UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Trophic interactions between rat thigh blood vessels and their innervation 1986

You don't seem to have a PDF reader installed, try download the pdf

Item Metadata

Download

Media
UBC_1986_A6_7 S34.pdf
UBC_1986_A6_7 S34.pdf
UBC_1986_A6_7 S34.pdf [ 6.79MB ]
Metadata
JSON: 1.0096806.json
JSON-LD: 1.0096806+ld.json
RDF/XML (Pretty): 1.0096806.xml
RDF/JSON: 1.0096806+rdf.json
Turtle: 1.0096806+rdf-turtle.txt
N-Triples: 1.0096806+rdf-ntriples.txt
Citation
1.0096806.ris

Full Text

TROPHIC INTERACTIONS BETWEEN RAT THIGH BLOOD VESSELS AND THEIR INNERVATION By NANCY LYNN SCHINDELHAUER Hons. B.Sc, McMaster University, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Anatomy, The University of British Columbia) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 1986 (c): Nancy Lynn Schindelhauer, 1986 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Z^-AJA-T"5/V-*/ The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date r W l l . 3 p ) f i 1Q\ ABSTRACT Much less work has been done on the denervation of smooth muscle compared with the extensive studies carried out on skeletal muscle. It was thought that denervation of smooth muscle produced few alterations in i t s morphology or physiology, especially since many blood vessels have vir t u a l l y no innervation, and therefore, they can survive without i t . Simple severing or excising a section of nerve trunk i s sufficient to denervate skeletal muscle, but this does not apply to smooth muscle. Therefore, denervation methods for smooth muscle have included those with widespread effects such as chemical and immunological sympathectomies, and superior cervical ganglionectomy. In this study, I have developed a a semi-permanent, localized denervation method for rat thigh vessels and have used this method to study the trophic interactions between blood vessels and their innervation. Female Wistar rats were denervated at 1-3 or 12 days of age, and examined at 30, 60, 90 and 120 days of age. The femoral nerve, which carries the vasomotor innervation to the thigh vessels, was severed in the thigh and brought inside the abdominal cavity. This method was necessary since preliminary experiments showed rapid re-innervation of the vessels i f any part of the proximal root remained in the thigh. Inside the abdominal cavity, the nerve was slipped into a plastic tube and heat sealed. The tubing further inhibited re-innervation by preventing collateral sprouting from the proximal stump. Samples of the dis t a l nerve stump, the proximal nerve stump, and from the femoral vein and saphenous artery were taken. In every animal, the contralateral side acted as a control. The distal and proximal nerve stumps showed marked evidence of degeneration. Fluorescence microscopy ( s p e c i f i c f or catecholamines) showed a s i g n i f i c a n t decrease i n the number of f l u o r e s c i n g dots around both the a r t e r y and v e i n . The presence of some fluorescencing dots around the denervated vessels may be from the nerves that were seen re-innervating the vessels at the time of sampling. These nerves came from aberrant areas. A r t e r i e s sampled at 90 days showed a s i g n i f i c a n t decrease i n the c r o s s - s e c t i o n a l area of the tunica media on the denervated sid e . The denervated femoral vein, i n s i t u , was seen to be grossly d i l a t e d compared to the c o n t r o l s i d e . Measurements of the luminal perimeter of sections of the vein showed that the denervated vein had a luminal area up to three times that of the c o n t r o l s . The d i f f e r e n c e i n wall thickness i n the femoral vein was not s i g n i f i c a n t at the p<0.05 l e v e l . My r e s u l t s i n d i c a t e that adrenergic nerves may not only have a trophic influence on a r t e r i e s , but may influence veins as w e l l . Therefore, i n t h i s study, trophic influences of nerves on blood vessels have been suggested by denervation causing 1) a thinner a r t e r i a l wall and by producing 2) a reproducible d i l a t i o n of the femoral v e i n . Also, trophic influences of blood vessels on nerves i s suggested by the presence of re-innervation from aberrant areas. TABLE OF CONTENTS Page Abstract i i List of Tables v i List of Figures v i i Acknowlegements ix Introduction 1 Introductory Statement 1 The Effects of Innervation on Skeletal Muscle 3 The Effects of Cardiac Muscle on Nerves 8 The Effects of Innervation on Smooth Muscle 10 Methods of Sympathetic Denervation 16 Problems with Denervation Methods 20 Thesis Topic 21 Materials and Methods 23 Animals 23 Denevation Procedure 23 Tissue Sampling and Processing 25 Sectioning 28 Fluorescence Microscopy =.->-' .,. 29 Analysis: Light Microscopy and Morphometries 30 Electron Microscopy 31 Fluorescence Microscopy 32 Statistics 32 Results 3^ Sampling for Light Microscopy 3^ Light Microscopic Appearance: Saphenous Artery 36 : Femoral Vein 37 : Nerve 37 E l e c t r o n Microscopy of the Abdominal Cavity Femoral Nerve 39 Fluorescence Microscopy: Gross Morphology of the Denervated Area 39 Description o f Sections under the Fluorescence Microscope 40 Analysis: Morphometries - Medial Area of the Saphenous Artery 41 - Vein Perimeter and Wall Thickness 42 - Fluorescence Counts 43 Discussion 44 Conclusions 62 Tables 63 Figures 69 Bibliography 94 vx LIST OF TABLES TABLE TITLE PAGE I Changes i n the Area of the Tunica Media of the Saphenous 63 Artery when Sampled at 60 Days of Age. I I Changes i n the Area of the Tunica Media of the Saphenous 64 Artery when Sampled at 90 Days of Age. I I I Comparison of the Perimeters of the Denervated and Con- 65 t r o l Femoral Vein. IV Comparison of Wall Thickness Between the Control And De- 66 nervated Femoral Vein. V Number of Fluorescing Areas Counted from the Control and 67 Denervated Saphenous A r t e r i e s and Femoral Veins V X 1 LIST OF FIGURES FIGURE TITLE PAGE 1 Denervation Procedure. 69 2 Denervation Procedure continued. 71 3 Diagram Showing the Cannulation Procedure. 73 4 Perfusion Pressure Recording 75 5 Light Micrographs of Control and Denervated Saphenous 77 Artery Walls. 6 Light Micrographs of Control and Denervated Femoral 79 Veins. 7 Light Micrographs of the Normal Saphenous Nerve taken from 81 the Control Side and the D i s t a l Stump from the Dener- vated Side 8 Light Micrographs of Femoral Nerves from Control 83 and Denervated Sides. 9 U l t r a s t r u c t u r a l l y , the Femoral Nerve i s Composed Mainly 85 of Collagen FIGURE TITLE v i i i PAGE 10 Fluorescence Microscopy of Control and Denervated 87 Saphenous Arteries. 11 Fluorescence Microscopy of Control and Denervated 89 Femoral Veins. 12 Per Cent Decrease in Medial Area of Denervated Saphe- 91 nous Arteries from Animals Sampled at 60 and 90 Days. 13 Per Cent Increase in Luminal Perimeter of Denervated 93 Femoral Veins. ACKNOWLEDGEMENTS I s i n c e r e l y thank Dr. M. E. Todd for her support and encouragement, e s p e c i a l l y when I lacked self-confidence. I t was her p o s i t i v e comments that kept me going. The assistance provided by Mrs. Connie Leung and Miss Fanny Chu was greatly appreciated. A s p e c i a l thanks goes to Dr. A. W. Vogl for two reasons: 1) h i s advice on sectioning and 2) for h i s mere presence i n the Anatomy Building l a t e i n the evenings. I would also l i k e to thank Fred McConnell and Bernie Cox who helped me with many te c h n i c a l d i f f i c u l t i e s . Special appreciation goes to Dr. J . Weinberg, to Mrs. He l l a Prochaska and e s p e c i a l l y to my very dear f r i e n d , Mrs. Sharon Bezio. Also, I thank my parents who encouraged me to continue my education. 1 INTRODUCTION Introductory Statement Nerves have a profound effect on the development and maintenance of muscle. This has been shown as early as the 1800s by studying alterations in human skeletal muscle histology caused by diseases which lead to the destruction of motor neurons or by lesions of the peripheral nerves (Pellegrino and Franzini, 1963; Sarnat, 1983). From biopsies, the time at which the alteration in human muscle takes place i s not always known; therefore, laboratory animals have been used to obtain a better correlation between the time of denervation and the onset of such alterations as atrophy. Cross-union experiments, which arose in the 1960s, took advantage of the normal condition seen in many mammalian laboratory animals which was that certain skeletal muscles are composed almost entirely of fibres of one type (Sarnat, 1983). These experiments, by producing changes in fu l l y mature adult muscle, have increased our insight into the pathologic reactions of denervated muscle because they have shown that a muscle's innervation determines, not only i t s development and maintenance, but also the physiologic, metabolic, histochemical and ultrastructural properties of individual muscle fibres (Sarnat, 1983). As a result, these additional alterations were looked for in future denervation studies. Since human diseases of skeletal muscle have been the impetus for studying the effects of denervation on muscle, much less work has been done on denervation studies involving cardiac and smooth muscle. In fact, i t was generally believed that denervation of smooth muscle in vivo produced few morphological or physiological changes (Chamley and Campbell, 1975). Another factor which may have deterred denervation 2 studies on smooth muscle i s the problems with the denervation method. While severing or removing a portion of the nerve is a common method for skeletal muscle, denervation which selectively destroys the sympathetic innervation has been problematic. Nevertheless, we are becoming aware that these nerves do appear to have a trophic influence on smooth muscle. In Stedman's Medical Dictionary, "trophic" i s defined as "relating to or dependent upon nutrition." Nerves and their target tissues are known to exhibit this "nutritive" dependency. The exact nature of neurotrophic interactions i s s t i l l unknown; however, nerves can influence their effector tissues by i t s impulse activity and by any neurotrophic factors the nerve c e l l may produce. Neurotrophic interactions, therefore, have been defined by Gutman (1976) as the "long-term maintenance regulation not mediated by nerve impulses." That i s , diffusable substances which are non-impulse related are thought to be the neurotrophic factors. Drachman (1976), on the other hand, believes that: " a definition of "neurotrophic" must be both broad and non-prejudicial: broad in the sense that i t includes a l l the relationships by which nerve cells and target cells alter each other's anatomy, chemistry or physiology, and non-prejudicial in the sense that i t must neither presup- pose nor exclude any particular mechanism of trophic inter- action. ... Thus, we may define as "neurotrophic" any long-term relationship in which nerve cells and target cells interact so as to influence the structure or func- tion of either member of the pair." Therefore, Drachman's definition of "neurotrophic" can include the effects of impulse activity. Even though the exact nature of the trophic interaction i s s t i l l unknown, the biochemistry, physiology and morphology of target tissues are indeed dependent on their nerve supply. These parameters have been looked at extensively i n skeletal muscle and less so in cardiac and smooth muscles. 3 The Effects of Innervation on Skeletal Muscle In skeletal muscle, binding studies have allowed the biochemical examination of the density of cholinergic receptors in mouse and rat muscles (Thesleff, 1974). In normally innervated hemidiaphragm muscles of the rat and mouse, the density of cholinergic receptors at the junctional site i s two thousand times greater than at extrajunctional sites (Thesleff, 1974). Following denervation of these muscles, the receptor density at the junctional site i s maintained, but the extrajunctional density i s increased by 20-200 times. As a result of this increase, one observes supersensitivity of the muscle membrane. This physiological parameter was seen in a 14-day denervated muscle fibre of the cat's tenuissimus muscle where a constant pulse of acetylcholine produced depolarization at each point of the membrane (Thesleff, 1974). In the innervated fibre, depolarizations to acetylcholine were produced only at the end-plate region. Luco and Eyaguirre (1955) found that the onset of hypersensitivity to acetylcholine in the cat tenuissmus muscle occurred earlier, and the onset of "spontaneous" f i b r i l l a r y potentials occurred sooner when the nerve was severed at a locus closer to the muscle (that i s , when the length of the distal nerve stump was shorter). In cultured triceps muscle of the newt, Triturus viridescens, the presence of a sensory ganglion prevented the loss of muscle cholin- esterase activity (Lentz, 1974). After three weeks in culture, the cholinesterase activity declined unless the muscle was cultured with the ganglion explant. Boiling of the ganglion for one minute abolished this maintenance effect. The effects of the ganglion were seen whether the ganglion was placed on the muscle surface or whether i t was placed in a separate chamber separated from the muscle by a Millipore f i l t e r . 4 Hoffman and T h e s l e f f (1972) studied the trophic influence of nerve on the physiology of the r a t extensor digitorum longus muscle. By i n j e c t i n g cholchicine i n t o the s c i a t i c nerve 2cm proximal to t h i s muscle, the organization of microtubules was destroyed, thereby preventing the proximo-distal movement of macromolecules and organelles. Measurements of the frequency and amplitude of the miniature end-plate p o t e n t i a l s and of the si n g l e twitch and tetan i c twitch tensions on the c o l c h i c i n e treated side did not d i f f e r from the corresponding measurements on the c o n t r a l a t e r a l control s i d e . Therefore, c o l c h i c i n e had no e f f e c t on nerve conduction. On the fourth day, a f t e r the c o l c h i c i n e i n j e c t i o n , e x t r a j u n c t i o n a l a c e t y l c h o l i n e s e n s i t i v i t y and tetrodotoxin-resistant a c t i o n p o t e n t i a l s were exhibited by the muscle (tetrodotoxin generally blocks action p o t e n t i a l generation). These e f f e c t s gradually subsided a f t e r the fourth day. When fi-bungarotoxin (a neurotoxin that blocks spontaneous neurotransmitter release) was subcutaneously i n j e c t e d into the a n t e r o l a t e r a l region of the r i g h t hind l e g (thereby paralysing i t ) , or when the extensor digitorum longus was denervated by sectioning the deep peroneal nerve close to the knee, ext r a j u n c t i o n a l s e n s i t i v i t y and tetr o d o t o x i n - r e s i s t a n t action p o t e n t i a l s were observed. However, when these l a t t e r two methods were used, the extrajunctional a c e t y l c h o l i n e s e n s i t i v i t y and tetrodotoxin-resistant a c t i o n p o t e n t i a l s developed e a r l i e r and remained f o r 3 days longer than i n the c o l c h i c i n e experiments (Hoffman and The s l e f f , 1972). That i s , i n comparing these two methods, the denervation changes observed following c o l c h i c i n e treatment were of a shorter duration and were l e s s pronounced. Since the consequences of nerve severing or of using B-bungarotoxin cannot be reproduced t o t a l l y by using c o l c h i c i n e , Hoffman and T h e s l e f f (1972) suggested that not only are trophic substances released by the nerve, but also neuromuscular 5 transmission and r e s u l t i n g muscle a c t i v i t y are cont r i b u t i n g trophic i n f l u e n c e s . This i s the same point that Drachman (1974) expressed i n h i s d e f i n i t i o n of "neurotrophic". Denervation methods often have been used to study the dependency of s k e l e t a l muscle morphology on i t s innervation. Lentz (1974) found that by severing the b r a c h i a l plexus i n the newt, the ju n c t i o n a l folds on the muscle surface decreased i n height and eventually f l a t t e n e d out once the traces of the axoplasm disappeared (21 days). The remaining Schwann c e l l s soon withdrew in t o the e x t r a c e l l u l a r space leaving the neuromuscular junction u n i d e n t i f i a b l e . He also found the same phenomenon occurring i n cultured newt s k e l e t a l muscle, only i n t h i s case, the changes occurred more r a p i d l y than _in vi v o . Since the cultured muscle maintained i t s j u n c t i o n a l folds i n the presence of a sensory ganglion, even though i t s own axon had degenerated, t h i s suggests that perhaps the maintenance of the neuromuscular junction might be dependent on a d i f f u s a b l e neurotrophic f a c t o r . U l t r a s t r u c t u r a l examinations and morphometric analyses have been c a r r i e d out on denervated mammalian muscle (Pellegrino and F r a n z i n i , 1963; Gauthier and Dunn, 1973; Engel and Stonnington, 1 9 7 4 ; ) . The most obvious change they saw was a decrease i n the s i z e of the muscle f i b r e . During the f i r s t 3 weeks following denervation, morphometric an a l y s i s of the r a t soleus (red) and the r a t gastrocnemius (white) muscles showed an 80% decrease i n the transverse mean f i b r e area (Engel and Stonnington, 1 9 7 4 ) . At the same time, the mean transverse m y o f i b r i l l a r area decreased proportionately to, or at a s l i g h t l y greater rate than the f i b r e area. Two weeks post-denervation, Pe l l e g r i n o and F r a n z i n i (1963) noted a d i s t i n c t reduction i n the number of m y o f i b r i l s i n the r a t soleus and gastrocnemius. They suggested that t h i s decrease i n m y o f i b r i l s accounted 6 for a large proportion of the weight loss measured in these muscles. At 2 weeks, the denervated muscle weighed only two-thirds that of controls and by 4 weeks, i t weighed only four-ninths that of controls. Engel and Stonnington (1974) noted that in the soleus and gastrocnemius, myofibrillar atrophy i n i t i a l l y began at the periphery of muscle fibres, and after a month, spread to the interior of the muscle. In contrast, Pellegrino and Franzini (1963) saw peripheral and interior myofibrillar atrophy in the soleus (red fibers) but only peripheral atrophy in the gastrocnemius (white fibers). I n i t i a l l y during the atrophy process, the Z-lines lost their straight line configuration across the f i b r i l s , becoming bent and "smeared" in appearance (Pellegrino and Franzini, 1963; Gauthier and Dunn, 1973) . Red fibres have a wider Z-line than white fibres (Gauthier and Dunn, 1973); however, 2 weeks after denervation, the difference in width of the Z-line between red and white fibres became less apparent (Engel and Stonnington, 1974) . Gauthier and Dunn (1973) contradict this. They maintain that differences in width remained even 14 days after denervation. One might expect their differing observations to be due to differences in the length of the distal stump. That i s , one might expect Gauthier and Dunn (1973) to have a much longer distal stump than Pellegrino and Franzini (1963); however, this was not the case. Gauthier and Dunn (1973) severed the sciatic nerve close to i t s contact with the muscle, whereas Pellegrino and Franzini (1963) severed the sc i a t i c nerve high in the thigh. Since both studies used adult albino rats, the differences may have occurred because Gauthier and Dunn (1973) studied the semitendinosus muscle, and Pellegrino and Franzini (1963) studied the soleus and gastrocnemius muscles. In their 60 day study, Pellegrino and Franzini (1963) found that eventually the Z-lines no longer showed any filamentous structure. They also discovered that a 7 single f i b r i l may show regions of disorganization at several points along i t s length with normal sarcomeres in between. One month post-dener- vation, peripheral filaments of myofibrils were found in the i n t e r f i b r i l l a r spaces where i t was assumed they were enzymatically destroyed by the lysosomes seen between the f i b r i l s (Pellegrino and Franzini, 1 9 6 3 ) . While fiber size and myofibrils decrease in size, there also occurs an absolute and relative increase in mitochondrial mass (Engel and Stonnington, 1974),; however, absolute mitochondrial mass decreases after a week and becomes proportionate to fibre size (Engel and Stonnington, 1974; Pellegrino and Franzini, 1 9 6 3 ) . Mitochondria, which normally are relatively uniformly distributed, tended to aggregate into small clusters, and change from being elongate in the transverse plane to being vi r t u a l l y parallel to the long-axis of the myofibril after denervation (Engel and Stonnington, 1974). The trend seen for mitochondrial mass i s the same for sarcotubular element concentration, only the decrease in sarcotubular elements (sarcoplasmic reticulum and transverse tubules) seen after the f i r s t week is less than the decrease seen in contractile elements; therefore, as a net result, the concentration of sarcotubular elements increases (Engel and Stonnington, 1 9 7 4 ) . Morphologically, the spatial arrangement of the sarcotubular elements becomes increasingly irregular, and sometimes the elements possess focal dilations (Engel and Stonnington, 1 9 7 4 ) . After one month of denervation, parallel arrays of tubular profiles representing proliferating components of the sarcoplasmic reticulum are seen (Engel and Stonnington, 1 9 7 4 ) . Morphological changes in other skeletal muscle organelles have also been noted. Lysosomes are present soon after denervation (Gauthier and 8 Dunn, 1973; P e l l e g r i n o and F r a n z i n i , 1963), and as the rate of degene- r a t i o n i n the f i b e r s increases, lysosomes become la r g e r and heavily loaded with material (Pellegrino and F r a n z i n i , 1963) . Lipofuscin granules and small autophagic v e s i c l e s were seen one week post-denervation by Engel and Stonnington (1974). Sometimes, c e n t r a l l y - located n u c l e i are found (Engel and Stonnington, 1974; P e l l e g r i n o and F r a n z i n i , 1963), and prominent Golgi networks are seen with moderate frequency i n the denervated muscle (Engel and Stonnington, 1974) . Gauthier and Dunn (1973) discovered an increase i n subsarcolemmal sarcoplasmic ribosomes i n denervated muscle, and they hypothesized that t h i s increase i n protein synthesising material could produce many new a c e t y l c h o l i n e receptors that might account for the increase i n a c e t y l c h o l i n e s e n s i t i v i t y of the s k e l e t a l muscle membrane. The E f f e c t s of Cardiac Muscle on Nerves Thus, denervation studies have shown that s k e l e t a l muscle i s dependent on i t s innervation to maintain i t s i n t e g r i t y . Muscles also appear to a f f e c t t h e i r nerves. Experiments using the supernatant from homogenized, denervated r a t cardiac muscle showed that the e f f e c t o r muscle t i s s u e can a f f e c t the s u r v i v a l of neurons (Kanakis et a l . , 1985) . Adult r a t s were denervated using an i n j e c t i o n of 6-hydroxydopamine (6-OHDA) which produces a chemical sypathectomy. The denervated cardiac muscle was tested to see how e f f e c t i v e 6-OHDA was at denervating t h i s muscle. The a c t i v i t y of tyrosine hydroxylase, an enzyme involved i n the r a t e - l i m i t i n g step i n the biosynthesis of noradrenaline, was used to assess t h i s r denervation. Although i t was found that denervation of the heart was not complete, i t was indeed e f f e c t i v e since treatment with 6-OHDA caused a s i g n i f i c a n t reduction i n tyrosine hydroxylase. Hearts were removed 4 9 days after injection and then homogenised. The heart extracts were then used to assess their a b i l i t y to promote the survival of dissociated 12-day old chick lumbar sympathetic ganglia. Sympathetic neuronal survival was significantly increased using the extracts of denervated heart when compared to the normal, control heart extracts. Extracts from the control and denervated hearts were then run through gels containing antibodies to Nerve Growth Factor (NGF). In normal tissue, NGF occurs in very low amounts. There was no significant difference in the effects on sympathetic neuronal survival between the treated and untreated control extracts. However, the effect of the anti-NGF-treated extract from denervated hearts on sympathetic neuronal survival was significantly decreased when compared to the untreated, denervated heart extract. This showed that NGF increases in denervated tissue and that NGF i s an important factor for the survival of sympathetic nerves. It was very interesting to note that in their experiments, Kanakis et a l . (1985) found that the anti-NGF-treated extracts from denervated hearts s t i l l promoted sympathetic neuronal survival better than untreated and anti-NGF-treated control extracts, although the difference was only significant at the 0.05 level with the treated control extract. This demonstrated that components (other than NGF) which have the a b i l i t y to enhance the survival of cultured sympathetic neurons may also be increased after denervation. As shown, many denervation studies on skeletal muscle have been carried out. Changes in the biochemistry resulting from denervation have been seen as an increase in the density of extrajunctional acetylcholine receptors. Supersensitivity of the muscle membrane and the presence of tetrodotoxin-resistant action potentials indicated changes in the physiology of the denervated muscle. Morphological changes include decreases in the junctional folds, decrease in size of muscle fibre, reduction of myofibrils and changes in the organelles. Also, with the work involving the denervated cardiac muscle extract, we can see that the effector tissue has a trophic influence on the nerve. The Effects of Innervation on Smooth Muscle There has been a tremendous amount of research carried out on the role of cholinergic innervation on skeletal muscle maintenance; an interest has also developed in the trophic interactions between smooth muscle and i t s adrenergic innervation. However, since not as much is known in this particular area, many preliminary hypotheses of trophic influences on smooth muscle have been derived by analogy to skeletal muscle. Nevertheless, skeletal muscle i s very different from smooth muscle, both in i t s ultrastructure and i t s innervation; thus, the analogies that have been made may not be correct. In vitro experiments, designed to study the trophic influences of sympathetic nerves on smooth muscle, have been carried out on smooth muscle from the guinea-pig vas deferens (Chamley and Campbell, 1975). Single c e l l suspensions of these cells were allowed to settle on collagen-coated glass coverslips, and then they were exposed to various substances. Afer 8 days in culture, smooth muscle ce l l s in control cultures had undergone intense proliferation and ultrastructurally, they looked dedifferentiated. That i s , they resembled embryonic smooth muscle. After 8 days in culture in the presence of sympathetic ganglion extract, dibutyryl cyclic AMP or theophylline, smooth muscle proliferation was prevented and the cells were maintained in their differentiated state. Spinal cord extract, l i v e r extract or noradre- naline resulted in smooth muscle cells that were intermediate in appearance between those j u s t described and the d e d i f f e r e n t i a t e d smooth muscle c e l l s of the control c u l t u r e s . However, a f t e r 8 days i n culture i n the presence of d i b u t y r y l c y c l i c AMP plus theophylline (a smooth muscle relaxant which increases i n t r a c e l l u l a r c y c l i c AMP) smooth muscle c e l l s appeared very w e l l - d i f f e r e n t i a t e d . In f a c t , they possessed few organelles, a c h a r a c t e r i s t i c of adult in vivo guinea-pig vas deferens. Chamley and Campbell (1975) suggested that the trophic substance or substances may act by s e l e c t i v e l y stimulating the enzyme adenyl cyclase (which converts ATP to c y c l i c AMP), a suggestion further exemplified by the a d d i t i o n of the substance, theophylline. Smooth muscles from d i f f e r e n t areas of the body respond d i f f e r e n t l y to s i m i l a r experimental treatments. For instance, smooth muscle from young r a b b i t thoracic aorta and ear a r t e r y were cultured and treated by Chamley and Campbell (1976) i n s i m i l a r ways as they treated the guinea-pig vas deferens smooth muscle just described. A f t e r 1-2 days i n c u l t u r e , the vascular smooth muscle consisted of 2 types of smooth muscle c e l l s d istinguished on the basis of morphology. They included (1) a ' d i f f e r e n t i a t e d ' c e l l type, which resembled the c e l l s of normal i n vivo r a b b i t media and only a small number underwent d i v i s i o n , and (2) an ' u n d i f f e r e n t i a t e d ' c e l l type, which underwent frequent d i v i s i o n and whose cytoplasm was f i l l e d with protein synthesizing machinery. The ' d i f f e r e n t i a t e d ' c e l l s maintained t h e i r morphology for only 4 days before changing to resemble the 'undifferentiated' c e l l type. In the presence of a sympathetic chain homogenate, the ' d i f f e r e n t i a t e d ' smooth muscle c e l l type maintained i t s d i f f e r e n t i a t e d appearance for at l e a s t 6 days. The presence of s p i n a l cord extract or noradrenaline made no diff e r e n c e to the cultured medial smooth muscle c e l l s ; that i s , these treated cultures did not d i f f e r from the control c u l t u r e s . In contrast, these 12 same substances did a f f e c t the cultured guinea-pig vas deferens smooth muscle c e l l s somewhat, producing a c e l l intermediate i n appearance between the ' d i f f e r e n t i a t e d ' and the 'de d i f f e r e n t i a t e d ' types. Smooth muscle from d i f f e r e n t areas of the body also may r e f l e c t t h e i r d ifferences by t h e i r a b i l i t y to a t t r a c t regenerating neurons. In normal sk i n , nonspecific cholinesterase and acetylcholinesterase a c t i v i t i e s are observed i n erector p i l i muscles and t h e i r nerves; however, fluorescent varicose adrenergic nerves were also also seen i n the erector p i l i muscles (Waris, 1 9 7 8 ) . In r a t ski n autographs, fluorescence microscopic techniques s p e c i f i c f o r catecholamines showed that the erector p i l i muscles were not re-innervated at the end of a 20 week period, but blood vessels i n t h i s g r a f t were p a r t i a l l y innervated at 16 and 20 weeks a f t e r transplantation (Waris, 1 9 7 8 ) . Hence, trophic i n t e r a c t i o n s between one type of smooth muscle and i t s sympathetic innervation should be compared with other studies that have used the same type of smooth muscle, and, i n studies of the c i r c u l a t o r y system, from the same part of the vascular t r e e . That vessels from d i f f e r e n t parts of the c i r c u l a t o r y system may r e f l e c t d ifferences i n t h e i r a b i l i t y to a t t r a c t regenerating neurons has been shown by transplantation experiments and tiss u e cultures of smooth muscle. Transplantation of the r a t femoral a r t e r y i n t o the a n t e r i o r chamber of an eye of a host r a t did not induce i r i d e a l nerve sprouting whereas the t a i l a r t e r y did (Todd, 1 9 8 6 ) . Also, smooth muscle cultures containing medial c e l l s from r a b b i t thoracic aorta or ear ar t e r y i l l u s t r a t e d a difference i n t h e i r i n t e r a c t i o n with r a b b i t sympathic ganglion explants (Chamley and Campbell, 1 9 7 6 ) . Nerve f i b e r s growing from the explants formed l o n g e r - l a s t i n g associations (up to 8 days) with the smooth muscle c e l l s from the ear ar t e r y than they did with c e l l s from 13 the thoracic aorta (1-2 hours). Therefore, r e s u l t s from one area of the vascular tree may not always be extrapolated to predict the r e s u l t s i n another part of the vascular tree, even within the same animal. Most of the i n vivo work i n v o l v i n g neurotrophic influences on vascular smooth muscle has been done on the r a b b i t ear a r t e r y by Bevan and her co-workers. In one study, Bevan (1975) denervated the l e f t r a b b i t ear a r t e r y by completely removing the l e f t superior c e r v i c a l ganglion i n 4 week old r a b b i t s . Two weeks postoperatively, a decrease i n the uptake of 3 H-Tdr by the vascular smooth muscle c e l l s i n the denervated l e f t ear a r t e r y was measured by s c i n t i l l a t i o n counting and by autoradiography. Denervated smooth muscle also appeared to have fewer m i t o t i c f i g u r e s . These r e s u l t s seem to i n d i c a t e that denervation i n h i b i t e d the p r o l i f e r a t i o n of the medial smooth muscle c e l l s . Bevan and Tsuru (1979) l a t e r postulated that since p r o l i f e r a t i v e growth decreased upon denervation, t h i s might create a smaller blood vessel which would be incapable of producing the maximum force o f contraction. Rabbits of 9 - H weeks of age underwent superior c e r v i c a l ganglionectomy. Eight weeks a f t e r the ganglionectomy, there was a noticeable decrease i n the wall thickness and i n the weight of the r a b b i t ear a r t e r y . The maximum force and the maximum tension were markedly decreased and i t was postulated that the r e s u l t s were due to a l o s s of smooth muscle mass and to a q u a l i t a t i v e change i n the c o n t r a c t i l e machinery, r e s p e c t i v e l y . S u p e r s e n s i t i v i t y to norepinephrine was also a consequence of denervation. An increased e l a s t i c modulus ( s t i f f e r wall) was evident as w e l l . This may r e f l e c t changes i n the e l a s t i c t i s s u e . Denervation i n t h i s case a f f e c t e d p r o l i f e r a t i o n and s e n s i t i v i t y of the smooth muscle c e l l s which, i n turn, a f f e c t e d the a r c h i t e c t u r e and mechanics of the blood v e s s e l . Bevan and Tsuru (1981) repeated the above procedure on three different age groups to see the effect i t may have on developing arteries. The three groups were (1) a growing group (3-4 weeks old), (2) a young adult group (9-11 weeks old) and (3) a mature group (16-20 weeks). When compared with their controls, significant decreases in cross sectional area of the media were seen in the f i r s t two denervated age groups. In the third group (mature animals), there was no significant decrease in the cross sectional area of the media after denervation. These studies on development carried out by Bevan and Tsuru (1981) correlate with those carried out by Rusterholz and Mueller (1982). Rusterholz and Mueller (1982) used the method of a unilateral superior cervical ganglionectomy on rabbits to evaluate the possible chronic influence that the sympathetic nerves might have on vascular resistance and to see i f the results were age-dependent. They studied denervation i n three separate groups which they termed (1) growing acute denervation (rabbits denervated at 4 weeks and studied approximately 9 days later), (2) growing chronic denervation (rabbits denervated at 4 weeks and studied 9 weeks later) and (3) adult chronic (rabbits that were denervated at 16 weeks and studied 10 weeks la t e r ) . Changes in vascular resistance were seen as changes in flow-perfusion pressure curves. Perfusion pressure was measured in maximally dilated vessels and this pressure measures the resistance of the vessels. Results from the vascular bed in denervated ears and the contralateral innervated ears were compared. A comparison between the denervated and innervated ears in the growing acute denervated group (ie. those studied only 9 days after denervation) showed no difference i n the perfusion pressure. The perfusion pressure of the denervated ear of the growing chronic denervated group (ie. those studied 9 weeks after denervation) was significantly lower than that from i t s contralateral control side. Therefore, i t seems that 9 days was an insufficient length of time after denervation for any noticeable changes to occur. No differences in the perfusion pressure curves were seen between the denervated ear and the contralateral innervated ear from the adult chronic denervated group. Thus, denervation does produce a decrease in vascular resistance in developing vessels but not in mature vessels and this decrease i s observed only after a substantial length of time following the ganglionectomy. Rusterholz and Mueller (1982) believe their results are compatible with the idea of the existence of an interaction between sympathetic nerves and blood vessels. Although the mechanism of this interaction i s unknown, they proposed that the decrease in vessel resistance was probably not exclusively the result of smooth muscle atrophy but also may have involved other factors such as an alteration in elastin/collagen ratio or an in alteration smooth muscle configuration. Bevan's report (1983) of an increase in arteriovenous anastomoses in the denervated rabbit ear artery may also be responsible for the observed decrease in perfusion pressure. Morphological studies on denervated blood vessels have been carried out by Branco et a l . (1984). They looked at the dog saphenous vein and the rabbit ear artery and found the wall of the denervated saphenous vein was thicker. Ultrastructurally, the smooth muscle ce l l s of the denervated saphenous vein had the appearance of dedifferentiated cells containing a l l the organelles characteristic of active protein synthesis. Their findings were similar in the denervated saphenous artery except the alterations i n the artery were restricted to the 2-3 smooth muscle layers closest to the adventitia. In both the artery and vein, the a l t e r a t i o n s were r e v e r s i b l e . Therefore, denervation studies on smooth muscle, p a r t i c u l a r l y vascular smooth muscle, are not as extensive as i n s k e l e t a l muscle. Nevertheless, changes i n vascular smooth muscle, as a consequence of denervation do occur. These changes include increased s u p e r s e n s t i v i t y to norepinephrine, decreases i n maximum force and maximum tension, a decrease i n smooth muscle c e l l p r o l i f e r a t i o n and a r e v e r s a l to a ded i f f e r e n t i a t e d u l t r a s t r u c t u r a l appearance. Methods of Sympathetic Denervation To study the denervation e f f e c t s on the ra b b i t ear arte r y , Bevan and Tsuru (1975; 1979; 1981), Branco et a l . (1984) and Rusterholz and Mueller (1982) used the method of u n i l a t e r a l sympathetic ganglionectomy. In the case of studying the ra b b i t ear ar t e r y , t h i s involves the removal of the en t i r e superior c e r v i c a l ganglion. Consequently, innervation to one h a l f of the head i s l o s t . Chemical sympathectomy involved the use of 6-hydroxydopamine (6-OHDA) as the denervating factor (Finch et a l . , 1 9 7 3 ) . Finch et a l . (1973) noted that administration of 6-OHDA in t o adult r a t s produced a s e l e c t i v e , temporary destruction of adrenergic nerve terminals. Regeneration of the adrenergic nerve terminals i n blood vessels was very f a s t and within j u s t a few days a f t e r i n j e c t i o n of 6-OHDA, and almost complete f u n c t i o n a l recovery was seen. Finch et a l . (1973) were therefore i n t e r e s t e d i n seeing i f 6-OHDA i n j e c t e d i n t o newborn r a t s would produce a complete and permanent destruction of the adrenergic nerves supplying the vascular system. In t h e i r study they compared the e f f e c t s of administering 6-OHDA in t o two d i f f e r e n t age groups of r a t s : one group was treated for the f i r s t 14 days d i r e c t l y a f t e r b i r t h and looked at 8 weeks l a t e r and the other group, the adult group, was given two injections, 7 days apart and looked on the next day after the last injection. To determine the effectiveness of denervation produced by 6-OHDA, stimulation of the entire sympathetic outflow was carried out on pithed rats which were previously adrenalectomized. The steel pithing rod was used as an electrode to stimulate spinal nerve roots. Sympathetic outflow was stimulated with supramaximal voltage and increasing frequencies. A rise in blood pressure with increasing stimulation frequency was seen in the control and newborn-treated animals although the newborn-treated pithed group was markedly lower (37 mmHg) than the controls (120 mmHg). No rise in blood pressure was seen in the adult-treated pithed group at any stimulation frequency. Since a small rise in blood pressure was observed in this latter age group in unpithed preparations, i t was possible that in the pithed rat, the steel rod used for stimulation did not excite a l l sympathetic nerves (Finch et a l . , 1973). They also found a depletion in norepinephrine levels in the newborn treated group, however, the percent depletion varied with the tissue type (eg. mesentery vascular bed had 50-60% of the norepinephrine content l e f t whereas norepinephrine in cardiac muscle was depleted to less than 5% of control levels). Since the levels of norepinephrine did not increase up to an age of 16 weeks, Finch et a l . (1973) considered the denervation to be permanent as a consequence of the destruction of c e l l bodies in the sympathetic ganglia by 6-OHDA. Using anaesthetized rats, Finch et a l . (1973) compared the extent of vasoconstriction of isolated renal artery preparations between the two groups. Periarterial nerve stimulation was carried out to observe the vasoconstrictor responses. Vasoconstrictor responses of the rats treated at birth did not differ from those of the controls. The adult-treated 18 r a t s showed reduced vasoconstrictor responses. Even though Finch et a l . (1973) showed i n t h e i r study that chemical sympathectomy i s a successful method of denervating c e r t a i n organs such as the heart, i t did not prove to be very promising as a means of achieving vascular denervation i n younger r a t s . In newborn-treated r a t s , t h i s method produced a permanent but incomplete denervation whereas i n the adult i t produced a v i r t u a l l y complete but non-permanent denervation. Immunological sympathectomy i s an a l t e r n a t i v e method of denervation. Histology, response to e l e c t r i c a l stimulation of lumbar sympathetic ganglia, increased s e n s i t i v i t y to norepinephrine and r e s u l t s of chemical stimulation of the sympathetic ganglia with 1,l-dimethyl -4-phenyl-pipe- razinium iodide (DMPP) are methods that have been used to determine the success of denervation (Brody,1964; Levi-Montalcini and A n g e l e t t i , 1966) . DMPP i s a white c r y s t a l l i n e substance that i s soluble i n water and i s not intended for therapeutic use (Chen et a l . , 1951) . When in j e c t e d intravenously i n t o animals, i t stimulates the sympathetic ganglia by a c t i n g at the n i c o t i n i c - l i k e receptors of the postganglionic synaptic membrane (Szekere, 1980) . This excites the post-synaptic neuron thereby causing an increase i n a r t e r i a l blood pressure and tachycardia. Immunological denervation has been achieved by i n j e c t i n g antiserum to Nerve Growth Factor i n t o r a t s and mice immediately a f t e r b i r t h . I n i t i a t i n g the i n j e c t i o n s immediately a f t e r b i r t h i s e s s e n t i a l to produce the most extensive denervation (Levi-Montalcini and A n g e l e t t i , 1966) . Denervation has been proven to be permanent by carrying out h i s t o l o g i c a l studies on mice two years a f t e r they were treated at b i r t h with the antiserum (Levi-Montalcini and A n g e l e t t i , 1966) . This method of denervation was shown to s u c c e s s f u l l y a b o lish vasomotor function since neither vasoconstrictor nor v a s o d i l a t i o n occurred when the lumbar chains of the immunized rat were el e c t r i c a l l y stimulated (Brody, 1964). Levi-Montalcini and Angeletti (1966) found that the average resting blood pressure in treated rats was only 70 mmHg compared to lOOmmHg in controls. Blood pressure seems to be proportional to the degree of sympathetic innervation. Another change found by Levi-Montalcini and Angeletti (1966) with this type of denervation was a severe decrease (but not total abolition) in the c e l l population of various ganglia - superior cervical (rats:residual c e l l population was 10-15$ of controls), celiac, stellate and thoracic chain ganglia. However, their results from pharmacological and physiological testing (ie. reactivity of the vascular system to chemical or ele c t r i c a l stimulation) suggest that the residual c e l l populations have very l i t t l e , i f any, functional activity. The view that blood pressure values are proportional to the degree of sympathetic innervation was also supported by Gerova et a l . (1974) who found that the maximum diameter reduction (compared to resting diameter as a percent) of the densely innervated femoral arteries in puppies, was much higher than than that found in adult dogs, whose innervation was less dense relatively. In their examination of the responses of an isolated vascular bed to sympathetic neurotransmitters and sympathetic nerve stimulation in order to determine the functions of vascular smooth muscle and i t s innervation in newborn dogs, Boatman et a l . (1965) used mongrel puppies aged 1 day and 1, 2, 4, and 8 weeks and adult dogs. They found that the vasoconstriction of the blood vessels in the hind limbs of dogs which was induced by nerve stimulation increased with age. They attributed this rise to the increase in functional maturity of the adrenergic vasomotor function. It was also noted that this onset and development of induced vasoconstriction coincided in age with the onset and development of the systemic blood pressure. This age-related increase i n blood pressure was also demonstrated on r a t s by L a i s et a l . (1977) as well as by G e r r i t y and C l i f f (1975) . These observations on blood pressure r e l a t e d i r e c t l y to the increase i n catecholamine fluorescence, to the presence of dense-cored v e s i c l e s and to the number of nerve processes i n r a t a r t e r i e s as they matured (Todd, 1980; Todd and Tokito, 1981) . Problems with Denervation Methods From these studies, i t i s evident that sympathetic innervation can a f f e c t vascular smooth muscle both s t r u c t u r a l l y and f u n c t i o n a l l y , and thereby may a l t e r the structure and function of the e n t i r e blood v e s s e l . The i i i vivo methods of denervation used so far have included s u r g i c a l ganglionectomy (Bevan and Tsuru, 1979; Bevan and Tsuru, 1981; Branco et a l . , 1984), chemical (Finch et a l . , 1973) and immunological (Levi-Montalcini and A n g e l e t t i , 1966) sympathectomies. Su r g i c a l sympathectomy involves the removal of an e n t i r e sympathetic ganglion such as the superior c e r v i c a l ganglion. This procedure eliminates the innervation to one h a l f of the head. Chemical sympathectomy involves using 6-hydroxydopamine (6-OHDA) which destroys adrenergic nerve terminals. This destruction i s s e l e c t i v e , but permanent i f administered to newborns, and complete, but non-permanent i f given to adults (Finch et a l . , 1 9 7 3 ) . Kanakis et a l . (1985) found that i n adult r a t s i t produced an e f f e c t i v e but incomplete denervation of the heart. To produce an immunosympathectomy, i n j e c t i o n s of antiserum to Nerve Growth Factor are given intravenously. I f given immediately a f t e r b i r t h , i t produces the most extensive and permanent denervation (Levi-Montalcini and A n g e l e t t i , 1966) . Thus, only when these three denervaton procedures are carried out on very young animals, are they the most permanent and do they produce the most noticeable changes in blood vessels (Levi-Montalcini and Angeletti, 1966; Finch et a l . , 1973; Bevan and Tsuru, 1979; 1981). These methods of denervation eliminate innervation from a large area in the body of the animal, that i s , the denervation i s not localized. These widespread denervations may produce unwanted, possibly toxic, effects which may have contributed to the results obtained by these past investigators. To avoid these aberrant effects, a localized denervation method i s necessary. This was attempted by Todd (1986) on rat thigh blood vessels, but was unsuccessful as a result of rapid regeneration. Severing, removing or repositioning the femoral nerve are not successful methods since re-innervation of the saphenous and superficial and epigastric arteries occurs in under 15 days (Todd, 1986). Rusterholz and Mueller (1982) found that denervation of young rabbit ear arteries produced a decrease in perfusion pressure 9 weeks post-denervation, but not at 9 days post-denervation. Hence, not only i s a localized' denervation method required, but also a permanent or semi-permanent denervation method since a sufficient post-operative length of time must past before changes are seen in the vessels. Thesis Topic The aim of my study, therefore, was two-fold. F i r s t , I wanted to develop a way of studying denervation effects on blood vessels under the most normal conditions possible, meaning, in vivo, leaving a l l of the nerves intact except for those innervating the vessels under investigation, and without having any side effects produced by c i r c u l a t i n g drugs or antibodies. Therefore, i t was necessary to develop a technique that would keep the blood vessels denervated for as long as p o s s i b l e . Second, I wanted to t e s t the hypothesis that blood vessels and t h e i r adrenergic innervation do exert trophic influences over each other. To achieve my o b j e c t i v e s , I developed a novel method o f denervating blood vessels i n the r a t thigh and measured changes i n vessel wall thicknesses and luminal perimeters. MATERIALS AND METHODS Animals Female Wistar r a t s from an inbred colony maintained i n the Department of Anatomy were denervated at 3 or 12 days of age. The mothers were fed Purina Lab Chow and water ad l i b i t u m . The pups were weaned at one month of age at which time they were fed the same d i e t as t h e i r mothers. They were housed i n pairs i n hanging cages i n a c o n t r o l l e d environment having a 12 hour dark, 12 hour l i g h t c y c l e . The denervated blood vessels were sampled when the pups were 3 0 , 60, 90 or 120 days of age. A t o t a l of 18 animals was used for the l i g h t and electron microscopic a n a l y s i s and 8 animals were sampled for fluorescence microscopy Denervation Procedure The animals were anaesthetized i n a glass desiccator i n which absorbent cotton was moistened with anhydrous ether (Fisher S c i e n t i f i c L t d . ) . Anaesthesia was maintained during the denervation procedure by using a nose cap containing absorbent cotton moistened with ether. The anaesthetized animals were placed on a small p l e x i g l a s s table, dorsal side down. Their f o r e - and hindlimbs were lo o s e l y pinned down under e l a s t i c bands and t h e i r abdomens were d i s i n f e c t e d with Savlon (Ayerst Laboratories). A small v e r t i c a l skin i n c i s i o n was made i n the r i g h t thigh and the r i g h t femoral, s u p e r f i c i a l e p i g a s t r i c and saphenous a r t e r i e s and femoral nerve exposed (Figure l a ) . The femoral nerve was traced back to the in g u i n a l ligament. The femoral nerve was mobilized and separated from 24 the femoral art e r y and s k e l e t a l muscle adjacent to the inguinal ligament. A length of black suture s i l k (8.0, Deknatel) was t i e d around the femoral nerve i n two places, as close to the inguinal ligament as possible, and then again more d i s t a l l y (Figure l b ) . The femoral nerve was severed just d i s t a l to the second knot (Figure 2a). The portion of femoral nerve d i s t a l to the cut was separated from the femoral a r t e r y and severed where the saphenous arte r y branches o f f the femoral art e r y (Figure 2a). Therefore, the length of femoral nerve from the second knot to the branch point was removed and discarded. Inside the abdominal c a v i t y , the femoral nerve runs c r a n i a l l y i n the po s t e r i o r wall of the abdominal c a v i t y . Therefore, a t i n y , v e n t r a l , paramedial i n c i s i o n was made i n the abdominal wall and the femoral nerve i n s i d e the abdominal c a v i t y was found. By gently p u l l i n g on the nerve here, the portion of the femoral nerve that was out i n the thigh was brought i n s i d e the abdominal c a v i t y . The black suture s i l k that was t i e d around the nerve was threaded through a length (approximately 1 cm) o f polyethylene tubing (PE-10, Clay Adams). The tube was s l i d over the nerve and the d i s t a l end was melted using a cautery gun (Figure 2b). The melted p l a s t i c sealed the tube and also secured the black suture s i l k when i t hardened, thereby securing the nerve i n s i d e the tube. The encased nerve was tucked back ins i d e the abdominal c a v i t y and the abdominal wall was sutured closed using 6.0 Deknatel suture s i l k . The sk i n i n c i s i o n was clamped with 7.5 mm wound c l i p s (Medicon) and cleaned of blood using Savlon. A f t e r regaining conciousness from the anaesthetic, the pups were sprinkled l i g h t l y with baby powder and returned to t h e i r mother. Vick's Vapo rub was inserted i n t o the mothers' n o s t r i l s when the pups were returned to them. Both, the Vick's Vapo rub and the baby powder were used to prevent cannibalism by the mothers. Tissue Sampling and Processing At the time of sampling, the animal's length, weight and t a i l cuff blood pressure were recorded. A programmed electro-sphygmomanometer (Narco Bio-Systems, Inc.), connected to a polygraph, was used for t a i l cuff pressure recordings. Following this, the animals were anaesthetized in the same manner as for denervation and then perfusion fixed using the following procedure (Todd et a l . , 1983). The ventral neck and abdominal regions were shaved and the areas were sponged with Savlon. A midline incision was made in the ventral neck region. The l e f t common carotid artery was found and cleared of i t s enveloping connective tissue. The vagus nerve was then carefully separated from the common carotid artery. The artery was ligated in two places using 4.0 Deknatel suture s i l k . The f i r s t ligature was tied tightly and as cranially as possible, and a second loose knot was made with this same ligature (Figure 3). The second ligature was tied approximately 1 cm caudal to the f i r s t . The latter knot was not tightened until the cannula was inserted, serving to hold i t in place. A small a r t e r i a l haemostat was clamped immediately proximal to the position of the second ligature. The cannula was a length of PE-50 polyethylene tubing (Clay Adams) bevelled at both ends, f i l l e d with a 1% heparin solution in saline and clamped at one end with a pair of hemostats. A tiny incision was made in the ventral wall of the artery and the cannula was inserted through the loose knot of the f i r s t ligature and then into the lumen of the artery. Once inside the lumen of the artery, the cannula was gently pushed caudally past the region of the second ligature. At this point, the second ligature and the second knot of the f i r s t ligature were securely tightened, and the small art e r i a l hemostat was released. The hemostats were removed from the cannula to see i f blood were being pumped through the cannula without being blocked. The cannula was then r e f i l l e d by injecting heparin and then connected through a T-tube to a syringe perfusion pump (Sage Instruments) and a pressure transducer (Statham Transducer, Gould). The pressure transducer was connected to a polygraph (Grass Instruments Co.) for blood pressure recordings, and was calibrated using a mercury manometer. The systolic, diastolic and mean blood pressures were recorded via the intra-arterial cannula (Figure 4) and then the perfusion was started. A 3% glutaraldehyde/2% paraformaldehyde solution (pH 7.3) made up in glucose-Krebs solution (Palaty, 1971) was perfused at the animal's mean blood pressure which was maintained with the continuously variable perfusion pump. Once the perfusion was started, the ti p of the t a i l was cut off and the skin on the ventral abdomen was cut and reflected back to expose the thigh vessels. The right and l e f t superficial epigastric veins were cut. Clear fixative was seen passing out of these veins and the t a i l shortly after perfusion started. Over a period of approximately 20 minutes, a total of 30-50 ml of fixative was perfused through each animal. A continuous recording was made of the perfusion pressure (Figure 4). In a l l animals, the right and l e f t saphenous arteries were removed. In some animals, samples of the right and l e f t saphenous and femoral nerves and veins were taken. Therefore, 18 samples of the right and l e f t saphenous arteries and 6 samples of right and l e f t femoral veins were removed. Also, femoral nerve samples were taken from inside the abdominal cavity. For the right femoral nerve, they were taken adjacent to the proximal end of the tube. The l e f t nerve was sampled from an equivalent area. Fixation: A l l tissues were processed the same way. They were fixed in the 3% glutaraldehyde/2% paraformaldehyde solution for a total of 1.5hr at room temperature (including perfusion time), followed by 0.5hr at 0°C. Since higher temperatures of this fixative promote the rate of penetration and maintain labile structures such as microtubules, and since artifacts due to polymerization of glutaraldehyde are minimized at lower temperatures, this sequence of aldehyde fixation acts as a compromise of the effects that occur at higher and lower temperatures. This was followed by two-5 minute washes in glucose-Krebs solution at 0°C and two-5 minute washes in 0.1M cacodylate buffer at 0°C. After washing, the tissues were fixed in 1% osmium tetroxide in 0.1M cacodylate buffer for 1.5 hr at 0°C. Following this, were four-5 minute washes in 0.1M cacodylate buffer at 0°C and one-5 minute wash in d i s t i l l e d water at 0°C in preparation for en bloc staining in saturated aqueous uranyl acetate (1 hr at 0°C). Before dehydration, the tissues were washed o again in d i s t i l l e d water for 5 minutes at 0 C. Dehydration: The tissues were put through an acetone dehydration sequence: 50%, 0°C, 5 min.; 75%, 0°C, 10 min.; 90%, 0°C, 10 min.; 100%, 0°C, 10 min.; 100%, room temperature, 5 min.; 100% room temperature, 5 min. Inf i l t r a t i o n : I n f i l t r a t i o n of the tissues using increasing ratios of Mollenhauer's (1961) embedding mixture (25 Epon 812 : 15 Araldite 502, Electron Microscopy Sciences): acetone solutions was done at room temperature. Tissues remained in the solutions of the ratios 1:3 and 1:2 for 30 min.. They were transferred to a 1:1 solution and the vials were put on an electric rotator (Labtronix Equipment) overnight. The next day, tissues were exposed to solutions of 2:1 followed by 3:1 ratios, 60 minutes in each. The tissues were then transferred to clean vials containing pure Epon-Araldite embedding mixture and placed in a vacuum r for approximately 1 hr. Following this, they were placed in pure, fresh embedding mixture again and put on the electric rotator overnight. In the morning, the embedding mixture was exchanged for fresh, and the open via l s were placed in a vacuum oven unt i l the length of time the tissue spent in pure Epon-Araldite embedding mixture totalled 24 hours. The tissues were embedded individually in freshly made, pure Mollenhauer•s embedding mixture. The vessels were placed in rubber molds and oriented so that they would be at right angles to the plane of sectioning. The resin was polymerized at 60°C for 48 hours and the blocks were further cured for 2 or more days at room temperature. Sectioning The vessels were aligned perpendicular to the knife edge so that complete transverse sections could be cut for light microscopy. Thick sections (0.5)im) for light microscopy were cut on a Reichert 0mU3 ultramicrotome with a glass knife. The sections were placed on a glass slide and heat fixed at a high temperature on a Sybron Thermolyne hot plate. They were stained with a 1:1 mixture of 1% azur II and 1% toluidine blue made up in 1% borax (Pease, D. C , 1964; Humphrey and Prittman, 1974). The stain was filt e r e d each day, just prior to use. Enough stain to cover the sections was placed on each slide with a dropper and l e f t to dry on the hot plate (approximately 85°C). The stain was rinsed off the slides with d i s t i l l e d water and slides were returned to the hot plate to dry. The slides were coverslipped using Histoclad (Clay Adams) as a mounting medium. Thick sections of the blood vessel and nerve tissue samples were cut from the 60 and 90 day old age groups only. Thin sections of one femoral nerve stump from the 60 day old age group were cut. A l l thin sections were cut on a Reichert 0mU3 29 ultramicrotome using a glass knife. Sections were collected on 200 and 300 mesh, rhubidium coated copper grids and stained for 15 minutes in 1% aqueous uranyl acetate (the rhubidium coat on the grids lessens the effect of surface tension at the time the grid i s breaking through the water's surface as the sections are being collected). After a thorough washing in d i s t i l l e d water, sections were stained with lead citrate for 15 minutes and then washed again. Fluorescence Microscopy A quick and consistent method for fluorescence of monoaminergic neurons and their axonal varicosities i s the sucrose-potassium phosphate-glyoxylic protocol as modified by De l a Torre (1980). This method of fluorescence microscopy was used to determine i f norepinephrine were present at the adventitial-medial borders in the denervated arteries and veins. Animals for fluorescence were terminated at 30, 60, 90 or 120 days. Animals were anaesthetized using sodium pentobarbitone (0.06g/ml) made up in 0.9% saline solution. The dosage given was lml/kg body weight. A 1% aqueous solution of Trypan blue injected into the l e f t femoral vein at a dose of 15mg/kg was l e f t to circulate for one hour. Trypan blue makes elastic tissue of the artery fluoresce red. This helps to differentiate the elastic tissue from the fluorescing norepinephrine-containing nerve varicosities (Mclnnes, 1977). Without trypan blue, elastic tissue and adrenergic nerve varicosities have almost the same blue-green fluorescence. Evans blue can also be used (De l a Lande and Waterson, 1968); however, Todd (1980) stated that trypan blue was the most effective. After one hour, the right and l e f t saphenous and femoral arteries and saphenous and.femoral veins were excised. Each vessel was immediately embedded with in Tissue Tek II O.C.T. Compound (Miles Laboratories, Inc.) 30 and frozen (-30°C). The vessels were embedded perpendicular to the cork stub so that complete transverse sections could be cut. The vessels were cryostat sectioned at -30°C and at a thickness of l8iim. Sections were picked up with a glass coverslip and exposed for 3 seconds to a pH 7.4 sucrose-potassium phosphate-glyoxylic acid solution (De l a Torre, 1980). The sections were put under an airstream un t i l the sucrose-potassium phosphate-glyoxylic acid solution had completely dried (30-45 min.). A drop of non-drying Type A immersion o i l (R.P. Cargille Laboraties, Inc.) was placed on each coverslip and the coverslips were put into a 90°C oven for 3.5 min.. The coverslips were mounted onto cleaned glass slides and the edges were sealed with melted dental wax. In almost a l l cases, there were four slides per vessel. For each vessel, the slides were numbered from 1 to 4, representing the order of the f i r s t to the last sections cut. ANALYSIS Light Microscopy and Morphometries Light microscopic photographs were taken of a l l the right and l e f t saphenous arteries in the 60 and 90 day age groups on a Leitz-Wetzlar Orthoplan light microscope at 10X (on 35mm film). The right and l e f t femoral veins that were sampled in these age groups were photographed at 6.3X. Montages of the veins were assembled. Three complete transverse sections from each art e r i a l and venous vessel were photographed. Black and white photographs were taken using 35mm Kodak Technical Pan (black and white) 2415 film. The film was developed using Kodak HC110 developer in a dilution of 1:15 for 7 minutes. The developer was discarded and the film was washed in d i s t i l l e d water containing a few drops of Kodak Stop Bath for 1 minute. The film was fixed for 5 minutes. Prints were made on Agfa-Gevaert Rapitone paper, using either grades 3 or 4 at fi n a l magnifications of 145-320X for the veins and 220-570X for the arteries. The prints were developed using a Rapidoprint electric print processor using the Agfa-Gevaert Rapidoprint activator and fixer. From each photograph, the entire cross-sectional area and the luminal area of the arteries were traced on an Apple II digitizing Tablet. The morphometric programme permitted the luminal area to be subtracted from the entire cross-sectional area to calculate the area of the tunica media (the area containing the smooth muscles c e l l s ) . Therefore, from the photographs of the denervated and control arteries, the medial area was calculated. The montages of the veins required tracing on a larger Talos CYBERGRAPH digitizing board interfaced with the University mainframe AMDIAHL 471/V8 computer. Only the luminal cross-sectional area was traced since the microscopic magnification for these photographs was too low to determine the outer limits of the tunica media. In order to measure wall thickness of the veins, four points from one of the montages of each vein were randomly chosen. These points were photographed on a Leiz-Wetzlar Orthoplan light microscope at 40X using 35mm Kodak Technical Pan black and white 2415 film. The film and prints were developed as described above. The higher magnification (40X) was necessary to identify the limits of the tunica media. The midpoint of each photograph was located, and at that point, the thickness of the tunica media was measured (in mm) using a ruler placed perpendicular to the endothelium. The four thicknesses of the tunica media for each vein were averaged together to produce the average medial thickness (in ym). Electron Microscopy The thin sections of a femoral nerve trunk were photographed on a Philips 300 electron microscope using 35mm film. The film was developed for 3 minutes in f u l l strength D19 developer followed by a wash in d i s t i l l e d water. The film was fixed for 5 minutes then washed with fi l t e r e d water for 45 minutes at 70°F. Fluorescence Microscopy The cryostat sections stained for fluorescence were examined with a Zeiss fluorescence microscope using a Zeiss ultraviolet f i l t e r H365 01 (FT 395, LP 397) that permitted wavelengths in the upper 400nm region to pass. Beginning in the upper l e f t corner, each coverslip was examined thoroughly to find the transverse sections of the saphenous arteries or femoral veins. Using a 40X Plan Neofluor objective, the number of fluorescing areas per section found at the advential-medial border of the vessel was counted. Counts were made for the right and l e f t saphenous arteries and femoral veins from 8 animals. From each animal, 14-35 sections of the saphenous artery were counted and 10-25 sections of the femoral vein were counted. The number of sections counted for each vessel represented the number of sections cut minus the few that were unsuitable due to their being folded or blurred by a bubble in the mounting medium (immersion o i l ) . Black and white photographs and coloured slides were taken of some of these vessels using 35mm Kodak T r i X black and white film and 35mm Kodak Ektachrome colour slide film (ASA 400). The black and white film was developed in Acufine developer for 5 minutes. After a rinse with d i s t i l l e d water, the film was fixed for 5 minutes followed by a 1 hour wash in fi l t e r e d water. Prints were developed in the same manner as for light microscopy. The colour film was developed commercially. Statistics The measurements of the tunica media of the saphenous arteries and femoral veins and of the lumen perimeter of the veins for both the denervated and control sides were compared using a paired Student's i t - t e s t . Results are shown as mean + SEM. The counts of the fluorescent areas from the saphenous a r t e r i e s and femoral veins are compared using three d i f f e r e n t analyses of variance (ANOVAs). ANOVAs were run on the other data as well except f o r the measurements of the medial thickness o f the femoral v e i n . RESULTS Sampling for Light Microscopy Three animals per group were sampled for light microscopy at 60 and 90 days of age, thus making a total of 12 animals (Tables I and II ) . At the time of sampling, the animals were checked for gross c l i n i c a l symptoms of denervation: dragging of the right foot when the animal walked; incomplete extension of the right leg when the animal i s suspended by i t s t a i l . Inside two animals, milky white scar tissue had to be removed from the denervated area to locate the positions of the femoral, the superficial epigastric and saphenous arteries. In the remaining animals, a more transparent connective tissue blanketed the vessels; therefore, the vessels were clearly v i s i b l e . There were no indications of infection in any of the animals. Generally, on the denervated side, there never appeared to be a healthy, glistening white saphenous nerve. Instead, the remnant of the nerve was so translucent that i t was not visible or i t was very thin and yellowish. The translucent nerve remnants had no form and were probably just connective tissue. This was indeed the case when these samples were examined under the light microscope. In four animals, re-innervation was noted and in two of these four cases, the re-innervation pathways were the same. That i s , in both cases, a very pale, translucent nerve, seemingly coming from the surrounding skeletal muscle of the thigh about half way along the femoral artery, coursed diagonally across the thigh in a caudal direction to reach the superficial epigastric artery a bit di s t a l l y to i t s branch point from the femoral artery. One of these animals was denervated at 1-3 days and sampled at 90 days and the other animal was denervated at 12 35 days and sampled at 60 days. In the third animal, re-innervation appeared to have come from the abdominal wall, and in the last animal, a nerve was found running parallel with the inguinal ligament, although i t s origin and destination were undetermined. Out of the six animals that were denervated at 1-3 days of age, only one had i t s tubed femoral nerve intact at the time of sampling (60 days of age). Because the nerves are so delicate at this stage (1-3 days), the nerve in three animals tore when pulling i t into the tube, therefore, three of the six animals had no tube at a l l . Despite this however, a large segment of the femoral nerve was removed from within the abdominal cavity in these three cases. Tubes from the remaining two animals were present, but were unattached to the rest of the femoral nerve and were found lying in a mass of adipose tissue inside the abdominal cavity. In these two cases, the femoral nerve stump was found and i t had a transparent yellowish appearance. One of these femoral nerve stumps seemed to be leading into the underlying skeletal muscle. A l l six animals denervated at 12 days of age had their tubes s t i l l attached to the femoral nerve. The tubes were encased in connective tissue and no nerves appeared to be growing out of the melted end of the tube. The portion of the femoral nerve just proximal to, as well as the portion within the tube was thin, pale and yellowish compared to the larger, glistening white femoral nerve on the control side. I t was not until sampling the remaining six animals in the experiment that a change in the denervated femoral vein was noticed. The femoral vein showed a dilation in the area of the branch points of the superficial epigastric and saphenous veins. This dilation continued proximally, gradually tapering back down to i t s normal diameter at approximately one centimeter from i t s branch points. A small segment at the branch points of the s u p e r f i c i a l e p i g a s t r i c and saphenous veins was s l i g h t l y d i l a t e d as w e l l . No anomalies of the femoral or saphenous a r t e r i e s were apparent at the gross l e v e l . LIGHT MICROSCOPIC APPEARANCE Saphenous Artery There was no remarkable diffe r e n c e i n the l i g h t microscopic appearance between the denervated and con t r o l saphenous a r t e r i e s (Figure 5). Both had a conspicuous endothelium with i t s many endothelial c e l l n u c l e i bulging i n t o the lumen. A prominent i n t e r n a l e l a s t i c lamina, a well developed tunica media and a t h i n external e l a s t i c lamina were c h a r a c t e r i s t i c of t h e i r h i s t o l o g i c a l appearance. The i n t e r n a l e l a s t i c lamina was co n s i s t e n t l y thicker (one-half to two t h i r d s thicker) than the external e l a s t i c lamina i n both the denervated and con t r o l v e s s e l s . In some of the denervated a r t e r i e s sampled at 60 days, the i n t e r n a l e l a s t i c lamina appeared to be s l i g h t l y thicker than the i n t e r n a l e l a s t i c lamina i n other denervated or con t r o l a r t e r i e s . Small breaks i n the i n t e r n a l and external e l a s t i c laminae i n con t r o l and denervated a r t e r i e s were apparent (Figure 5). In one con t r o l a r t e r y , the connective t i s s u e from the media appeared to be continuous with connective tis s u e of the a d v e n t i t i a through one of the breaks i n the external e l a s t i c lamina. There were no differences i n the tunica media between the denervated and control sides at the l i g h t microscopic l e v e l . Both had l o n g i t u d i n a l and c r o s s - s e c t i o n a l p r o f i l e s of smooth muscle c e l l s . Consequently, the p r o f i l e s were of many d i f f e r e n t shapes and s i z e s . Some of the l o n g i t u d i n a l p r o f i l e s were long slender spindle shapes or long, t h i n and f l a t . Cross-sectional p r o f i l e s included square, t r i a n g u l a r , round, oval and polygonal shapes. Each c e l l was c l e a r l y separated from i t s neighbours by a t h i n unstained area, thereby emphasizing the highly i r r e g u l a r c e l l boundaries. In both the denervated and co n t r o l a r t e r i e s , the p r o f i l e s of the smooth muscle n u c l e i were also of various shapes and s i z e s because of the d i f f e r e n t planes of s e c t i o n at which these c e l l s were cut. However, the shape of nuclear p r o f i l e s did not ne c e s s a r i l y conform to the shape of the c e l l p r o f i l e , and, o c c a s i o n a l l y , the n u c l e i were e c c e n t r i c a l l y located. The n u c l e i were very euchromatic, there were none that were p i c n o t i c . The a d v e n t i t i a , i n both cases, appeared the same. From v i s u a l assessment,the a d v e n t i t i a of the denervated a r t e r y did not appear to contain more or fewer connective tis s u e c e l l s than the a d v e n t i t i a of the c o n t r o l a r t e r i e s . Femoral Vein Although the lumen of the denervated veins was d i l a t e d , t h e i r walls did not d i f f e r i n t h e i r l i g h t microscopic appearance from those of the c o n t r o l veins (Figure 6 ) . The d e f i n i t i v e layers so c h a r a c t e r i s t i c of blood vessels were not e a s i l y i d e n t i f i a b l e i n the veins (Figure 6 ) . In a cross-section, an endothelium and a d v e n t i t i a were always present around the e n t i r e vein, but d i s t i n c t i v e e l a s t i c laminae and media were not. Connective t i s s u e occupied most of the area under the endothelium, and i t was dotted with small groups of or i n d i v i d u a l smooth muscle c e l l s . I t was d i f f i c u l t to always p o s i t i v e l y i d e n t i f y the i s o l a t e d c e l l s as smooth muscle c e l l s at t h i s l e v e l of microscopy; some may have been f i b r o b l a s t s . Nerve Figures 7(a) and 8(a) c l e a r l y show the l i g h t microscopic appearance of normal saphenous and femoral nerve t i s s u e taken r e s p e c t i v e l y from the c o n t r a l a t e r a l c o n t r o l side. The m u l t i p l i c i t y of myelin sheaths are packed t i g h t l y together, each one surrounded by a t h i n layer of endoneurium. The p r o f i l e s of myelin sheaths ranged from large to very small. Schwann c e l l and f i b r o b l a s t i c n u c l e i , small blood vessels, and unmyelinated nerve p r o f i l e s are interspersed amongst the myelinated axons. D i s t a l Stump of Saphenous Nerve from the Denervated Side Four samples of the d i s t a l stump (that part d i s t a l to the point of the segment removed) of the saphenous nerve were thick sectioned and stained, and the l i g h t microscopic appearance of one of these samples i s seen i n Figure 7(b). The nerve bundle i s surrounded by a thick perineurium encapsulating a highly compact mass of c e l l s and connective t i s s u e . Some of the c e l l s were large and pale s t a i n i n g while others were t h i n and more f i b r o b l a s t - l i k e . The c e l l s are so c l o s e l y packed together that c e l l s boundaries are u n i d e n t i f i a b l e . The other samples had very s i m i l a r appearances. In one of these sections, a macrophage was seen, and i n another, there were s t i l l myelinated axons present, however, these were very small, were few i n number and were surrounded by a thick endoneurium. A l l of these samples were well v a s c u l a r i z e d . Abdominal Cavity Nerve from the Denervated Side Thick sections from one tube showed the l i g h t microscopic appearance o f the encased nerve (data not shown). A very thick capsule of c i r c u l a r l y arranged, highly c e l l u l a r dense connective t i s s u e surrounded a core of denser connective t i s s u e . This core was also very c e l l u l a r , well vascularized and contained macrophages. The three samples of that portion of the femoral nerve j u s t proximal to the tube had s l i g h t l y d i f f e r e n t l i g h t microscopic appearances. Figure 8(b) i l l u s t r a t e s " the appearance of one such sample. Here, a t h i c k , c i r c u l a r l y arranged coat of connective t i s s u e surrounded an inner core of connective t i s s u e . The connective t i s s u e core consisted of many n u c l e i belonging to cells whose boundaries were not distinguishable. Some nuclei were f l a t and fusiform, whereas others were square or round. The core was well vascularized and in the very centre were tiny profiles of myelinated axons (Figure 8, insert), much smaller than those from the control nerve. The number of these axons varied with each sample. For example, the sample in Figure 8(b) had only a few whereas the other two samples had more. Even though two of the samples had many profiles of myelinated axons, the number was not comparable to the control side. Moreover, a l l the profiles were very small and they were surrounded by a very thick endoneurium. Although a positive identification of the cells in the core cannot be made at this level of microscopy, perhaps they were a mixture of fibroblasts and Schwann c e l l s . Electron Microscopy of the Abdominal Cavity Femoral Nerve The electron microscopic appearance of one sample was examined (Figure 9). The bulk of the tissue consisted of collagen fibres scattered amongst a few fibroblasts. The tissue also exhibited signs of degeneration. Occasionally, myelinated and unmyelinated nerve profiles were seen (Figure 9, insert), but only in the very centre of the nerve's core. FLUORESCENCE MICROSCOPY Altogether, eight animals were sampled for fluorescence, thus, 2 animals were sampled at 30, 60, 90 and 120 days of age. A l l exhibited positive c l i n i c a l symptoms of denervation at the time of sampling. Gross Morphology of the Denervated Area In half of the rats sampled, a dilation of the femoral vein was noted. Also in four of the animals, the denervated saphenous and superficial epigastric arteries and veins branched from the femoral a r t e r y i n very close proximity to each other. In some cases, they branched o f f side by side, and i n others, the s u p e r f i c i a l e p i g a s t r i c a r t e r y branched from the saphenous arte r y rather than from the femoral a r t e r y . On the con t r o l side, i n a l l cases except f o r one, the branch points of the saphenous and s u p e r f i c i a l e p i g a s t r i c a r t e r i e s and veins were approximately 0.5 cm apart. Even though most of the animals sampled had the tube s t i l l attached to the femoral nerve, re-innervation of the saphenous a r t e r y was seen i n h a l f of the animals. The re-innervating nerves were very pale and translucent, one being s i m i l a r i n appearance to an empty a r t e r i o l e . Re-innervation came from aberrant areas such as the abdominal w a l l , the s k e l e t a l muscle underneath the vessels, and from within the scar t i s s u e blanketing the area. A l l l e d to the branch point of the saphenous a r t e r y . In two animals, sprouting of the femoral nerve i n s i d e the abdominal c a v i t y was v i s i b l e . In one of these animals, the tube had detached, and i n the other animal, sprouting of the femoral nerve was proximal to i t entering the tube. I was unable to determine the destinations of these axonal sprouts because the contents of the abdominal c a v i t y were blue from the trypan blue. Description of the Sections under the Fluorescence Microscope Figures 10 and 11 show the appearance of the denervated and con t r o l vessels of the saphenous arte r y and saphenous vein, r e s p e c t i v e l y . A f t e r i n j e c t i o n with trypan blue, the external e l a s t i c lamina always fluoresced i n the red range, however, the i n t e r n a l e l a s t i c lamina d i d not fluoresce red as c o n s i s t e n t l y . Although s p e c i f i c adrenergic fluorescence was concentrated at the adventitial-medial border, occ a s i o n a l l y i t was seen i n the a d v e n t i t i a . In some of the veins, the fluorescent nerves extended beyond the a d v e n t i t i a l border towards the endothelium. The fluorescent areas varied in size from very tiny to large dots. Sometimes, the smaller dots had a "beads on a string" appearance. Generally, fluorescent dots were relatively uniform in their distribution around the circumference of the control arteries, whereas the veins had patches of fluorescent dots irregularly spaced around their circumference. The identity of a re-innervating adrenergic nerve that had the appearance of an arteriole was verified by fluorescence. Cross sections of this nerve contained many small, pale fluorescent dots. ANALYSIS Morphometries Medial Area of the Saphenous Artery From light micrographs, morphometries were carried out on the saphenous artery and femoral vein. The raw data as well as the means are lis t e d in Tables I and II. For the arteries, a Student's t-test was carried out on the data from each table separately. When the arteries were sampled at 60 days, the results of the t-tests show that there was a tendency for a thinner media, but this was not significant at the p<0.05 level (Table I ) . However, i f each sample i s looked at individually, the measurements do show that the area of the tunica media was decreased in four of the six animals. The remaining two animals show a very slight increase in the media of the denervated arteries; therefore there was virtua l l y no change in the media of these two samples. Animals sampled at 90 days showed a significant decrease in the wall on the denervated side (Table I I ) . A two-factor and a three-factor ANOVA were carried out on the data from Tables I and II. The two-factor ANOVA of age of denervation (1-3 or 12 days) versus condition (denervated or control) was carried out separately on the 60 and the 90 day old groups. This ANOVA verified the i - t e s t results, but i t also showed that the age at which the animals were denervated was not significant, and that there was no interaction between the two variables. The three-factor ANOVA included, in addition to the two factors just mentioned, the age at time of sampling. From this, sampling age did make a significant difference (P=0.024). Figure 12 summarizes the data from Tables I and II as per cent decreases in the media, where the total mean area of the control side represents lOOifc. The graph shows that both sampling age groups show a 15% decrease. Vein Perimeter and Wall Thickness From light microscopic cross-sections of the femoral vein, the luminal perimeter and the wall thickness were measured for each of the denervated and contralateral control veins. The raw measurements of the luminal perimeter and their means are l i s t e d in Table III. Both a Student's % -test and an ANOVA were carried out on these means, and both tests confirmed that the increase in luminal perimeter of the denervated veins was significant. The ANOVA also showed that denervating the femoral vein at either 3 or 12 days had no significance, and there was no interaction between the two factors tested, these being, age at denervation and condition (denervated or control). The percent increase i s shown in Figure 13. The results of measuring the wall thickness of the denervated and control femoral veins are recorded in Table IV. The p-test results indicate that the difference in wall thickness between the denervated and control femoral veins i s not significant. Also, the raw data shows that the wall thicknesses from both sides were quite variable. Fluorescence Counts The number of fluorescing dots found around the denervated and control saphenous arteries and femoral veins was counted. The counts are li s t e d i n Table V. Two, two-factor and one, three-factor ANOVAs were carried out on the data. The two, two-factor ANOVAS showed that there was a significant decrease in the number of fluorescent dots both for the artery and for the vein. The age at denervation was not significant for denervating the femoral vein. The second two-factor ANOVA (condition versus age of sampling) was designed to see i f the sampling age had any effect. That i s , by ignoring the effect of condition (denervated or control), the ANOVA showed that the effect of age of sampling on the fluorescence counts was significant for the arteries (P=0.019) and for the veins (P=0.048). The additional information gained from the three-factor ANOVA confirms a point which may be self-evident; that i s , there was a significant difference in fluoresence between the two blood vessel types. DISCUSSION In t h i s study, I have developed a denervation technique to study the trophic influences between nerves and blood vessels i n the r a t t h i g h . This technique was designed to be as l o c a l i z e d and as permanent as possible f or the following reasons: (1) sympathetic ganglionectomy and chemical and immunological sympathectomies destroy the adrenergic innervation to such a large area of the body that i t i s important to determine whether the denervation e f f e c t s seen with these methods are due s o l e l y to the absence of innervation on the blood vessel or whether other denervated structures had changed and thereby contributed to the r e s u l t s , and (2) simple crushing, severing or r e p o s i t i o n i n g the nerve are unsuitable methods for denervating blood vessels since re-innervation occurs within a few days (Todd, 1986) - a period of time which may be too short for denervation changes to occur. After determining the success of t h i s technique, I then used the method to determine whether depriving blood vessels of t h e i r vasomotor innervation s i g n i f i c a n t l y a l t e r s t h e i r a r c h i t e c t u r e . In t h i s study, the area of the tunica media of the r a t saphenous a r t e r y decreased and the femoral vein was d i l a t e d . The experiments also reconfirmed that i t i s extremely d i f f i c u l t to prevent aberrant re-innervation of the thigh v e s s e l s . In previous studies, the success of denervation on blood vessels was determined by a v a r i e t y of ways. S u p e r s e n s i t i v i t y of the smooth muscle to exogenously applied norepinephrine, stimulation of the e n t i r e sympathetic outflow using a s t e e l p i t h i n g rod as the electrode (6-hydroxydopamine denervation), h i s t o l o g i c a l studies, and chemical and e l e c t r i c a l stimulation of s p e c i f i c ganglia (immunological sympathectomy) are j u s t a few of these ways (Levi-Montalcini and A n g e l e t t i , 1966; Finch et a l . , 1973; Bevan and Tsuru, 1979; 1 9 8 1 ) . Although the femoral nerve was torn i n 3 out of the 6 animals that I denervated at 1-3 days of age, I s t i l l regarded them as being s u c c e s s f u l l y denervated. The nerves tore while pushing the tube on; therefore, the r e s u l t i n g proximal stump was very short ( i e . close to the sympathetic chain) i n the 3 day old animal. I t has been reported that the closer the i n j u r y i s to the c e l l body, the greater the damage to the c e l l body (Gabella, 1976; Barr and Kiernan, 1 9 8 3 ) . Thus, I assumed that the nerves torn i n the 3 day old animals tore close enough to t h e i r nerve c e l l bodies to most l i k e l y cause c e l l death. Since ( 1 ) atrophy and degeneration of c e l l bodies can p e r s i s t months a f t e r axotomy (Gabella, 1 9 7 6 ) , ( 2 ) destruction of adrenergic c e l l bodies abolishes vasomotor c o n t r o l , and ( 3 ) c e l l bodies i n newborn animals are more susceptible to denervation techniques (Levi-Montalcini and A n g e l e t t i , 1 9 6 6 ; Finch et a l . , 1973)> I assume i n my experiment that the three animals who had the torn femoral nerve were s u c c e s s f u l l y denervated, even at time of sampling. For a l l animals, those with or without a tube, the degree of success of my denervation technique on the saphenous arte r y and femoral vein was determined. The methods used for t h i s included showing that the proximal and d i s t a l stumps of the femoral nerve had a degenerated appearance using l i g h t and electron microscopy (electron microscopy was c a r r i e d out only on the proximal nerve stump). In a d d i t i o n , evidence for denervation was obtained by examining the gross morphology of the thigh for the presence of regenerating nerves, by l i g h t microscopic examination of the a d v e n t i t i a of the vessels, and by a fluorescence technique s p e c i f i c for catecholamines. The l i g h t microscopic appearance of the d i s t a l and proximal stumps from my study showed d e f i n i t e signs of degeneration. When a peripheral nerve i s cl e a n l y transected, proper axonal regeneration requires appositioning the two cut ends and suturing through the epineurium (Barr and Kiernan, 1983). This surgical repair i s not necessary i f the axons have been transected by a crush because the connective tissues in the nerve remain intact (Barr and Kiernan, 1983). Axonal transection, therefore, produces permanent degeneration of the distal adrenergic fibres and in temporary degeneration of the c e l l body (Burnstock and Costa, 1975). Distal to the site of injury, the detached portion of the peripheral nerve undergoes Wallerian degeneration whereas the proximal portion undergoes the axon reaction which is best displayed in the c e l l bodies as chromatolysis (Barr and Kiernan, 1983). Wallerian degeneration is characterized by the axon i n i t i a l l y becoming swollen and then breaking up into fragments. Accompanying these axonal changes are changes in the myelin sheath. It i s broken into short ellipsoidal segments and then gradually disintegrates. The Schwann cells multiply, f i l l i n g the cylindrical area enclosed by the endoneurium. The remnants of motor neurons (the axon and i t s myelin sheath) and the axons of unmyelinated fibres are phagocytosed, with the distal stump being composed of columns of Schwann ce l l s (bands of von Bungner). The content of norepinephrine disappears anywhere from 18 to 48 hours after damage to the nerve (Burnstock and Costa, 1975); which may explain why terminal nerve transmission i s not immediately stopped following damage (Gabella, 1976). These results vary between organs and species. Blood vessels of the rabbit ear s t i l l responded to nerve stimulation three days post-severing (Gabella, 1976). The general structure of sympathetic axons appears normal for several days (Burnstock and Costa, 1975), and the length of time for degeneration i s probably directly proportional to the axon length (Burnstock and Costa, 1975; Gabella, 1976). That i s , a short dis t a l stump degenerates faster than a longer one. Since application of colchine produces degeneration of a nerve distal to i t s application, this suggests that transport of substances i s v i t a l for the survival of the axons (Burnstock and Costa, 1975). Therefore, the light microscopic appearance of the dis t a l portion of the saphenous nerve in my study indicates that i t was probably devoid of adrenergic fibres, and the ce l l s that were there were most l i k e l y Schwann cells arranged in the bands of von Bungner. The distal stump of the saphenous nerve closely resembled the distal stump of the cat t i b i a l nerve described by Pellegrino and Spencer (1985). Seven weeks post-denervation, the cat t i b i a l stump i s virtu a l l y devoid of myelin debris, and 89% of the nuclei in a cross-section represent bundles of Schwann cells which are separated by a lot of collagen and elastin fibres, scattered fibroblasts and patent blood vessels. In addition, Pellegrino and Spencer (1985) found that 3 seven weeks post-denervation the uptake of H-thymidine into the f i r s t 9.5cm of the distal t i b i a l nerve stump i s linear over a 3-hour period and does not differ along the length of the nerve whereas i f this 7 week denervated distal stump i s joined end-to-end to a newly severed proximal 3 stump of the peroneal nerve, an increase in H-thymidine uptake i s seen within the f i r s t 6cm of the coapted distal stump. Beyond these f i r s t 6cm, the H-thymidine uptake i s similar to that along the entire 9.5cm length of the 7 week denervated (non-joined) dis t a l stump. Pellegrino and Spencer (1985) found that the f i r s t 2-6.5cm distal to the site where the nerves were joined i s an area of axon-Schwann c e l l contact. This suggests the presence of axons i s mitogenic thereby stimulating the myelination process. The results of Pellegrino and Spencer (1985) also suggest that in my study, the Schwann cells remained as bundles. That i s , since direct re-innervation of the dis t a l saphenous nerve stump by i t s proximal stump was prevented by encasing the proximal end of the 48 femoral nerve in a tube, the Schwann cells in the distal stump had no stimulus to divide and form myelin, Changes in the proximal portion of a severed nerve may vary depending on the type of neuron; therefore, some neurons may totally disappear whereas others may not be significantly altered (Barr and Kiernan, 1983). Large motor neurons supplying skeletal muscle exhibit the cytological details of the classical axon reaction, the description of which follows. The most significant alteration in the severed axon occurs immediately adjacent to the cut. The remainder of the axon i s not altered appreciably. Coarse clumping of Nissl substance can appear in the c e l l body as soon as 6 hours after section. The nucleus becomes eccentrically located (Barr and Kiernan, 1983; Gabella, 1976) and flattens out, later on becoming indented (Gabella, 1976); this process reaches a maximum at 10-20 days after injury (Barr and Kiernan, 1983)• Organelles become somewhat disorganized (Barr and Kiernan, 1983). At the end of 6 weeks, 40-50? of the cells may s t i l l show signs of chromatolysis (Gabella, 1976), or some may persist for months (Barr and Kiernan, 1983; Gabella, 1976). In my study, the proximal end of the femoral nerve was encased in a polyethylene tube which may have contributed to the increased spread of degeneration along the length of this nerve. A similar appearance to my observations was seen in the nerve repair studies by Colin et a l . (1984). In their study, a 5-7mm portion of rat t i b i a l nerve was excised from one side. The rats were divided into 2 groups. To induce the formation of a fibrovascular sheath in one group, a l l rats had their proximal and distal nerve stumps connected by a silicone rod whereas, in the other group, the 2 ends were l e f t separated. Four weeks later, a l l the silicone rods were removed, leaving a fibrovascular sheath behind. These rats were divided into 3 subgroups, and further experimental treatment was carried out on 2 of these subgroups while the third acted as a control. The same protocol was applied to the group without the rod inserted, that i s , the unsheathed group. The animals were l e f t for 3 months. One of the experimental treatments carried out on a subgroup involved connecting the two free ends of the nerves with a collagen tube. The results were interesting since both the sheathed and unsheathed collagen-tube-encased nerves exhibited the thick fibrosed epineurium which I found on my plastic tube-encased femoral nerve. However, the cores of the nerves in the study by Colin et a l . (1984) had a normal histological appearance. Therefore, the collagen tube may have induced the thick, fibrosed epineurium but i t also facilitated the normal regeneration of the nerve by guiding the axonal sprouts into the distal segment. The core of the femoral nerve in my study resembled most that of the unsheathed control group in Colin et al.'s study. That i s , the core had a fibrosed internal milieu with a few diffuse mini-fasicles, the axons of which were small in diameter with thin myelin sheaths. From these comparisons, i t can be seen that the proximal and distal stumps in my study definitely had a degenerated appearance. By examining the gross morphology of the thigh area, I was able to see very thin, translucent, unmyelinated nerves re-innervating the saphenous and superficial epigastric arteries in some of the denervated animals. Although I did not see any nerves in the adventitia of the denervated vessels with light microscopy, the fluorescence technique did reveal the presence of some catecholamines at the adventitial-medial border. These results indicate that re-innervation did occur. As in humans (Williams and Warwick, 1975), the femoral nerve of the rat carries the adrenergic innervation to the saphenous and femoral arteries and veins (Todd, 1986). Therefore, adrenergic regulation of the vasomotor activity of these vessels ceases by severing the femoral nerve. However, after denervation, vascular smooth muscle exhibits automaticity which i s the a b i l i t y of a blood vessel to maintain a basal tone without a coordinating nerve supply (Page and McCubbin, 1965). Under a r t i f i c i a l conditions in restricted areas (in hamster cheek pouch or web of frog's foot) two phenomena have been observed after denervation. These include the maintenance of a basal tone and rhythmic changes in tone (Page and McCubbin, 1965). Page and McCubbin (1965) state that certain blood vessels, such as those that supply the skin, are much more dependent on the nervous system for coordination than blood vessels supplying other areas. Denervation of the blood vessels of the brain, heart, l i v e r and kidneys does not appear to alter the organs' blood supply, suggesting that these blood vessels depend on other controlling factors such as myogenic activity or the local chemical environment (Page and McCubbin, 1965). I f the dependency of a blood vessel on i t s neural coordination may reflect that vessels' a b i l i t y to stimulate i t s re-innervation, then the occurrence of re-innervation in my study and in Todd's (1986) experiments suggest that the blood vessels in the rat thigh are highly dependent on adrenergic control. Since the femoral nerve does not appear to be the source of these re-innervating fibres, the axonal sprouting from aberrant areas in the v i c i n i t y of the denervated vessels suggests that vascular smooth muscle cells may have a widespread and powerful mechanism which prevents permanent denervation from occurring. Being so widespread, this mechanism might be a diffusible chemotrophic or chemotactic factor. Nerve density around a blood vessel may be directly proportional to a vessel's a b i l i t y to induce sprouting of neighbouring nerves. This i s supported by transplantation experiments c a r r i e d out by Todd ( 1 9 8 6 ) . In her study, segments of the densely innervated r a t t a i l a r t e r y and the r a t femoral ar t e r y , which has v i r t u a l l y no innervation, were transplanted separately i n t o the anterior eye chamber of a host r a t . Only the t a i l a r t e r y was capable of inducing i r i d i a l nerve sprouting. Although these r e s u l t s suggest i t i s the blood vessel i t s e l f that i s the trophic inducer, P o l i t i s et a l . (1982) provides evidence that the transected d i s t a l nerve stump contains d i f f u s i b l e f a c t ors which can a t t r a c t regenerating axons. Re-innervation of blood vessels from aberrant areas i s also seen i n denervation experiments where the e n t i r e superior c e r v i c a l ganglion i s removed (R. D. Bevan, personal communication with M. E. Todd; Kobayashi et a l . , 1 9 8 3 ) . Bevan states that re-innervation of the r a b b i t ear vessels occurs between 6 - 8 weeks i n the denervated r a b b i t whereas Kobayashi et a l . (1983) report nerves regenerating between H-6 weeks and reach a maximum between 9 and 12 months i n denervated Wistar r a t s . However, the maximum number of regenerating nerves i s approximately h a l f of the normal number. In other cases, nerve regeneration a f t e r denervation has been reported to occur i n under 15 days (Todd, 1 9 8 6 ; Dyck and Hopkins, 1 9 7 2 ) . Todd ( 1 9 8 6 ) t r i e d four d i f f e r e n t methods of s u r g i c a l l y denervating the saphenous and s u p e r f i c i a l e p i g a s t r i c a r t e r i e s . They included severing the femoral nerve, removal of a segment of the femoral nerve, c a u t e r i z i n g the proximal stump a f t e r a segment was removed and r e p o s i t i o n i n g the severed femoral nerve by suturing i t to the abdominal w a l l . In a l l of these cases, re-innervation occurred within 15 days. Axonal sprouts were seen 5-15 days a f t e r crushing the c e r v i c a l sympathetic trunk by Dyck and Hopkins ( 1 9 7 2 ) . Since the re-innervating f i b r e s i n these two studies sprouted from the severed or crushed nerve, rather than from aberrant areas as in studies involving more drastic methods of denervation, this may account for the short regeneration time. Another method I used to analyze the degree of success of my denervation procedure was fluorescence. The presence of catecholamines in some of the denervated vessels suggested that these vessels had been re-innervated. In arteries, adrenergic nerve varicosities occur at the adventitial-medial border (Burnstock and Costa, 1975; Todd, 1980) rather than penetrating into the media as they do in veins. Since resolution of the light microscope i s limited, the fluorescing dots are more l i k e l y to represent cluster of axonal varicosities rather than individual varicosities. However, the "beads on a string" appearance i s characteristic of the series of adrenergic varicosities of a single nerve ending (Ham and Cormack, 1979) and has been shown by Burnstock and Costa (1975) using fluorescence. A rapid and consistent histofluorescence method specific for the visualization of catecholamines was modified by De l a Torre (1980). Since he has standardized this method, a very consistent intensity of monoaminergic neurons and their axonal varicosities from one section to the next has been achieved (De l a Torre, 1980). Therefore, differences in counts should truly reflect differences in catecholamines rather than inconsistencies produced by the method. By injecting trypan blue into the animal before sampling, any confusion between the fluorescence of the external elastic lamina and that of nerve varicosities i s eliminated, thereby ensuring that only the dots produced by the fluorescence of catecholamines were counted. The significant decrease in fluorescence obtained in this study, represented a decrease in the amount of norepinephrine. Whether this represented an absence or a decrease in 53 u n m y e l i n a t e d n e r v e t e r m i n i a l s a w a i t s f u r t h e r i n v e s t i g a t i o n . T h e f l u o r e s c e n c e d a t a i n d i c a t e t h a t s o m e n e r v e s w e r e p r e s e n t a r o u n d t h e d e n e r v a t e d v e s s e l s . T h e s e c o u n t s p r o b a b l y r e p r e s e n t t h e n o r e p i n e p h r i n e c o n t e n t o f t h e r e - i n n e r v a t i n g n e r v e s . A s p r e v i o u s l y d i s c u s s e d , r e - i n n e r v a t i o n d i d o c c u r s o m e t i m e s . T h e r e - i n n e r v a t i n g n e r v e s h a d a v e r y t r a n s l u c e n t g r o s s m o r p h o l o g y ; t h e r e f o r e , t h e a c t u a l n u m b e r p r e s e n t p r o b a b l y e x c e e d e d t h a t v i e w e d . T h i s w o u l d a c c o u n t f o r t h e f l u o r e s c e n c e s e e n i n a l l t h e v e s s e l s s a m p l e d w h e r e a s b y g r o s s o b s e r v a t i o n , r e - i n n e r v a t i o n w a s o n l y r e p o r t e d i n h a l f o f t h e a n i m a l s . I n s p i t e o f h o w w e l l t h e s u r g i c a l t e c h n i q u e p r e v e n t s t h e p r o x i m a l e n d o f a s e v e r e d n e r v e f r o m s p r o u t i n g a n d r e - i n n e r v a t i n g i t s t a r g e t b l o o d v e s s e l , i t s e e m s t h a t t h e i r r e - i n n e r v a t i o n b y a d r e n e r g i c n e r v e s c a n n o t b e p r e v e n t e d . O n c e t h e d e g r e e o f s u c c e s s o f my d e n e r v a t i o n t e c h n i q u e w a s e s t a b l i s h e d , m o r p h o l o g i c a l a n d m o r p h o m e t r i c a l a n a l y s e s w e r e c a r r i e d o u t o n t h e s a p h e n o u s a r t e r y a n d f e m o r a l v e i n s a m p l e s . C h a n g e s i n t h e m o r p h o l o g y o f a r t e r i a l w a l l s h a s b e e n r e c o r d e d i n o n l y a f e w o t h e r d e n e r v a t i o n s t u d i e s ( B r a n c o e t a l . , 1984; T o d d , 1986). T h e e x t e r n a l e l a s t i c l a m i n a o f t h e s u r g i c a l l y d e n e r v a t e d s u p e r f i c i a l e p i g a s t r i c a r t e r y b e c o m e s i r r e g u l a r a n d b r o k e n a s s e e n u l t r a s t r u c t u r a l l y b y T o d d (1986). I n t h e p r e s e n t s t u d y , i t a p p e a r e d f r o m 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 t h a t t h e i n t e r n a l a n d e x t e r n a l e l a s t i c l a m i n a e may h a v e b e e n m o r e b r o k e n o n t h e d e n e r v a t e d s i d e c o m p a r e d t o t h e c o n t r a l a t e r a l c o n t r o l s ; h o w e v e r , t h e b r e a k s w e r e n o t c o u n t e d a n d c o m p a r e d s t a t i s t i c a l l y . A l t h o u g h t h e i n t e r n a l e l a s t i c l a m i n a l o o k e d s o m e w h a t t h i c k e r u n d e r t h e l i g h t m i c r o s c o p e a n d i n p r e l i m i n a r y e l e c t r o n m i c r o s c o p i c e x a m i n a t i o n s , f u r t h e r u l t r a s t r u c t u r a l i n v e s t i g a t i o n a n d m e a s u r e m e n t s o f t h e i n t e r n a l e l a s t i c l a m i n a i s n e e d e d t o d e t e r m i n e t h i s c o n c l u s i v e l y . Branco et a l . ( 1 9 8 4 ) showed that the smooth muscle c e l l s i n the denervated dog saphenous vein and the denervated r a b b i t ear a r t e r y appear d e d i f f e r e n t i a t e d with c h a r a c t e r i s t i c s of a c t i v e protein synthesis. That i s , they are l a r g e r , have larger euchromatic n u c l e i with prominent n u c l e o l i and the cytoplasm i s r i c h i n ribosomes and has well developed rough endoplasmic reticulum. Although a l l the smooth muscle c e l l s i n the venous wall show these morphological changes, only 2 - 3 smooth muscle c e l l l a y e rs adjacent to the adventitial-medial junction i n the r a b b i t ear a r t e r y showed these changes. The increases i n the diameter of smooth muscle regresses towards normal values a f t e r 1 2 0 days i n the vein and 3 5 days i n the a r t e r y (Branco et a l . , 1 9 8 4 ) . The d e d i f f e r e n t i a t e d appearance of smooth muscle c e l l s was seen i n v i t r o by Chamley and Campbell ( 1 9 7 6 ) and t h i s d e d i f f e r e n t i a t i o n was prevented for a few extra days by the presence of sympathetic ganglion extract. Contrary to the findings of Branco et a l . ( 1 9 8 4 ) , other i n v e s t i g a t i o n s i n v o l v i n g the denervation of the r a b b i t ear a r t e r y v i a superior c e r v i c a l sympathectomy have suggested that denervation creates a thinner tunica media (Bevan and Tsuru, 1 9 7 9 ; Bevan and Tsuru, 1 9 8 1 ; Bevan et a l . , 1 9 8 3 ) . Branco et a l . ( 1 9 8 4 ) sampled the r a b b i t ear a r t e r y 1 5 and 3 5 days post-denervation whereas Bevan and Tsuru ( 1 9 8 1 ) sampled t h e i r s 8 weeks l a t e r which i s well past the time where Branco et a l . ( 1 9 8 4 ) saw regression of the a r t e r y back to normal; therefore, i t may be that the d i f f e r e n c e i n the sampling age i s what produced these opposing observations. Todd ( 1 9 8 6 ) saw no s t r u c t u r a l d i f f e r e n c e i n the media at e i t h e r the l i g h t or electron microscopic l e v e l . A change i n the media was not obvious by l i g h t microscopic examination i n my study e i t h e r . My preliminary electron microscopic i n v e s t i g a t i o n s of the denervated media also did not show any apparent differences from the c o n t r a l a t e r a l c o n t r o l side although f i b r o b l a s t s and f i b r o b l a s t i c processes abutted the adventitial-medial border of the denervated v e s s e l s . This observation may have been the r e s u l t of the lack of nerves i n the area since Branco et a l . (1984) found that f i b r o b l a s t s were more numerous i n the denervated vessels and showed c h a r a c t e r i s e s of s y n t h e t i c a l l y a c t i v e c e l l s . Although a decrease i n the tunica media of the denervated saphenous a r t e r y did not appear markedly thinner by l i g h t microscopic observation, act u a l measurements of the media demonstrated a decrease. Decreases i n the wall thickness of the denervated r a b b i t ear art e r y have been shown by Bevan and Tsuru ( 1 9 7 9 ) , and decreases i n weights of denervated middle and posterior r a b b i t cerebral a r t e r i e s was shown by Bevan et a l . ( 1 9 8 3 ) . Bevan and Tsuru (1979) suggested that denervation creates a smaller w a l l . This appears to be true i n my study as w e l l . Contrary to these fi n d i n g s , however, are the r e s u l t s of Branco et a l . (1984) who found an increase i n wall thickness i n the denervated r a b b i t ear a r t e r y . In my study, a preliminary electron microscopic i n v e s t i g a t i o n was c a r r i e d out. No change i n the u l t r a s t r u c t u r e of the media between the denervated and co n t r o l saphenous a r t e r i e s was apparent. This suggests that the decrease i n the media may have been the consequence of changes i n the e x t r a c e l l u l a r matrix (Rusterholz and Mueller, 1 9 8 2 ) . More extensive e l e c t r o n microscopic studies of t h i s denervated smooth muscle should be c a r r i e d out to investigate t h i s p o s s i b i l i t y . Small changes i n c e l l s i z e (volume) that are not dis t i n g u i s h a b l e by q u a l i t a t i v e examination might also contribute. For example, i f each medial smooth muscle c e l l had a s l i g h t decrease i n s i z e or change i n shape, the a d d i t i v e e f f e c t s of these i n d i v i d u a l decreases may possibly produce a measureable decrease i n the media. Morphometric a n a l y s i s , such as that developed by Todd (1983) for vascular smooth muscle, should also be carried out to see i f c e l l size actually contributes to the medial decrease. Differences in measurements were expected between the two ages of denervation (1-3 days and 12 days). Todd (1980) reported that neurotransmitter in nerves did not appear around the sapheous artery u n t i l 3 days of age, as determined by the presence of fluorescence in the developing innervation. With this in mind, the intent of my study was to denervate the artery before the neurotransmitter appeared and had any influence over the developing artery. Also, two peaks in the number of nerves per unit area were seen by Todd (1980), one at 5 days of age and the other at 12-15 days of age. The greater the density of the adrenergic plexuses, the greater the potential for neurogenic muscular tone (Bevan and Su, 1973)• Perhaps, then, a greater nerve density also might mean a greater trophic influence on the smooth muscle c e l l s . Hence, the denervation at the two ages, in this study. However, the s t a t i s t i c s proved that there was no difference in denervating at 1-3 or 12 days of age. This may be a question of whether or not the neurons are functional before 12 days of age. Thus, even though there may be a greater density of nerves at 12 days, their effect on the smooth muscle would be no different than that at 3 days i f the nerves have not f u l l y developed, that i s i f they are not generating an action potential and transmitting, and not producing any trophic factors. Ultrastructural nerve profiles very different in appearance from profiles of mature nerves are seen at 11 days (Todd and Tokito, 1981). Even though adrenergic terminals have been identified in the hind limb of the dog by one week of age (Dolezel et a l . , 1974), Boatman et a l . , (1965) have reported that vascular adrenergic innervation in the hind limb i s nonfunctional un t i l after two weeks of age. Even though I could f i n d no abnormality i n the gross morphology of the saphenous arte r y i t s e l f , other a l t e r a t i o n s i n the gross arrangement of the denervated vessels were noticed. When sampling during the fluorescence procedure, the estimated distance of 0.5cm between the superior e p i g a s t r i c and saphenous branch points from the femoral artery and vein was noticeably decreased on the denervated side only. Also, a d i l a t i o n i n the denervated femoral vein at the area of i t s branches was very obvious. Such gross s t r u c t u r a l changes have not been mentioned i n the l i t e r a t u r e ; however, Bevan (1984) does mention an increase i n arteriovenous anastomoses i n the denervated r a b b i t ear a r t e r y . These changes i n the pattern may be secondary e f f e c t s to a l o c a l a l t e r a t i o n i n blood pressure r e s u l t i n g from denervation. The r e s u l t s from the denervated vein, where the vessel became grossly d i l a t e d , may be due to s t r u c t u r a l changes i n the wall for two reasons. F i r s t , other studies (Bevan and Tsuru, 1979; 1981) and t h i s one have indicated that wall thickness decreases following denervation. Since denervation causes a decrease i n the m i t o t i c index of the smooth muscle c e l l s from the media of the ra b b i t ear ar t e r y (Bevan and Tsuru, 1975) t h i s suggests that denervation may indeed cause a thinner w a l l . Also, maximum tension ( f o r c e / c r o s s - s e c t i o n a l area) was l e s s i n the denervated r a b b i t ear artery; therefore, Bevan and Tsuru (1979) a t t r i b u t e d t h i s decrease to a q u a l i t a t i v e change i n the c o n t r a c t i l e machinery. Therefore, changes i n smooth muscle mass, i n smooth muscle number and i n the c o n t r a c t i l e machinery may produce a weaker wall r e s u l t i n g i n the d i l a t e d veins reported here. Secondly, changes i n the connective t i s s u e f i b e r s ( e l a s t i n and collagen) may a f f e c t the resistance of the blood vessel w a l l . Aneurysms and varicose veins are c l i n i c a l examples of d i l a t i o n s i n blood v e s s e l s . 58 Although aneurysms are mainly associated with the ar t e r i a l side of the circulatory system, aneurysms in veins, such as the portal vein, do exist (Ohnishi et a l . , 1984). The etiologies of these two c l i n i c a l examples may involve changes in the connective tissue fibre components of blood vessel walls (Grobety et a l . , 1977; Niebes et a l . , 1977; Crissman, 1984; Dobrin et a l . , 1984). Rusterholz and Mueller (1982) suggested that the decrease in perfusion pressure (representing vascular resistance) of the denervated rabbit ear vascular bed may not be exclusively due to a decrease in smooth muscle mass, but may be the result of alterations in the collagen or elastin. Therefore, the importance of collagen and elastin in the blood vessel wall should not be overlooked. Fibroblasts synthesize collagen and elastin as do smooth muscle c e l l s ; however, the fibroblasts are only found in the adventitia of blood vessels. Also, when Chamley and Campbell (1975) cultured smooth muscle cells from the guinea pig vas deferens, they described the ultrastructure of fibroblasts present in the culture after 1-2 days in culture. However, in their study, they did not follow through on the appearance of the fibroblasts; therefore, any effect that norepinephrine, sympathetic ganglion extract and cyclic AMP might have had on these c e l l s was not mentioned. Dobrin et a l . (1984) have looked at the importance of these two types of fibres in canine and human arteries. Treatment of canine common carotid arteries and human external, internal and common i l i a c arteries with elastase produced dilation of these vessels, with a decrease in compliance seen at higher pressures. The integrity of these elastase-treated vessels was always maintained. With collagenase treatment, the vessels were less dilated than the elastase-treated vessels, although they were significantly different from the controls. Also, the collagenase-treated vessels leaked uncontrollably and 59 eventually ruptured. Thus, collagen i s needed to maintain the integrity of the vessel whereas elastin i s needed to maintain the normal shape of the wall, but not the integrity. The normal shape of the denervated femoral vein in this study was significantly dilated, but i t did not rupture or leak. The fact that the femoral vein did not rupture or leak does not prove that collagen was not changed at a l l in amount; however, the fact that the normal shape of the vessel was changed suggests that the elastin was affected somehow. For instance, the normal content of elastin might not have been attained during the development of the denervated vein thereby disturbing the elastic/collagen ratio. An alteration in the elastic/ccllagen ratio as a consequence of denervation was suggested by Rusterholz and Mueller (1982) . Changes in the structure of the elastic fibres themselves or in their orientation may have been affected by the denervation procedure. The three-dimensional network of elastic fibres in canine saphenous veins has been studied by Crissman (1984) . In normal saphenous vein, he found that the internal elastic lamina, the media and the external elastic lamina each had their own unique elastic fibre organization. The elastic fibres from each of the three layers merged with the adjacent layer, forming a continuous network through the entire thickness of the wall. The single layer of elastic fibres in the internal elastic lamina consisting of large longitudinally oriented (at a slight angle from the true longitudinal) branching fibres intersected by finer fibres i s thought to distribute stress around and along the longitudinal surface. The media also had two sets of fibres, both larger than those of internal elastic lamina, but arranged somewhat the same. However, the longitudinal thicker fibres were angled tangentially, either directed externally or internally, traversing different levels of the media and did not form s t r a t i f i e d layers of fibres. This organization i s thought to distribute tension throughout the venous wall. The external elastic lamina was formed by several parallel layers of wide ribbons of closely opposing thick elastic fibres. Ribbons within the same level as well as those in adjacent levels were interconnected by the thick and thin elastic fibres. It was suggested that the arrangement and thickness of this layer would increase i t s r i g i d i t y thereby maintaining the shape of the vein and keeping the lumen open when external pressures were exerted on the venous wall by external organs. Thus, Crissman (1984) believes that the architecture of the elastic network would contribute to vascular integrity and f l e x i b i l i t y as well as aid in the distribution of stress throughout the venous wall. With this in mind, i t i s possible that denervation may have disrupted the elastic architecture in the wall of the rat femoral vein in my study. Since Todd (1986) found ultrastructural changes in the external elastic lamina, this layer may be the one most affected. Changes in the collagen content and other connective components were seen in varicose veins (Grobety et a l . , 1977; Niebes et a l . , 1977). Light microscopic studies carried out on human saphenous varicose veins showed a marked increase of i n t e r s t i t i a l staining with Alcian blue and Toluidine blue as well as an increase in PAS positive material (Grobety et a l . , 1977), which widely separated the bundles of smooth muscle. Electron microscopy confirmed the increased i n t e r s t i t i a l space and also showed the loss of normal organization and structure of the i n t e r s t i t i a l connective tissue (Grobety et a l . , 1977). These histochemical results coincided with the biochemical results obtained by Niebes et a l . (1977). They found that the insoluble collagen content was significantly less in the varicose veins, but the total amount of glycosaminoglycans and 61 glycoproteins was s i g n i f i c a n t l y greater. Since no va r i a t i o n s were i n the smooth muscle or energetic metabolism (Niebes et a l . , 1977), the combined h i s t o l o g i c a l and biochemical findings i n d i c a t e that the main abnormalities of varicose veins are i n the connective t i s s u e components. I t appears from past studies that connective t i s s u e components play an important r o l e i n maintaining the shape of the blood vessel w a l l . I f the collagen component i s changed by lack of innervation, then the wall may weaken and a d i l a t i o n such as that seen i n the femoral vein i n t h i s study may occur. Not many denervation studies have been done on veins. Denervation of the r a t p o r t a l vein (Aprigliano, 1983) produced s u p e r s e n s i t i v i t y of the smooth muscle c e l l membrane to norepinephrine. Morphological studies of the denervated dog saphenous vein were c a r r i e d out by Branco et a l . (1984). They found that the wall thickness of the denervated saphenous vein i n the dog was greater than that of the co n t r o l s . In t h e i r study, t h i s thicker wall persisted i n the vein even at 120 days post-denervation. The smooth muscle c e l l s i n the denervated dog saphenous vein had a de d i f f e r e n t i a t e d appearance, a c h a r a c t e r i s t i c of which was lar g e r smooth muscle c e l l s . Since I found a decrease i n medial thickness of the saphenous art e r y , I assumed that, i n my study, the media of the veins would be thinner too, e s p e c i a l l y since the vein was so d i l a t e d . However, t h i s was not the case. Some cross-sections o f the d i l a t e d r a t femoral vein showed that some areas of the wall were very t h i n and tenuous. Although, when four randomly chosen points were measured and averaged together, the wall of the denervated femoral vein i n my study was not thinner than that of the control s i d e . In f a c t , the t o t a l mean wall thickness of the denervated femoral vein tended to be higher than the mean of the con t r o l v e i n . This d i f f e r e n c e was not s i g n i f i c a n t at the P<0.05 l e v e l (Table IV). I t i s possible, therefore, that the d i l a t i o n may not be the r e s u l t of a decrease i n the e l a s t i n content as implied by the work of Dobrin et a l . (1984), but rather a change i n the arch i t e c t u r e of the e l a s t i c f i b r e s (Crissman, 1984) . No other morphological differences between the denervated r a t femoral v e i n and i t s c o n t r a l a t e r a l c o n t r o l side were seen at the l i g h t microscopic l e v e l i n t h i s present study. The explanation given for the lack of d i f f e r e n c e between denervating the saphenous a r t e r y at the two d i f f e r e n t age groups (that i s , at 1-3 or 12 days) probably applies to the femoral v e i n . CONCLUSIONS The method of s u r g i c a l denervation i n t h i s study i s as successful as superior c e r v i c a l ganglionectomy, although my method i s far more l o c a l i z e d . Although re-innervation from aberrant areas does occur, my method of denervation does produce a l t e r a t i o n s i n the blood v e s s e l s . The decreases i n the medial area of the a r t e r y do p e r s i s t long a f t e r denervation (78-87 days); however, whether the decrease i s due to a reduction i n smooth muscle c e l l s i z e or a reduction i n the p a r a c e l l u l a r matrix remains to be determined. Also, t h i s denervation technique leads to a reproducible d i l a t i o n of the femoral vein, the mechanism by which t h i s d i l a t i o n occurs i s unknown. TABLE I: Changes in the Area of the Tunica Media of the Saphenous Artery when Sampled at 60 Days of Age. Age at Denervation (Days old) 1-3 12 Age Sampled At (Days old) 60 60 60 60 60 60 Area of the Tunica Media of the Saphenous Artery Control Side (u 2) 34481.6 34859.5 34337.2 41183.1 40846.5 40676.9 37182.5 37633.1 37321.7 36068.1 36361.7 37007.6 25176.9 25125.3 24506.3 47077.6 46928.9 46629.6 TOTAL MEAN +SEM P=0.10 n=6 Mean 34559.4 40902.2 37379.1 36479.1 24936.2 46878.7 36855.8 +2969.8 Denervated Side (u 2) 24534.7 24054.2 24155.9 40783.2 41534.0 41599.0 34244.1 34720.0 34315.0 19305.9 19842.9 19844.6 26254.4 26180.6 26040.5 42412.7 42360.2 47769.8 Mean 24248.3 41305.4 34426.6 19664.5 26158.5 44180.9 31664.0 +4027.2 GO TABLE I I : Changes i n the Area of the Tunica Media of the Saphenous Artery When Sampled at 90 Days of Age Age at Denervation (Days old) 1-3 12 Age Sampled At (Days old) 90 90 90 90 90 90 Area of the Tunica Media of the Saphenous Artery Control Side (u 2) 41634.3 42159.2 41924.0 42103.4 41638.5 41401.2 41926.8 42529.4 43237.4 44045.5 44412.5 45182.8 43897.9 48085.9 47745.3 47627.3 47393.8 50364.2 50675.9 50664.5 TOTAL MEAN +SEM Mean 41905.8 41714.4 42564.5 44384.7 47713.1 50568.2 44808.4 +1470.0 Denervated Side ( P 2 ) 34345.0 35579.2 34795.3 35665.2 36092.9 35826.8 45722.4 46054.6 45718.2 42659.4 43784.8 43069.5 43017.1 34849.4 34287.4 32486.1 33269.2 37405.1 38171.1 36789.6 Mean 34906.5 35861.6 45831.7 43132.7 33698.0 37455.3 38481.0 +4883.4 P=0.05 TABLE I I I : Comparison of the Perimeters of the Denervated and Control Femoral Vein Age at Denervation Age Sampled At (Days old) (Days old) 1 2 6 0 60 1 - 3 9 0 9 0 1 2 9 0 9 0 Perimeter of Femoral Vein Control Mean Denervated Mean Side Side ("2) (u2) 3 8 0 8 6 8 9 5 3 7 9 6 3 7 9 7 6 8 7 8 6 8 7 4 3 7 9 2 6 8 5 0 3 3 1 7 4 4 6 1 3 3 2 7 3 3 2 4 4 4 2 7 4 4 3 9 3 3 2 8 4 4 3 0 4 4 2 5 7 2 0 6 4 2 7 3 4 2 3 7 7 2 6 8 7 2 2 4 4 2 1 3 7 1 9 8 2 8 0 9 4 8 4 4 2 7 0 9 2 7 9 3 4 9 0 9 4 8 8 8 2 8 6 2 4 9 1 0 4 5 1 2 6 7 5 1 HH88 4 4 9 0 6 7 7 6 6 7 5 7 4 4 7 1 6 7 4 3 4 3 1 8 6 8 0 1 4 2 1 6 4 2 8 9 6 7 6 6 6 8 1 7 4 3 3 3 6 8 8 5 TOTAL MEAN 3 8 2 2 6 1 6 7 +SEM +267 + 4 8 3 P=0.0005 n=6 66 TABLE IV: Comparison of Wall Thickness between the Denervated and Control Femoral Vein. Age at Denervation Age at Sampling Mean Thickness of (days old) (days,old) Femoral Vein Wall Control Side (M ) Denervated Side (u) 12 60 6.02 5-37 12 60 4.60 5.93 1-3 90 7.67 13.57 1-3 90 2.58 3.56 12 90 2.86 4.14 12 90 5.93 3.20 TOTAL MEAN +SEM 4.94 +0.81 5.96 +1.58 T-TEST RESULTS n=6 P=0.25 TABLE V: Number of Fluorescing Areas Counted from the Control and Denervated Saphenous Arteries and Femoral Veins Age at Age Sampled Number of +Average Number Number of +Average Number *DN At (days old) Sections: per Saphenous Artery Sections: per Femoral Vein (days Arteries Control Denervated Veins Control Denervated ild) Side Side Side Side 3 30 10 15 5 - - - 12 30 14 28 3 10 48 0 12 60 32 40 2 19 220 27 12 60 32 49 25 13 105 54 3 90 25 75 27 15 169 62 3 90 36 63 18 30 168 91 12 120 45 68 6 25 101 3 12 120 36 113 30 31 105 26 ANOVA Results P: =0.008 P: =0.004 n=8 n=7 *DN = denervation + refers to the average number of fluorescing areas. Figure 1: Denervation Procedure, (a) and (b) show the r a t thigh with the skin r e f l e c t e d . The ( i ) femoral nerve, ( i i ) the femoral arte r y and vein, ( i i i ) the s u p e r f i c i a l e p i g a s t r i c a r t e r y and vein and ( i v ) the saphenous a r t e r y and vein can be seen. In (b), black suture s i l k has been t i e d around the femoral nerve i n two places.  Figure 2: Denervation Procedure continued, (a) In the thigh, the ( i ) femoral nerve i s severed j u s t d i s t a l to the second knot (arrow). The d i s t a l portion of the femoral nerve i s separated from the ( i i ) femoral artery and vein up to the branch points of the ( i i i ) s u p e r f i c i a l e p i g a s t r i c and ( i v ) saphenous a r t e r i e s and veins. The nerve i s then severed again at the branch points (arrowhead), (b) Inside the abdominal c a v i t y , the femoral nerve i s threaded through the tube and the end i s heat sealed (arrowhead) The a s t e r i s k i n d i c a t e s the abdominal w a l l .  Figure 3: Diagram showing the cannulation procedure. The c a r o t i d a r t e r y i s t i e d o f f c r a n i a l l y with suture s i l k ( i ) and the cannula i s inserted through the loose knot ( i i ) and then i n t o a small i n c i s i o n i n the c a r o t i d a r t e r y . Once the cannula i s i n s i d e the ar t e r y , then the small a r t e r i a l clamp ( i i i ) i s released so the cannula can be pushed i n more caudally. The second knot ( i v ) , the one most caudally, and the loose, more c r a n i a l knot ( i i ) are tightened.  Figure 4; Perfusion Pressure Recording. The (a) mean, (b) s y s t o l i c and (c) d i a s t o l i c blood pressures are recorded, (d) Perfusion i s c a r r i e d out at the mean blood pressure. 75 Figure 5: Light micrographs of (a) control and (b) denervated saphenous artery walls. The three tunics are well defined: (i) tunica interna, ( i i ) tunica media and ( i i i ) tunica externa. The breaks in the thicker internal elastic lamina (arrows) and thinner internal elastic lamina (arrowheads) are seen in both (a) and (b). There i s no remarkable difference between the denervated and i t s contralateral control side. These samples were taken from an animal denervated at 3 days and sampled at 60 days of age. Magnification for (a) and (b) i s 670X.  Figure 6: Light micrographs of the (a) c o n t r o l and (b) denervated femoral veins. The three tunics so c h a r a c t e r i s t i c of blood vessels are not so e a s i l y i d e n t i f i a b l e i n these veins. In (b), the endothelium has a granular appearance (arrows). Magnification for (a) and (b) i s 6 9 0 X . 79 b Figure 7: Light micrographs of the (a) normal saphenous nerve taken from the control side and (b) the distal stump from the denervated side. These tissue samples came from an animal denervated at 3 days and sampled at 60 days, and were taken adjacent to the saphenous artery. The overall histological appearance has changed, and there i s a marked decrease in size following denervation. Magnification for (a) i s 415X and for (b) i t i s 660X.  Figure 8: Light micrographs of femoral nerves from (a) control (125X) and (b) denervated sides (125X). Samples were taken from within the abdominal cavity. Sample (b) was taken just proximal to the tube and therefore represents the proximal nerve stump. There i s a marked decrease in size and a noticeable change in the histology of the nerve that i s tubed. The insert (1050X) shows an enlarged portion of the core with few very small myelinated axons remaining. Sample (a) was taken from a corresponding area. Samples were taken from an animal denervated at 3 days and sampled at 60 days.  Figure 9- U l t r a s t r u c t u r a l l y , the femoral nerve (same sample as seen i n Figure 8(b)) i s composed mainly of collagen (5610X). F i b r o b l a s t s (arrows) can be seen i n the midst of a l l the collagen. Membrane-lined areas of degeneration (arrowheads) are present. A c a p i l l a r y i s located i n the centre of the micrograph. The i n s e r t i l l u s t r a t e s a small myelinated nerve ( a s t e r i s k ) and an unmyelinated nerve (arrow) observed i n the core of t h i s sample ( 1 6 0 3 0 X ) . 85 Figure 10: Fluorescence microscopy of con t r o l and denervated saphenous a r t e r i e s . Trypan blue causes the e l a s t i c t i s s u e to fluoresce i n the red range, (a) Catecholamine fluorescence from the adrenergic terminals i s seen at the adventitial-medial border of the cont r o l saphenous art e r y , (b) V i r t u a l l y no fluorescence o f catecholamines i s seen i n the denervated a r t e r y .  Figure 11: Fluorescence microscopy o f co n t r o l and denervated femoral v e i n s . Dense innervation i s seen i n the (a) con t r o l versus the (b) denervated saphenous v e i n .  Figure 12: Per cent decrease i n medial area of denervated saphenous a r t e r i e s from animals sampled at 60 and 90 days of age. The con t r o l side represents 100?. • C o n t r o l H D e n e r v a t e d % 1 0 0 8 0 6 0 4 0 2 0 I I 6 0 days 9 0 days Saphenous A r t e r y F i g u r e 13: Per cent increase In luminal perimeter of e n e r v a t e d f e m o r a l veins. The c c n t r c l side represents 100*. F e m o r a l V e i n BIBLIOGRAPHY Aprigliano, 0. (1983) Neural Influences and Norepinephrine Sensitivity in the Rat Portal Vein. Federation Proc. 42:257-262. Barr, M.L. and Kiernan, J.A. (1983) The Human Nervous System. An Anato- mical Viewpoint. 4^n Ed. Harper and Row, Publishers, Philadelphia. Bevan, J.A. and Su, C. (1973) Sympathetic Mechanisms in Blood Vessels: Nerve and Muscle Relationships, Annual Review of Pharmacology. 1 3 : 2 6 9 - 2 8 5 . Bevan, R.D. (1984) Trophic Effects of Peripheral Adrenergic Nerves on Vascular Structure. Hypertension 6[Suppl. III]:III-9 - 111-26. Bevan, R.D. (1975) Effect of Sympathetic Denervation on Smooth Muscle Cell Proliferation in the Growing Ear Artery. Circ. Res. 37:14-19. Bevan, R.D., Tsuru, H and Bevan, J.A. (1983) Cerebral Artery Mass in the Rabbit i s Reduced by Chronic Sympathetic Denervation. Stroke l4(3):393-396. Bevan, R.D. and Tsuru, H. (1979) Long-Term Denervation of Vascular Smooth Muscle causes not only Functional but Structural Change. Blood Vessels 16:109-112. Bevan, R.D. and Tsuru, H. (1981) Functional and Structural Changes in the Rabbit Ear Artery after Sympathetic Denervation. Circ. Res. 49:478-485. Boatman, D.L., Shaffer, R.A., Dixon, R.L. and Brody, M.J. (1965) Function of Vascular Smooth Muscle and i t s Sympathetic Innervation in the Newborn Dog. J. of C l i n i c a l Investigations 44:241-246. Branco, D., Albino Teixeira, A., Azevedo, I., and Osswald, W. (1984) Structural and Functional Alterations caused at the Extraneuronal Level by Sympathetic Denervation of Blood Vessels. Naunyn-Schmie- deberg's Arch. Pharmacol.326:302-312 Brody, M.J. (1964) Cardiovascular Responses Following Immunological Sympa- thectomy. Circ. Res. 15:161-167. Burnstock, G. and Costa, M. (1975) Adrenergic Neurons. Chapman & Hall, Ltd., London. Chamley, J.H. and Campbell, G.R. (1975) Trophic Influences of Sympathetic Nerves and Cyclic AMP on Differentiation and Proliferation of Isolated Smooth Muscle Cells in Culture. Cell Tissue Research 161:407-510. Chamley, J.H. and Campbell, G.R. (1976) Tissue Culture: Interaction Bet- ween Sympathetic Nerves and Vascular Smooth Muscle. In Vascular Neuro- effector Mechanisms, edited by J.A. Bevan, G. Burnstock, B. Johansson, R.A. Maxwell and O.A. Nedergaard. Basil, Karger, pp 10-18. Chen, G., Portraan, R. and Wickel, A. (1951) Pharmacology of 1,1-dimethyl- 4-phenyl piperazinium iodide, a Ganglionic Stimulating Agent. J. Phar- macology 103:330 Colin, W. and Donoff, R.B. (1984) Nerve Regeneration Through Collagen Tubes. J. Dent. Res. 63(7):987-993. Crissman, R.S. (1984) The Three-Dimensional Configuration of the Elastic Fibre Network in Canine Saphenous Vein. Blood Vessels 21:156-170. De l a Lande, I.S. and Waterson, J.G. (1968) Modification of Autofluores- cence in the Formaldehyde-treated Rabbit Ear Artery by Evans Blue. J. Histochem., Cytochem. 16:281-282. De l a Torre, J.C. (1980) An Improved Approach to Histofluorescence using the SPG Method for Tissue Monoamines. J. Neurosci. Methods 3:1-5. Dobrin, P.B., Baker, W.H. and Gley, W.C. (1984) Elastolytic and Collageno- l y t i c Studies of Arteries. Arch. Surg. 119:405-409. Dolezel, S., Gerova, M. and Gero, J. (1974) Postnatal development of the sympathetic innervation in skeletal muscles of the dog. Physiol. Bohe- moslov. 2 3 : 1 3 8 - 1 3 9 . Drachman, D.B. (1974) Trophic Actions of the Neuron: An Introduction. In Trophic Functions of the Neuron, edited by D.B. Drachman, Annals New York Acad. Sci. 228:3-5. Dyck, P.J. and Hopkins, A.P. (1972) Electron Microscopic Observations of Degeneration and Regeneration of Unmyelinated Fibres. Brain 95:223-234. 96 Engel, A.G. and Stonnington, H.H. (1974) Morphological E f f e c t s of Dener- vation of Muscle. A Q u a l i t a t i v e U l t r a s t r u c t u r a l Study. In Trophic Functions of the Neuron, edited by D.B. Drachman, Annals New York Acad. S c i . 228:68-88. Finch, L., Haeusler, G. and Thoenen, H. (1973) A Comparison of the E f f e c t s of Chemical Sympathectomy by 6-Hydroxydopamine i n Newborn and Adult Rats. Br. J . Pharm. 47:249-260. Gabella, G. (1976) Structure of the Autonomic Nervous System. Chapman and H a l l , Ltd., London. Gauthier, G.F. and Dunn, R.A. (1973) U l t r a s t r u c t u r a l and Cytochemical Features of Mammalian S k e l e t a l Muscle Fibers Following Denervation. J . C e l l S c i . 12:525-547. Gerova, M., Gero, J . , Dolesel, S. and Konecny, M. (1974) Postnatal Deve- lopment of Sympathetic Control i n Canine Fermoral Artery. Physiologia Bohemoslovaca 23:289-295. G e r r i t y , R.G. and C l i f f , W.J. (1975) The A o r t i c Tunica Media of the Developing Rat. Laboratory Investigation 32:585-600. Grobety, J . and Bouvier, C A . (1977) Studies on Normal and Varicose Human Saphenous Veins I: S t r u c t u r a l Differences, Histochemical and Electron Microscope Investigations. 9 t n Europ. Conf. M i c r o c i r c u l a t i o n , Antwerp, 1976. B i b l . Anat. 16:298-300. Gutman, E. (1976) Neurotrophic Relations. Annu. Rev. P h y s i o l . 38:177-216. Ham, A.W. and Cormack, D.H. (1979) Histology 8 t n Ed., J.B. L i p p i n c o t t Company Philadelphia and Toronto, p. 575. Hayat, M.A. (1970) P r i n c i p l e s and Techniques of El e c t r o n Microscopy: Bio- l o g i c a l Applications V o l . 1. Van Nostrand Reinhold Company, New York. Hoffman, W.W. and Thesleff, S. (1972) Studies on the Trophic Influence of Nerve on S k e l e t a l Muscle. Eur. J . Pharmac. 20:256-260. Humphrey, C. and Prittman, F. (1974) A Simple Methylene blue-Azure II Basic Fuchsin for Epoxy-Embedded Tissue Sections. Stain Tech. 49:9-14. I l l u s t r a t e d Stedman's Medical Dictionary, 24 t" Ed. (1982) Williams and Wilkins, Baltimore, MD. Kanakis, S.J., H i l l , C.E., Hendry, I.A. and Watters, D.J. (1985) Sympa- t h e t i c Neuronal Su r v i v a l Factors Change a f t e r Denervation. Dev. Brain Res. 20:197-202. Kobayashi, S., Tsukahara, S., T s u j i , T., Sugita, K. and Nagata, T. (1983) Histochemical Studies on Regeneration of Aminergic Nerves i n Rat Cere b r a l Artery a f t e r Superior C e r v i c a l Ganglionectomy Histochemistry 77:57-62. L a i s , L.T., Rios, L.L., Boutelle, S., DiBona, G.F. and Brody, M.J. (1977) A r t e r i a l Pressure Development i n Neonatal and Young Spontaneously Hypertensive Rats. Blood Vessels 14:277-284. Lentz, T.L. (1974) Neurotrophic Regulation at the Neuromuscular Junction. Ann. New York Acad. S c i . 228:323-337. Levi-Montalcini, R. and A n g e l e t t i , P.U. (1966) Immunosympathectomy. Pharmac. Rev. 18:619-628. Luco, J.V. and Eyzaguirre, C. (1955) F i b r i l l a t i o n and Hypersensitivity to Ach i n Denervated Muscle. E f f e c t of Length of Degenerating Nerve F i b r e s . J . Neurophysiol. 18:65-73- Mclnnes, A. (1977) Modification of the F a l c k - H i l l a r p Technique with Intra v i t a l Trypan blue to D i f f e r e n t i a t e E l a s t i c Fibres from Noradrenergic Endings. J . P h y s i o l . 268:22P-23P. Mollenhauer, H.H. (1964) P l a s t i c Embedding Mixture for use i n Electron Microscopy. Stain Tech. 39:111-114. Niebes, P., Engels, E. and jegerlehner, M. (1977) Studies on Normal V a r i - cose Human Saphenous Veins I I : Differences i n the Compostion of Collagen and Glycosaminoglycans. 9 t h Europ. Conf. M i c r o c i r c u l a t i o n , Antwerp, 1976. B i b l . Anat. 16:301-303. Ohnishi, K., Nakayama, T., Saito, M., Nomura, F., Koen, H., Tamaru, J . , Iwasaki, I . and Okuda, K. (1984) Aneurysm of the Intrahepatic Branch of the P o r t a l Vein. Gastroenterology 8 6 : 1 6 9 - 1 7 3 . Page, I.H. and McCubbin, J.W. (1965) Chapter 61: The Physiology of Arte- r i a l Hypertension. In Handbook of Physiology. Section 2, C i r c u l a t i o n . V o l . I I , American P h y s i o l o g i c a l Society, Wash., DC. Palaty, V. (1971) D i s t r i b u t i o n of Magnesium i n the A r t e r i a l Wall. J.Physiol. 218:353-368. Pease, D.C. (1964) Histological Techniques for Electron Microscopy. 2 Ed. Academic Press Inc., New York and London. Pellegrino, C. and Franzini, C. (1963) An Electron Microscope Study of Denervation Atrophy in Red and White Skeletal Muscle Fibers. J. Cell B i o l . 17:327-349. Pellegrino, R.G. and Spencer, P.S. (1985) Schwann Cell Mitosis in Response to Regenerating Peripheral Axons in vivo. Brain Res. 341:16-25. P o l i t i s , M.J., Ederle, K. and Spencer, P.S. (1982) Tropism Nerve Regene- ration in vivo. Attraction of Regenerating Axons by Diffusible Factors Derived from Cells in Distal Stumps of Transected Peripheral Nerves. Brain Res. 253:1-12. Rusterholz, D.B. and Mueller, S.M. (1982) Sympathetic Nerves Exert a Chronic Influence on the Intact Vasculature that i s Age Related. Ann. Neurol. 11:365-371. Sarnat, H.B. (1983) Ch. 3. Denervation and Reinnervation of Muscle. In Muscle Pathology and Histochemistry. American Society of Cl i n i c a l Pathologists Press, Chicago, pp. 35-43. Szekere, L. (ed.) (1980) Adrenergic Activators and Inhibitors. Handbook of Experimental Pharmacology 54:1, Springer-Verlag, Berlin. Thesleff, S. (1974) Physiological Effects of Denervation of Muscle. In Trophic Functions of the Neuron, edited by D.B. Drachman. Ann. New York Acad. Sci. 228:89-104. Todd, M.E. ( 1980) Development of Adrenergic Innervation in Rat Peripheral Vessels: A Fluorescence Microscopic Study. J. Anat. 1 3 1 : 1 2 1 - 1 3 3 . Todd, M.E., Laye, C.G. and Osborne, D.N. ( 1 9 8 3 ) The Dimensional Charac- ter i s t i c s of Smooth Muscle in Rat Blood Vessels: A Computer-Assisted Analysis. Circ. Res. 5 3 : 3 1 9 - 3 3 1 . Todd, M.E. and Tokito, M.K. (1981) An Ultrstructural Investigation of Developing Vasomotor Innervation in Rat Peripheral Vessels. Amer. J. Anat. 160:195-212. Todd, M.E. (1986) Trophic Interactions Between Rat Nerves and Blood Vessels i n Denervated Peripheral A r t e r i e s and i n Anterior Eye Chamber Transplants. C i r c . Res. 58: In Press. Waris, T. (1978) Re-inneration of Free Skin Autografts i n the Rat. Scand. J . P l a s t . Reconstr. Surg. 12:85-93. Williams, P.L. and Warwick, R. (1975) Functional Neuroanatomy of Man. Phil a d e l p h i a , W.B. Saunders.

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
United States 3 8
United Kingdom 2 0
Canada 2 0
Japan 2 0
China 1 10
City Views Downloads
Surrey 2 0
Unknown 2 1
Ashburn 2 0
Tokyo 2 0
Mountain View 1 8
Beijing 1 0

{[{ mDataHeader[type] }]} {[{ month[type] }]} {[{ tData[type] }]}

Share

Share to:

Comment

Related Items