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F o x a n d J a c o b s o n , 1969a, b). T h e a u t h o r s m e n t i o n e d u s e d u n s p e c i f i c m e t h o d s , e.g. t h e o s m i u m t e t r o x i d e - z i n c i o d i d e t e c h n i q u e b y C h a m p y - C o u j a r d a n d m e t h y l e n e b l u e t e c h n i q u e s . I n a d d i t i o n , N a d k a r n i (1970) u s i n g t h e e l e c t r o n m i c r o s c o p e , s t a t e s t h a t m y e l i n a t e d n e r v e s are p r e s e n t in t h e h u m a n u m b i l i c a l cord. T h e p r e s e n t s t u d y s h o w s t h e d i s t r i b u t i o n of a d r e n e r g i c n e r v e s in d i f f e r e n t s e c t i o n s of t h e u m b i l i c a l vessels in g u i n e a - p i g s . F u r t h e r m o r e t h e s t r u c t u r e of t h e w a l l of d i f f e r e n t vessels (and t h e i r d i f f e r e n t sections) c h a r a c t e r i z e d b y d i f f e r e n t p a t t e r n s of i n n e r v a t i o n h a s b e e n a n a l y z e d . F o r c o m p a r i s o n t h e u m b i l i c a l c o r d of man, mouse and rabbit was investigated.
Materials and Methods Fetuses from 38 pregnant guinea-pigs (Cavia cobaya) were obtained close to term by sectio eaesarea in nembutal anaesthesia. The umbilical cords were divided in two halves. In addition the following intrafetal portions of blood vessels were excised: the free intrafetal portion of the umbilical vein including ductus venosus, vena cava inferior, abdominal aorta and the intrafetal portion of the umbilical arteries. The extra- and intrafetal sections of the umbilical vessels from a few guinea-pig fetuses were proceeded in one block preparation. Furthermore small tissue pieces from both extremities and from the middle of the human umbilical cord were dissected immediately after the delivery of the placenta. The specimens mentioned and umbilical cords of the mouse and rabbit were proceeded according to the Falck-Hillarp procedure. The specimens were placed and oriented on small boxes of copper tin, quenched in liquid propane (about --175 ~ C) and freeze dried. The freeze dried specimens were reacted with paraformaldehyde (for 1 hr at + 80 ~ C)and embedded in paraffin in vacuo (Falck, 1962; Falck and Owman, 1965; Baumgarten, 1971). Sections were cut at 10 ~ and sticked to slides with albumen glycerine, covered with liquid paraffin and studied in a fluorescence microscope Standard Universal M by Zeiss (excitation filter Sehott BG 12, barrier filter K 470 and K 500). With this filter set noradrenaline containing structures exhibit a yellow-greenftuoreseence color, which is abolished either by continuous irradiation or by watering (see discussion, p. 132). Afterwards the sections were deparaffinized and counterstained according to Goldner, with haematoxyline-eosine, resorcine-fuchsine or resorcine-fuchsine-picrie acid-thiazine-red (Romeis, 1968). Stretch preparations from the intrafetal portion of the vena umbilicalis of the guinea-pig were prepared as follows: the vessel was sliced open, placed on a strip of copper tin with the intima facing the metal, stretched gently with two forceps, quenched, freeze dried and formaldehyde treated as outlined above. Photographs were taken on Ilford F P 4, exposure time 1 min, and developed in Microphen. The following specimens were dissected from 4 guinea-pig fetuses and 8 rabbit fetuses near term: 1. whole umbilical cord, 2. both umbilical arteries (intrafetal portions from the anulus umbilicalis to the upper circumference of the urinary bladder), 3. umbilical vein (intrafetal portion, from the anulus umbilicalis to the caudal margin of the liver). The specimens were frozen in liquid nitrogen and the noradrenaline concentration determined according to the method of Bertler, Carlsson, Rosengren and Waldeck ( 1958 ) as modified by H~ggendal (1963). 1
Observations
I. Structure o/the Wall o/the Umbilical Vessels in the Guinea-Pig T h e u m b i l i c a l a r t e r i e s a r e b r a n c h e s of t h e i n t e r n a l iliacal a r t e r i e s w h i c h b o r d e r t h e u r i n a r y b l a d d e r , c o n v e r g e f r o m t h e u p p e r p o l e of t h e b l a d d e r a n d r u n a l o n g t h e i n n e r a s p e c t of t h e v e n t r a l a b d o m i n a l w a l l to e n t e r t h e a n u l u s u m b i l i c a l i s . 1 For laboratory facilities I am indebted to Prof. E. Rosengren, Lund, supported by a grant from Ford Foundation (No. 68-383), New York.
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Fig. 1. Longitudinal section from the intrafetal umbilical artery at the level of the urinary bladder. Thick membrana elastica interna from which elastic lamellae radiate in an archlike manner into the media. Very fine elastic fibres in the adventitia, but no membrana elastiea externa. Staining of elastic tissue with resorcine-fuchsine. Freeze dried section. • 92 I n its i n i t i a l segments, still in the pelvic cavity, the umbilical a r t e r y does n o t differ in s t r u c t u r e from t h e a r t e r i a iliaca interna. The solid m e m b r a n a clastica i n t e r n a is h e a v i l y u n d u l a t e d (Fig. 1). The m e d i a of the vessel is d i v i d e d in layers b y s y s t e m s of elastic fibres a n d m e m b r a n e s which enclose bundles of p r e d o m i n a n t l y circularly a r r a n g e d s m o o t h muscle cells. The a d v e n t i t i a consists of collagen tissue a n d contains v a s a v a s o r u m a n d s c a t t e r e d elastic fibres. The construction p a t t e r n of t h e m n b i l i c a l a r t e r y is g r a d u a l l y changed when i t a p p r o a c h e s t h e umbilicus: t h e m e m b r a n a elastica i n t e r n a becomes rarefied, the n u m b e r a n d thickness of elastic lamellae in between t h e s m o o t h muscle bundles of the m e d i a is r e d u c e d a n d t h e a d v e n t i t i a contains b u t few l o n g i t u d i n a l l y oriented elastic fibres a n d numerous v a s a v a s o r u m . The s m o o t h muscle bundles of the t u n i c a m e d i a are a r r a n g e d in spiral t o u r s which are s e p a r a t e d from each o t h e r b y t h i n l a y e r s of collagen tissue a n d elastic fibres. The m o s t p e r i p h e r a l bundles of s m o o t h m u s c u l a t u r e run steeply, n e a r l y parallel to t h e l o n g i t u d i n a l axis of t h e a r t e r y . This so ealled outer l o n g i t u d i n a l l a y e r is n o t v e r y distinct. Closer to t h e i n t i m a the angle of i n d i v i d u a l spiral t o u r s becomes more f l a t t e n e d t h u s s i m u l a t i n g a "circular" s m o o t h muscle l a y e r which, in reality, is f o r m e d from obliquely a r r a n g e d muscle bundles which cross each o t h e r a t s h a r p angles. Below t h e l a m i n a elastiea i n t e r n a extensions from t h e obliquely a r r a n g e d s m o o t h muscle bundles b e n d into a more longit u d i n a l direction. A f t e r passing t h e anulus umbiliealis t h e umbilical arteries lose their a d v e n t i t i a . The m o s t peripheral, s t e e p l y oriented o u t e r ends of muscle spirals border
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Wharton's jelly which lacks elastic fibres. The number of steeply arranged inner components of the muscle spirals decreases with increasing distance from the umbilicus. The compact intermediate layer of more or less circularly bending smooth muscle spirals remains unaltered. Vasa vasorum follow the arteries to a level just outside the anulus umbilicalis. More dis~al portions of the umbilical vessels in the cord are devoid of vasa vasorum. The vena umbflicalis of the umbilical cord is a thick walled vessel the lumen of which shows irregularities in width because of irregular contractions of its smooth muscle after birth. The endothelium is bordered by a well developed internal elastic membrane and a broad media which passes over into Wharton's jelly without an intervening advential connective tissue layer. The extrafetal umbilical vein lacks vasa vasorum. No distinct arrangement into layers of smooth muscle cells with heterogenous courses has been recognized. The smooth muscle cells are arranged in bundles separated from each other by masses of collagen tissue (Fig. 2). An individual bundle consists of three to six smooth muscle cells (as revealed in longitudinal sections) the nuclei of which are often grouped into a row. The bundles are arranged in spirals which embrace the lumen in opposite directions, showing steep inner and outer ends and flat intermediate "circular" tours. After entering the anulus umbilicalis the structure of the umbilical vein changes. The endothelium is bordered by a lamina elastica interna composed of closely packed, longitudinally oriented bundles of elastic fibres some of which extend into the media. The muscle cells are aggregated into bundles separated from each other by large amounts of collagen tissue. The helical tours of muscle bundles embracing the lumen in opposite direction take a more steep course in the intrafetal section (in between the umbilicus and the liver) than in the extrafctal portion.The media passes on into the adventitia without a limiting outer'elastic lamina. The adventitia is made up of loose collagen tissue without elastic fibres but many fibrocytes and numerous vasa vasorum running in the longitudinal axis of the vein. The construction pattern of the wall of the umbilical vein changes after entering the liver. The number of smooth muscle cells increase at the expense of decreasing collagen tissue. Distally the total thickness of the tunica media decreases gradually. Elastic lamellae issue from the thick internal elastic membrane subdividing the media into three to five separate layers. The adventitia consists of bundles of collagen tissue with interspersed elastic fibres. Branches of the intrahepatic portion of the umbilical vein show the same principle of construction as the vena umbfliealis. The terminal branch of the umbilical vein, the ductus venosus, reaches the vena cava inferior without further ramification.
11. The Adrenergic Innervation o/the Umbilical Cord 1. Guinea-Pig. No structures exhibiting a specific, formaldehyde induced fluorescence have been detected in the wall of the umbilical cord vessels and the surrounding connective tissue layer (Wharton's jelly). Some scanty nerves are, however, present in the wall of the umbilical artery of the most proximal cord segment (up to 1 cm from the umbilicus z adumbilical portion). I n the adumbilical section of the cord some large ramifying bundles of nerve fibres which fluoresce
Fig. 2. Tangentially cut section from the wall of the umbilical cord vein of the guinea-pig. The smooth muscle bundles are arranged in spirals which embrace the lumen running in opposite directions, showing steep inner and outer ends and flat intermediate circular tours. The smooth muscle bundles consist of 4 to 6 smooth muscle cells, the nuclei of which are arranged in para]lel rows. H.E.-staining. Freeze dried section. X 68
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Fig. 3. Longitudinal section from the initial portion of the umbilical cord of the guinea-pig. Thick ramifying bundles of nerve fibres without relation to an umbilical vessel. Some fibres are varicosed. Some mast cells are visible (arrow). • 450
with m e d i u m to high i n t e n s i t y are r e g u l a r l y o b s e r v e d (Fig. 3). T h e y c o n t a i n in p a r t varicose axons. Most adrenergic nerve fascicles a c c o m p a n y r e p e a t e d l y b r a n c h h l g "terminal" vessels which are f r e q u e n t in W h a r t o n ' s jelly of t h e i n i t i a l cord section close to t h e umbilicus. The vessels m a y belong to a b e r r a n t c u t a n e o u s vessels. I n t h e a d u m b i l i c a l cord section m a s t cells are f r e q u e n t l y o b s e r v e d which show a strong, yellow autofluorescence, b o u n d to c y t o p l a s m i c granules. 2. Man, Rabbit and Mouse. Similar to t h e conditions in t h e g u i n e a - p i g umbilical cord no fluorescent s t r u c t u r e s h a v e been verified in t h e cord of t h e o t h e r species m e n t i o n e d .
III. The Adrenergic Innervation o/ the Intra/etal Portions o/the Umbilical Artery in the Guinea Pig T h e i n t r a f e t a l sections of t h e u m b i l i c a l a r t e r y possess a strong adrenergic i n n e r v a t i o n . Smooth, l i g h t l y u n d u l a t i n g nerve fibre bundles course in t h e o u t e r a d v e n t i t i a , m a i n l y r u n n i n g in a l o n g i t u d i n a l direction, occasionally in c o m p a n y with v a s a v a s o r u m . These bundles are c h a r a c t e r i z e d b y a w e a k specific fluorescence w i t h o u t signs of varicose structures. T h e y give off small bundles of nerve fibres which p e n e t r a t e deeper layers of t h e a d v e n t i t i a a n d which fluoresce m o r e intensely. These bundles p a r t i c i p a t e in f o r m i n g a n a r r o w m e s h e d plexus of i n t e n s e l y fluorescent adrenergic nerves l o c a t e d a t t h e b o r d e r of t h e a d v e n t i t i a
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Fig. 4. Tangential section from the intrafetal umbilical artery. Thick undulating bundles of axons in the adventitia issuing fibres which form a dense adrenergic plexus around the artery. The plexus forming fibres follow the course of smooth muscle bundles. Fluorescent mast cells in the adventitia. X 220 Fig. 5. Longitudinal section from the intrafetal umbilical artery. Compare to Fig. 4. The plexus forming fibres penetrate the outer third of the media, x 220
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and media. Individual strands of this plexus follow the course of smooth muscle bundles, i.e. more peripheral components of this plexus are arranged in a longitudinal and inner components are oriented in an oblique or circular fashion (Fig. 4). Regularly some fibres of this plexus penetrate the outer third of the media which mainly contains circularly arranged smooth muscle bundles (Fig. 5). No adrenergic nerves are seen in between smooth muscle bundles of the inner two thirds of the media. I n the adventitia mast cells are common in varying amounts showing an unspecific yellowish fluorescence as described above (Fig. 4). The pattern of adrenergic innervation outlined above is typical for the umbilical artery in between the upper pole of the urinary bladder and the anulus umbilicalis (narrow meshed adrenergic plexus confined to the outer third of the media). The density of this plexus decreases in the umbilical artery when it passes the navel (Fig. 6) ; the plexus shows irregular meshes and dissolves rapidly in the initial segment of the cord. I n 5-7 cm C.R. length embryos there is no elaborate plexus of adrenergic nerves in the wall of the umbilical artery. Thick, strongly fluorescent, smoothly contoured bundles of nerve fibres run straight in the adventitia, parallel to its longitudinal axis. Only rarely do thin, non-varicose fibres switch off from these bundles to reach the media (Fig. 7).
IV. The Adrenergic Innervation o/ the Intra/etal Portions o/ the Umbilical Vein, the Ductus Venous and the Vena Cava In/erior o/the Guinea-Pig Adrenergic nerves are present in the wall of the free intrafetal portion of the umbilical vein in between the caudal margin of the liver and the navel. I n contrast to the conditions found in the artery nerve fibre bundles in the adventitia of the umbilical vein regularly accompany vasa vasorum. In perpendicularly cut sections from the umbilical vein thin walled vasa vasorum are recognized in the adventitia which run in the direction of the vessel's longitudinal axis and which are accompanied by strongly fluorescent partly varieosed nerve fibres (Fig. 8). Stretched whole mount preparations of the umbilical vein reveal the vasa vasorum to be supplied 1. by a plexus of adrenergic nerves rich in varicosities and superimposed to the media, and 2. by thin, weakly fluorescent, partly varicosed "fibres", embedded in the surrounding connective tissue (Fig. 9). The last mentioned "fibres" are indeed small bundles of axons, a fact which is clearly indicated at places where single axons diverge from the bundles (Fig. 9). These bundles issue smoothly contured thin fascicles which form a wide meshed adrenergic network around the umbilical vein. Individual strands of this plexus are rich in varicosities. Only rarely do small bundles of green fluorescent axons penetrate into deeper sections of the media. The adrenergic innervation changes in correspondence with the sudden change of the architecture of the wall of the umbilial vein after its entrance into the liver (Fig. 10). Thick bundles of intensely fluorescent, lightly undulating, non-varicose nerve fibres course in a longitudinal direction in the outer sections of the adventitia which is rich in vasa vasorum (Fig. 11). These bundles do not ramify very often and run independently (without attachement to vasa vasorum) in the adventitia. The vasa vasorum are well innervated by adrenergic nerves. The media of the intrahepatic portion of the vena umbilicalis contains abundant strongly fluorescent varicose nerves which
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Fig. 6. Tangential section from the extrafetal umbilical artery just outside the anulus umbilicalis. Only rarely do plexus-forming varicose fibres branch off from the nerve fibre bundles running in a longitudinal direction. X 220 Fig. 7. Longitudinal section from the intrafetal umbilical artery of a guinea-pig fetus of 5-7 cm C.R.-length. Thick remarkably brightly fluorescent, smoothly contoured bundles of nerve fibres run parallel to the artery. Occasional thin fibres with sparse varicosities enter the media, x 220
i n v a d e deeper sections of the muscle layer in loose c o m p a n y with elastic lamellae (Fig. 11). I n d i v i d u a l fibres m a y reach the internal elastic m e m b r a n e . The h e p a t i c a r t e r y possesses a n a r r o w meshed, well developed adrenergic plexus confined to the border of the media and a d v e n t i t i a . This border is defined by the external elastic lamina. The p a t t e r n of the adrenergic i n n e r v a t i o n of the ductus
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Fig. 8. Longitudinal section from the intrafetal umbilical vein. The adventitia contains longitudinally oriented vasa vasorum accompanied b y thin varieosed adrenergic nerves. Fibres running to the media are seen just above the centre of the picture. • 220 Fig. 9. Whole m o u n t stretch preparation from the intrMetal umbilical vein. I n the lower left corner of the picture a vas vasorum is seen surrounded b y an adrenergic plexus superimposed to the media. Fibres forming a wide-meshed network around the umbilical vein issue from more peripheral preterminal bundles of axons which run parallel to the longitudinal axis of the umbilical vein. Along their course smooth axons suddenly diverge from the minute bundles (arrow). • 220
v e n o s u s c o r r e s p o n d s t o t h a t d e s c r i b e d f o r t h e i n t r a h e p a t i c p o r t i o n of t h e u m b i l i c a l v e i n . T h e a m o u n t of a d r e n e r g i c n e r v e s d e c r e a s e s i n t h e d u c t u s v e n o s u s w h e n i t l e a v e s t h e l i v e r (Fig. 12). T h u s t h e i n n e r v a t i o n d e n s i t y i n t h e d u c t u s v e n o s u s b e c o m e s g r a d u a l l y a d j u s t e d t o t h a t of t h e v e n a c a v a i n f e r i o r w h i c h i n t u r n r e s e m b l e s t h e w i d e m e s h e d a d r e n e r g i c p l e x u s of t h e p r o x i m a l s e c t i o n o~ t h e intrafetal umbilical vein. 9
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Fig. 10. Longitudinal section from the umbilical vein shortly after its entrance into the liver. Autofluorescent elastic lamellae appear in the media (arrow) at the point where the adrenergic innervation increases. • 150
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Table. Noradrenaline concentration in the umbilical vessels o] guinea-pigs and rabbits n a
Weight of tissue analyzed (g)
Noradrenaline content (~g)
NAcone. (~g/g)
Guinea-pig: Whole umbilical cord Vena umbilicalis, intrafetal Arteria umbilicalis, intrafetal
4 4 4
0,7020 0,1485 0.0639
0.0400 0.0285 0.0378
0.06 0.19 0.59
Rabbit: Whole umbilical cord Vena umbilicalis, intrafetal Arteria umbilicalis, intrafetal
8 8 8
0.4651 0.0826 0.0677
0.0158 0.0123 0.0112
0.03 0.15 0.17
a Number of specimens pooled.
V. Noradrenaline Content o/ the Umbilical Vessels o/ the Guinea-Pig and Rabbit The results of the fluorimetric n o r a d r e n a l i n e estimations performed on different sections of the umbilical vessels i n guinea-pigs a n d r a b b i t s are presented in the Table. Very low c o n c e n t r a t i o n s are measured i n the umbilical cord of b o t h species. The traces of n o r a d r e n a l i n e most p r o b a b l y derive from the nerve cont a i n i n g a d u m b i l i e a l sections of the cord which have been included i n the specimens. The a m o u n t of n o r a d r e n a l i n e f o u n d i n the i n t r a f e t a l portions of the u m b i lical vessels is high i n both species i n accordance with the regional differences in the d i s t r i b u t i o n of adrenergic nerves (see fluorescence microscopical observations). The three-fold higher n o r a d r e n a l i n e c o n c e n t r a t i o n in the umbilical a r t e r y compared to the umbilical vein in the guinea-pig nicely corresponds to the differences i n the a m o u n t of fluorescent nerves detected i n b o t h vessels. Surprisingly, the i n t r a fetal sections of the arteria a n d v e n a umbilicalis from the r a b b i t c o n t a i n nearly equal a m o u n t s of noradrenaline. These findings c a n n o t be c o m m e n t e d u p o n because the umbilical vessels of the r a b b i t have n o t been i n v e s t i g a t e d b y fluorescence microscopy. Discussion The architecture of the wall of the umbilical vessels i n the guinea-pig has n o t been i n v e s t i g a t e d comprehensively u p to now. I n 1902 H e n n e b e r g i n v e s t i g a t e d the structure of the wall of the extra- a n d i n t r a f e t a l sections of the h u m a n u m bilical a r t e r y a n d vein. I t is well established (v. H a y e k , 1936; K. Goerttler, 1951)
Fig. I 1. Longitudinal section from the intrahepatic portion of the umbilical vein. Non-varicose undulating bundles of nerve fibres in the outer adventitia. Fibres from the adrenergic plexus extend up to the membrana elastica interna. • 210 Fig. 12. Longitudinal section from the ductus venosus where it leaves the liver (L). Well developed plexus of adrenergic axons showing the same architecture as the adrenergie network in the intrahepatic portion of the umbilical vein. • 210 9*
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that the smooth muscle coat of the human umbilical cord artery consists of a system of crossing spiral tours. My results obtained in guinea-pigs are in good agreement with the findings of Goerttler and Henneberg. The green fluorescence induced in the freeze dried specimens by formaldehyde treatment was bound to nerves, reached maximum intensity after 1 hour's formaldehyde condensation at 80 ~ C, could be abolished either by prolonged irradiation or by treatment with water. The results of the histochemical and histophysical techniques applied to the tissues permit the conclusions that the formaldehyde induced green fluorescence is caused by a primary catecholamine, e.g. dopamine or noradrenaline. Secondary catecholamines, like adrenaline, reach maximum fluorescence intensity only after two to three hour's condensation. Indolamines, e.g. scrotonin, require longer formaldehyde condensation and exhibit a yellowish fluorescence extremely sensitive to irradiation (for review see Corrodi and Jonsson, 1967). Only noradrenaline has been detected in significant amounts in the peripheral nervous system of mammals (v. Euler, 1961). Therefore, the conclusion seems justified that the green fluorescent structures contain noradrenaline. The results of the chemical estimations support this assumption. The findings obtained in man, guinea-pig, mouse and rabbit which prove a lack of adrenergic nerves in practically the whole umbilical cord (except the most proximal part) are in good agreement with the results of light microscopical studies by certain authors (Spivack, 1943 ; Pearson and Sauter, 1968, 1969, 1970). Ehinger et al. (1968) using the Falck-Hillarp method detected nerves in the umbilical cord of man only in cord sections close to the navel. More distal sections of the umbilical cord were devoid of adrenergic and cholinesterase containing nerves. Walker and McLean (1971) failed to demonstrate adrenergic nerves in the human umbilical cord and placenta vessels after incubation with alpha-methyl-noradrenaline. In my opinion, the small amount of noradrenaline found in the umbilical cord of the guinea-pig and rabbit supports the idea of a lack of an adrenergic innervation. Davignon et al. (1965) assayed still lower concentrations of noradrenaline in the umbilical cord of man since they could not include the proximal nerve containing section of the umbilical cord in their specimens for assay. The findings of Fox and Jacobson (1969) which indicate the presence of nerves in all sections of the umbilical cord of man were obtained with the unspecific methylene blue method in thick sections. Fox and Jacobson claim that the structures stained were irregularly distributed all over the cord. Part of their pictures said to demonstrate nerves are not convincing. The lack of nerves in the umbilical cord of man and guinea-pig is substantiated by the results of electron microscopical investigations carried out by Boyd and Hamilton (1970) and Hfilsemann (1971). I t is well established that the intrafetal sections of the umbilical vessels are innervated (Schott, 1836; Spivack, 1943; Pearson and Sauter, 1969, 1970). Spivack found no differences in the pattern of the innervation of the umbilical artery and umbilical vein using methylene blue and silver methods. The present study disclosed that the density of adrenergic nerves is much higher in the umbilical artery than in the umbilical vein, a finding which is substantiated by the
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noradrenaline estimations (see the Table). According to H~ilsemann (1971) the amount of cholinergic nerves supplying the umbilical vein is higher than the cholinergic inflow to the artery. This fact seems to offer an explanation for the discrepancy between the results of Spivack and of myself since methylene blue is supposed to stain adrenergic and cholinergic nerves simultaneously. The pattern of distribution of adrenergic nerves is different for the umbilical artery and the umbilical vein. Vascular nerves in the adventitia of the umbilical vein stick to the vasa vasorum and the main terminal plexus originating from this nerves is wide-meshed. Only rarely do single nerves penetrate into the media of the vein. Contrary to that the nerves in the adventitia of the artery are not supported by vasa vasorum. They form a dense plexus confined to the border of the media and adventitia which follows the course of individual smooth muscle bundles and some branches of which penetrate into the outer third of the media. The innervation pattern described above which differs for both vessels compared has been confirmed in Hiilsemann's electron microscopical study. Ehinger et al. (1968) did not detect a terminal adrenergic plexus in the wall of the umbilical vein from human fetuses of the 20th to the 23rd week of gestation. The umbilical artery has not been investigated up to now by fluorescence microscopy. A short circuit exists between the vena umbilicalis and vena cava inferior, known as the ductus venosus. Poor knowledge exists about the presence of a ductus venosus in terms of comparative anatomy. According to van Gclderen (1933) a ductus venosus exists in rodents, carnivores, ruminants and primates with occasional exceptions from the rule, however. A sphincter-like thickening in the transition of the vena umbilicalis and ductus venosus has been described by Barron (1942, 1944) in human and sheep fetuses. Ehinger et al. detected a sudden increase in the number of plexus-forming adrenergic nerves at this site which they interpreted as indicating a sphincter-mechanism. According to the present findings in the guinea-pig the ductus venosus is but an intrahepatic branch of the umbilical vein with which it shares the innervation and construction pattern. No adrenergically innervated sphincter exists at the beginning of the ductus venosus in this small mammal. The increase in the density of the adrenergic innervation of the umbilical vein after its entrance in the liver is paralleled by changes in the architecture of the wall. I n the free, intrafetal section in between the umbilicus and the liver the smooth muscle cells in the media of the vena umbilicalis are aggregated to bundles. These bundles are obviously innervated as units. This type of innervation requires only a restricted number of nerves. Hfilsemann (1971) discovered numerous junctions between smooth muscle cells in these bundles which serve as impulse transmitting structures (Prosser, Burnstock and Kahn, 1960; Holman, 1970). The media of the intrahepatic portion of the vena umbilicalis is divided in several layers by elastic lamellae. The adrenergic nerves do not form a condensed plexus but penetrate into deeper layers of the media. Hardly anything is known about the influence of elastic membranes on the diffusion rate of the adrenergic transmitter, but they may very well impede diffusion of noradrenaline (Bevan and TSrSk, 1970). Tight junctions between neighbouring smooth muscle cells which are necessary for electrotonic coupling have not yet been demonstrated in the intrahepatic portion of the umbilical vein. Paule (1963) and Silva and Ikeda
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(1971) have shown that peculiar forms of contacts between smooth muscle cells occur in the aorta of the young rat and in the ductus arteriosus of the fetal lamb. I n these vessels the innervation is restricted to the media-adventitia border and the outer third of the media respectively. A pattern of innervation with adrenergic nerves penetrating up to the lamina elastica interna similar to the conditions in the intrahepatic portion of the vena umbilicalis the media of which is subdivided into layers by elastic tissue has been described for the vena cutanea dorsalis penis (Ehinger, Falck, and Sporrong, 1968; Baumgarten, Falck, and Lange, 1969) and for mesenterie veins (Loizou and Tindall, 1969). An ultrastructural study of these vessels with a careful search for close contacts between smooth muscle cells seems desirable. The present findings justify to consider the umbilical cord as a model of a nerve-free effector organ. The smooth muscle cells of these vessels respond to the application of different substances with a contraction. Serotonin and angiotensin (Dyer, 1970) and oxytocin (Somlyo, Woo, and Somlyo, 1965) have been found to be exceptionally effective. Species differences in drug response seem to exist. The effect of catecholamines is said to be less than that of the drugs mentioned above. The few quantitative details that are available in literature, which deal with the effect of vasoactive substances, have been obtained on strip preparations with differences in the experimental approach, thus permitting no comparison with the dose-response curves of other innervated vessels. Under the condition that the umbihcal cord vessels possess the same pattern of distribution of receptors and the same pattern of equipment with enzymes as adrenergically innervated vessels, an enhanced sensitivity of umbihcal cord vessels to catecholamines should be expected. The enhanced sensitivity of non-innervated vascular smooth muscle is explained by the lack of noradrcnaline inactivating sites (i.e. the catecholamine-uptake and -reuptake meehnisms of adrenergic synapses, see Iversen, 1967). Nearly every muscular artery with its adrenergic nerves confined to the most peripherally located smooth muscle cells of the media serves as a physiological example of the differences in sensitivity caused by the topographical arrangement of adrenergic nerves: Graham and Keatinge (1971) demonstrated that the inner segment of the media of the arteria carotis in the sheep is by fare more sensitive towards noradrenaline than the outer segment, located close to the adventitia and rich in adrenergic nerves. The lack of an enhanced sensitivity of the non-innervated vessels of the umbilical cord against catecholamines could otherwise be explained only by the assumption that the smooth muscle cells in the umbilical cord possess properties which differ from those of other vessels. A supersensitivity of the umbilical cord vessels has not been proved up to now. Gennser and v. Studnitz (1969) found a low activity of monoamine oxidase and catechol-o-methyl transferase in the human umbilical cord, a finding which would indeed favour the idea of an enhanced effect of catecholamines. More quantitative data on the effect of catecholamines and their antagonists on non-innervated umbilical vessels and, for comparison, on innervated blood vessels are needed before statements on the distribution of receptors in the smooth muscle cells of umbilical cord vessels can be made.
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References
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Henneberg, B.: Beitr/ige zur feineren Struktur, Entwicklungsgeschichte und Ptlysiologie der Umbiliealgef/~$e des Menschen. Anat. H. 19, 523-568 (1902). Holman, M. E. : J u n c t i o n potentials in smooth muscle. In: Smooth muscle, ed. by E. Biilbring, A. F. Brading, A. W. Jones and T. Tomita, p. 244-288. London: Edward Arnold 1970. Hfilsemann, M.: l n n e r v a t i o n of the umbilical vessels. Electron microscopic studies on the guinea-pig. Z. Zellforsch. 120, 137-150 (1971). Iversen, L. L.: The uptake and storage of noradrenaline in sympathetic nerves. Cambridge: University Press 1967. Jacobson, H. N., Chapler, F. K. : intrinsic innervation of the h u m a n placenta. Nature (Lond.) 214, 103-104 (1967). Loizou, L., Tindall, A. R. : The monoaminergic innervation of mesenteric veins. J. Physiol. (Lond.) 201, 1 9 P - 2 0 P (1969). Nadkarni, B. B.: Innervation of the h u m a n umbilical artery. An electron microscope study. Amer. J. Obstet. Gynec. 107, 303-312 (1970). Paule, W. J.: Electron microscopy of the newborn rat aorta. J. Ultrastruct. Res. 8, 219-235 (1963). Pearson, A. A., Sauter, R. W.: Observations on the innervation of the umbilical vessels in h u m a n embryos and fetuses. Anat. Rec. 160, 4 0 6 4 0 7 (1968). - - - - The innervation of the umbilical vein in h u m a n embryos and fetuses. Amer. J. Anat. 126, 345 352 (1969). - - - Nerve contributions to the pelvic plexus and the umbilical cord. Amer. J. Anat. 128, 485~t~98 (1970). Prosser, L. L., Burnstock, G., Kahn, J. : Conduction in smooth muscle : comparitive structural properties. Amer. J. Physiol. 19`9, 545-552 (1960). Romeis, B. : Mikroskopische Technik. Mfinchen und Wien: R. Oldenbourg 1968. Schmermund, H. J., Rodegra, H., Soehring, K. : l~ber die Beteiligung yon 5-Hydroxytryptamin (Serotonin) beim physiologischen VerschluB der Nabelschnurgef/il~e post partum. Arch. Gyn/~k. 1,91, 4 5 7 4 6 2 (1959). Schott, J. A. C.: Die Controverse fiber die Nerven des Nabelstrangs und seiner Gef/~13e. F r a n k f u r t a. M. : F. Wilmans 1836. Silva, D. G., Ikeda, M. : Ultrastructural and acetylcholine-esterase studies on the innervation of the ductus arteriosus, pulmonary t r u n k and aorta of the fetal lamb. J. Ultrastruct. Res. 34, 358-374 (1971). Somlyo, A. V., Woo, C. u Somlyo, A. P.: Responses of nerve-free vessels to vasoactive amines and polypeptides. Amer. J. Physiol. 208, 748-753 (1965). Spivack, M.: On the presence or absence of nerves in the umbilical blood vessels of man and guinea-pig. Anat. Rec. 86, 85 109 (1943). ten Berge, B. S. : Nervenelemente in Placenta und Nabelschnur. Gynaecologia (Basel) 166, 4953 (1963). Walker, D. W., McLean, J. R. : Absence of adrenergic nerves in the h u m a n placenta. Nature (Lond.) 229, 344-345 (1971). Dr. L. Lachenmayer Institute of Anatomy University of Hamburg BRD-2000 Hamburg 1V[artinistralle 52 Germany