UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Trophic interactions between rat thigh blood vessels and their innervation Schindelhauer, Nancy Lynn 1986

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

Item Metadata

Download

Media
[if-you-see-this-DO-NOT-CLICK]
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
Original Record: 1.0096806 +original-record.json
Full Text
1.0096806.txt
Citation
1.0096806.ris

Full Text

TROPHIC INTERACTIONS BETWEEN RAT THIGH  BLOOD VESSELS AND THEIR INNERVATION By NANCY LYNN SCHINDELHAUER Hons. B . S c , 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 B r i t i s h Columbia)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA April  1986  (c): Nancy Lynn Schindelhauer, 1986  In  presenting  requirements of  British  it  freely  agree for  this f o r an  available  that  understood  that  financial  by  his  at  the  Library  shall  for reference  and  study.  I  for extensive  or  be  her or  shall  copying  granted  by  not  be  of  Z^-AJA-T"5/V-*/  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  1Q\  r W l l .  Columbia  3 p ) f i  make  further this  thesis  head o f  this  It  my  is  thesis  a l l o w e d w i t h o u t my  permission.  Department of  the  of  representatives. publication  the  University  the  copying  gain  f u l f i l m e n t of  that  p u r p o s e s may  or  degree  I agree  permission  scholarly  in partial  advanced  Columbia,  department  for  thesis  written  ABSTRACT  Much l e s s work has been done on the denervation  of smooth muscle  compared with the extensive studies c a r r i e d out on s k e l e t a l muscle. was  thought that denervation  It  of smooth muscle produced few a l t e r a t i o n s i n  i t s morphology or physiology,  e s p e c i a l l y since many blood vessels have  v i r 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 s u f f i c i e n t to denervate s k e l e t a l muscle, but t h i s does not apply to smooth muscle. Therefore,  denervation  methods for smooth muscle have included those with  widespread e f f e c t s such as chemical and immunological sympathectomies, and superior c e r v i c a l ganglionectomy. a semi-permanent, l o c a l i z e d denervation  In t h i s study, I have developed a method for r a t 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 examined at 30, 60, 90 and 120 days of age.  or 12 days of age,  and  The femoral nerve, which  c a r r i e s the vasomotor innervation to the thigh vessels, was the thigh and brought inside the abdominal c a v i t y .  severed i n  This method  was  necessary since preliminary experiments showed rapid re-innervation of the vessels i f any part of the proximal root remained i n the thigh. Inside the abdominal cavity, the nerve was and heat sealed.  slipped into a p l a s t i c tube  The tubing further i n h i b i t e d re-innervation by  preventing c o l l a t e r a l sprouting from the proximal stump.  Samples of the  d i s t a l nerve stump, the proximal nerve stump, and from the femoral vein and saphenous artery were taken.  In every animal, the c o n t r a l a t e r a l side  acted as a c o n t r o l . The d i s t a l and proximal nerve stumps showed marked evidence of  degeneration.  Fluorescence  microscopy ( s p e c i f i c f o r c a t e c h o l a m i n e s )  showed a s i g n i f i c a n t decrease i n the number o f f l u o r e s c i n g dots around both the a r t e r y and  vein.  The  presence o f some f l u o r e s c e n c i n g  around the denervated v e s s e l s may  be  from the nerves t h a t were seen  r e - i n n e r v a t i n g the v e s s e l s a t the time o f s a m p l i n g . from a b e r r a n t  areas.  dots  A r t e r i e s sampled a t 90  These nerves came  days showed a  significant  decrease i n the c r o s s - s e c t i o n a l a r e a o f the t u n i c a media on  the  denervated s i d e . The  denervated femoral v e i n , i n s i t u , was  compared t o the c o n t r o l s i d e .  seen to be g r o s s l y  Measurements o f the l u m i n a l p e r i m e t e r  s e c t i o n s o f the v e i n showed t h a t the denervated v e i n had up  to three  i n the  times t h a t o f the c o n t r o l s .  femoral v e i n was  The  a luminal  difference i n wall  not s i g n i f i c a n t a t the p<0.05 l e v e l .  r e s u l t s i n d i c a t e that adrenergic i n f l u e n c e on a r t e r i e s , but may  dilated  nerves may  of  area  thickness  My  not o n l y have a t r o p h i c  i n f l u e n c e v e i n s as w e l l .  Therefore,  in  t h i s s t u d y , t r o p h i c i n f l u e n c e s o f nerves on b l o o d v e s s e l s have been suggested by d e n e r v a t i o n  causing  p r o d u c i n g 2) a r e p r o d u c i b l e  1) a t h i n n e r a r t e r i a l w a l l and  d i l a t i o n o f the femoral v e i n .  Also,  by trophic  i n f l u e n c e s o f blood v e s s e l s on nerves i s suggested by the presence o f r e - i n n e r v a t i o n from a b e r r a n t  areas.  TABLE OF CONTENTS  Page Abstract  i i  L i s t of Tables  vi  L i s t of Figures  vii  Acknowlegements  ix 1  Introduction Introductory Statement  1  The E f f e c t s of Innervation on Skeletal Muscle  3  The E f f e c t s of Cardiac Muscle on Nerves  8  The E f f e c t s 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 .,.  29  Analysis: Light Microscopy and Morphometries  30  Fluorescence Microscopy  = . > '  Electron Microscopy  31  Fluorescence Microscopy  32  Statistics  32  Results Sampling for Light Microscopy  3^ 3^  L i g h t M i c r o s c o p i c Appearance: Saphenous A r t e r y  36  : Femoral V e i n  37  : Nerve  37  E l e c t r o n M i c r o s c o p y o f the Abdominal C a v i t y Femoral Nerve  39  F l u o r e s c e n c e M i c r o s c o p y : Gross Morphology o f the Denervated Area  39  D e s c r i p t i o n o f S e c t i o n s under the Fluorescence Microscope A n a l y s i s : Morphometries - M e d i a l A r e a o f the Saphenous A r t e r y  40 41  - V e i n P e r i m e t e r and W a l l T h i c k n e s s  42  - F l u o r e s c e n c e Counts  43  Discussion  44  Conclusions  62  Tables  63  Figures  69  Bibliography  94  vx LIST OF TABLES  TABLE  I  TITLE  Changes i n the Area o f the T u n i c a Media o f t h e Saphenous A r t e r y when Sampled  II  Comparison  63  a t 60 Days o f Age.  Changes i n the Area o f the T u n i c a Media o f t h e Saphenous A r t e r y when Sampled  III  PAGE  64  a t 90 Days o f Age.  o f the P e r i m e t e r s o f t h e Denervated and Con-  65  t r o l Femoral V e i n .  IV  Comparison  o f W a l l T h i c k n e s s Between the C o n t r o l And De-  66  n e r v a t e d Femoral V e i n .  V  Number o f F l u o r e s c i n g Areas Counted  from the C o n t r o l and  Denervated Saphenous A r t e r i e s and Femoral V e i n s  67  VX1  LIST OF FIGURES  FIGURE  TITLE  PAGE  1  Denervation Procedure.  69  2  D e n e r v a t i o n Procedure c o n t i n u e d .  71  3  Diagram  73  4  P e r f u s i o n Pressure Recording  75  5  L i g h t Micrographs o f C o n t r o l and Denervated Saphenous  77  Showing the C a n n u l a t i o n P r o c e d u r e .  Artery Walls.  6  L i g h t Micrographs o f C o n t r o l and Denervated Femoral  79  Veins.  7  L i g h t Micrographs o f the Normal Saphenous Nerve taken from the  81  C o n t r o l S i d e and the D i s t a l Stump from the Dener-  vated Side  8  L i g h t Micrographs o f Femoral Nerves from C o n t r o l and Denervated  9  Ultrastructurally, of Collagen  83  Sides.  the Femoral Nerve i s Composed M a i n l y  85  viii FIGURE  TITLE  PAGE  10  Fluorescence Microscopy of Control and Denervated  87  Saphenous A r t e r i e s .  11  Fluorescence Microscopy of Control and Denervated  89  Femoral Veins.  12  Per Cent Decrease i n Medial Area of Denervated Saphe-  91  nous Arteries from Animals Sampled at 60 and 90 Days.  13  Per Cent Increase i n Luminal Perimeter of Denervated Femoral Veins.  93  ACKNOWLEDGEMENTS  I s i n c e r e l y thank D r . M. E . Todd f o r her s u p p o r t and encouragement, e s p e c i a l l y when I l a c k e d s e l f - c o n f i d e n c e . t h a t kept me g o i n g .  I t was her p o s i t i v e comments  The a s s i s t a n c e p r o v i d e d by Mrs. Connie Leung and  M i s s Fanny Chu was g r e a t l y a p p r e c i a t e d .  A s p e c i a l thanks goes t o Dr. A.  W. V o g l f o r two r e a s o n s : 1) h i s a d v i c e on s e c t i o n i n g and 2) f o r h i s mere presence i n the Anatomy B u i l d i n g l a t e i n the e v e n i n g s . t o thank F r e d McConnell difficulties. H e l l a Prochaska Bezio.  like  and B e r n i e Cox who h e l p e d me w i t h many t e c h n i c a l  S p e c i a l a p p r e c i a t i o n goes t o Dr. J . Weinberg, t o Mrs. and e s p e c i a l l y t o my v e r y dear f r i e n d , Mrs. Sharon  A l s o , I thank my p a r e n t s who encouraged  education.  I would a l s o  me t o c o n t i n u e my  1  INTRODUCTION  Introductory Statement Nerves have a profound e f f e c t on the development and maintenance of muscle.  This has been shown as early as the 1800s by studying  a l t e r a t i o n s i n human s k e l e t a l muscle histology caused by diseases which lead to the destruction of motor neurons or by lesions of the peripheral nerves (Pellegrino and F r a n z i n i , 1963; Sarnat, 1983).  From biopsies, the  time at which the a l t e r a t i o n i n human muscle takes place i s not always known; therefore, laboratory animals have been used to obtain a better c o r r e l a t i o n between the time of denervation and the onset of such a l t e r a t i o n s as atrophy. Cross-union experiments, which arose i n the 1960s, took advantage of the normal condition seen i n many mammalian laboratory animals which was that certain s k e l e t a l muscles are composed almost e n t i r e l y of f i b r e s of one type (Sarnat, 1983).  These experiments,  by producing changes i n f u 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 u l t r a s t r u c t u r a l properties of i n d i v i d u a l muscle f i b r e s (Sarnat,  1983). As a r e s u l t , these a d d i t i o n a l a l t e r a t i o n s were looked  for i n future denervation studies. Since human diseases of s k e l e t a l muscle have been the impetus for studying the e f f e c t s 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 i n 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 i s a common method for s k e l e t a l muscle, denervation which s e l e c t i v e l y 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 " r e l a t i n g to or dependent upon n u t r i t i o n . " to  Nerves and their target tissues are known  exhibit this " n u t r i t i v e " dependency.  The exact nature of neurotrophic  interactions i s s t i l l unknown; however, nerves can influence their e f f e c t o r tissues by i t s impulse a c t i v i t y and by any neurotrophic factors the nerve c e l l may produce. been defined by Gutman  Neurotrophic interactions, therefore, have  (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 f a c t o r s . Drachman (1976), on the other hand, believes that: " a d e f i n i t i o n o f "neurotrophic" must be both broad and non-prejudicial: broad i n the sense that i t includes a l l the relationships by which nerve c e l l s and target c e l l s a l t e r each other's anatomy, chemistry or physiology, and non-prejudicial i n the sense that i t must neither presuppose nor exclude any p a r t i c u l a r mechanism of trophic i n t e r action. ... Thus, we may define as "neurotrophic" any long-term r e l a t i o n s h i p i n which nerve c e l l s and target c e l l s i n t e r a c t so as to influence the structure or funct i o n of either member o f the pair."  Therefore, Drachman's d e f i n i t i o n of "neurotrophic" can include the e f f e c t s o f 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 s k e l e t a l muscle and less so i n cardiac and smooth muscles.  3 The E f f e c t s of Innervation on Skeletal Muscle In s k e l e t a l muscle, binding studies have allowed the biochemical examination  of the density of cholinergic receptors i n mouse and r a t  muscles (Thesleff, 1974).  In normally innervated hemidiaphragm muscles  of the r a t and mouse, the density of cholinergic receptors at the junctional s i t e i s two thousand times greater than at extrajunctional s i t e s (Thesleff, 1974).  Following denervation of these muscles, the  receptor density at the junctional s i t e i s maintained, but the extrajunctional density i s increased by 20-200 times. As a r e s u l t of this increase, one observes supersensitivity of the muscle membrane.  This physiological parameter was seen i n a 14-day  denervated muscle f i b r e 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 f i b r e , depolarizations to  acetylcholine were produced only at the end-plate region.  Luco and  Eyaguirre (1955) found that the onset of hypersensitivity to acetylcholine i n the cat tenuissmus muscle occurred e a r l i e r , 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 d i s t a l nerve stump was shorter). In cultured triceps muscle of the newt, Triturus viridescens, the presence of a sensory ganglion prevented esterase a c t i v i t y (Lentz, 1974).  the loss of muscle c h o l i n -  After three weeks i n culture, the  cholinesterase a c t i v i t y declined unless the muscle was cultured with the ganglion explant. maintenance e f f e c t .  B o i l i n g of the ganglion for one minute abolished this The e f f e c t s of the ganglion were seen whether the  ganglion was placed on the muscle surface or whether i t was placed i n a separate chamber separated from the muscle by a M i l l i p o r e  filter.  4 Hoffman and T h e s l e f f the  (1972) s t u d i e d  t h e t r o p h i c i n f l u e n c e o f nerve on  p h y s i o l o g y o f the r a t e x t e n s o r d i g i t o r u m longus muscle.  By i n j e c t i n g  cholchicine  i n t o t h e s c i a t i c nerve 2cm p r o x i m a l t o t h i s muscle, t h e  organization  o f m i c r o t u b u l e s was d e s t r o y e d , thereby p r e v e n t i n g t h e  proximo-distal  movement o f macromolecules and o r g a n e l l e s .  Measurements  o f the frequency and amplitude o f the m i n i a t u r e e n d - p l a t e p o t e n t i a l s and o f the s i n g l e t w i t c h  and t e t a n i c t w i t c h  treated side d i d not d i f f e r  on the c o l c h i c i n e  from t h e c o r r e s p o n d i n g measurements on t h e  contralateral control side. conduction.  tensions  T h e r e f o r e , c o l c h i c i n e had no e f f e c t on nerve  On t h e f o u r t h day, a f t e r t h e c o l c h i c i n e  extrajunctional  acetylcholine  injection,  s e n s i t i v i t y and t e t r o d o t o x i n - r e s i s t a n t  a c t i o n p o t e n t i a l s were e x h i b i t e d by t h e muscle ( t e t r o d o t o x i n blocks action potential generation). a f t e r the f o u r t h day.  When  fi-bungarotoxin  spontaneous n e u r o t r a n s m i t t e r r e l e a s e ) the a n t e r o l a t e r a l r e g i o n  These e f f e c t s g r a d u a l l y (a n e u r o t o x i n t h a t  generally subsided blocks  was s u b c u t a n e o u s l y i n j e c t e d i n t o  o f the r i g h t hind  l e g (thereby p a r a l y s i n g i t ) ,  or when the e x t e n s o r d i g i t o r u m longus was denervated by s e c t i o n i n g the deep p e r o n e a l nerve c l o s e t o t h e knee, e x t r a j u n c t i o n a l tetrodotoxin-resistant  s e n s i t i v i t y and  a c t i o n p o t e n t i a l s were o b s e r v e d .  t h e s e l a t t e r two methods were used, the e x t r a j u n c t i o n a l s e n s i t i v i t y and t e t r o d o t o x i n - r e s i s t a n t  action potentials  However, when acetylcholine developed  e a r l i e r and remained f o r 3 days l o n g e r than i n t h e c o l c h i c i n e experiments (Hoffman and T h e s l e f f , 1972). the  That i s , i n comparing these two methods,  d e n e r v a t i o n changes observed f o l l o w i n g c o l c h i c i n e treatment were o f a  shorter  duration  nerve s e v e r i n g using  and were l e s s pronounced.  S i n c e t h e consequences o f  o r o f u s i n g B - b u n g a r o t o x i n cannot be reproduced t o t a l l y by  c o l c h i c i n e , Hoffman and T h e s l e f f  t r o p h i c substances r e l e a s e d  (1972) suggested t h a t n o t o n l y a r e  by t h e n e r v e , b u t a l s o neuromuscular  5 t r a n s m i s s i o n and r e s u l t i n g muscle a c t i v i t y a r e c o n t r i b u t i n g  trophic  T h i s i s the same p o i n t t h a t Drachman ( 1 9 7 4 ) expressed i n h i s  influences.  d e f i n i t i o n of "neurotrophic". D e n e r v a t i o n methods o f t e n have been used t o study the dependency o f s k e l e t a l muscle morphology on i t s i n n e r v a t i o n .  Lentz (1974)  found  that  by s e v e r i n g the b r a c h i a l p l e x u s i n the newt, the j u n c t i o n a l f o l d s on  the  muscle s u r f a c e decreased i n h e i g h t and e v e n t u a l l y f l a t t e n e d out once the t r a c e s o f the axoplasm  d i s a p p e a r e d (21  days).  The r e m a i n i n g Schwann  c e l l s soon withdrew i n t o the e x t r a c e l l u l a r space l e a v i n g the neuromuscular  junction unidentifiable.  He a l s o found the same phenomenon  o c c u r r i n g i n c u l t u r e d newt s k e l e t a l muscle, changes o c c u r r e d more r a p i d l y than _in v i v o .  o n l y i n t h i s c a s e , the S i n c e the c u l t u r e d muscle  m a i n t a i n e d i t s j u n c t i o n a l f o l d s i n the presence o f a s e n s o r y g a n g l i o n , even though  i t s own  axon had degenerated,  maintenance o f the neuromuscular  t h i s s u g g e s t s t h a t perhaps  the  j u n c t i o n might be dependent on a  diffusable neurotrophic factor. U l t r a s t r u c t u r a l examinations and morphometric a n a l y s e s have been c a r r i e d out on denervated mammalian muscle ( P e l l e g r i n o and 1963;  G a u t h i e r and Dunn, 1973;  o b v i o u s change they saw was D u r i n g the f i r s t  E n g e l and S t o n n i n g t o n , 1 9 7 4 ; ) .  fibre.  3 weeks f o l l o w i n g d e n e r v a t i o n , morphometric a n a l y s i s o f (white) muscles  decrease i n the t r a n s v e r s e mean f i b r e a r e a (Engel and  1974).  The most  a decrease i n the s i z e o f the muscle  the r a t s o l e u s ( r e d ) and the r a t gastrocnemius 80%  Franzini,  showed an  Stonnington,  At the same time, the mean t r a n s v e r s e m y o f i b r i l l a r a r e a decreased  p r o p o r t i o n a t e l y t o , o r a t a s l i g h t l y g r e a t e r r a t e than the f i b r e a r e a . Two  weeks p o s t - d e n e r v a t i o n , P e l l e g r i n o and F r a n z i n i  ( 1 9 6 3 ) noted a  d i s t i n c t r e d u c t i o n i n the number o f m y o f i b r i l s i n the r a t s o l e u s and gastrocnemius.  They suggested t h a t 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 i n 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 i n the soleus and gastrocnemius, m y o f i b r i l l a r atrophy i n i t i a l l y began at the periphery of muscle f i b r e s , and a f t e r a month, spread to the i n t e r i o r of the muscle. Pellegrino and Franzini (1963) saw  In contrast,  peripheral and i n t e r i o r m y o f i b r i l l a r  atrophy i n the soleus (red f i b e r s ) but only peripheral atrophy i n the gastrocnemius (white f i b e r s ) .  I n i t i a l l y during the atrophy process, the  Z-lines l o s t t h e i r s t r a i g h t l i n e configuration across the  fibrils,  becoming bent and "smeared" i n appearance (Pellegrino and F r a n z i n i , 1963; Gauthier and Dunn, 1 9 7 3 ) .  Red  f i b r e s have a wider Z-line than white  f i b r e s (Gauthier and Dunn, 1 9 7 3 ) ; however, 2 weeks a f t e r denervation, the difference i n width of the Z-line between red and white f i b r e s became l e s s apparent (Engel and Stonnington, 1 9 7 4 ) . contradict t h i s .  Gauthier and Dunn (1973)  They maintain that differences i n width remained even  14 days a f t e r denervation.  One might expect t h e i r d i f f e r i n g  to be due to differences i n the length of the d i s t a l stump.  observations That i s , one  might expect Gauthier and Dunn (1973) to have a much longer d i s t a l stump than Pellegrino and Franzini ( 1 9 6 3 ) ; however, t h i s was  not the case.  Gauthier and Dunn (1973) severed the s c i a t i c nerve close to i t s contact with the muscle, whereas Pellegrino and Franzini (1963) severed the s c i a t i c nerve high i n the thigh. r a t s , the differences may  Since both studies used adult albino  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 F r a n z i n i (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 i n between.  One month post-dener-  vation, peripheral filaments of myofibrils were found i n 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 Franzini,  (Pellegrino and  1963).  While f i b e r size and myofibrils decrease i n s i z e , there also occurs an absolute and r e l a t i v e increase i n mitochondrial mass (Engel and Stonnington,  1974),; however, absolute mitochondrial mass decreases a f t e r  a week and becomes proportionate to f i b r e size (Engel and  1974; Pellegrino and F r a n z i n i ,  1963).  Stonnington,  Mitochondria, which normally  are  r e l a t i v e l y uniformly d i s t r i b u t e d , tended to aggregate into small c l u s t e r s , and change from being elongate i n the transverse plane to being v i r t u a l l y p a r a l l e l to the long-axis of the m y o f i b r i l a f t e r (Engel and Stonnington,  denervation  1974).  The trend seen for mitochondrial mass i s the same for sarcotubular element concentration, only the decrease i n sarcotubular elements (sarcoplasmic reticulum and transverse tubules) seen a f t e r the f i r s t week i s less than the decrease seen i n c o n t r a c t i l e elements; therefore, as a net r e s u l t , the concentration of sarcotubular elements increases (Engel and Stonnington,  1974).  Morphologically, the s p a t i a l arrangement of the  sarcotubular elements becomes increasingly i r r e g u l a r , and sometimes the elements possess focal d i l a t i o n s (Engel and Stonnington,  1974).  After  one month of denervation, p a r a l l e l arrays of tubular p r o f i l e s representing p r o l i f e r a t i n g components of the sarcoplasmic reticulum are seen (Engel and Stonnington,  1974).  Morphological changes i n other s k e l e t a l muscle organelles have also been noted.  Lysosomes are present soon a f t e r 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 , 1 9 6 3 ) , and as the r a t e o f degener a t i o n i n the f i b e r s i n c r e a s e s , lysosomes loaded with m a t e r i a l  become l a r g e r and  ( P e l l e g r i n o and F r a n z i n i , 1 9 6 3 ) .  heavily  Lipofuscin  g r a n u l e s and s m a l l a u t o p h a g i c v e s i c l e s were seen one week p o s t - d e n e r v a t i o n by E n g e l and S t o n n i n g t o n ( 1 9 7 4 ) .  Sometimes, c e n t r a l l y -  l o c a t e d n u c l e i a r e found ( E n g e l and S t o n n i n g t o n , 1974; P e l l e g r i n o F r a n z i n i , 1 9 6 3 ) , and prominent  and  G o l g i networks a r e seen w i t h moderate  f r e q u e n c y i n the denervated muscle ( E n g e l and S t o n n i n g t o n , 1 9 7 4 ) . G a u t h i e r and Dunn (1973) d i s c o v e r e d an i n c r e a s e i n s a r c o p l a s m i c ribosomes  subsarcolemmal  i n denervated muscle, and they h y p o t h e s i z e d t h a t  t h i s i n c r e a s e i n p r o t e i n s y n t h e s i s i n g m a t e r i a l c o u l d produce many  new  a c e t y l c h o l i n e r e c e p t o r s t h a t might account f o r the i n c r e a s e 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 o f the s k e l e t a l muscle membrane.  The E f f e c t s o f C a r d i a c Muscle on  Nerves  Thus, d e n e r v a t i o n s t u d i e s have shown t h a t s k e l e t a l muscle i s dependent on i t s i n n e r v a t i o n t o m a i n t a i n i t s i n t e g r i t y . a f f e c t t h e i r nerves.  Experiments  Muscles a l s o appear  to  u s i n g the s u p e r n a t a n t from homogenized,  denervated r a t c a r d i a c muscle showed t h a t the e f f e c t o r muscle t i s s u e a f f e c t the s u r v i v a l o f neurons  (Kanakis e t a l . , 1 9 8 5 ) .  can  A d u l t r a t s were  denervated u s i n g an i n j e c t i o n o f 6-hydroxydopamine (6-OHDA) which produces a c h e m i c a l sypathectomy. t e s t e d t o see how  The denervated c a r d i a c muscle  e f f e c t i v e 6-OHDA was  was  a t d e n e r v a t i n g t h i s muscle.  The  a c t i v i t y o f t y r o s i n e h y d r o x y l a s e , an enzyme i n v o l v e d i n the r a t e - l i m i t i n g s t e p i n the b i o s y n t h e s i s o f n o r a d r e n a l i n e , was  used t o a s s e s s t h i s r  denervation. complete,  Although i t was  i t was  found t h a t d e n e r v a t i o n o f the h e a r t was  indeed e f f e c t i v e s i n c e treatment w i t h 6-OHDA caused a  s i g n i f i c a n t reduction i n tyrosine hydroxylase.  H e a r t s were removed 4  not  9 days a f t e r i n j e c t i o n and then homogenised.  The heart extracts were then  used to assess their a b i l i t y to promote the s u r v i v a l of dissociated 12-day o l d chick lumbar sympathetic ganglia.  Sympathetic neuronal  s u r v i v a l was s i g n i f i c a n t l y 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). very low amounts.  In normal tissue, NGF occurs i n  There was no s i g n i f i c a n t difference i n the effects on  sympathetic neuronal s u r v i v a l between the treated and untreated control extracts.  However, the e f f e c t o f the anti-NGF-treated extract from  denervated hearts on sympathetic neuronal s u r v i v a l was s i g n i f i c a n t l y decreased when compared to the untreated, denervated heart extract.  This  showed that NGF increases i n denervated tissue and that NGF i s an important factor for the s u r v i v a l of sympathetic nerves.  I t was very  i n t e r e s t i n g to note that i n 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 s u r v i v a l better than untreated and anti-NGF-treated control extracts, although the difference was only s i g n i f i c a n t at the 0.05 l e v e l with the treated control extract. demonstrated  This  that components (other than NGF) which have the a b i l i t y to  enhance the s u r v i v a l o f cultured sympathetic neurons may also be increased a f t e r denervation. As shown, many denervation studies on s k e l e t a l muscle have been carried out.  Changes i n the biochemistry r e s u l t i n g from denervation have  been seen as an increase i n the density o f extrajunctional acetylcholine receptors.  Supersensitivity of the muscle membrane and the presence o f  tetrodotoxin-resistant action potentials indicated changes i n the physiology o f the denervated muscle.  Morphological changes include  decreases i n the junctional folds, decrease i n s i z e o f muscle f i b r e , reduction o f myofibrils and changes i n the organelles.  Also, with the  work involving the denervated cardiac muscle extract, we can see that the e f f e c t o r tissue has a trophic influence on the nerve.  The E f f e c t s o f Innervation  on Smooth Muscle  There has been a tremendous amount o f research  carried out on the r o l e  of cholinergic innervation on s k e l e t a l muscle maintenance; an i n t e r e s t has also developed i n the trophic interactions between smooth muscle and i t s adrenergic innervation.  However, since not as much i s known i n t h i s  p a r t i c u l a r area, many preliminary hypotheses o f trophic influences on smooth muscle have been derived by analogy to s k e l e t a l muscle. Nevertheless, s k e l e t a l muscle i s very d i f f e r e n t from smooth muscle, both i n i t s ultrastructure and i t s innervation; thus, the analogies that have been made may not be correct. In v i t r o 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 c e l l s were allowed to s e t t l e on collagen-coated substances.  glass coverslips, and then they were exposed to various  Afer 8 days i n culture, smooth muscle c e l l s i n control  cultures had undergone intense p r o l i f e r a t i o n and u l t r a s t r u c t u r a l l y , they looked dedifferentiated. muscle.  That i s , they resembled embryonic smooth  After 8 days i n culture i n the presence of sympathetic ganglion  extract, dibutyryl c y c l i c AMP or theophylline, smooth muscle p r o l i f e r a t i o n was prevented and the c e l l s were maintained i n t h e i r d i f f e r e n t i a t e d state.  Spinal cord extract, l i v e r extract or noradre-  naline resulted i n smooth muscle c e l l s that were intermediate i n  appearance between those j u s t d e s c r i b e d muscle c e l l s o f the c o n t r o l c u l t u r e s . i n the presence o f d i b u t y r y l c y c l i c AMP muscle r e l a x a n t which i n c r e a s e s  and  the d e d i f f e r e n t i a t e d smooth  However, a f t e r 8 days i n c u l t u r e plus theophylline  i n t r a c e l l u l a r c y c l i c AMP)  c e l l s appeared v e r y w e l l - d i f f e r e n t i a t e d .  smooth muscle  In f a c t , they possessed  o r g a n e l l e s , a c h a r a c t e r i s t i c o f a d u l t in v i v o g u i n e a - p i g Chamley and  (a smooth  vas  few  deferens.  Campbell (1975) suggested t h a t the t r o p h i c substance o r  s u b s t a n c e s may  a c t by s e l e c t i v e l y s t i m u l a t i n g the enzyme a d e n y l  (which c o n v e r t s  ATP  t o c y c l i c AMP),  the a d d i t i o n o f the s u b s t a n c e ,  a suggestion  cyclase  further exemplified  by  theophylline.  Smooth muscles from d i f f e r e n t a r e a s o f the body respond d i f f e r e n t l y s i m i l a r experimental treatments. r a b b i t t h o r a c i c a o r t a and and  For i n s t a n c e ,  smooth muscle from young  ear a r t e r y were c u l t u r e d and  t r e a t e d by Chamley  Campbell (1976) i n s i m i l a r ways as they t r e a t e d the g u i n e a - p i g  d e f e r e n s smooth muscle j u s t d e s c r i b e d .  A f t e r 1-2  days i n c u l t u r e ,  v a s c u l a r smooth muscle c o n s i s t e d o f 2 types o f smooth muscle d i s t i n g u i s h e d on the b a s i s o f morphology. 'differentiated' cell r a b b i t media and  (1)  They i n c l u d e d  o n l y a s m a l l number underwent d i v i s i o n , and type, which underwent f r e q u e n t  a  (2)  an  d i v i s i o n and  f i l l e d w i t h p r o t e i n s y n t h e s i z i n g machinery.  changing t o resemble the  'undifferentiated' c e l l  o f a sympathetic c h a i n homogenate, the  The  the  cells  type.  whose  The  ' d i f f e r e n t i a t e d ' c e l l s m a i n t a i n e d t h e i r morphology f o r o n l y 4 days  cell  vas  type, which resembled the c e l l s o f normal i n v i v o  'undifferentiated' c e l l c y t o p l a s m was  to  In the  before  presence  ' d i f f e r e n t i a t e d ' smooth muscle  type m a i n t a i n e d i t s d i f f e r e n t i a t e d appearance f o r a t l e a s t 6 days. presence o f s p i n a l c o r d e x t r a c t o r n o r a d r e n a l i n e  to the c u l t u r e d m e d i a l smooth muscle c e l l s ;  made no  difference  t h a t i s , these t r e a t e d  c u l t u r e s d i d not d i f f e r from the c o n t r o l c u l t u r e s .  In c o n t r a s t ,  these  12  same substances d i d a f f e c t t h e c u l t u r e d g u i n e a - p i g  vas d e f e r e n s smooth  muscle c e l l s somewhat, p r o d u c i n g a c e l l i n t e r m e d i a t e  i n appearance  between the ' d i f f e r e n t i a t e d ' and t h e ' d e d i f f e r e n t i a t e d ' t y p e s . Smooth muscle from d i f f e r e n t a r e a s o f the body a l s o may r e f l e c t d i f f e r e n c e s by t h e i r a b i l i t y t o a t t r a c t r e g e n e r a t i n g  neurons.  their  I n normal  s k i n , n o n s p e c i f i c c h o l i n e s t e r a s e and a c e t y l c h o l i n e s t e r a s e a c t i v i t i e s a r e observed i n e r e c t o r p i l i muscles and t h e i r n e r v e s ; however, f l u o r e s c e n t varicose adrenergic  nerves were a l s o a l s o seen i n the e r e c t o r  muscles (Waris, 1 9 7 8 ) .  I n r a t s k i n autographs, f l u o r e s c e n c e  pili microscopic  t e c h n i q u e s s p e c i f i c f o r c a t e c h o l a m i n e s showed t h a t the e r e c t o r muscles were n o t r e - i n n e r v a t e d  a t t h e end o f a 20 week p e r i o d , b u t b l o o d  v e s s e l s i n t h i s g r a f t were p a r t i a l l y i n n e r v a t e d t r a n s p l a n t a t i o n (Waris, 1 9 7 8 ) .  a t 16 and 20 weeks a f t e r  Hence, t r o p h i c i n t e r a c t i o n s between one  type o f smooth muscle and i t s sympathetic i n n e r v a t i o n s h o u l d with other  pili  be compared  s t u d i e s t h a t have used the same type o f smooth muscle, and, i n  s t u d i e s o f the c i r c u l a t o r y system, from t h e same p a r t o f the v a s c u l a r tree. That v e s s e l s from d i f f e r e n t p a r t s o f the c i r c u l a t o r y system may r e f l e c t differences 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 t r a n s p l a n t a t i o n experiments and t i s s u e c u l t u r e s o f smooth muscle.  Transplantation  o f the r a t f e m o r a l a r t e r y i n t o the a n t e r i o r  chamber o f an eye o f a h o s t r a t d i d n o t induce i r i d e a l nerve whereas t h e t a i l a r t e r y d i d (Todd, 1 9 8 6 ) .  sprouting  A l s o , smooth muscle c u l t u r e s  c o n t a i n i n g m e d i a l c e l l s from r a b b i t t h o r a c i c a o r t a o r e a r a r t e r y i l l u s t r a t e d a d i f f e r e n c e i n t h e i r i n t e r a c t i o n w i t h r a b b i t sympathic ganglion  explants  from the e x p l a n t s  (Chamley and Campbell, 1 9 7 6 ) .  Nerve f i b e r s growing  formed l o n g e r - l a s t i n g a s s o c i a t i o n s  (up t o 8 days) w i t h  the smooth muscle c e l l s from the e a r a r t e r y than they d i d w i t h c e l l s  from  13  the  thoracic aorta  vascular  t r e e may  (1-2 not  hours).  always be e x t r a p o l a t e d  another p a r t o f the v a s c u l a r Most o f the  a r t e r y by  area of  t r e e , even w i t h i n  been done on  In one  the same a n i m a l .  Two  vascular her  (1975) denervated the l e f t r a b b i t ear  c o m p l e t e l y removing the l e f t s u p e r i o r  week o l d r a b b i t s .  on  the r a b b i t ear a r t e r y by Bevan and  s t u d y , Bevan  the  t o p r e d i c t the r e s u l t s i n  i n v i v o work i n v o l v i n g n e u r o t r o p h i c i n f l u e n c e s  smooth muscle has co-workers.  T h e r e f o r e , r e s u l t s from one  weeks p o s t o p e r a t i v e l y ,  c e r v i c a l ganglion i n 4  a decrease i n the uptake o f  3  H-Tdr by  the v a s c u l a r  a r t e r y was  smooth muscle c e l l s i n the  measured by s c i n t i l l a t i o n c o u n t i n g and  denervated l e f t by  autoradiography.  Denervated smooth muscle a l s o appeared to have fewer m i t o t i c These r e s u l t s seem t o i n d i c a t e t h a t d e n e r v a t i o n i n h i b i t e d p r o l i f e r a t i o n o f the m e d i a l smooth muscle Bevan and  Tsuru  figures.  the  cells.  (1979) l a t e r p o s t u l a t e d  that since p r o l i f e r a t i v e  growth decreased upon d e n e r v a t i o n , t h i s might c r e a t e  a smaller  blood  v e s s e l which would be i n c a p a b l e o f p r o d u c i n g the maximum f o r c e Rabbits of 9 - H  contraction.  ganglionectomy. noticeable  The  decreased and  maximum f o r c e and  i t was  postulated  smooth muscle mass and  cervical  i n the weight o f the  t h a t the r e s u l t s were due  An  T h i s may  increased  to a l o s s  of  r e f l e c t changes i n the e l a s t i c  smooth muscle c e l l s which, i n t u r n , a f f e c t e d vessel.  also a  e l a s t i c modulus ( s t i f f e r  D e n e r v a t i o n 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  mechanics o f the b l o o d  rabbit  the maximum t e n s i o n were markedly  S u p e r s e n s i t i v i t y t o n o r e p i n e p h r i n e was  consequence o f d e n e r v a t i o n . as w e l l .  and  a  t o 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 .  evident  underwent s u p e r i o r  of  E i g h t weeks a f t e r the ganglionectomy, t h e r e was  decrease i n the w a l l t h i c k n e s s  ear a r t e r y .  was  weeks o f age  ear  tissue.  s e n s i t i v i t y of  the a r c h i t e c t u r e  wall)  and  the  Bevan and Tsuru (1981) repeated the above procedure on three d i f f e r e n t age groups to see the e f f e c t i t may have on developing arteries.  The three groups were (1) a growing group (3-4  weeks o l d ) , (2)  a young adult group (9-11 weeks old) and (3) a mature group (16-20 weeks).  When compared with their controls, s i g n i f i c a n t decreases i n  cross sectional area of the media were seen i n the f i r s t two age groups.  In the t h i r d group (mature animals), there was  denervated no  s i g n i f i c a n t decrease i n the cross sectional area of the media a f t e r 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 u n i l a t e r a l superior c e r v i c a l ganglionectomy on rabbits to evaluate the possible chronic influence that the sympathetic nerves might have on vascular resistance and to see i f the r e s u l t s 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 l a t e r ) , (2) growing chronic denervation (rabbits denervated at 4 weeks and studied 9 weeks l a t e r ) and (3) adult chronic (rabbits that were denervated at 16 weeks and studied 10 weeks l a t e r ) .  Changes i n vascular  resistance were seen as changes i n flow-perfusion pressure curves. Perfusion pressure was measured i n maximally d i l a t e d vessels and t h i s pressure measures the resistance of the vessels.  Results from the  vascular bed i n denervated ears and the c o n t r a l a t e r a l innervated ears were compared.  A comparison between the denervated and innervated ears  i n the growing acute denervated group ( i e . those studied only 9 days a f t e r denervation) showed no difference i n the perfusion pressure. perfusion pressure of the denervated ear of the growing chronic  The  denervated group ( i e . those studied 9 weeks a f t e r denervation) was s i g n i f i c a n t l y lower than that from i t s c o n t r a l a t e r a l control side. Therefore,  i t seems that 9 days was an i n s u f f i c i e n t length of time a f t e r  denervation  for any noticeable changes to occur.  No differences i n the  perfusion pressure curves were seen between the denervated ear and  the  c o n t r a l a t e r a l innervated ear from the adult chronic denervated group. Thus, denervation  does produce a decrease i n vascular resistance i n  developing vessels but not i n mature vessels and t h i s decrease i s observed only a f t e r a substantial length of time following the ganglionectomy.  Rusterholz and Mueller  (1982) believe their r e s u l t s are  compatible with the idea of the existence of an i n t e r a c t i o n between sympathetic nerves and blood vessels.  Although the mechanism of this  i n t e r a c t i o n i s unknown, they proposed that the decrease i n vessel resistance was  probably not exclusively the r e s u l t of smooth muscle  atrophy but also may  have involved other factors such as an a l t e r a t i o n i n  elastin/collagen r a t i o or an i n a l t e r a t i o n smooth muscle configuration. Bevan's report (1983) of an increase i n arteriovenous denervated rabbit ear artery may decrease i n perfusion Morphological  anastomoses i n the  also be responsible for the observed  pressure.  studies on denervated blood vessels have been c a r r i e d  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.  U l t r a s t r u c t u r a l l y , the smooth muscle c e l l s of the  denervated saphenous vein had the appearance of dedifferentiated c e l l s containing a l l the organelles c h a r a c t e r i s t i c of active protein synthesis.  Their findings were s i m i l a r i n the denervated saphenous  artery except the a l t e r a t i o n s i n the artery were r e s t r i c t e d to the smooth muscle layers closest to the a d v e n t i t i a .  2-3  In both the artery and  v e i n , t h e a l t e r a t i o n s were r e v e r s i b l e . Therefore,  denervation  s t u d i e s on smooth muscle, p a r t i c u l a r l y  smooth muscle, a r e n o t as e x t e n s i v e  as i n s k e l e t a l muscle.  vascular  Nevertheless,  changes i n v a s c u l a r smooth muscle, as a consequence o f d e n e r v a t i o n do occur.  These changes i n c l u d e i n c r e a s e d s u p e r s e n s t i v i t y t o  norepinephrine,  decreases i n maximum f o r c e and maximum t e n s i o n , 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 t o a d e d 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 o f Sympathetic D e n e r v a t i o n To study t h e d e n e r v a t i o n T s u r u (1975; 1979;  e f f e c t s on t h e r a b b i t e a r a r t e r y , Bevan and  1 9 8 1 ) , Branco e t a l . (1984) and R u s t e r h o l z  and M u e l l e r  (1982) used t h e method o f u n i l a t e r a l sympathetic ganglionectomy.  In the  case o f s t u d y i n g t h e r a b b i t e a r a r t e r y , t h i s i n v o l v e s t h e removal o f t h e entire superior c e r v i c a l ganglion.  Consequently, i n n e r v a t i o n t o one h a l f  o f t h e head i s l o s t . Chemical sympathectomy i n v o l v e d t h e use o f 6-hydroxydopamine (6-OHDA) as t h e d e n e r v a t i n g  factor (Finch e t a l . , 1 9 7 3 ) .  F i n c h e t a l . (1973) noted  t h a t a d m i n i s t r a t i o n o f 6-OHDA i n t o a d u l t r a t s produced a s e l e c t i v e , temporary d e s t r u c t i o n o f a d r e n e r g i c adrenergic  nerve t e r m i n a l s .  Regeneration o f t h e  nerve t e r m i n a l s i n blood v e s s e l s was v e r y f a s t and w i t h i n  just  a few days a f t e r i n j e c t i o n o f 6-OHDA, and almost complete f u n c t i o n a l recovery  was seen.  F i n c h e t a l . (1973) were t h e r e f o r e i n t e r e s t e d i n  s e e i n g 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 d e s t r u c t i o n o f t h e a d r e n e r g i c system.  nerves s u p p l y i n g  the v a s c u l a r  I n t h e i r study they compared t h e e f f e c t s o f a d m i n i s t e r i n g 6-OHDA  i n t o two d i f f e r e n t age groups o f r a t s : one group was t r e a t e d f o r t h e 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 l o o k e d  a t 8 weeks l a t e r and t h e  other group, the adult group, was given two i n j e c t i o n s , 7 days apart and looked on the next day a f t e r the l a s t i n j e c t i o n . To determine the effectiveness o f denervation produced by 6-OHDA, stimulation o f the entire sympathetic outflow was carried out on pithed r a t s which were previously adrenalectomized.  The s t e e l pithing rod was used as an  electrode to stimulate s p i n a l nerve roots.  Sympathetic outflow was  stimulated with supramaximal voltage and increasing frequencies.  A rise  i n blood pressure with increasing stimulation frequency was seen i n the control and newborn-treated animals although the newborn-treated pithed group was markedly lower (37 mmHg) than the controls (120 mmHg).  No r i s e  i n blood pressure was seen i n the adult-treated pithed group a t any stimulation frequency.  Since a small r i s e i n blood pressure was observed  i n t h i s l a t t e r age group i n unpithed preparations,  i t was possible that  i n the pithed r a t , the s t e e l rod used for stimulation did not excite a l l sympathetic nerves (Finch et a l . , 1973).  They also found a depletion i n  norepinephrine l e v e l s i n the newborn treated group, however, the percent depletion varied with the tissue type (eg. mesentery vascular bed had 50-60% o f the norepinephrine content l e f t whereas norepinephrine i n cardiac muscle was depleted  to l e s s than 5% o f control l e v e l s ) .  Since  the l e v e l s o f norepinephrine did not increase up to an age o f 16 weeks, Finch et a l . (1973) considered  the denervation to be permanent as a  consequence o f the destruction o f c e l l bodies i n the sympathetic ganglia by 6-OHDA. Using anaesthetized  r a t s , Finch et a l . (1973) compared the extent o f  vasoconstriction o f i s o l a t e d renal artery preparations groups.  between the two  P e r i a r t e r i a l nerve stimulation was carried out to observe the  vasoconstrictor responses.  Vasoconstrictor responses o f the r a t s treated  at b i r t h did not d i f f e r from those o f the controls.  The adult-treated  18  r a t s showed reduced (1973)  v a s o c o n s t r i c t o r responses.  Even though F i n c h e t a l .  showed i n t h e i r study t h a t c h e m i c a l sympathectomy i s a s u c c e s s f u l  method o f d e n e r v a t i n g c e r t a i n organs such as t h e h e a r t , i t d i d n o t prove t o be v e r y p r o m i s i n g a s a means o f a c h i e v i n g v a s c u l a r d e n e r v a t i o n i n younger r a t s .  I n newborn-treated r a t s , t h i s method produced a permanent  but incomplete  d e n e r v a t i o n whereas i n t h e a d u l t i t produced a v i r t u a l l y  complete b u t non-permanent d e n e r v a t i o n . Immunological sympathectomy i s an a l t e r n a t i v e method o f d e n e r v a t i o n . H i s t o l o g y , response  t o e l e c t r i c a l s t i m u l a t i o n o f lumbar  ganglia, increased  s e n s i t i v i t y t o n o r e p i n e p h r i n e and r e s u l t s o f c h e m i c a l  s t i m u l a t i o n o f t h e sympathetic  g a n g l i a with  sympathetic  1,l-dimethyl-4-phenyl-pipe-  r a z i n i u m i o d i d e (DMPP) a r e methods t h a t have been used t o determine t h e success o f denervation 1966).  DMPP i s a white  (Brody,1964;  L e v i - M o n t a l c i n i and A n g e l e t t i ,  c r y s t a l l i n e substance  t h a t i s s o l u b l e i n water  and i s n o t i n t e n d e d f o r t h e r a p e u t i c use (Chen e t a l . , 1 9 5 1 ) . i n j e c t e d i n t r a v e n o u s l y i n t o animals,  When  i t s t i m u l a t e s t h e sympathetic  g a n g l i a by a c t i n g a t t h e n i c o t i n i c - l i k e r e c e p t o r s o f t h e p o s t g a n g l i o n i c s y n a p t i c membrane (Szekere, 1 9 8 0 ) .  T h i s e x c i t e s t h e p o s t - s y n a p t i c neuron  thereby c a u s i n g an i n c r e a s e i n a r t e r i a l b l o o d p r e s s u r e and t a c h y c a r d i a . Immunological d e n e r v a t i o n has been a c h i e v e d by i n j e c t i n g a n t i s e r u m t o Nerve Growth F a c t o r i n t o r a t s and mice immediately I n i t i a t i n g t h e i n j e c t i o n s immediately the most e x t e n s i v e d e n e r v a t i o n Denervation  after  birth.  a f t e r b i r t h i s e s s e n t i a l t o produce  ( L e v i - M o n t a l c i n i and A n g e l e t t i ,  1966).  has been proven t o be permanent by c a r r y i n g o u t h i s t o l o g i c a l  s t u d i e s on mice two y e a r s a f t e r they were t r e a t e d a t b i r t h w i t h t h e antiserum  ( L e v i - M o n t a l c i n i and A n g e l e t t i , 1 9 6 6 ) .  T h i s method o f  d e n e r v a t i o n was shown t o s u c c e s s f u l l y a b o l i s h vasomotor f u n c t i o n s i n c e n e i t h e r v a s o c o n s t r i c t o r nor v a s o d i l a t i o n o c c u r r e d when t h e lumbar c h a i n s  of the immunized r a t were e l e c t r i c a l l y stimulated  (Brody, 1964).  Levi-Montalcini and Angeletti (1966) found that the average r e s t i n g blood pressure i n treated r a t s was only 70 mmHg compared to lOOmmHg i n controls.  Blood pressure seems to be proportional to the degree o f  sympathetic innervation.  Another change found by Levi-Montalcini and  Angeletti (1966) with this type of denervation  was a severe decrease (but  not t o t a l a b o l i t i o n ) i n the c e l l population of various ganglia - superior c e r v i c a l ( r a t s : r e s i d u a l c e l l population was 10-15$ of c o n t r o l s ) , c e l i a c , s t e l l a t e and thoracic chain ganglia.  However, their r e s u l t s from  pharmacological and physiological t e s t i n g ( i e . r e a c t i v i t y o f the vascular system to chemical or e l e c t r i c a l stimulation) suggest that the r e s i d u a l c e l l populations have very l i t t l e , i f any,  functional a c t i v i t y .  The view that blood pressure values are proportional to the degree o f sympathetic innervation was also supported by Gerova et a l . (1974) who found that the maximum diameter reduction (compared to r e s t i n g diameter as a percent) of the densely innervated  femoral a r t e r i e s i n puppies, was  much higher than than that found i n adult dogs, whose innervation was l e s s dense r e l a t i v e l y . In their examination of the responses o f an i s o l a t e d vascular bed to sympathetic neurotransmitters  and sympathetic nerve stimulation i n order  to determine the functions o f vascular smooth muscle and i t s innervation i n 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 i n the hind limbs of dogs which was induced by nerve stimulation increased with age.  They attributed t h i s  r i s e to the increase i n functional maturity o f the adrenergic vasomotor function.  I t was also noted that this onset and development o f induced  vasoconstriction coincided i n age with the onset and development of the  systemic blood pressure.  This age-related increase i n blood pressure  was  a l s o demonstrated on r a t s by L a i s e t a l . (1977) as w e l l as by G e r r i t y  and  Cliff  (1975).  These o b s e r v a t i o n s on b l o o d p r e s s u r e r e l a t e d i r e c t l y to  the i n c r e a s e i n catecholamine dense-cored  v e s i c l e s and  f l u o r e s c e n c e , t o the presence  of  to the number o f nerve p r o c e s s e s i n r a t a r t e r i e s  as they matured (Todd, 1980; Todd and T o k i t o , 1 9 8 1 ) .  Problems w i t h D e n e r v a t i o n  Methods  From these s t u d i e s , i t i s e v i d e n t t h a t sympathetic a f f e c t v a s c u l a r smooth muscle both s t r u c t u r a l l y and thereby may The  a l t e r the s t r u c t u r e and  i n n e r v a t i o n can  functionally,  f u n c t i o n o f the e n t i r e b l o o d  and vessel.  i i i v i v o methods o f d e n e r v a t i o n used so f a r have i n c l u d e d s u r g i c a l  ganglionectomy (Bevan and Tsuru, 1979; Bevan and Tsuru, 1981; Branco e t a l . , 1984),  chemical  ( F i n c h e t a l . , 1973)  ( L e v i - M o n t a l c i n i and A n g e l e t t i , 1966)  and  immunological  sympathectomies.  S u r g i c a l sympathectomy i n v o l v e s the removal o f an e n t i r e g a n g l i o n such as the s u p e r i o r c e r v i c a l g a n g l i o n .  This  sympathetic  procedure  e l i m i n a t e s the i n n e r v a t i o n t o one h a l f o f the head. Chemical  sympathectomy i n v o l v e s u s i n g 6-hydroxydopamine (6-OHDA) which  d e s t r o y s a d r e n e r g i c nerve t e r m i n a l s . permanent i f a d m i n i s t e r e d  This destruction i s s e l e c t i v e ,  but  to newborns, and complete, but non-permanent i f  given to a d u l t s (Finch et a l . , 1 9 7 3 ) .  Kanakis  e t a l . (1985) found t h a t i n  a d u l t r a t s i t produced an e f f e c t i v e but incomplete  d e n e r v a t i o n o f the  heart. To produce an immunosympathectomy, i n j e c t i o n s o f a n t i s e r u m Growth F a c t o r a r e g i v e n i n t r a v e n o u s l y . it  I f g i v e n immediately  t o Nerve after  birth,  produces the most e x t e n s i v e and permanent d e n e r v a t i o n ( L e v i - M o n t a l c i n i  and A n g e l e t t i , 1 9 6 6 ) .  Thus, o n l y when these t h r e e denervaton  procedures  are carried out on very young animals, are they the most permanent and do they produce the most noticeable changes i n blood vessels (Levi-Montalcini and Angeletti, 1966; Tsuru, 1979;  Finch et a l . , 1973;  Bevan and  1981).  These methods of denervation eliminate innervation from a large area i n the body of the animal, that i s , the denervation i s not l o c a l i z e d . These widespread denervations may  produce unwanted, possibly toxic,  e f f e c t s which may have contributed to the r e s u l t s obtained by these past investigators. To avoid these aberrant e f f e c t s , a l o c a l i z e d denervation method i s necessary.  This was attempted by Todd (1986) on r a t thigh blood vessels,  but was unsuccessful as a r e s u l t of rapid regeneration.  Severing,  removing or repositioning the femoral nerve are not successful methods since re-innervation of the saphenous and s u p e r f i c i a l and e p i g a s t r i c a r t e r i e s occurs i n under 15 days (Todd, 1986).  Rusterholz and Mueller  (1982) found that denervation of young rabbit ear a r t e r i e s produced a decrease i n 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 s u f f i c i e n t post-operative length of time must past before changes are seen i n 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 e f f e c t s on blood vessels under the most normal conditions possible, meaning, i n vivo, leaving a l l of the nerves i n t a c t except for those innervating the vessels under i n v e s t i g a t i o n , and without having any side e f f e c t s produced by  c i r c u l a t i n g drugs o r a n t i b o d i e s .  Therefore,  i t was  n e c e s s a r y to d e v e l o p  a t e c h n i q u e t h a t would keep the blood v e s s e l s denervated f o r as l o n g possible.  Second, I wanted t o t e s t  t h e i r adrenergic other.  i n n e r v a t i o n do e x e r t  To a c h i e v e my  denervating  the h y p o t h e s i s t h a t b l o o d v e s s e l s  and  and  t r o p h i c i n f l u e n c e s over each  o b j e c t i v e s , I developed a n o v e l method o f  blood v e s s e l s i n the r a t t h i g h and  wall thicknesses  as  luminal  perimeters.  measured changes i n v e s s e l  MATERIALS AND METHODS  Animals Female W i s t a r r a t s from an i n b r e d c o l o n y maintained o f Anatomy were denervated a t 3 o r 12 days o f age. P u r i n a Lab Chow and water ad l i b i t u m .  i n the Department  The mothers were f e d  The pups were weaned a t one month  o f age a t which time they were f e d the same d i e t as t h e i r mothers. were housed i n p a i r s i n hanging cages i n a c o n t r o l l e d environment a 12 hour dark, 12 hour l i g h t c y c l e . sampled when the pups were 3 0 , a n i m a l s was used  They having  The d e n e r v a t e d b l o o d v e s s e l s were  60, 90 o r 120 days o f age. A t o t a l o f 18  f o r the l i g h t and e l e c t r o n m i c r o s c o p i c a n a l y s i s and 8  a n i m a l s were sampled f o r f l u o r e s c e n c e microscopy  Denervation  Procedure  The a n i m a l s were a n a e s t h e t i z e d i n a g l a s s d e s i c c a t o r i n which absorbent c o t t o n was moistened Ltd.).  w i t h anhydrous e t h e r ( F i s h e r  Scientific  A n a e s t h e s i a was m a i n t a i n e d d u r i n g the d e n e r v a t i o n procedure by  u s i n g a nose cap c o n t a i n i n g absorbent c o t t o n moistened  with ether.  a n a e s t h e t i z e d animals were p l a c e d on a s m a l l p l e x i g l a s s t a b l e , s i d e down.  The  dorsal  T h e i r f o r e - and h i n d l i m b s were l o 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 w i t h Savlon ( A y e r s t Laboratories). A s m a l l v e r t i c a l s k i n i n c i s i o n was made i n the r i g h t t h i g h and the r i g h t f e m o r a l , 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 f e m o r a l nerve  exposed ( F i g u r e l a ) . The femoral nerve was t r a c e d back t o the  i n g u i n a l ligament.  The f e m o r a l nerve was m o b i l i z e d and  separated  from  24 the femoral a r t e r y and s k e l e t a l muscle a d j a c e n t t o the ligament.  A length of black suture s i l k  the f e m o r a l nerve  i n two  Deknatel) was  t i e d around  p l a c e s , as c l o s e t o the i n g u i n a l ligament  p o s s i b l e , and then a g a i n more d i s t a l l y was  (8.0,  inguinal  (Figure l b ) .  s e v e r e d j u s t d i s t a l to the second knot  femoral nerve d i s t a l t o the c u t was  The  femoral  ( F i g u r e 2a).  The  as  nerve  portion of  s e p a r a t e d from the femoral a r t e r y  s e v e r e d where the saphenous a r t e r y branches  and  o f f the femoral a r t e r y  ( F i g u r e 2a).  T h e r e f o r e , the l e n g t h o f femoral nerve from the second  t o the branch  p o i n t was  knot  removed and d i s c a r d e d .  I n s i d e the abdominal c a v i t y , the femoral nerve runs c r a n i a l l y i n the p o s t e r i o r w a l l o f the abdominal c a v i t y . paramedial  i n c i s i o n was  Therefore, a tiny,  made i n the abdominal w a l l and the femoral  i n s i d e the abdominal c a v i t y was  found.  By g e n t l y p u l l i n g on the  h e r e , the p o r t i o n o f the femoral nerve t h a t was brought  i n s i d e the abdominal c a v i t y .  around the nerve was  threaded through  melted  the d i s t a l end was  nerve  out i n the t h i g h  was  a l e n g t h ( a p p r o x i m a t e l y 1 cm) The  tube was  melted u s i n g a c a u t e r y gun  s l i d over  abdominal w a l l was s k i n i n c i s i o n was  tucked back i n s i d e the abdominal c a v i t y and s u t u r e d c l o s e d u s i n g 6.0 clamped with 7.5  o f blood u s i n g Savlon.  mm  The the  (Medicon) and  A f t e r r e g a i n i n g conciousness  from  The  silk  Deknatel s u t u r e s i l k .  wound c l i p s  of  the  p l a s t i c s e a l e d the tube and a l s o s e c u r e d the b l a c k s u t u r e  nerve was  tied  ( F i g u r e 2b).  when i t hardened, thereby s e c u r i n g the nerve i n s i d e the tube. encased  nerve  The b l a c k s u t u r e s i l k t h a t was  p o l y e t h y l e n e t u b i n g (PE-10, C l a y Adams). nerve and  ventral,  The  cleaned  the  a n a e s t h e t i c , the pups were s p r i n k l e d l i g h t l y w i t h baby powder and r e t u r n e d to t h e i r mother.  V i c k ' s Vapo rub was  n o s t r i l s when the pups were r e t u r n e d to them. and  i n s e r t e d i n t o the mothers' Both, the V i c k ' s Vapo rub  the baby powder were used t o prevent c a n n i b a l i s m 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 c u f f pressure recordings.  Following t h i s , the animals were anaesthetized  i n 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. i n c i s i o n was made i n the ventral neck region.  A midline  The l e f t common carotid  artery was found and cleared o f i t s enveloping connective t i s s u e .  The  vagus nerve was then c a r e f u l l y separated from the common carotid artery. The artery was l i g a t e d i n two places using 4.0 Deknatel suture s i l k .  The  f i r s t l i g a t u r e was t i e d t i g h t l y and as c r a n i a l l y as possible, and a second loose knot was made with this same l i g a t u r e (Figure 3). The second l i g a t u r e was t i e d approximately 1 cm caudal to the f i r s t  . The  l a t t e r knot was not tightened u n t i l the cannula was inserted, serving to hold i t i n place.  A small a r t e r i a l haemostat was clamped  immediately  proximal to the position of the second l i g a t u r e . The cannula was a length of PE-50 polyethylene tubing (Clay Adams) bevelled at both ends,  filled  with a 1% heparin solution i n saline and clamped at one end with a pair of hemostats.  A tiny i n c i s i o n was made i n the ventral wall of the artery  and the cannula was inserted through the loose knot of the f i r s t and then into the lumen o f the artery.  ligature  Once inside the lumen of the  artery, the cannula was gently pushed caudally past the region of the second l i g a t u r e .  At t h i s point, the second l i g a t u r e and the second knot  of the f i r s t l i g a t u r e were securely tightened, and the small a r t 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 i n j e c t i n g 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.) f o r blood pressure recordings, and was c a l i b r a t e d using a mercury manometer. The s y s t o l i c , d i a s t o l i c and mean blood pressures were recorded v i a the i n t r a - a r t e r i a l cannula (Figure 4) and then the perfusion was started.  A 3% glutaraldehyde/2% paraformaldehyde solution (pH 7.3) made  up i n glucose-Krebs solution (Palaty, 1971) was perfused at the animal's mean blood pressure which was maintained with the continuously variable perfusion pump. cut  Once the perfusion was started, the t i p o f the t a i l was  o f f and the skin on the ventral abdomen was cut and r e f l e c t e d back to  expose the thigh vessels. veins were cut.  The r i g h t and l e f t s u p e r f i c i a l epigastric  Clear f i x a t i v e was seen passing out of these veins and  the t a i l shortly a f t e r perfusion started.  Over a period of approximately  20 minutes, a t o t a l of 30-50 ml o f f i x a t i v e was perfused through each animal.  A continuous recording was made o f the perfusion pressure  (Figure 4 ) . In a l l animals, the r i g h t and l e f t saphenous a r t e r i e s were removed. In some animals, samples of the r i g h t and l e f t saphenous and femoral nerves and veins were taken.  Therefore, 18 samples o f the r i g h t and l e f t  saphenous a r t e r i e s and 6 samples o f r i g h t and l e f t femoral veins were removed.  Also, femoral nerve samples were taken from inside the  abdominal c a v i t y .  For the r i g h t femoral nerve, they were taken adjacent  to the proximal end o f 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  i n the 3% glutaraldehyde/2% paraformaldehyde solution for a t o t a l of 1.5hr  at room temperature (including perfusion time), followed by 0.5hr  at 0°C.  Since higher temperatures of t h i s f i x a t i v e promote the rate of  penetration and maintain l a b i l e structures such as microtubules, and since a r t i f a c t s due to polymerization of glutaraldehyde are minimized at lower temperatures,  t h i s sequence of aldehyde f i x a t i o n acts as a  compromise of the effects that occur at higher and lower  temperatures.  This was followed by two-5 minute washes i n glucose-Krebs  solution at  0°C and two-5 minute washes i n 0.1M cacodylate buffer at 0°C. After washing, the tissues were fixed i n 1% osmium tetroxide i n 0.1M cacodylate buffer for 1.5 hr at 0°C.  Following t h i s , were four-5 minute washes i n  0.1M cacodylate buffer at 0°C and one-5 minute wash i n d i s t i l l e d water at 0°C i n preparation for en bloc staining i n saturated aqueous uranyl acetate (1 hr at 0°C).  Before dehydration, the tissues were washed o  again i n 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. Infiltration:  I n f i l t r a t i o n of the tissues using increasing r a t i o s of  Mollenhauer's (1961) embedding mixture  (25 Epon 812 : 15 Araldite 502,  Electron Microscopy Sciences): acetone solutions was done at room temperature.  Tissues remained i n the solutions of the r a t i o s 1:3 and 1:2  for 30 min..  They were transferred to a 1:1 solution and the v i a l s were  put on an e l e c t r i c rotator (Labtronix Equipment) overnight.  The next  day, tissues were exposed to solutions of 2:1 followed by 3:1 r a t i o s , 60 minutes i n each.  The tissues were then transferred to clean v i a l s  containing pure Epon-Araldite embedding mixture and placed i n a vacuum  r for approximately 1 hr.  Following t h i s , they were placed i n pure, fresh  embedding mixture again and put on the e l e c t r i c rotator overnight. In the morning, the embedding mixture was exchanged for fresh, and the open v i a l s were placed i n a vacuum oven u n t i l the length of time the tissue spent i n pure Epon-Araldite embedding mixture t o t a l l e d 24 hours. The tissues were embedded i n d i v i d u a l l y i n freshly made, pure Mollenhauer•s embedding mixture.  The vessels were placed i n rubber molds  and oriented so that they would be at r i g h t angles to the plane o f sectioning.  The r e s i n was polymerized a t 60°C for 48 hours and the  blocks were further cured for 2 or more days a t room temperature. Sectioning The vessels were aligned perpendicular to the knife edge so that complete transverse sections could be cut for l i g h t microscopy.  Thick  sections (0.5)im) for l i g h t microscopy were cut on a Reichert 0mU3 ultramicrotome with a glass k n i f e .  The sections were placed on a glass  s l i d e and heat fixed at a high temperature on a Sybron Thermolyne hot plate.  They were stained with a 1:1 mixture o f 1% azur I I and 1%  t o l u i d i n e blue made up i n 1% borax (Pease, D. C , 1964; Humphrey and Prittman, 1974).  The s t a i n was f i l t e r e d each day, just prior to use.  Enough s t a i n to cover the sections was placed on each s l i d e with a dropper and l e f t to dry on the hot plate (approximately 85°C). The s t a i n was rinsed o f f the s l i d e s with d i s t i l l e d water and s l i d e s were returned to the hot plate t o dry.  The s l i d e s were coverslipped using  Histoclad (Clay Adams) as a mounting medium.  Thick sections o f the blood  vessel and nerve tissue samples were cut from the 60 and 90 day o l d age groups only. Thin sections o f 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 k n i f e .  Sections were collected on 200  and  300 mesh, rhubidium coated copper grids and stained for 15 minutes i n 1% aqueous uranyl acetate (the rhubidium  coat on the grids lessens the  e f f e c t of surface tension at the time the g r i d i s breaking through the water's surface as the sections are being c o l l e c t e d ) .  After a thorough  washing i n d i s t i l l e d water, sections were stained with lead c i t r a t e for 15 minutes and then washed again. Fluorescence  Microscopy  A quick and consistent method for fluorescence of monoaminergic neurons and their axonal v a r i c o s i t i e s 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 i n the denervated and veins. days.  Animals for fluorescence were terminated at 30, 60, 90 or 120  Animals were anaesthetized using sodium pentobarbitone  made up i n 0.9% saline s o l u t i o n . weight.  arteries  (0.06g/ml)  The dosage given was lml/kg body  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 c i r c u l a t e for one hour. Trypan blue makes e l a s t i c tissue of the artery fluoresce red.  This helps  to d i f f e r e n t i a t e the e l a s t i c tissue from the f l u o r e s c i n g norepinephrine-containing nerve v a r i c o s i t i e s (Mclnnes, 1977). Without trypan blue, e l a s t i c tissue and adrenergic nerve v a r i c o s i t i e s 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 e f f e c t i v e . After one hour, the r i g h t and l e f t saphenous and femoral a r t e r i e s and  saphenous and.femoral veins were excised.  Each vessel was  immediately  embedded with i n 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 a t -30°C and a t a thickness of l8iim. Sections were picked up with a glass coverslip and exposed f o r 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 u n t i l the  sucrose-potassium phosphate-glyoxylic acid solution had completely dried (30-45 min.).  A drop o f non-drying Type A immersion o i l (R.P. C a r g i l l e  Laboraties, Inc.) was placed on each c o v e r s l i p and the coverslips were put into a 90°C oven f o r 3.5 min..  The coverslips were mounted onto  cleaned glass s l i d e s and the edges were sealed with melted dental wax. In almost a l l cases, there were four s l i d e s per v e s s e l .  For each vessel,  the s l i d e s were numbered from 1 to 4, representing the order of the f i r s t to the l a s t sections cut. ANALYSIS Light Microscopy and Morphometries Light microscopic photographs were taken of a l l the r i g h t and l e f t saphenous a r t e r i e s i n the 60 and 90 day age groups on a Leitz-Wetzlar Orthoplan l i g h t microscope a t 10X (on 35mm f i l m ) .  The r i g h t and l e f t  femoral veins that were sampled i n these age groups were photographed a t 6.3X. Montages o f the veins were assembled.  Three complete transverse  sections from each a r t 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 f i l m .  The f i l m was developed using Kodak HC110 developer  i n a d i l u t i o n of 1:15 for 7 minutes.  The developer was discarded and the  f i l m was washed i n d i s t i l l e d water containing a few drops of Kodak Stop Bath f o r 1 minute.  The f i l m was fixed f o r 5 minutes.  Prints were made  on Agfa-Gevaert Rapitone paper, using either grades 3 or 4 a t f i n a l  magnifications o f 145-320X for the veins and 220-570X for the a r t e r i e s . The prints were developed using a Rapidoprint e l e c t r i c p r i n t processor using the Agfa-Gevaert Rapidoprint a c t i v a t o r and f i x e r . From each photograph, the e n t i r e cross-sectional area and the luminal area o f the a r t e r i e s were traced on an Apple I I d i g i t i z i n g Tablet. The morphometric programme permitted the luminal area to be subtracted from the e n t i r e cross-sectional area to calculate the area o f the tunica media (the area containing the smooth muscles c e l l s ) .  Therefore, from the  photographs o f the denervated and control a r t e r i e s , the medial area was calculated.  The montages o f the veins required t r a c i n g on a larger Talos  CYBERGRAPH d i g i t i z i n g 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 l i m i t s o f the tunica media. In order to measure wall thickness o f the veins, four points from one of the montages o f each vein were randomly chosen.  These points were  photographed on a Leiz-Wetzlar Orthoplan l i g h t microscope at 40X using 35mm Kodak Technical Pan black and white 2415 f i l m . were developed as described above.  The f i l m and p r i n t s  The higher magnification (40X) was  necessary to i d e n t i f y the l i m i t s o f the tunica media.  The midpoint o f  each photograph was located, and at that point, the thickness o f the tunica media was measured ( i n mm) using a r u l e r placed perpendicular to the endothelium.  The four thicknesses o f the tunica media for each vein  were averaged together to produce the average medial thickness ( i n ym). Electron Microscopy The thin sections of a femoral nerve trunk were photographed on a P h i l i p s 300 electron microscope using 35mm f i l m . for 3 minutes i n f u l l strength D19 developer  The f i l m was developed  followed by a wash i n  d i s t i l l e d water.  The f i l m was fixed for 5 minutes then washed with  f i l t e r e d water f o r 45 minutes a t 70°F. Fluorescence  Microscopy  The cryostat sections stained f o r fluorescence were examined with a Zeiss fluorescence microscope using a Zeiss u l t r a v i o l e t f i l t e r H365 01 (FT 395, LP 397) that permitted wavelengths i n the upper 400nm region to pass.  Beginning i n the upper l e f t corner, each coverslip was examined  thoroughly to find the transverse sections o f the saphenous a r t e r i e s or femoral veins.  Using a 40X Plan Neofluor objective, the number o f  fluorescing areas per section found at the advential-medial border o f the vessel was counted.  Counts were made for the r i g h t and l e f t saphenous  a r t e r i e s and femoral veins from 8 animals.  From each animal, 14-35  sections o f the saphenous artery were counted and 10-25 sections o f the femoral vein were counted.  The number o f sections counted for each  vessel represented the number o f sections cut minus the few that were unsuitable due to their being folded or blurred by a bubble i n the mounting medium (immersion o i l ) . Black and white photographs and coloured s l i d e s were taken o f some o f these vessels using 35mm Kodak T r i X black and white f i l m and 35mm Kodak Ektachrome colour s l i d e f i l m (ASA 400). The black and white f i l m was developed  i n Acufine developer for 5 minutes.  After a rinse with  d i s t i l l e d water, the f i l m was fixed f o r 5 minutes followed by a 1 hour wash i n f i l t e r e d water. l i g h t microscopy.  P r i n t s were developed  i n the same manner as f o r  The colour f i l m was developed  commercially.  Statistics The measurements o f the tunica media o f the saphenous a r t e r i e s and femoral veins and o f the lumen perimeter o f the veins f o r both the denervated and control sides were compared using a paired Student's  it-test. areas  R e s u l t s a r e shown a s mean + SEM.  The counts o f t h e f l u o r e s c e n t  from the saphenous a r t e r i e s and femoral v e i n s a r e compared u s i n g  t h r e e d i f f e r e n t a n a l y s e s o f v a r i a n c e (ANOVAs). o t h e r data a s w e l l except the femoral v e i n .  ANOVAs were r u n on the  f o r the measurements o f t h e medial  thickness o f  RESULTS Sampling for Light Microscopy Three animals per group were sampled for l i g h t microscopy at 60 and 90 days of age, thus making a t o t a l of 12 animals (Tables I and I I ) .  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 r i g h t foot when the animal walked; incomplete extension of the r i g h t l e g when the animal i s suspended by i t s tail. Inside two animals, milky white scar tissue had to be removed from the denervated area to locate the positions of the femoral, the 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 .  In the remaining animals, a more  transparent connective tissue blanketed the vessels; therefore, the vessels were c l e a r l y v i s i b l e .  There were no indications of i n f e c t i o n i n  any of the animals. Generally, on the denervated side, there never appeared to be a healthy, g l i s t e n i n g white saphenous nerve.  Instead, the remnant of the  nerve was so translucent that i t was not v i s i b l e or i t was very thin and yellowish.  The translucent nerve remnants had no form and were probably  just connective t i s s u e . This was indeed the case when these samples were examined under the l i g h t microscope. In four animals, re-innervation was noted and i n two of these four cases, the re-innervation pathways were the same.  That i s , i n both  cases, a very pale, translucent nerve, seemingly coming from the surrounding s k e l e t a l muscle of the thigh about h a l f way along the femoral artery, coursed diagonally across the thigh i n a caudal d i r e c t i o n to reach the s u p e r f i c i a l epigastric artery a b i t d i 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 t h i r d animal, re-innervation  appeared to have come from the abdominal wall, and i n the l a s t animal, a nerve was found running p a r a l l e l with the inguinal ligament, although i t s o r i g i n and destination were undetermined. Out of the s i x animals that were denervated at 1-3 days o f age, only one had i t s tubed femoral nerve i n t a c t at the time of sampling (60 days of age).  Because the nerves are so delicate at t h i s stage (1-3 days),  the nerve i n three animals tore when p u l l i n g i t into the tube, therefore, three of the s i x animals had no tube at a l l .  Despite t h i s however, a  large segment o f the femoral nerve was removed from within the abdominal cavity i n these three cases.  Tubes from the remaining two animals were  present, but were unattached to the rest of the femoral nerve and were found l y i n g i n 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 s k e l e t a l muscle. A l l s i x animals denervated at 12 days o f age had their tubes s t i l l attached to the femoral nerve.  The tubes were encased i n connective  tissue and no nerves appeared to be growing out of the melted end o f the tube.  The portion o f the femoral nerve just proximal to, as well as the  portion within the tube was t h i n , pale and yellowish compared to the larger, g l i s t e n i n g white femoral nerve on the control side. I t was not u n t i l sampling the remaining s i x animals i n the experiment that a change i n the denervated femoral vein was noticed.  The femoral  vein showed a d i l a t i o n i n the area of the branch points o f the s u p e r f i c i a l epigastric and saphenous veins.  This d i l a t i o n 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 p o i n t s o f the s u p e r f i c i a l e p i g a s t r i c and saphenous v e i n s was  s l i g h t l y d i l a t e d as w e l l . No anomalies o f the f e m o r a l o r saphenous a r t e r i e s were apparent a t the g r o s s l e v e l . LIGHT MICROSCOPIC APPEARANCE Saphenous A r t e r y There was no remarkable d i f f e r e n c e i n the l i g h t m i c r o s c o p i c appearance between t h e denervated and c o n t r o l saphenous a r t e r i e s  (Figure 5 ) .  had a conspicuous endothelium w i t h i t s many e n d o t h e l i a l c e l l b u l g i n g i n t o t h e lumen.  A prominent  Both  nuclei  i n t e r n a l e l a s t i c lamina, a w e l l  developed t u n i c a media and a t h i n e x t e r n a l e l a s t i c lamina were c h a r a c t e r i s t i c o f t h e i r h i s t o l o g i c a l appearance. l a m i n a was c o n s i s t e n t l y t h i c k e r  The i n t e r n a l  elastic  ( o n e - h a l f t o two t h i r d s t h i c k e r ) than the  e x t e r n a l e l a s t i c lamina i n both t h e denervated and c o n t r o l v e s s e l s .  In  some o f the denervated a r t e r i e s sampled a t 60 days, the i n t e r n a l  elastic  lamina appeared t o be s l i g h t l y t h i c k e r than the i n t e r n a l e l a s t i c  lamina  i n o t h e r denervated o r c o n t r o l a r t e r i e s .  S m a l l breaks i n the i n t e r n a l  and e x t e r n a l e l a s t i c laminae i n c o n t r o l and denervated a r t e r i e s were apparent the  ( F i g u r e 5 ) . I n one c o n t r o l a r t e r y , t h e c o n n e c t i v e t i s s u e  media appeared  from  t o be c o n t i n u o u s w i t h c o n n e c t i v e t i s s u e o f t h e  a d v e n t i t i a through one o f the breaks i n the e x t e r n a l e l a s t i c l a m i n a . There were no d i f f e r e n c e s i n the t u n i c a media between the denervated and c o n t r o l s i d e s a t the l i g h t m i c r o s c o p i c 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 o f smooth muscle c e l l s . p r o f i l e s were o f many d i f f e r e n t shapes and s i z e s .  Consequently, t h e  Some o f the  l o n g i t u d i n a l p r o f i l e s were l o n g s l e n d e r s p i n d l e shapes o r l o n g , t h i n and flat.  C r o s s - s e c t i o n a l p r o f i l e s i n c l u d e d square, t r i a n g u l a r , round,  and p o l y g o n a l shapes.  Each c e l l was c l e a r l y s e p a r a t e d from i t s  n e i g h b o u r s by a t h i n u n s t a i n e d a r e a , t h e r e b y emphasizing the h i g h l y  oval  i r r e g u l a r c e l l boundaries. the  In both the denervated and c o n t r o l a r t e r i e s ,  p r o f i l e s o f the smooth muscle  n u c l e i were a l s o o f v a r i o u s shapes  and  s i z e s because o f the d i f f e r e n t p l a n e s o f s e c t i o n a t which  these c e l l s  were c u t .  necessarily  However, the shape o f n u c l e a r p r o f i l e s d i d not  conform t o the shape o f 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 , were e c c e n t r i c a l l y l o c a t e d .  the n u c l e i  The n u c l e i were v e r y euchromatic, t h e r e were  none t h a t were p i c n o t i c . The a d v e n t i t i a , assessment,the  i n both c a s e s , appeared  the same.  From v i s u a l  a d v e n t i t i a o f the denervated a r t e r y d i d not appear  to  c o n t a i n more o r fewer c o n n e c t i v e t i s s u e c e l l s than the a d v e n t i t i a o f the control arteries. Femoral V e i n Although the lumen o f the denervated v e i n s was  dilated, their walls  d i d not d i f f e r i n t h e i r l i g h t m i c r o s c o p i c appearance c o n t r o l veins (Figure blood vessels cross-section, the  6).  from those o f the  The d e f i n i t i v e l a y e r s so c h a r a c t e r i s t i c o f  were not e a s i l y i d e n t i f i a b l e i n the v e i n s ( F i g u r e  6).  In a  an endothelium and a d v e n t i t i a were always p r e s e n t around  e n t i r e v e i n , but d i s t i n c t i v e e l a s t i c laminae and media were n o t .  C o n n e c t i v e t i s s u e o c c u p i e d most o f the a r e a under  the endothelium, and i t  was  d o t t e d w i t h s m a l l groups o f o r i n d i v i d u a l smooth muscle c e l l s .  was  difficult  muscle  It  t o 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  c e l l s a t t h i s l e v e l o f microscopy; some may  have been f i b r o b l a s t s .  Nerve F i g u r e s 7(a) and 8(a) c l e a r l y show the l i g h t m i c r o s c o p i c appearance normal saphenous and f e m o r a l nerve t i s s u e taken r e s p e c t i v e l y contralateral control side.  from the  The m u l t i p l i c i t y o f m y e l i n sheaths a r e  packed t i g h t l y t o g e t h e r , each one surrounded by a t h i n l a y e r o f endoneurium.  The p r o f i l e s o f m y e l i n sheaths ranged from l a r g e t o v e r y  of  small.  Schwann c e l l and  unmyelinated  nerve  fibroblastic  profiles  n u c l e i , s m a l l b l o o d v e s s e l s , and  a r e i n t e r s p e r s e d amongst the m y e l i n a t e d  axons.  D i s t a l Stump o f Saphenous Nerve from the Denervated S i d e Four samples o f the d i s t a l stump ( t h a t p a r t d i s t a l to the p o i n t o f the segment removed) o f the saphenous nerve were t h i c k s e c t i o n e d and s t a i n e d , and  the l i g h t m i c r o s c o p i c appearance o f one  Figure 7(b).  The  o f these samples i s seen i n  nerve bundle i s surrounded  by a t h i c k  e n c a p s u l a t i n g a h i g h l y compact mass o f c e l l s and o f the c e l l s were l a r g e and fibroblast-like. boundaries  In one  endoneurium.  The o t h e r samples had v e r y  o f these s e c t i o n s , a macrophage was  i n number and were surrounded  the encased nerve arranged,  c o r e o f denser  to  thick  Side  ( d a t a not shown). highly cellular  A very t h i c k capsule o f  dense c o n n e c t i v e t i s s u e surrounded  T h i s c o r e was  a l s o very c e l l u l a r ,  just  well  proximal  s l i g h t l y d i f f e r e n t l i g h t m i c r o s c o p i c appearances.  i l l u s t r a t e s " the appearance o f one  circularly  a  c o n t a i n e d macrophages.  t h r e e samples o f t h a t p o r t i o n o f the femoral nerve  the tube had  8(b)  by a  tube showed the l i g h t m i c r o s c o p i c appearance  connective t i s s u e .  v a s c u l a r i z e d and The  seen, and i n  A l l o f these samples were w e l l v a s c u l a r i z e d .  T h i c k s e c t i o n s from one  circularly  similar  axons p r e s e n t , however, these were  Abdominal C a v i t y Nerve from the Denervated  of  Some  c e l l s a r e so c l o s e l y packed t o g e t h e r t h a t c e l l s  t h e r e were s t i l l m y e l i n a t e d  v e r y s m a l l , were few  connective t i s s u e .  p a l e s t a i n i n g w h i l e o t h e r s were t h i n and more  are u n i d e n t i f i a b l e .  appearances. another,  The  perineurium  arranged  connective t i s s u e .  such sample.  Here, a t h i c k ,  c o a t o f c o n n e c t i v e t i s s u e surrounded The  Figure  an i n n e r c o r e o f  c o n n e c t i v e t i s s u e c o r e c o n s i s t e d o f many n u c l e i  belonging to c e l l s whose boundaries were not distinguishable.  Some  n u c l e i were f l a t and fusiform, whereas others were square or round.  The  core was well vascularized and i n the very centre were tiny p r o f i l e s o f myelinated axons (Figure 8, i n s e r t ) , much smaller than those from the control nerve.  The number o f these axons varied with each sample. For  example, the sample i n Figure 8(b) had only a few whereas the other two samples had more.  Even though two of the samples had many p r o f i l e s o f  myelinated axons, the number was not comparable to the control side. Moreover, a l l the p r o f i l e s were very small and they were surrounded by a very thick endoneurium.  Although a positive i d e n t i f i c a t i o n of the c e l l s  i n the core cannot be made at t h i s l e v e l of microscopy, perhaps they were a mixture of f i b r o b l a s t s and Schwann c e l l s . Electron Microscopy o f 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 f i b r o b l a s t s . degeneration.  The tissue also exhibited signs o f  Occasionally, myelinated and unmyelinated nerve p r o f i l e s  were seen (Figure 9, i n s e r t ) , but only i n the very centre o f 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 h a l f of the rats sampled, a d i l a t i o n of the femoral vein was noted.  Also i n four of the animals, the denervated saphenous and  s u p e r f i c i a l epigastric a r t e r i e s and veins branched from the femoral  a r t e r y i n v e r y c l o s e p r o x i m i t y t o each o t h e r .  I n some c a s e s , they  branched o f f s i d e by s i d e , and i n o t h e r s , the s u p e r f i c i a l  epigastric  a r t e r y branched from t h e saphenous a r t e r y r a t h e r than from t h e f e m o r a l artery.  On t h e c o n t r o l s i d e , i n a l l c a s e s except f o r one, t h e branch  p o i n t s o f t h e 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 v e i n s were a p p r o x i m a t e l y 0.5 cm a p a r t . Even though most o f the a n i m a l s sampled had the tube s t i l l a t t a c h e d t o the f e m o r a l nerve, r e - i n n e r v a t i o n o f the saphenous a r t e r y was seen i n h a l f o f the a n i m a l s .  The r e - i n n e r v a t i n g n e r v e s were v e r y p a l e and  t r a n s l u c e n t , one b e i n g s i m i l a r i n appearance t o an empty a r t e r i o l e . R e - i n n e r v a t i o n came from a b e r r a n t a r e a s such as the abdominal w a l l , the s k e l e t a l muscle underneath the v e s s e l s , and from w i t h i n the s c a r b l a n k e t i n g the a r e a .  tissue  A l l l e d t o t h e branch p o i n t o f t h e saphenous a r t e r y .  In two a n i m a l s , s p r o u t i n g o f the f e m o r a l nerve i n s i d e t h e abdominal c a v i t y was v i s i b l e .  I n one o f t h e s e a n i m a l s , t h e tube had detached, and  i n the o t h e r a n i m a l , s p r o u t i n g o f t h e f e m o r a l nerve was p r o x i m a l t o i t e n t e r i n g the tube.  I was unable t o determine t h e d e s t i n a t i o n s o f t h e s e  a x o n a l s p r o u t s because t h e c o n t e n t s o f t h e abdominal c a v i t y were b l u e from the t r y p a n b l u e . D e s c r i p t i o n o f t h e S e c t i o n s under the F l u o r e s c e n c e Microscope F i g u r e s 10 and 11 show the appearance o f the denervated and c o n t r o l v e s s e l s o f the saphenous a r t e r y and saphenous v e i n , r e s p e c t i v e l y . i n j e c t i o n w i t h t r y p a n b l u e , the e x t e r n a l e l a s t i c lamina always  After  fluoresced  i n t h e r e d range, however, the i n t e r n a l e l a s t i c lamina d i d n o t f l u o r e s c e r e d as c o n s i s t e n t l y .  Although s p e c i f i c a d r e n e r g i c f l u o r e s c e n c e was  c o n c e n t r a t e d a t t h e a d v e n t i t i a l - m e d i a l b o r d e r , o c c 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 . beyond  I n some o f the v e i n s , t h e f l u o r e s c e n t nerves extended  t h e a d v e n t i t i a l border towards t h e endothelium.  The f l u o r e s c e n t  areas varied i n size from very tiny to large dots.  Sometimes, the  smaller dots had a "beads on a s t r i n g " appearance.  Generally,  fluorescent dots were r e l a t i v e l y uniform i n their d i s t r i b u t i o n around the circumference o f the control a r t e r i e s , whereas the veins had patches o f fluorescent dots i r r e g u l a r l y spaced around their circumference. The i d e n t i t y of a re-innervating adrenergic nerve that had the appearance of an a r t e r i o l e was v e r i f i e d by fluorescence.  Cross sections  of t h i s nerve contained many small, pale fluorescent dots. ANALYSIS Morphometries Medial Area of the Saphenous Artery From l i g h t micrographs, morphometries were carried out on the saphenous artery and femoral v e i n . l i s t e d i n Tables I and I I .  The raw data as well as the means are  For the a r t e r i e s , a Student's t - t e s t was  carried out on the data from each table separately. When the a r t e r i e s were sampled at 60 days, the r e s u l t s of the t-tests  show that there was a  tendency for a thinner media, but t h i s was not s i g n i f i c a n t at the p<0.05 l e v e l (Table I ) . However, i f each sample i s looked at i n d i v i d u a l l y , the measurements do show that the area o f the tunica media was decreased i n four of the s i x animals.  The remaining two animals show  a very s l i g h t increase i n the media of the denervated a r t e r i e s ; therefore there was v i r t u a l l y no change i n the media of these two samples.  Animals  sampled at 90 days showed a s i g n i f i c a n t decrease i n 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 I I .  The two-factor ANOVA o f 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 v e r i f i e d the  i - t e s t r e s u l t s , but i t also showed that the age at which the animals were denervated was not s i g n i f i c a n t , and that there was no i n t e r a c t i o n between the two v a r i a b l e s . The three-factor ANOVA included, i n addition to the two factors just mentioned, the age at time of sampling.  From t h i s , sampling age did make  a s i g n i f i c a n t difference (P=0.024). Figure 12 summarizes the data from Tables I and II as per cent decreases i n the media, where the t o t a l 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 l i g h t microscopic cross-sections of the femoral vein, the luminal perimeter and the wall thickness were measured for each of the denervated and c o n t r a l a t e r a l control veins.  The raw measurements of the luminal  perimeter and their means are l i s t e d i n Table I I I . Both a Student's % - t e s t and an ANOVA were carried out on these means, and both tests confirmed that the increase i n luminal perimeter of the denervated veins was s i g n i f i c a n t .  The ANOVA also showed that denervating the femoral vein  at either 3 or 12 days had no s i g n i f i c a n c e , and there was no i n t e r a c t i o n between the two factors tested, these being, age at denervation and condition (denervated or c o n t r o l ) .  The percent increase i s shown i n  Figure 13. The r e s u l t s of measuring the wall thickness of the denervated control femoral veins are recorded i n Table IV.  The p-test  and  results  indicate that the difference i n wall thickness between the denervated control femoral veins i s not s i g n i f i c a n t .  and  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 a r t e r i e s and femoral veins was counted. i n Table V.  The counts are l i s t e d  Two, two-factor and one, three-factor ANOVAs were carried  out on the data.  The two, two-factor ANOVAS showed that there was a  s i g n i f i c a n t decrease i n the number o f fluorescent dots both for the artery and f o r the v e i n .  The age at denervation was not s i g n i f i c a n t f o r  denervating the femoral v e i n . The second two-factor ANOVA (condition versus age of sampling) was designed to see i f the sampling age had any e f f e c t .  That i s , by ignoring  the e f f e c t of condition (denervated or c o n t r o l ) , the ANOVA showed that the e f f e c t o f age of sampling on the fluorescence counts was s i g n i f i c a n t for the a r t e r i e s (P=0.019) and f o r 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 s i g n i f i c a n t difference i n fluoresence between the two blood vessel types.  DISCUSSION  In t h i s study, I have developed  a d e n e r v a t i o n technique t o study  the  t r o p h i c i n f l u e n c e s between nerves and b l o o d v e s s e l s i n the r a t t h i g h . T h i s t e c h n i q u e was  designed  t o be as l o c a l i z e d and as permanent as  p o s s i b l e f o r the f o l l o w i n g r e a s o n s : c h e m i c a l and  immunological  (1) sympathetic  ganglionectomy  sympathectomies d e s t r o y the a d r e n e r g i c  i n n e r v a t i o n t o such a l a r g e a r e a o f the body t h a t i t i s important determine  and  to  whether the d e n e r v a t i o n e f f e c t s seen with these methods a r e  due  s o l e l y t o the absence o f i n n e r v a t i o n on the b l o o d v e s s e l o r whether o t h e r denervated and  s t r u c t u r e s had changed and t h e r e b y c o n t r i b u t e d t o the  (2) s i m p l e c r u s h i n g , s e v e r i n g o r r e p o s i t i o n i n g the nerve  results,  are  u n s u i t a b l e methods f o r d e n e r v a t i n g b l o o d v e s s e l s s i n c e r e - i n n e r v a t i o n o c c u r s w i t h i n a few days (Todd, 1986)  - a p e r i o d o f time which may  s h o r t f o r d e n e r v a t i o n changes t o o c c u r . t h i s t e c h n i q u e , I then used  the method t o determine  whether d e p r i v i n g their  In t h i s s t u d y , the a r e a o f the t u n i c a media o f the r a t  saphenous a r t e r y decreased and the femoral v e i n was experiments  too  A f t e r d e t e r m i n i n g the s u c c e s s o f  b l o o d v e s s e l s o f t h e i r vasomotor i n n e r v a t i o n s i g n i f i c a n t l y a l t e r s architecture.  be  a l s o r e c o n f i r m e d t h a t i t i s extremely  dilated.  The  d i f f i c u l t to prevent  a b e r r a n t r e - i n n e r v a t i o n o f the t h i g h v e s s e l s . In p r e v i o u s s t u d i e s , the s u c c e s s o f d e n e r v a t i o n on b l o o d v e s s e l s determined  by a v a r i e t y o f ways.  to exogenously sympathetic  was  S u p e r s e n s i t i v i t y o f the smooth muscle  a p p l i e d n o r e p i n e p h r i n e , s t i m u l a t i o n o f the  entire  o u t f l o w u s i n g a s t e e l p i t h i n g r o d as the e l e c t r o d e  (6-hydroxydopamine d e n e r v a t i o n ) , h i s t o l o g i c a l s t u d i e s , and c h e m i c a l e l e c t r i c a l s t i m u l a t i o n o f s p e c i f i c g a n g l i a (immunological  and  sympathectomy)  a r e j u s t a few o f these ways ( L e v i - M o n t a l c i n i and A n g e l e t t i , 1966;  Finch  a l . , 1973;  et was I  Bevan and T s u r u , 1 9 7 9 ;  1981).  Although  the femoral nerve  t o r n i n 3 out o f the 6 a n i m a l s t h a t I denervated a t 1 - 3  still  r e g a r d e d them as b e i n g s u c c e s s f u l l y d e n e r v a t e d .  days o f age,  The nerves  w h i l e p u s h i n g the tube on; t h e r e f o r e , the r e s u l t i n g p r o x i m a l stump  tore was  c h a i n ) i n the 3 day o l d a n i m a l .  v e r y s h o r t ( i e . c l o s e t o the sympathetic  I t has been r e p o r t e d t h a t the c l o s e r the i n j u r y i s t o the c e l l body, the g r e a t e r the damage t o the c e l l body ( G a b e l l a , 1 9 7 6 ; 1983).  B a r r and K i e r n a n ,  Thus, I assumed t h a t the nerves t o r n i n the 3 day o l d a n i m a l s  t o r e c l o s e enough to t h e i r nerve c e l l b o d i e s t o most l i k e l y cause death.  Since (1)  a t r o p h y and d e g e n e r a t i o n o f c e l l  months a f t e r axotomy ( G a b e l l a , 1 9 7 6 ) ,  b o d i e s can  persist  (2) destruction of adrenergic c e l l  b o d i e s a b o l i s h e s vasomotor c o n t r o l , and  ( 3 ) c e l l b o d i e s i n newborn  a n i m a l s a r e more s u s c e p t i b l e t o d e n e r v a t i o n t e c h n i q u e s and A n g e l e t t i , 1 9 6 6 ; F i n c h e t a l . , 1973)> the t h r e e animals who  cell  (Levi-Montalcini  I assume i n my  experiment  that  had the t o r n f e m o r a l nerve were s u c c e s s f u l l y  d e n e r v a t e d , even a t time o f s a m p l i n g . For o f my  a l l a n i m a l s , those w i t h o r w i t h o u t a tube, the degree o f s u c c e s s d e n e r v a t i o n t e c h n i q u e on the saphenous a r t e r y and  determined. and d i s t a l light  was  The methods used f o r t h i s i n c l u d e d showing t h a t the p r o x i m a l stumps o f the f e m o r a l nerve had a degenerated  and e l e c t r o n microscopy  on the p r o x i m a l nerve stump). o b t a i n e d by examining of  femoral v e i n  ( e l e c t r o n microscopy was  appearance  using  c a r r i e d out o n l y  In a d d i t i o n , e v i d e n c e f o r d e n e r v a t i o n  the g r o s s morphology o f the t h i g h f o r the  was  presence  r e g e n e r a t i n g n e r v e s , by l i g h t m i c r o s c o p i c examination o f the  a d v e n t i t i a o f the v e s s e l s , and by a f l u o r e s c e n c e t e c h n i q u e s p e c i f i c f o r catecholamines.  The l i g h t m i c r o s c o p i c appearance  p r o x i m a l stumps from my  o f the d i s t a l  and  study showed d e f i n i t e s i g n s o f d e g e n e r a t i o n .  When a p e r i p h e r a l nerve i s c l e a n l y t r a n s e c t e d , proper a x o n a l  regeneration requires appositioning the two cut ends and suturing through the epineurium (Barr and Kiernan,  1983).  This s u r g i c a l repair i s not  necessary i f the axons have been transected by a crush because the connective tissues i n the nerve remain i n t a c t (Barr and Kiernan,  1983).  Axonal transection, therefore, produces permanent degeneration o f the d i s t a l adrenergic f i b r e s and i n temporary degeneration o f the c e l l body (Burnstock and Costa,  1975).  D i s t a l to the s i t e o f injury, the detached  portion o f the peripheral nerve undergoes Wallerian degeneration whereas the proximal portion undergoes the axon reaction which i s best displayed i n the c e l l bodies as chromatolysis (Barr and Kiernan,  1983). Wallerian  degeneration i s characterized by the axon i n i t i a l l y becoming swollen and then breaking up into fragments. changes i n the myelin sheath.  Accompanying these axonal changes are  I t i s broken into short e l l i p s o i d a l  segments and then gradually disintegrates.  The Schwann c e l l s multiply,  f i l l i n g the c y l i n d r i c a l area enclosed by the endoneurium.  The remnants  of motor neurons (the axon and i t s myelin sheath) and the axons o f unmyelinated  f i b r e s are phagocytosed, with the d i s t a l stump being  composed o f columns o f Schwann c e l l s (bands of von Bungner).  The content  of norepinephrine disappears anywhere from 18 to 48 hours a f t e r 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 r e s u l t s vary between organs and species.  Blood vessels o f  the rabbit ear s t i l l responded to nerve stimulation three days post-severing (Gabella,  1976).  The general structure o f sympathetic  axons appears normal for several days (Burnstock and Costa,  1975), and  the length o f time for degeneration i s probably d i r e c t l y proportional to the axon length (Burnstock and Costa,  1975; Gabella, 1976).  short d i s t a l stump degenerates faster than a longer one.  That i s , a  Since  a p p l i c a t i o n of colchine produces degeneration of a nerve d i s t a l to i t s a p p l i c a t i o n , t h i s suggests that transport of substances i s v i t a l for the s u r v i v a l of the axons (Burnstock and Costa, 1975).  Therefore, the l i g h t  microscopic appearance of the d i s t a l portion of the saphenous nerve i n my study indicates that i t was probably devoid of adrenergic f i b r e s , and the c e l l s that were there were most l i k e l y Schwann c e l l s arranged i n the bands of von Bungner.  The d i s t a l stump of the saphenous nerve closely  resembled the d i s t a l 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 v i r t u a l l y devoid of myelin debris, and 89% of the n u c l e i i n a cross-section represent bundles of Schwann c e l l s which are separated by a l o t of collagen and e l a s t i n f i b r e s , scattered f i b r o b l a s t s 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 d i s t a l t i b i a l nerve stump i s l i n e a r over a 3-hour period and does not d i f f e r along the length of the nerve whereas i f t h i s 7 week denervated d i s t a l stump i s joined end-to-end  to a newly severed proximal 3  stump of the peroneal nerve, an increase i n  H-thymidine  within the f i r s t 6cm of the coapted d i s t a l stump. 6cm,  the  uptake i s seen  Beyond these f i r s t  H-thymidine uptake i s s i m i l a r to that along the entire 9.5cm  length of the 7 week denervated (non-joined) d i s t a l stump.  Pellegrino  and Spencer (1985) found that the f i r s t 2-6.5cm d i s t a l to the s i t e 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 r e s u l t s of Pellegrino and Spencer (1985) also  suggest that i n my study, the Schwann c e l l s remained as bundles.  That  i s , since d i r e c t re-innervation of the d i s t a l saphenous nerve stump by i t s proximal stump was prevented by encasing the proximal end of the  48 femoral nerve i n a tube, the Schwann c e l l s i n the d i s t a l stump had no stimulus to divide and form myelin, Changes i n the proximal portion of a severed nerve may vary depending on the type of neuron; therefore, some neurons may  t o t a l l y disappear  whereas others may not be s i g n i f i c a n t l y altered (Barr and Kiernan, 1983).  Large motor neurons supplying s k e l e t a l muscle exhibit the  c y t o l o g i c a l d e t a i l s of the c l a s s i c a l axon reaction, the description of which follows.  The most s i g n i f i c a n t a l t e r a t i o n i n the severed axon  occurs immediately adjacent to the cut. a l t e r e d appreciably.  The remainder of the axon i s not  Coarse clumping of N i s s l substance can appear i n  the c e l l body as soon as 6 hours a f t e r section.  The nucleus becomes  e c c e n t r i c a l l y located (Barr and Kiernan, 1983; Gabella, 1976) f l a t t e n s out, l a t e r on becoming indented (Gabella, 1976);  and  t h i s process  reaches a maximum at 10-20 days a f t e r injury (Barr and Kiernan, 1983)• Organelles become somewhat disorganized (Barr and Kiernan, 1983). end of 6 weeks, 40-50? of the c e l l s may (Gabella, 1976),  or some may  At the  s t i l l show signs of chromatolysis  p e r s i s t for months (Barr and Kiernan, 1983;  Gabella, 1976). In my study, the proximal end of the femoral nerve was encased i n a polyethylene tube which may have contributed to the increased spread of degeneration along the length of t h i s nerve.  A s i m i l a r appearance to my  observations was seen i n the nerve repair studies by Colin et a l . (1984).  In t h e i r study, a 5-7mm portion of r a t t i b i a l nerve was excised  from one s i d e .  The rats were divided into 2 groups.  To induce the  formation of a fibrovascular sheath i n one group, a l l rats had t h e i r proximal and d i s t a l nerve stumps connected by a s i l i c o n e rod whereas, i n the other group, the 2 ends were l e f t separated.  Four weeks l a t e r , a l l  the s i l i c o n e rods were removed, leaving a fibrovascular sheath behind.  These r a t s were divided into 3 subgroups, and further experimental treatment was carried out on 2 o f these subgroups while the t h i r d acted as a c o n t r o l .  The same protocol was applied to the group without the rod  inserted, that i s , the unsheathed group. months.  The animals were l e f t f o r 3  One of the experimental treatments carried out on a subgroup  involved connecting the two free ends of the nerves with a collagen tube.  The r e s u l t s were i n t e r e s t i n g since both the sheathed and  unsheathed  collagen-tube-encased nerves exhibited the thick fibrosed  epineurium which I found on my p l a s t i c tube-encased  femoral nerve.  However, the cores o f the nerves i n the study by Colin et a l . (1984) had a normal h i s t o l o g i c a l appearance.  Therefore, the collagen tube may have  induced the thick, fibrosed epineurium but i t also f a c i l i t a t e d the normal regeneration of the nerve by guiding the axonal sprouts into the d i s t a l segment.  The core of the femoral nerve i n my study resembled most that  of the unsheathed  control group i n Colin et a l . ' s study.  That i s , the  core had a fibrosed i n t e r n a l milieu with a few d i f f u s e m i n i - f a s i c l e s , the axons o f which were small i n diameter with thin myelin sheaths. these comparisons,  From  i t can be seen that the proximal and d i s t a l stumps i n  my study d e f i n i t e l y had a degenerated  appearance.  By examining the gross morphology o f the thigh area, I was able to see very t h i n , translucent, unmyelinated nerves re-innervating the saphenous and s u p e r f i c i a l epigastric a r t e r i e s i n some of the denervated animals. Although I did not see any nerves i n the adventitia o f the denervated vessels with l i g h t microscopy, the fluorescence technique did reveal the presence o f some catecholamines at the adventitial-medial border.  These  r e s u l t s indicate that re-innervation d i d occur. As i n humans (Williams and Warwick, 1975), the femoral nerve o f the r a t c a r r i e s the adrenergic innervation to the saphenous and femoral  a r t e r i e s and veins (Todd, 1986).  Therefore, adrenergic regulation of the  vasomotor a c t i v i t y of these vessels ceases by severing the femoral nerve.  However, a f t e r 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 i n r e s t r i c t e d areas ( i n hamster cheek pouch or web of frog's foot) two phenomena have been observed a f t e r denervation.  These include the maintenance of a basal tone and rhythmic  changes i n 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 a l t e r the organs' blood supply, suggesting that these blood vessels depend on other c o n t r o l l i n g factors such as myogenic a c t i v i t y or the l o c a l chemical environment  (Page and McCubbin, 1965).  I f the dependency of a blood  vessel on i t s neural coordination may r e f l e c t that vessels' a b i l i t y to stimulate i t s re-innervation, then the occurrence of re-innervation i n my study and i n Todd's (1986) experiments suggest that the blood vessels i n the r a t thigh are highly dependent on adrenergic c o n t r o l .  Since the  femoral nerve does not appear to be the source of these re-innervating f i b r e s , the axonal sprouting from aberrant areas i n the v i c i n i t y of the denervated vessels suggests that vascular smooth muscle c e l l s may have a widespread and powerful mechanism which prevents permanent denervation from occurring.  Being so widespread, t h i s mechanism might be a  d i f f u s i b l e chemotrophic or chemotactic factor. Nerve density around a blood vessel may be d i r e c t l y proportional to a vessel's a b i l i t y to induce sprouting of neighbouring nerves.  This i s  s u p p o r t e d by t r a n s p l a n t a t i o n experiments  c a r r i e d o u t by Todd ( 1 9 8 6 ) .  In  her study, segments o f t h e d e n s e l y i n n e r v a t e d r a t t a i l a r t e r y and t h e r a t f e m o r a l a r t e r y , which has v i r t u a l l y no i n n e r v a t i o n , were t r a n s p l a n t e d s e p a r a t e l y i n t o the a n t e r i o r eye chamber o f a h o s t r a t .  Only t h e t a i l  a r t e r y was capable o f i n d u c i n g i r i d i a l nerve s p r o u t i n g .  Although  r e s u l t s suggest  these  i t i s the blood v e s s e l i t s e l f that i s the t r o p h i c  i n d u c e r , P o l i t i s e t a l . ( 1 9 8 2 ) p r o v i d e s e v i d e n c e t h a t the t r a n s e c t e d d i s t a l nerve stump c o n t a i n s d i f f u s i b l e f a c t o r s which can a t t r a c t r e g e n e r a t i n g axons. R e - i n n e r v a t i o n o f b l o o d v e s s e l s from a b e r r a n t a r e a s i s a l s o seen i n d e n e r v a t i o n experiments  where t h e e n t i r e s u p e r i o r c e r v i c a l g a n g l i o n i s  removed (R. D. Bevan, p e r s o n a l communication w i t h M. E. Todd; et  al.,  1983).  Bevan s t a t e s t h a t r e - i n n e r v a t i o n o f the r a b b i t e a r  v e s s e l s o c c u r s between 6 - 8 weeks i n t h e denervated Kobayashi  Kobayashi  r a b b i t whereas  e t a l . ( 1 9 8 3 ) r e p o r t nerves r e g e n e r a t i n g between H-6 weeks and  r e a c h a maximum between 9 and 12 months i n denervated  Wistar  rats.  However, t h e maximum number o f r e g e n e r a t i n g nerves i s a p p r o x i m a t e l y of  the normal number. In  to  half  o t h e r c a s e s , nerve r e g e n e r a t i o n a f t e r d e n e r v a t i o n has been r e p o r t e d  o c c u r i n under 15 days (Todd, 1 9 8 6 ; Dyck and Hopkins, 1 9 7 2 ) .  (1986) t r i e d  Todd  f o u r d i f f e r e n t methods o f s u r g i c a l l y d e n e r v a t i n g t h e  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 i n c l u d e d s e v e r i n g  the femoral nerve, removal o f a segment o f the femoral nerve,  cauterizing  the p r o x i m a l stump a f t e r a segment was removed and r e p o s i t i o n i n g t h e s e v e r e d f e m o r a l nerve by s u t u r i n g i t t o t h e abdominal w a l l .  In a l l o f  these c a s e s , r e - i n n e r v a t i o n o c c u r r e d w i t h i n 15 days.  s p r o u t s were  seen 5 - 1 5 days a f t e r c r u s h i n g the c e r v i c a l sympathetic Hopkins ( 1 9 7 2 ) .  Axonal  trunk by Dyck and  S i n c e the r e - i n n e r v a t i n g f i b r e s i n these two s t u d i e s  sprouted from the severed or crushed nerve, rather than from aberrant areas as i n studies involving more d r a s t i c methods of denervation, t h i s 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 i n some of the denervated vessels suggested that these vessels had been re-innervated.  In a r t e r i e s , adrenergic nerve v a r i c o s i t i e s occur at the  adventitial-medial border (Burnstock and Costa, 1975; Todd, 1980) rather than penetrating into the media as they do i n veins.  Since resolution of  the l i g h t microscope i s l i m i t e d , the fluorescing dots are more l i k e l y to represent cluster of axonal v a r i c o s i t i e s rather than i n d i v i d u a l varicosities.  However, the "beads on a s t r i n g " appearance i s  c h a r a c t e r i s t i c of the series of adrenergic v a r i c o s i t i e s 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 s p e c i f i c for the v i s u a l i z a t i o n of catecholamines was modified by De l a Torre (1980). Since he has standardized t h i s method, a very consistent i n t e n s i t y of monoaminergic neurons and t h e i r axonal v a r i c o s i t i e s from one section to the next has been achieved (De l a Torre, 1980).  Therefore, differences  i n counts should t r u l y r e f l e c t differences i n catecholamines rather than inconsistencies produced by the method.  By i n j e c t i n g trypan blue into  the animal before sampling, any confusion between the fluorescence of the external e l a s t i c lamina and that of nerve v a r i c o s i t i e s i s eliminated, thereby ensuring that only the dots produced by the fluorescence of catecholamines were counted.  The s i g n i f i c a n t decrease i n fluorescence  obtained i n this study, represented a decrease i n the amount of norepinephrine.  Whether t h i s represented an absence or a decrease i n  53 unmyelinated The the  nerve  terminials  fluorescence data  denervated  indicate  vessels.  norepinephrine  These  further  that  counts  probably  the  re-innervating  discussed,  re-innervation  did  occur  had  translucent  present  probably  gross  fluorescence  seen  observation,  re-innervation  spite  of  severed i t  in  how w e l l nerve  seems  a l l  the  the  their  nerves.  This  was o n l y  would  reported  surgical technique  in  by  the previously  the  by  half  nerves  number  for  the  gross  of  the  its  adrenergic  actual  account  prevents  and r e - i n n e r v a t i n g  re-innervation  around  The r e - i n n e r v a t i n g  therefore,  viewed.  present  As  v e s s e l s sampled whereas  from sprouting  that  were  represent  sometimes.  morphology;  exceeded that  investigation.  some n e r v e s  of  a very  content  awaits  the  animals.  proximal  target  nerves  end  blood  cannot  In  of  a  vessel,  be  prevented. Once  the  degree  established, on  the  of  denervation  success  morphological  saphenous  morphology  of  artery  my d e n e r v a t i o n  and morphometrical  and  femoral  arterial walls  studies  of  (Branco  has et  vein  been  a l . ,  recorded  1984;  of  the  s u r g i c a l l y denervated  becomes  irregular  and  broken  In  the  present  the  internal  the  denervated  breaks  were  internal  study,  and  counted  elastic  microscope  and  ultrastructural lamina  is  lamina  in  needed  elastic  compared and  to  preliminary  laminae  the  determine  only  a  1986).  carried in  few The  other external  Todd  artery  (1986).  microscopic examination  may h a v e  been more b r o k e n  statistically. t h i c k e r under  however,  Although the  measurements  of  conclusively.  the  that on the  the  light  electron microscopic examinations,  this  out  the  superficial epigastric  contralateral controls;  somewhat  i n v e s t i g a t i o n and to  from l i g h t  compared  looked  Changes  seen u l t r a s t r u c t u r a l l y by  appeared  external  side  not  i t  in  Todd,  lamina  was  a n a l y s e s were  samples.  elastic  as  technique  internal  further  elastic  Branco e t a l . denervated dog  (1984)  showed t h a t the  saphenous v e i n and  smooth muscle c e l l s i n  the  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 o f a c t i v e p r o t e i n synthesis. is,  That  they a r e l a r g e r , have l a r g e r euchromatic n u c l e i w i t h prominent  n u c l e o l i and  the c y t o p l a s m i s r i c h i n ribosomes and  rough endoplasmic r e t i c u l u m .  Although a l l the  has  well  developed  smooth muscle c e l l s i n  venous w a l l show these m o r p h o l o g i c a l changes, o n l y  2 - 3 smooth muscle  l a y e r s a d j a c e n t t o the a d v e n t i t i a l - m e d i a l  i n the r a b b i t  a r t e r y showed these changes.  The  junction  increases  the cell  ear  i n the diameter o f smooth  muscle r e g r e s s e s towards normal v a l u e s a f t e r 1 2 0 days i n the v e i n and 3 5 days i n the a r t e r y  (Branco e t a l . ,  1984).  appearance o f smooth muscle c e l l s was Campbell days by  (1976)  the  and  The  dedifferentiated  seen i n v i t r o by Chamley  t h i s d e d i f f e r e n t i a t i o n was  prevented f o r a few  presence o f sympathetic g a n g l i o n e x t r a c t .  f i n d i n g s o f Branco e t a l .  (1984),  and extra  Contrary to  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  the  d e n e r v a t i o n o f the r a b b i t ear a r t e r y v i a s u p e r i o r  cervical  have suggested t h a t d e n e r v a t i o n c r e a t e s  t u n i c a media (Bevan  Tsuru,  1979;  (1984)  sampled the r a b b i t ear a r t e r y  Bevan and  whereas Bevan and p a s t the  Tsuru  Tsuru,  (1981)  Bevan e t a l . , 15  and  35  sampled t h e i r s  time where Branco e t a l .  back t o normal; t h e r e f o r e , age  1981;  a thinner  i t may  (1984)  be  that  saw  8  weeks l a t e r which i s w e l l  regression  s t r u c t u r a l d i f f e r e n c e i n the media a t e i t h e r the  m i c r o s c o p i c examination i n my  Branco e t a l .  o f the  the d i f f e r e n c e i n the  A change i n the media was study e i t h e r .  Todd  (1986)  l i g h t or  preliminary  from the  sampling no  electron  not o b v i o u s by My  artery  saw  light electron  m i c r o s c o p i c i n v e s t i g a t i o n s o f the denervated media a l s o d i d not apparent d i f f e r e n c e s  and  days p o s t - d e n e r v a t i o n  i s what produced these opposing o b s e r v a t i o n s .  microscopic l e v e l .  1983).  sympathectomy  show  c o n t r a l a t e r a l c o n t r o l side although  any  f i b r o b l a s t s and f i b r o b l a s t i c p r o c e s s e s a b u t t e d the a d v e n t i t i a l - m e d i a l border o f the denervated v e s s e l s .  T h i s o b s e r v a t i o n may  have been the  r e s u l t o f the l a c k o f nerves i n the a r e a s i n c e Branco e t a l . ( 1 9 8 4 )  found  t h a t f i b r o b l a s t s were more numerous i n the denervated v e s s e l s and showed characterises of synthetically active  cells.  Although a decrease i n the t u n i c a media o f the denervated saphenous a r t e r y d i d not appear markedly  t h i n n e r by l i g h t m i c r o s c o p i c o b s e r v a t i o n ,  a c t u a l measurements o f the media demonstrated  a decrease.  Decreases i n  the w a l l t h i c k n e s s o f the denervated r a b b i t ear a r t e r y have been shown by Bevan and T s u r u ( 1 9 7 9 ) , and d e c r e a s e s i n weights o f denervated middle p o s t e r i o r r a b b i t c e r e b r a l a r t e r i e s was  and  shown by Bevan e t a l . ( 1 9 8 3 ) .  Bevan and T s u r u ( 1 9 7 9 ) suggested t h a t d e n e r v a t i o n c r e a t e s a s m a l l e r wall.  T h i s appears t o be t r u e i n my  study as w e l l .  Contrary to these  f i n d i n g s , however, a r e the r e s u l t s o f Branco e t a l . ( 1 9 8 4 ) who  found  an  i n c r e a s e i n w a l l t h i c k n e s s i n the denervated r a b b i t ear a r t e r y . In  my  study, a 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 i n v e s t i g a t i o n  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 o f the media between the  denervated and c o n t r o l saphenous a r t e r i e s was t h a t the decrease i n the media may in  was  apparent.  T h i s suggests  have been the consequence o f changes  the e x t r a c e l l u l a r m a t r i x ( R u s t e r h o l z and M u e l l e r , 1 9 8 2 ) .  More  e x t e n s i v e e l e c t r o n m i c r o s c o p i c s t u d i e s o f t h i s denervated smooth muscle s h o u l d be c a r r i e d out t o i n v e s t i g a t e t h i s p o s s i b i l i t y . cell  size  (volume) t h a t a r e not d i s t i n g u i s h a b l e by  examination might a l s o c o n t r i b u t e .  Small changes i n  qualitative  For example, i f each m e d i a l smooth  muscle c e l l had a s l i g h t decrease i n s i z e o r change i n shape, the a d d i t i v e e f f e c t s o f these i n d i v i d u a l d e c r e a s e s may measureable  decrease i n the media.  Morphometric  p o s s i b l y produce  a  a n a l y s i s , such as t h a t  developed by Todd ( 1 9 8 3 ) f o r v a s c u l a r smooth muscle,  should a l s o  be  carried out to see i f c e l l s i z e a c t u a l l y contributes to the medial decrease. Differences i n measurements were expected between the two ages of denervation  (1-3 days and 12 days).  neurotransmitter  Todd (1980) reported that  i n nerves did not appear around the sapheous artery  u n t i l 3 days of age, as determined by the presence of fluorescence i n the developing innervation.  With t h i s i n mind, the intent of my study was  denervate the artery before the neurotransmitter appeared and had influence over the developing a r t e r y .  any  Also, two peaks i n the number of  nerves per unit area were seen by Todd (1980), one at 5 days of age the other at 12-15  days of age.  to  and  The greater the density of the  adrenergic plexuses, the greater the p o t e n t i a l 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, i n t h i s study.  However, the  s t a t i s t i c s proved that there was no difference i n denervating at 1-3 12 days of age.  This may  or  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 e f f e c t on the smooth muscle would be no d i f f e r e n t 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 f a c t o r s .  Ultrastructural  nerve p r o f i l e s very d i f f e r e n t i n appearance from p r o f i l e s of mature nerves are seen at 11 days (Todd and Tokito, 1981).  Even though  adrenergic terminals have been i d e n t i f i e d i n 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 i n the hind limb i s nonfunctional u n t i l after two weeks of age.  Even though I c o u l d f i n d no a b n o r m a l i t y i n the g r o s s morphology o f the saphenous a r t e r y i t s e l f , o t h e r a l t e r a t i o n s i n t h e g r o s s arrangement o f the denervated  v e s s e l s were n o t i c e d .  f l u o r e s c e n c e procedure,  When sampling  the e s t i m a t e d d i s t a n c e o f  s u p e r i o r e p i g a s t r i c and saphenous branch  0.5cm between t h e  p o i n t s from t h e femoral  and v e i n was n o t i c e a b l y decreased on the denervated d i l a t i o n i n the denervated very obvious.  during the  side only.  artery Also, a  femoral v e i n a t the a r e a o f i t s branches was  Such g r o s s s t r u c t u r a l changes have n o t been mentioned i n  the l i t e r a t u r e ; however, Bevan  (1984) does mention an i n c r e a s e i n  a r t e r i o v e n o u s anastomoses i n t h e denervated changes i n t h e p a t t e r n may be secondary  r a b b i t ear a r t e r y .  These  effects to a local alteration i n  b l o o d p r e s s u r e r e s u l t i n g from d e n e r v a t i o n . The r e s u l t s from the denervated  v e i n , where the v e s s e l became g r o s s l y  d i l a t e d , may be due t o s t r u c t u r a l changes i n the w a l l f o r two r e a s o n s . F i r s t , o t h e r s t u d i e s (Bevan and T s u r u , 1979; 1981) and t h i s one have i n d i c a t e d t h a t w a l l t h i c k n e s s decreases d e n e r v a t i o n causes a decrease  following denervation.  Since  i n the m i t o t i c index o f the smooth muscle  c e l l s from t h e media o f the r a b b i t e a r a r t e r y  (Bevan and T s u r u ,  1975)  t h i s suggests t h a t d e n e r v a t i o n may indeed cause a t h i n n e r w a l l .  Also,  maximum t e n s i o n ( f o r c e / c r o s s - s e c t i o n a l a r e a ) was l e s s i n the denervated r a b b i t e a r a r t e r y ; t h e r e f o r e , Bevan and Tsuru decrease  (1979) a t t r i b u t e d t h i s  t o a q u a l i t a t i v e change i n t h e c o n t r a c t i l e machinery.  T h e r e f o r e , 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 w a l l r e s u l t i n g i n t h e d i l a t e d v e i n s reported here. Secondly,  changes i n the c o n n e c t i v e t i s s u e f i b e r s  ( e l a s t i n and  c o l l a g e n ) may a f f e c t the r e s i s t a n c e o f the b l o o d v e s s e l w a l l .  Aneurysms  and v a r i c o s e v e i n s a r e c l i n i c a l examples o f d i l a t i o n s i n b l o o d v e s s e l s .  58 Although aneurysms are mainly associated with the a r t e r i a l side of the c i r c u l a t o r y system, aneurysms i n veins, such as the portal vein, do e x i s t (Ohnishi et a l . , 1984).  The e t i o l o g i e s o f these two c l i n i c a l examples  may involve changes i n the connective  tissue f i b r e 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  decrease i n perfusion pressure  (1982) suggested that the  (representing vascular resistance) of the  denervated rabbit ear vascular bed may not be exclusively due to a decrease i n smooth muscle mass, but may be the r e s u l t o f a l t e r a t i o n s i n the collagen or e l a s t i n .  Therefore,  the importance of collagen and  e l a s t i n i n the blood vessel wall should not be overlooked.  Fibroblasts  synthesize collagen and e l a s t i n as do smooth muscle c e l l s ; however, the f i b r o b l a s t s are only found i n the adventitia of blood vessels.  Also,  when Chamley and Campbell (1975) cultured smooth muscle c e l l s from the guinea pig vas deferens,  they described the u l t r a s t r u c t u r e of f i b r o b l a s t s  present i n the culture a f t e r 1-2 days i n culture.  However, i n their  study, they did not follow through on the appearance o f the f i b r o b l a s t s ; therefore, any e f f e c t that norepinephrine,  sympathetic ganglion extract  and c y c l i c 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 f i b r e s i n canine and human a r t e r i e s . Treatment o f canine common c a r o t i d a r t e r i e s and human external, i n t e r n a l and common i l i a c a r t e r i e s with elastase produced d i l a t i o n of these vessels, with a decrease i n compliance seen a t higher pressures.  The i n t e g r i t y o f these  elastase-treated vessels was always maintained.  With  collagenase  treatment, the vessels were l e s s d i l a t e d than the elastase-treated vessels, although they were s i g n i f i c a n t l y d i f f e r e n t from the controls. Also, the collagenase-treated vessels leaked uncontrollably and  59 eventually ruptured.  Thus, collagen i s needed to maintain the i n t e g r i t y  of the vessel whereas e l a s t i n i s needed to maintain the normal shape of the wall, but not the i n t e g r i t y . femoral vein i n t h i s study was rupture or leak.  The normal shape of the denervated  s i g n i f i c a n t l y d i l a t e d , but i t did not  The f a c t that the femoral vein did not rupture or leak  does not prove that collagen was  not changed at a l l i n amount; however,  the fact that the normal shape of the vessel was the e l a s t i n was  affected somehow.  changed suggests that  For instance, the normal content of  e l a s t i n might not have been attained during the development of the denervated vein thereby disturbing the e l a s t i c / c o l l a g e n r a t i o .  An  a l t e r a t i o n i n the e l a s t i c / c c l l a g e n r a t i o as a consequence of denervation was  suggested by Rusterholz and Mueller ( 1 9 8 2 ) .  Changes i n the  structure of the e l a s t i c f i b r e s themselves or i n t h e i r orientation  may  have been affected by the denervation procedure. The three-dimensional network of e l a s t i c f i b r e s i n canine saphenous veins has been studied by Crissman ( 1 9 8 4 ) .  In normal saphenous vein, he  found that the i n t e r n a l e l a s t i c lamina, the media and the e l a s t i c lamina each had t h e i r own  external  unique e l a s t i c f i b r e organization.  The  e l a s t i c f i b r e s 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 e l a s t i c f i b r e s i n the i n t e r n a l e l a s t i c lamina  consisting of large l o n g i t u d i n a l l y oriented  (at a s l i g h t angle from the  true longitudinal) branching f i b r e s intersected by f i n e r f i b r e s i s thought to d i s t r i b u t e stress around and along the longitudinal surface. The media also had two sets of f i b r e s , both larger than those of i n t e r n a l e l a s t i c lamina, but arranged somewhat the same.  However, the  longitudinal thicker f i b r e s were angled tangentially, either directed externally or i n t e r n a l l y , traversing d i f f e r e n t l e v e l s of the media and  did not form s t r a t i f i e d layers o f f i b r e s .  This organization i s thought  to d i s t r i b u t e tension throughout the venous w a l l .  The external e l a s t i c  lamina was formed by several p a r a l l e l layers o f wide ribbons of c l o s e l y opposing thick e l a s t i c f i b r e s . those i n adjacent elastic fibres.  Ribbons within the same l e v e l as well as  l e v e l s were interconnected  by the thick  and t h i n  I t was suggested that the arrangement and thickness o f  t h i s layer would increase i t s r i g i d i t y thereby maintaining  the shape o f  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 e l a s t i c network would contribute to vascular i n t e g r i t y and f l e x i b i l i t y as well as a i d i n the d i s t r i b u t i o n o f stress throughout the venous w a l l . denervation  With t h i s i n mind, i t i s possible that  may have disrupted the e l a s t i c architecture i n the wall o f  the r a t femoral vein i n my study.  Since Todd (1986) found  u l t r a s t r u c t u r a l changes i n the external e l a s t i c lamina, t h i s layer may be the one most affected. Changes i n the collagen content and other connective  components were  seen i n 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 o f i n t e r s t i t i a l s t a i n i n g with Alcian blue and Toluidine blue as well as an increase i n PAS p o s i t i v e 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 l o s s 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).  coincided with the biochemical  These histochemical r e s u l t s  r e s u l t s obtained by Niebes et a l . (1977).  They found that the insoluble collagen content was s i g n i f i c a n t l y l e s s i n the varicose veins, but the t o t a l amount o f glycosaminoglycans and  61 g l y c o p r o t e i n s was s i g n i f i c a n t l y g r e a t e r .  S i n c e no v a r i a t i o n s were i n the  smooth muscle o r e n e r g e t i c metabolism (Niebes e t a l . , 1977), t h e combined h i s t o l o g i c a l and b i o c h e m i c a l abnormalities  f i n d i n g s i n d i c a t e t h a t t h e main  o f v a r i c o s e veins a r e i n the connective  I t appears from past s t u d i e s t h a t c o n n e c t i v e important r o l e i n m a i n t a i n i n g  t i s s u e components.  t i s s u e components p l a y an  t h e shape o f t h e b l o o d  vessel wall.  I f the  c o l l a g e n component i s changed by l a c k o f i n n e r v a t i o n , then the w a l l may weaken and a d i l a t i o n such a s t h a t seen i n the femoral v e i n i n t h i s  study  may o c c u r . Not  many d e n e r v a t i o n  the r a t p o r t a l v e i n  s t u d i e s have been done on v e i n s .  Denervation o f  ( A p r i g l i a n o , 1983) produced s u p e r s e n s i t i v i t y o f t h e  smooth muscle c e l l membrane t o n o r e p i n e p h r i n e .  Morphological  studies o f  the denervated dog saphenous v e i n were c a r r i e d o u t by Branco e t a l . (1984).  They found t h a t the w a l l t h i c k n e s s  v e i n i n t h e dog was g r e a t e r  o f the denervated saphenous  than t h a t o f t h e c o n t r o l s .  In t h e i r  study,  t h i s t h i c k e r w a l l p e r s i s t e d i n the v e i n even a t 120 days post-denervation.  The smooth muscle c e l l s i n the denervated dog  saphenous v e i n had a d e 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 o f which was l a r g e r smooth muscle c e l l s . thickness  Since  I found a decrease i n m e d i a l  o f the saphenous a r t e r y , I assumed t h a t , i n my study, t h e media  o f t h e v e i n s would be t h i n n e r t o o , e s p e c i a l l y s i n c e t h e v e i n was s o dilated.  However, t h i s was n o t the c a s e .  Some c r o s s - s e c t i o n s o f t h e  d i l a t e d r a t femoral v e i n showed t h a t some a r e a s o f t h e w a l l were v e r y t h i n and tenuous.  A l t h o u g h , when f o u r randomly chosen p o i n t s were  measured and averaged t o g e t h e r ,  the w a l l o f the denervated femoral v e i n  i n my study was n o t t h i n n e r than t h a t o f the c o n t r o l s i d e . t o t a l mean w a l l t h i c k n e s s higher  I n f a c t , the  o f the denervated femoral v e i n tended t o be  than the mean o f the c o n t r o l v e i n .  T h i s d i f f e r e n c e was n o t  s i g n i f i c a n t a t the P<0.05 l e v e l t h a t the d i l a t i o n may  (Table I V ) .  I t i s possible, therefore,  not be the r e s u l t o f a decrease  i n the  c o n t e n t as i m p l i e d by the work o f D o b r i n e t a l . ( 1 9 8 4 ) , change i n the a r c h i t e c t u r e o f the e l a s t i c  fibres  elastin  but r a t h e r a  (Crissman,  1984).  No o t h e r m o r p h o l o g i c a l d i f f e r e n c e s between the denervated 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 s i d e were seen a t the  microscopic l e v e l i n t h i s present study.  r a t femoral  light  The e x p l a n a t i o n g i v e n f o r the  l a c k o f d i f f e r e n c e between d e n e r v a t i n g the saphenous a r t e r y a t the d i f f e r e n t age groups ( t h a t i s , a t 1-3  two  o r 12 days) p r o b a b l y a p p l i e s t o the  femoral v e i n .  CONCLUSIONS The method o f s u r g i c a l d e n e r v a t i o n i n t h i s study i s as s u c c e s s f u l as s u p e r i o r c e r v i c a l ganglionectomy, localized.  Although  a l t h o u g h my  method i s f a r more  r e - i n n e r v a t i o n from a b e r r a n t a r e a s does o c c u r ,  my  method o f d e n e r v a t i o n does produce a l t e r a t i o n s i n the b l o o d v e s s e l s . decreases  The  i n the m e d i a l a r e a o f the a r t e r y do p e r s i s t l o n g a f t e r  d e n e r v a t i o n (78-87  d a y s ) ; however, whether the decrease i s due t o a  r e d u c t i o n i n smooth muscle c e l l s i z e or a r e d u c t i o n i n the m a t r i x remains t o be determined.  paracellular  A l s o , t h i s denervation technique  leads  t o a r e p r o d u c i b l e d i l a t i o n o f the femoral v e i n , the mechanism by which t h i s d i l a t i o n o c c u r s i s unknown.  TABLE I: Changes i n the Area of the Tunica Media of the Saphenous Artery when Sampled at 60 Days of Age. Age at Denervation (Days old)  Age Sampled At (Days old)  Area of the Tunica Media of the Saphenous Artery Control Mean Denervated Mean Side Side (u ) (u ) 2  1-3  60  60  60  12  60  60  60  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  2  34559.4  40902.2  37379.1  36479.1  24936.2  46878.7  36855.8 +2969.8  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  24248.3  41305.4  34426.6  19664.5  26158.5  44180.9  31664.0 +4027.2  P=0.10 n=6 GO  TABLE I I : Changes i n the Area o f t h e T u n i c a Media o f t h e Saphenous A r t e r y When Sampled a t 90 Days o f Age Age a t Denervation (Days o l d )  Age Sampled A t (Days o l d )  Area o f the T u n i c a Media o f the Saphenous A r t e r y Control Mean Mean Denervated Side Side (u ) ( P ) 2  2  1-3  90  90  90  12  90  90  90  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  P=0.05  41905.8  41714.4  42564.5  44384.7  47713.1  50568.2  44808.4 +1470.0  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  34906.5  35861.6  45831.7  43132.7  33698.0  37455.3  38481.0 +4883.4  TABLE I I I : Comparison o f the P e r i m e t e r s o f the Denervated and C o n t r o l Femoral V e i n  Age  a t Denervation (Days o l d )  12  Age Sampled At (Days o l d )  Control Side ("2)  60  Perimeter Mean  3808 3796  6895 3797  3792 60  1-3  3317 3327 3328  90  90  12  2809 2709 2862  90  3324  2793  4439  7268 7198 4844 4909 4910  7224  4888  6776  6757  6743  4318 4216  4461 4427 4430  6751 4490  4471  90  6874  7206 4237  4512 HH88  6878 6850  4425 4273 4213  o f Femoral V e i n Denervated Mean Side (u2)  6801 4289  4333  6766  6817  6885  TOTAL MEAN  3822  6167  +SEM  +267  +483  P=0.0005 n=6  66  TABLE IV: Comparison of Wall Thickness between the Denervated and Control Femoral Vein. Age at Denervation (days old)  Age at Sampling (days,old)  Mean Thickness of Femoral Vein Wall Denervated Control Side ( M ) 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  4.94 +0.81  5.96 +1.58  TOTAL MEAN +SEM 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 *DN (days ild)  Age Sampled At (days old)  Number of Sections: Arteries  +  Average Number per Saphenous Artery Control Denervated Side Side  +Average Number per Femoral Vein Control Denervated 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 n=8  *DN = denervation r e f e r s to the average number of fluorescing areas.  +  Number o f Sections: Veins  P:=0.004 n=7  Figure  1: D e n e r v a t i o n Procedure,  w i t h the  skin reflected.  a r t e r y and and  vein,  The  (b) show the r a t  ( i ) femoral n e r v e , ( i i ) the  thigh femoral  ( 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  ( i v ) the saphenous a r t e r y and  s u t u r e s i l k has  (a) and  v e i n can  been t i e d around the  be s e e n .  In ( b ) ,  f e m o r a l nerve i n two  vein black  places.  F i g u r e 2: D e n e r v a t i o n Procedure c o n t i n u e d ,  (a) In the t h i g h , the  ( i ) f e m o r a l nerve i s s e v e r e d j u s t d i s t a l t o the second knot (arrow).  The d i s t a l p o r t i o n o f the f e m o r a l nerve i s s e p a r a t e d from  the ( i i ) f e m o r a l a r t e r y and v e i n up t o the branch p o i n t s o f 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 v e i n s . The nerve i s then s e v e r e d a g a i n a t the branch p o i n t s  (arrowhead),  (b) I n s i d e the abdominal c a v i t y , the f e m o r a l nerve i s threaded through the tube and the end i s heat s e a l e d a s t e r i s k i n d i c a t e s the abdominal  wall.  (arrowhead)  The  F i g u r e 3: Diagram showing  the c a n n u l a t i o n procedure.  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  The  carotid  ( i ) and the c a n n u l a i s  i n s e r t e d through the l o o s e knot ( i i ) and then i n t o a s m a l l 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 c a n n u l a i s i n s i d e the a r t e r y ,  then  the s m a l l a r t e r i a l clamp ( i i i ) i s r e l e a s e d so the cannula can be pushed  i n more c a u d a l l y .  The second knot ( i v ) , the one most  c a u d a l l y , and the l o o s e , more c r a n i a l knot  ( i i ) are t i g h t e n e d .  F i g u r e 4; and  P e r f u s i o n Pressure Recording.  The (a) mean, (b) s y s t o l i c  (c) d i a s t o l i c blood pressures are recorded,  carried  o u t a t t h e mean b l o o d p r e s s u r e .  (d) P e r f u s i o n i s  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. breaks i n the thicker i n t e r n a l e l a s t i c lamina (arrows) and  The thinner  i n t e r n a l e l a s t i c lamina (arrowheads) are seen i n both (a) and (b). There i s no remarkable difference between the denervated and i t s c o n t r a l a t e r a l control side.  These samples were taken from an animal  denervated at 3 days and sampled at 60 days of age. for (a) and (b) i s 670X.  Magnification  Figure  6: L i g h t micrographs o f the  femoral v e i n s . a r e not  so e a s i l y  endothelium has (a) and  The  (a) c o n t r o l and  t h r e e t u n i c s so c h a r a c t e r i s t i c  i d e n t i f i a b l e i n these v e i n s .  a granular  (b) i s 6 9 0 X .  appearance ( a r r o w s ) .  In  (b) denervated of blood (b),  vessels  the  Magnification  for  79  b  Figure 7: Light micrographs of the (a) normal saphenous nerve taken from the control side and (b) the d i s t a l 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 o v e r a l l h i s t o l o g i c a l appearance has changed, and there  i s a marked decrease i n 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). within the abdominal cavity.  Samples were taken from  Sample (b) was taken just proximal to  the tube and therefore represents the proximal nerve stump.  There  i s a marked decrease i n size and a noticeable change i n the histology of the nerve that i s tubed.  The i n s e r t (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.  F i g u r e 9- U l t r a s t r u c t u r a l l y , the f e m o r a l nerve  (same sample as seen  i n F i g u r e 8(b)) i s composed m a i n l y o f c o l l a g e n  (5610X).  Fibroblasts  (arrows) can be seen i n the midst o f a l l the c o l l a g e n . Membrane-lined a r e a s o f d e g e n e r a t i o n (arrowheads) a r e p r e s e n t . c a p i l l a r y i s l o c a t e d i n the c e n t r e o f the micrograph.  The  A  insert  i l l u s t r a t e s a s m a l l m y e l i n a t e d nerve ( a s t e r i s k ) and an unmyelinated nerve  (arrow) observed i n the c o r e o f t h i s sample  (16030X).  85  F i g u r e 10: F l u o r e s c e n c e microscopy o f c o n t r o l and d e n e r v a t e d saphenous a r t e r i e s .  Trypan b l u e causes the e l a s t i c t i s s u e t o  f l u o r e s c e i n the r e d range,  (a) Catecholamine f l u o r e s c e n c e from the  a d r e n e r g i c t e r m i n a l s i s seen a t the a d v e n t i t i a l - m e d i a l border o f the c o n t r o l saphenous a r t e r y ,  (b) V i r t u a l l y no f l u o r e s c e n c e o f  c a t e c h o l a m i n e s i s seen i n the denervated a r t e r y .  F i g u r e 11: F l u o r e s c e n c e microscopy veins.  o f c o n t r o l and denervated  femoral  Dense i n n e r v a t i o n i s seen i n the (a) c o n t r o l v e r s u s the (b)  denervated  saphenous v e i n .  F i g u r e 12: Per cent decrease i n m e d i a l a r e a o f denervated a r t e r i e s from a n i m a l s sampled a t 60 and 90 days o f age. s i d e r e p r e s e n t s 100?.  saphenous The  control  100 80 %  •  Control  H  Denervated  I  I  60 40 20 6 0 days Saphenous  9 0 days Artery  F i g u r e  13:  Per cent i n c r e a s e I n l u m i n a l p e r i m e t e r  f e o r a l veins. m  The  c c n t r c l side represents  100*.  of  enervated  Femoral  Vein  BIBLIOGRAPHY  Aprigliano, 0. ( 1 9 8 3 ) Neural Influences and Norepinephrine S e n s i t i v i t y i n the Rat Portal Vein. Federation Proc. 42:257-262.  Barr, M.L. and Kiernan, J.A. ( 1 9 8 3 ) The Human Nervous System. An Anatomical Viewpoint. 4^ Ed. Harper and Row, Publishers, Philadelphia. n  Bevan, J.A. and Su, C. ( 1 9 7 3 ) Sympathetic Mechanisms i n Blood Vessels: Nerve and Muscle Relationships, Annual Review of Pharmacology. 13:269-285.  Bevan, R.D. (1984) Trophic E f f e c t s of Peripheral Adrenergic Nerves on Vascular Structure. Hypertension 6[Suppl. III]:III-9 - 111-26.  Bevan, R.D. (1975) E f f e c t of Sympathetic Denervation on Smooth Muscle C e l l P r o l i f e r a t i o n i n the Growing Ear Artery. C i r c . Res. 37:14-19.  Bevan, R.D., Tsuru, H and Bevan, J.A. (1983) Cerebral Artery Mass i n 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 i n the Rabbit Ear Artery a f t e r Sympathetic Denervation. C i r c . 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 i n the Newborn Dog. J . of C l i n i c a l Investigations 44:241-246.  Branco, D., Albino T e i x e i r a , A., Azevedo, I., and Osswald, W. (1984) Structural and Functional Alterations caused at the Extraneuronal Level by Sympathetic Denervation of Blood Vessels. Naunyn-Schmiedeberg's Arch. Pharmacol.326:302-312  Brody, M.J. (1964) Cardiovascular Responses Following Immunological thectomy. C i r c . Res. 15:161-167.  Sympa-  Burnstock, G. and Costa, M. (1975) Adrenergic Neurons. Chapman & H a l l ,  Ltd., London.  Chamley, J.H. and Campbell, G.R. (1975) Trophic Influences of Sympathetic Nerves and C y c l i c AMP on D i f f e r e n t i a t i o n and P r o l i f e r a t i o n of Isolated Smooth Muscle C e l l s i n Culture. C e l l Tissue Research 161:407-510.  Chamley, J.H. and Campbell, G.R. (1976) Tissue Culture: Interaction Between Sympathetic Nerves and Vascular Smooth Muscle. In Vascular Neuroeffector Mechanisms, edited by J.A. Bevan, G. Burnstock, B. Johansson, R.A. Maxwell and O.A. Nedergaard. B a s i l , Karger, pp 10-18.  Chen, G., Portraan, R. and Wickel, A. (1951) Pharmacology of 1,1-dimethyl4-phenyl piperazinium iodide, a Ganglionic Stimulating Agent. J . Pharmacology 103:330  C o l i n , W. and Donoff, R.B. (1984) Nerve Regeneration Tubes. J . Dent. Res. 63(7):987-993.  Through Collagen  Crissman, R.S. (1984) The Three-Dimensional Configuration of the E l a s t i c Fibre Network i n Canine Saphenous Vein. Blood Vessels 21:156-170.  De l a Lande, I.S. and Waterson, J.G. (1968) Modification of Autofluorescence i n 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) E l a s t o l y t i c and l y t i c Studies of A r t e r i e s . Arch. Surg. 119:405-409.  Collageno-  Dolezel, S., Gerova, M. and Gero, J . ( 1 9 7 4 ) Postnatal development of the sympathetic innervation i n s k e l e t a l muscles of the dog. Physiol. Bohemoslov.  23:138-139.  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. S c i . 228:3-5.  Dyck, P.J. and Hopkins, A.P. (1972) Electron Microscopic Observations Degeneration and Regeneration of Unmyelinated F i b r e s . Brain 95:223-234.  of  96 E n g e l , A.G. and S t o n n i n g t o n , H.H. (1974) M o r p h o l o g i c a l E f f e c t s o f Denerv a t i o n o f 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 T r o p h i c F u n c t i o n s o f the Neuron, e d i t e d by D.B. Drachman, Annals New York Acad. S c i . 228:68-88.  F i n c h , L., H a e u s l e r , G. and Thoenen, H. (1973) A Comparison o f the E f f e c t s o f Chemical Sympathectomy by 6-Hydroxydopamine i n Newborn and A d u l t R a t s . B r . J . Pharm. 47:249-260.  G a b e l l a , G. (1976) S t r u c t u r e o f the Autonomic Nervous System. Chapman and H a l l , L t d . , London.  G a u t h i e r , G.F. and Dunn, R.A. (1973) U l t r a s t r u c t u r a l and C y t o c h e m i c a l F e a t u r e s o f Mammalian S k e l e t a l Muscle F i b e r s F o l l o w i n g D e n e r v a t i o n . J . C e l l S c i . 12:525-547.  Gerova, M., Gero, J . , D o l e s e l , S. and Konecny, M. (1974) P o s t n a t a l Development o f Sympathetic C o n t r o l i n Canine F e r m o r a l A r t e r y . P h y s i o l o g i a 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 T u n i c a Media o f the D e v e l o p i n g Rat. L a b o r a t o r y I n v e s t i g a t i o n 32:585-600.  G r o b e t y , J . and B o u v i e r , C A . (1977) S t u d i e s on Normal and V a r i c o s e Human Saphenous V e i n s I : S t r u c t u r a l D i f f e r e n c e s , H i s t o c h e m i c a l and E l e c t r o n Microscope I n v e s t i g a t i o n s . 9 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. t n  Gutman, E. (1976) N e u r o t r o p h i c R e l a t i o n s . Annu. Rev. P h y s i o l .  Ham,  A.W. and Cormack, D.H. (1979) H i s t o l o g y 8 Company P h i l a d e l p h i a and T o r o n t o , p. 575.  t n  38:177-216.  Ed., J.B. L i p p i n c o t t  Hayat, M.A. (1970) P r i n c i p l e s and Techniques o f E l e c t r o n Microscopy: B i o l o g i c a l A p p l i c a t i o n s V o l . 1. Van Nostrand R e i n h o l d Company, New York. Hoffman, W.W. and T h e s l e f f , S. (1972) S t u d i e s on the T r o p h i c I n f l u e n c e o f Nerve on S k e l e t a l Muscle. E u r . J . Pharmac. 20:256-260.  Humphrey, C. and P r i t t m a n , F. (1974) A Simple Methylene blue-Azure I I B a s i c F u c h s i n f o r Epoxy-Embedded T i s s u e S e c t i o n s . S t a i n Tech. 49:9-14.  t  I l l u s t r a t e d Stedman's M e d i c a l D i c t i o n a r y , 2 4 " E d . (1982) W i l l i a m s and W i l k i n s , B a l t i m o r e , MD.  K a n a k i s , S.J., H i l l , C.E., Hendry, I.A. and Watters, D.J. (1985) Sympat h e t i c Neuronal S u r v i v a l F a c t o r s Change a f t e r D e n e r v a t i o n . Dev. B r a i n Res. 20:197-202.  Kobayashi, S., Tsukahara, S., T s u j i , T., S u g i t a , K. and Nagata, T. (1983) H i s t o c h e m i c a l S t u d i e s on Regeneration o f Aminergic Nerves i n Rat Cere b r a l A r t e r y a f t e r S u p e r i o r C e r v i c a l Ganglionectomy H i s t o c h e m i s t r y 77:57-62.  L a i s , L.T., R i o s , L.L., B o u t e l l e , S., DiBona, G.F. and Brody, M.J. ( 1 9 7 7 ) A r t e r i a l P r e s s u r e Development i n Neonatal and Young Spontaneously H y p e r t e n s i v e R a t s . Blood V e s s e l s 14:277-284.  L e n t z , T.L. (1974) N e u r o t r o p h i c R e g u l a t i o n a t the Neuromuscular J u n c t i o n . Ann. New York Acad. S c i . 228:323-337.  L e v i - M o n t a l c i n i , R. and A n g e l e t t i , P.U. Pharmac. Rev. 18:619-628.  (1966) Immunosympathectomy.  Luco, J.V. and E y z a g u i r r e , C. (1955) F i b r i l l a t i o n and H y p e r s e n s i t i v i t y t o Ach i n Denervated Muscle. E f f e c t o f Length o f Degenerating Nerve F i b r e s . J . N e u r o p h y s i o l . 18:65-73-  Mclnnes, A. (1977) M o d i f i c a t i o n o f the F a l c k - H i l l a r p Technique w i t h I n t r a v i t a l Trypan b l u e t o D i f f e r e n t i a t e E l a s t i c F i b r e s from N o r a d r e n e r g i c E n d i n g s . J . P h y s i o l . 268:22P-23P.  Mollenhauer, H.H. (1964) P l a s t i c Embedding M i x t u r e f o r use i n E l e c t r o n M i c r o s c o p y . S t a i n Tech. 39:111-114.  Niebes, P., E n g e l s , E. and j e g e r l e h n e r , M. (1977) S t u d i e s on Normal V a r i cose Human Saphenous V e i n s I I : D i f f e r e n c e s i n the Compostion o f C o l l a g e n and G l y c o s a m i n o g l y c a n s . 9 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. t h  O h n i s h i , K., Nakayama, T., S a i t o , M., Nomura, F., Koen, H., Tamaru, J . , Iwasaki, I . and Okuda, K. ( 1 9 8 4 ) Aneurysm o f the I n t r a h e p a t i c Branch o f the P o r t a l V e i n . G a s t r o e n t e r o l o g y 8 6 : 1 6 9 - 1 7 3 .  Page, I.H. and McCubbin, J.W. (1965) Chapter 61: The P h y s i o l o g y o f A r t e r i a l H y p e r t e n s i o n . In Handbook o f P h y s i o l o g y . S e c t i o n 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 S o c i e t y , Wash., DC.  P a l a t y , V.  (1971) D i s t r i b u t i o n o f Magnesium i n the A r t e r i a l W a l l .  J.Physiol. 218:353-368.  Pease, D.C. (1964) H i s t o l o g i c a l Techniques for Electron Microscopy. Ed. Academic Press Inc., New York and London.  2  Pellegrino, C. and F r a n z i n i , C. (1963) An Electron Microscope Study of Denervation Atrophy i n Red and White Skeletal Muscle F i b e r s . J . C e l l B i o l . 17:327-349.  Pellegrino, R.G. and Spencer, P.S. (1985) Schwann C e l l Mitosis i n Response to Regenerating Peripheral Axons i n v i v o . Brain Res. 341:16-25.  P o l i t i s , M.J., Ederle, K. and Spencer, P.S. (1982) Tropism Nerve Regener a t i o n i n vivo. A t t r a c t i o n of Regenerating Axons by D i f f u s i b l e Factors Derived from C e l l s i n D i s t a l 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 C l i n i c a l Pathologists Press, Chicago, pp. 35-43.  Szekere, L. (ed.) (1980) Adrenergic Activators and I n h i b i t o r s . Handbook of Experimental Pharmacology 54:1, Springer-Verlag, B e r l i n .  Thesleff, S. (1974) P h y s i o l o g i c a l E f f e c t s of Denervation of Muscle. In Trophic Functions of the Neuron, edited by D.B. Drachman. Ann. New York Acad. S c i . 228:89-104.  Todd, M.E. ( 1 9 8 0 ) Development of Adrenergic Innervation i n 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 Charact e r i s t i c s of Smooth Muscle i n Rat Blood Vessels: A Computer-Assisted Analysis. C i r c . Res. 5 3 : 3 1 9 - 3 3 1 .  Todd, M.E. and Tokito, M.K. (1981) An U l t r s t r u c t u r a l Investigation of Developing Vasomotor Innervation i n Rat Peripheral Vessels. Amer. J . Anat. 160:195-212.  Todd, M.E. (1986) T r o p h i c I n t e r a c t i o n s Between Rat Nerves and Blood V e s s e l s i n Denervated P e r i p h e r a l A r t e r i e s and i n A n t e r i o r Eye Chamber T r a n s p l a n t s . C i r c . Res. 58: In P r e s s .  Waris, T. (1978) R e - i n n e r a t i o n o f Free S k i n A u t o g r a f t s i n the Rat. J . P l a s t . Reconstr. Surg. 12:85-93.  W i l l i a m s , P.L. and Warwick, R. (1975) F u n c t i o n a l Neuroanatomy o f P h i l a d e l p h i a , W.B. Saunders.  Scand.  Man.  

Cite

Citation Scheme:

    

Usage Statistics

Country Views Downloads
China 8 10
United States 7 8
Germany 4 2
United Kingdom 3 0
Japan 2 0
Canada 2 0
Taiwan 1 0
City Views Downloads
Unknown 7 2
Shenzhen 7 10
Ashburn 6 0
Surrey 2 0
Tokyo 2 0
Mountain View 1 8
Beijing 1 0
Taipei 1 0

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

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0096806/manifest

Comment

Related Items