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Establishment of the autonomic neuroanatomy to the vulval erectile tissues Penhale, Shona Marie Louise 2003

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E S T A B L I S H M E N T OF T H E A U T O N O M I C N E U R O A N A T O M Y TO THE V U L V A L ERECTILE TISSUES  by SHONA MARIE LOUISE P E N H A L E B . S c , The University of British Columbia, 2000 A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U 1 R M E N T S FOR T H E D E G R E E OF M A S T E R S OF S C I E N C E  in F A C U L T Y OF G R A D U A T E STUDIES ( D E P A R T M E N T OF A N A T O M Y )  We accept this thesis as conforming to the required standard  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A September 2003 © Shona Marie Louise Penhale  U B C Rare Books and Special Collections - Thesis Authorisation F o r m  Page 1 o f 1  In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t copying o r p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver, Canada  http://www.library.ubc.ca/spcoll/thesauth.html  07/10/2003  ABSTRACT  E r e c t i l e tissues are the v a s c u l a r structures w i t h i n the v u l v a that fill w i t h b l o o d a n d are an integral c o m p o n e n t o f the w o m a n ' s s e x u a l response. D e s p i t e the i m p o r t a n c e o f these structures to her s e x u a l f u n c t i o n , the d e t a i l e d n e u r o a n a t o m i c a l p a t h w a y s o f the a u t o n o m i c n e r v e s u n d e r l y i n g t h i s m e c h a n i s m o f v a s c u l a r c o n g e s t i o n , h a v e not b e e n  elucidated.  T h e r e f o r e , this research h a d been d e s i g n e d to identify the m i s s i n g c o m p o n e n t s to answer the q u e s t i o n : " W h a t is the p a t h w a y o f the nerves r u n n i n g f r o m the f e m a l e p e l v i c p l e x u s to the external g e n i t a l i a ? " . I n the m a l e , the a u t o n o m i c i n n e r v a t i o n o f the erectile tissue i n the penis has been f u l l y d e s c r i b e d s u c h that damage f r o m surgery can be a v o i d e d or m i n i m i z e d w h e n e v e r p o s s i b l e . T h e c a v e r n o s a l nerves arise f r o m the prostatic p l e x u s a n d pierce the u r o g e n i t a l d i a p h r a g m ( U G D ) to s u p p l y the erectile tissue o f the c a v e r n o s a l bodies. I n contrast, the d e s c r i p t i o n o f the i n n e r v a t i o n o f the female erectile tissue is m u c h less complete a n d there are n o descriptions o f the nerves b e t w e e n the v a g i n a l p l e x u s and the " a n t e r i o r parts". S p e c i f i c a l l y , their o r i g i n f r o m the v a g i n a l p l e x u s and their precise path to the erectile structures o f the v u l v a are u n k n o w n a n d any s i m i l a r i t i e s to m a l e c a v e r n o s a l nerves are u n k n o w n . A d d i t i o n a l l y , whether or not there are i n d i v i d u a l branches to the specific erectile structures, w h i c h i n c l u d e the right a n d left c l i t o r a l c o r p o r a , bisected vestibular b u l b a n d periurethral tissues is also unclear. U t i l i z i n g cadaver tissue, the complete p a t h w a y o f the cavernous nerve i n the female w a s e x a m i n e d u s i n g several methods. U s i n g gross d i s s e c t i o n o f intact p e l v i s e s I i d e n t i f i e d p r e v i o u s l y w e l l d o c u m e n t e d structures o f the r e p r o d u c t i v e organs, major p e l v i c vasculature, the p e l v i c p r o m o n t o r y , the sacral nerves, the sympathetic c h a i n g a n g l i a , the superior h y p o g a s t r i c p l e x u s ( S H P ) , the i n f e r i o r hypogastric p l e x u s , and the p e l v i c s p l a n c h n i c nerves. T h e putative cavernous nerve was then i d e n t i f i e d o n the lateral w a l l o f the v a g i n a at the l e v e l o f the v e s i c o u t e r i n e p o u c h . A t this p o i n t it c o u r s e d i n f e r i o r l y a l o n g w i t h the v a g i n a a n d  11  urethra towards the muscular urogenital diaphragm ( U G D ) . During the inferior course it moved anteriorly into the dense connective tissue between the urethra and vagina and ended up 5 m m lateral to the urethra at the level o f the U G D . Previously the cavernous nerve was described as a single, large, easily dissected nerve within the erectile tissue that surrounds the periurethral sponge. In contrast, serial sectioning followed by histological analysis allowed me to determine that the cavernous nerve was most commonly a single nerve bundle that branched into 2-5 smaller nerves upon piercing the U G D . Several branches then coursed laterally, came within 1 m m o f the pudendal nerve, and innervated the crua; more medial branches carried on in their orientation to feed the vestibular bulbs. In both serial sectioning and marco dissection I was able to identify differences from previously documented structural anatomy o f the bisected vestibular bulbs . W e found all our specimens to have much more erectile tissue o f the bulbs continue around the anterior wall o f the urethra in the shape o f an inverted " u " . N o evidence o f the 'bisected' nature o f the bulbs was found, as well the bulbs had more fullness o f depth than previously documented. This research has provided a more detailed description o f the pathway o f the autonomic innervation as it passes through the pelvis to the external genitals than had been previously described in the literature. In particular the course o f the cavernous nerve as it passes along the lateral wall o f the vagina to pierce the U G D and branch to feed the various erectile tissues. This detailed description should enable the necessary immunohistochemical studies to confirm nerve content. It has also raised questions about the previously established bisected structure o f the vestibular bulbs which may have significant implications for engorgment in sexual arousal.  in  T A B L E  O F  C O N T E N T S  ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST OF T A B L E S  v  LIST OF FIGURES  vi  LIST OF A B B R E V I A T I O N S  vii  LIST OF APPENDICES  viii  ACKNOWLEDGMENTS  ix  INTRODUCTION  1  HISTORICAL REVIEW  1  PELVIC A N A T O M Y  5  Bone and muscle structure  5  Reproductive tract  6  Perineum  7  Innervation  9  F I G U R E S 1-7  12-24  THESIS OBJECTIVES A N D R A T I O N A L E  25  METHODS AND MATERIALS  26  TISSUE P R E P A R A T I O N  26  GROSS A N A T O M I C A L DISSECTION  26  HISTOLOGICAL PROCESSING  27  3D M O D E L  29  TABLE 1  32  F I G U R E 8-13  34-44  RESULTS  45  MACROSCOPIC  45  MICROSCOPIC  47  F I G U R E S 14-20  51-75  DISCUSSION  76  REFERENCES  83  iv  APPENDIX A-B  88-89 LIST O F T A B L E S  Table 1.  Table o f variety o f specimens and techniques used  V  32  LIST OF FIGURES Figure #  Page #  Fig. 1  Representative course o f the cavernous nerve in the male  16  Fig. 2  Pelvic bone structure  18  Fig. 3  Muscles o f the pelvic diaphragm  20  Fig. 4  Suspension o f the pelvic organs  22  Fig. 5  Urogenital and anal triangles  24  Fig. 6  Urogenital diaphragm  26  Fig. 7  Innervation o f the pelvic viscera  28  Fig. 8  Illustration o f removal/access o f abdomen and pelvis  34  Fig. 9  Mid-sagittal pelvis with rectal branches  36  Fig. 10  Sample o f tissue blocks removed for processing  38  Fig 11  Serial section used in 3 D reconstruction  40  Fig. 12  Schematic o f 3D image development  42  Fig. 13  Different views o f 3 D image  44  Fig. 14  Gross anatomical dissection o f pelvis  51  Fig. 15  Lateral pelvic wall dissection  53  Fig. 16  Lateral pelvic wall with I H P visualization  55  Fig. 17  Lateral view o f dissected U G D  57  Fig. 18  Histological cross-section o f cavernous nerve origin  59  Fig. 19  Histological cross-section U G D  61  Fig. 20  Histological cross-section o f external genitalia  63  Fig. 21  Histological sections o f U G D and area - specimen 7  65  Fig. 22  Histological sections o f U G D and area - specimen 22  67  Fig. 23  Histological sections o f U G D and area - specimen 24  69  Fig. 24  Histological sections o f U G D and area - specimen 9  71  Fig. 25  Histological whole mount section o f U G D  73  Fig. 26  Illustration o f vestibular bulb structure and orientation  75  vi  LIST OF ABBREVIATIONS ANS  Autonomic Nervous System  CNS  Central Nervous System  H&E  Haematoxylin and Eosin  IHP  Inferior Hypogastric Plexus  L2  Lumbar Nerve 2  PNS  Parasympathetic Nervous System  S2  Sacral Nerve 2  S4  Sacral Nerve 4  SHP  Superior Hypogastric Plexus  SNS  Sympathetic Nervous System  Til  Thoracic Nerve 11  UGD  Urogenital Diaphragm  vii  LIST OF APPENDICES Appendix A - Permission from Elsevier for journal figure use  88  Appendix B - Ethics Approval from U B C  89  viii  ACKNOWLEDGMENTS A s I write this, the last page o f my work, I find myself truly inspired by and grateful for the experiences I have had i n this department. Thus thanks must first go to everyone involved with the Roskelley lab. Thanks for your patience and for helping me navigate the grad studies maze. To the faculty and staff in the Department o f Anatomy: Our Friday afternoons together were a great source o f enjoyment and enlightenment for me. I won't miss the smell but I w i l l miss Charlie's perspective on things, Wayne's enthusiasm, and the smiles o f Betty, Louise and Donna which always greeted me. Bruce, I can never repay you for the countless times you lent me your ear. Thanks to all o f you for your never ending support and encouragement.... A very special thank you to K i m Fredriksen and Elizabeth Wellwood. Both o f you gave time well beyond what was required o f you. Y o u saved me countless hours and always help me stay focussed. Cameron and Karen, your dedication to perfection took my data from ordinary to extraordinary. What started as a research project has culminated in a friendship that w i l l last a lifetime. To my committee: Drs. Sydney Thompson, C a i Roskelley, Wayne V o g l and Rosemary Basson. Dr. Thompson, thank you not only for participating at my defence but also for your encouragement along the way. Cai, you took a chance on being my supervisor and have given me a second chance to live my dream. "Thank y o u " seems so lame by comparison. Wayne, your enthusiasm for anatomy is an inspiration to all those around you; thank you for instilling it in me. Rosemary, you are everything one wishes for in a mentor. Y o u have given me such big shoes to fill. To my husband Bob: thank you for your constant support in every area o f our lives together. Despite the difficulties, it is the growing we have both done as individuals that makes us so strong as a couple. To the rest o f my two families, thank you. Y o u r unconditional support o f Bob and me was a crucial factor i n the completion o f this work. Finally, I would like to dedicate this thesis to my beautiful daughters Grace and Katherine. When things got crazy you were always there to bring me back to what is important in life. Some day I hope you w i l l truly understand not only the joy you have brought into my life but that it was the two o f you that inspired my belief that anything is possible, you just have to use your imagination. M a y your desire to learn and grow as individuals throughout your lifetime be as enduring as my love for you.  IX  INTRODUCTION  HISTORICAL REVIEW The study o f functional neuroanatomy has a long history. Claudius Galen o f Pergammon ( A D 129-199) was a talented anatomic dissector who provided the earliest known description o f visceral innervation (the ganglionated sympathetic trunk and its neuronal distributions). H e also discovered the anatomic and physiologic basis for the peripheral nervous system after experimental observations that injury to a peripheral nerve produced consequences that included a loss o f muscle activity or skin sensitivity. This work resulted in Galen being credited with the discovery o f the scientific method. The sophistication o f his work becomes all the more remarkable when one recognizes that it was done close to 2,000 years ago. Vesalius, who wrote De Humanis Corporis Fabrica, the first complete anatomy textbook, and many other scientist continued to enunciate the mysteries o f the human body and by the 1600's neuroanatomical studies began to accelerate. In 1664 Thomas W i l l i s coined the term "neurology" with the first reasonably accurate drawings o f the sympathetic trunk branches, white and grey rami, splanchnic nerves, prevertebral ganglia, and visceral plexuses. H i s research gave the first physical representation to the theories o f innervation o f the body (qtd.in Feindel, 1970). In the late 1700's anatomy continued to evolve into what is commonly considered the "classical" form. In general this was marked by efforts to more precisely portray the general anatomical structures that were already known. F o r example, Walter, 1804 described spinal nerve outflow and the nerve plexus organization o f the thorax, abdomen, and pelvis o f the human male in remarkable detail. H i s illustrations are probably the first to explicitly represent the nerve supply to the visceral structures in the pelvis. Tiedemann, Beck, and Lee (1822) provided the first modern descriptions o f the abdominal and pelvic sympathetic chain ganglia that supply the uterus and adjacent  structures in the female. O f particular note is the work o f Frankenhauser, (1865) who went to great lengths to describe the pelvic origins o f the sympathetic nervous system as it related to uterine anatomy (qtd. in Davis, 1933). Commensurate with the efforts to describe the physical layout o f the human nervous system were experiments designed to determine function. However, it wasn't until the mid 1800s that Claude Bernard, whose series o f nerve transection experiments in rabbits, confirmed the existence o f efferent and afferent pathways that carry out visceral spinal cord reflexes (qtd. in Olmsted, 1970). In 1831 Michael Faraday invented the electrical generator and thus enabled the study o f electrophysiology. Its first known application to pelvic innervation research was by Budge (1858), who demonstrated that electrical stimulation o f the inferior mesenteric ganglion or hypogastric nerves in rabbits produced contractile responses in the vas deferens and seminal vesicles. Just five years later Conrad Eckhard established the role o f the visceral rami o f the sacral nerves by demonstrating that their stimulation led to penile erection in the dog (Ekhart, 1863)(qtd in Davis, 1933).  John Newport Langley later expanded his studies  into neuropharmacology and his studies o f the effects o f nicotine enabled h i m to map out preganglionic and postganglionic neurons (Langley, 1916); terminology that is still in use today. Other terms that he coined include "autonomic nervous system" during his study o f the male pelvis (Langley et al., 1895) and "parasympathetic" based on his studies using nicotine and curare (Langley, 1906). The groundwork laid by Langley and his contemporaries allowed for rapid advancement o f knowledge in local functions o f the nervous system in various parts o f the body, including the pelvis. From this arose the concepts o f antagonistic, parasympathetically-based cholinergic and sympathetically-based adrenergic control o f visceral functions; concepts which were the core o f autonomic neurobiologic activity for greater than half a century.  2  A t the beginning o f the last century, the neuroanatomical and neurochemical functioning o f the pelvis began to receive great scrutiny. Hashimoto, 1904, Latarjet et al., 1913, Delmas, 1933 and Curtis et al., 1942 all carried out microscopic dissections that revealed the spinal derivations, morphology, anatomic relationships, and neuronal interconnections o f the pelvic plexuses. They also documented the anatomic course and regional terminations o f peripheral nerves. The net result o f this work was one o f the first detailed descriptions o f the hypogastric plexus. The hypogastric plexus is a considerably sized, widely innervational, three-dimensional structure situated laterally adjacent to the deep pelvic viscera that, is composed o f an interlacement o f nerve fibers containing numbers o f extremely small, almost microscopic, ganglia (Davis, 1933). Walsh and Donker's work (1982) built on the earlier concepts o f autonomic function but focused strictly on male genitourinary system and pelvis. B y serial sectioning tissue from male fetuses and newborns, they confirmed that autonomic innervation o f the pelvic organs and external genitalia arises from the pelvic plexus. They also demonstrated that the parasympathetic visceral fibers arise from the sacral center (S2-S4) and attach to the pelvic plexus v i a the pelvic nerve while sympathetic fibers from the thoracolumbar center (TI 1-L2) flow into the pelvis v i a the sympathetic trunk to the inferior hypogastric nerve to the plexus. The pelvic plexus forms a fenestrated plate retroperitoneally beside the rectum and extends from the sacrum, ventrally as high as the rectouterine pouch in the male newborn. The plexus provides visceral branches that innervate the bladder, ureter, seminal vesicles, prostate, rectum, urethra and corpora cavernosa. These autonomic fibers also innervate pelvic vasculature and contain motor sensory fibers that extend to the levator ani, coccygeus and striated urethral musculature. Despite the detail in Walsh's work, the precise pathway o f the autonomic innervation distal o f the prostate was not delineated due to the rich interconnections among fetal nerve bundles making identification difficult. The importance o f the innervation o f this erectile tissue is paramount to understanding not just penile and clitoral erection but also sexual  3  arousal in general. In 1993, Paick et al. published their study which focused on the cavernous nerves distal to the prostate. Building on Walsh and Donkers work, this study helped to complete the picture o f the autonomic nerve pathway from the pelvis to the penis. They found that the cavernous nerve divides into several branches approximately 10 mm superior to the urogenital diaphragm. These branches then course anterolateral^, remain lateral to the urethra as they pierce the urogenital diaphragm and turn medially to branch to the corpora cavernosa. This branching and lateral course may help explain why some men do not lose sexual functioning despite prostectomy's or traumatic injury to the U G diaphragm (Paick et al.,1993)(Fig. 1). Despite such findings, the knowledge o f human pelvic anatomy is still not complete. While work in male pelvic anatomy has lead to surgical advances that include nerve sparing prostectomy's and colo-rectal surgeries, we have not made the same anatomical advances to apply to female pelvic surgery. In 1956 Krantz sectioned vagina and external genitalia o f tissue varying from a 8 month fetus to a 55 year old. This extensive serial section study focused on categorizing the type o f nerve endings in the female genitals. Quantitative analysis showed the presence o f touch, pressure and pain nerve endings i n the mons, labia, clitoris and hymeneal ring with only the occasional pain nerve ending within the vagina. Unfortunately Krantz did not describe any o f the pathways o f these nerves. Smith and Ballantyne (1968) investigated the  innervation to the pelvic contents after Lewington, 1956,  and Bowers, Moeckel, Yates and Wesson (1957) both reported functional changes to the bladder after total and radical hysterectomy's. They found the autonomic nerves associated with the bladder course through the pelvis v i a the inferior hypogastric plexus and move medial just below the uterine artery, to branch to the vagina and bladder. While there was a lack o f published research regarding the anatomical basis o f sexual arousal in the 70's and 80's it once again gained prominence i n the m i d 90's. In 1998 O ' C o n n e l l et al. did extensive work on the 3-D anatomical relationships o f the structures o f the external genitals. The work highlighted evidence o f significant post-menopausal atrophy  o f erectile tissue as well as defining relationship between erectile structures. The relationship o f the cavernous nerve to surrounding tissue is described as lying lateral to the urethra within the deep perineal membrane. Baskin et al, 1999 serially sectioned fetal material focusing on the innervation o f the human clitoris. In particular, he demonstrated detailed branching o f the dorsal nerve and correlated it to nerve sparing technique for feminizing genitoplasty with plastic surgery. Butler-Manuel et al, 2000 detailed the nerve content differences in the uterosacral and cardinal ligaments at the level o f division used in radical and simple hysterectomies. Their work, re-confirmed by Kato et al., 2002 demonstrated that both ligaments are more than just supports for the uterus they are also conduits for autonomic nerves to reach the pelvic organs. Specifically, both found that nerve contents varied along the length with and increase in the middle to lateral thirds toward their origin to the pelvic side wall.  Both sets o f researchers also confirmed that the autonomic nerve content in the  uterosacral ligaments were much higher than that in the cardinal ligaments. A s the knowledge o f neuroanatomy is necessary to guide surgeons operating for benign and malignant disease this study was designed to document the neural pathways through the urogenital diaphragm ( U G D ) supplying the female erectile tissue. It completes the missing component o f the path o f the cavernous nerve. Specifically: the direction and course o f the cavernous nerve as it branches off the pelvic plexus, orientates to the vagina and urethra and pierces the U G diaphragm to innervate the external genitals.  FEMALE PELVIC ANATOMY The female pelvis plays important roles in variety o f bodily functions.  The  significant interactions between organs and systems within the bony pelvis make understanding basic anatomy.and anatomical relationships important. Specifically for this research, the basic anatomy and positional relationships o f organs w i l l be employed to describe the pathway o f nerves as they enter the pelvis until their terminal branches within the external genitals.  Bone Anatomy  A pair of curved bones (os coxae) commonly known as the pelvic bones form the pelvic girdle. Each os coxa develops from ossification centers of three embryonic bones that fuse in the adult. The names of the three bones are retained for the corresponding regions in the adult pelvic girdle: the ilium, the ischium and the pubis. This girdle articulates solidly with the sacrum. It supports the mass of the upper body and distributes it to the legs. Several anatomic regions are recognized in association with the pelvic girdle. The os coxae and the sacrum enclose a space called the pelvic cavity. The brim of the pelvis is formed by a circular line passing along the upper edge of the pubic bones and sacral promontory. The pelvic brim encircles the superior entrance into the true pelvis and is called the pelvic inlet. More importantly for this research is the fact that the sympathetic innervation to the pelvis passes directly over the promontory to enter the pelvis. The pelvic outlet is the inferior opening of the pelvis, and is bounded by the coccyx posteriorly and the ischial and pubic bones anterolaterally. (Fig. 2). Muscles of the pelvic floor form in an upright funnel shape and support the organs that project into the pelvisfromthe abdominal cavity. Two main muscles are involved in this task: the levator ani and the coccygeus. The levator ani, the larger and more important of the two, is itself comprised of three smaller muscles: the pubococcygeus, puborectalis, and iliococcygeus. The pubococcygeus forms a "u" shaped sling when attached to its partner. The puborectalis forms the main portion of the levator ani and the iliococcygeus forms the posterior portion. These three muscles form a muscular sling that supports abdominopelvic viscera as well as helping to resist increases in intra-abdominal pressure. The posterolateral portion of the pelvic floor is supported by the coccygeus muscle. In addition to support, these muscles contract voluntarily during defecation, urination and sexual intercourse as well as involuntarily during orgasm (Moore et al., 1999)(Fig. 3). Reproductive tract The adult female reproductive tract consists of the ovaries, the oviducts, the uterus, the vagina and the external genitalia. The ovaries are almond shaped, paired organs located  on either side o f the pelvic wall..  The oviducts, also known as uterine tubes, extend from the  horns o f the body o f the uterus and extend laterally to open adjacent to the ovaries. The lateral ends o f the oviducts consist o f finger like projections called fimbrae which are involved in 'catching' ova once released (Moore et al., 1999)(Fig 4). The uterus is a hollow, pear-shaped, thick-walled sac that rests on the floor o f the abdominopelvic cavity between the bladder and the rectum. Anatomically it is typically divided into two parts: the body and the cervix. The body o f the uterus is held loosely in place by broad, round and ovarian (suspensory) ligaments.  The broad ligament, which  attaches the uterus and oviducts to the lateral pelvic walls and floor is derived from the double layered peritoneum and is also a conduit for vessels and nerves to pass from the lateral wall to the reproductive organs.  The round ligament attaches near the uterine horns  and runs lateral to the inguinal canal and eventually the labia majora. The ligament o f the ovary is the superiplateral extension o f the broad ligament which also attaches to the lateral pelvic wall (Maas et al, 1999)(Fig 4). The hollow uterus connects to the outside through the cervical and vaginal canals. The cervix is a narrow neck-like extension o f the uterus that protrudes into the vagina. Although the broad ligament does not cover the cervical portion o f the uterus, the cervix is well supported by two other ligaments: the cardinal and the uterosacral (Moore et al, 1999). The cardinal ligaments extend from the cervix to the lateral pelvic wall. The uterosacral ligaments also start at the cervix however they then course posterior to attach to the sacrum. Both o f these ligaments provide a pathway for nerves to reach the pelvic organs (Maas et al, 1999; Butler-Manuel et al., 2000; Kato et al, 2002). Given the contents o f these ligaments particular attention w i l l be to dissection o f them and in the surrounding area. The vagina provides a passage from the uterus to the outside o f the body. It is largely a fibromuscular tube that collapses on itself except at its superior end where it attaches to the cervix. This superior portion o f the vagina is called the vagina vault and it is slightly widened and allows for sperm accumulation during sexual intercourse. The external genitalia are discussed below with the perineum.  Perineum The inferior end o f the vagina passes through a muscular diaphragm and opens to the outside o f the body where it is surrounded by the external genitalia. The area collectively called the perineum includes both the external genitals and the muscular diaphragm by which it is attached to the body. It is a diamond shaped area extending laterally to the ischial tuberosities, anteriorly to the pubic symphysis and posteriorly to the coccyx. The diamond shaped area is typically divided into two by drawing a line between the tuberosities. The anterior portion is known as the urogenital triangle and the posterior portion is the anal triangle (Moore et al, 1999)(Fig. 5). The anal triangle contains skeletal muscle, the terminal portion o f the large intestine and shares innervation with the urogenital triangle. The urogenital triangle consists o f both a musculofacial diaphragm called the urogenital diaphragm ( U G D ) and the structures o f the external genitalia (Moore et al, 1999). Both the U G D and the external genitals contain organs/tissues o f interest for this study. The U G D , which is a sandwich o f fascia over muscles with orifices for both the urethra and vagina, is known as the deep perineal space.  The muscles involved are the deep  transverse perineal and the urethral sphincter. A l o n g with the muscles o f the pelvic diaphragm, the U G D provides both support for the internal structures and attachment for structures which sit inferior to it (Moore et al, 1999)(Fig. 6). The U G D is a key focus area for this research. Specifically, the path o f the autonomic cavernous nerve, through the U G D and its subsequent innervation o f the tissues o f the gentials involved in sexual arousal. The inferior fascia o f the U G D , also known as the deep perineal fascia, consists o f a fatty layer and subcutaneous connective tissue (Moore et al, 1999). It is continuous over the labia majora and it attaches medially to the pubic symphysis and laterally to the pubic body. The U G D inferior fascia is also the attachment point for the superficial perineal muscles; the superficial transverse perineal, the ischiocavernosus and the bulbospongiosus. The female external genitalia structures, collectively called the vulva, consists o f the mons pubis, labia  majora and minora, the clitoris, the bulb o f the vestibule and the greater and lesser vestibular glands (Moore et al, 1999).  Innervation The human nervous system consists o f complex networks o f neurons that carry information to and from the central nervous system ( C N S ) . The central nervous system develops from the neural plate, a thickened slipper shaped area o f embryonic ectoderm. It is the notochord and paraxial mesoderm that induce the overlying ectoderm to differentiate into the neural plate (Larsen, 1993). The neural plate eventually differentiates into the neural tube and crest. The neural tube differentiates into the C N S , consisting o f brain and spinal cord while the neural crest cells gives rise to form the majority o f the peripheral nervous system (Larsen, 1993). The peripheral nervous system and its central pathways are traditionally divided into two systems. The somatic nervous system is responsible for carrying conscious sensations and for innervating the voluntary muscles Of the body. The autonomic nervous system ( A N S ) is primarily concerned with involuntary processes and is further subdivided into two divisions: parasympathetic and sympathetic.  The parasympathetic nervous system  (PNS) is considered very generally to be responsible for the visceral activities characteristic o f periods o f peace and relaxation. The sympathetic nervous system ( S N S ) controls the involuntary activities that occur under stressful "flight or fight" conditions. O f course, like all parts o f the body there are always exceptions. This is particularly true in the case o f sexual arousal. The parasympathetic system is responsible for the arousal phase, which is anything but peaceful and relaxing and the sympathetics are responsible for the post-orgasmic detumescence.  ^'  -  The pelvis is innervated by the autonomic sympathetic nerves o f the thoracolumbar sympathetic trunk via the superior hypogastric plexus, and the parasympathetics o f the sacral trunk. The superior hypogastric plexus (SHP) is a network o f sympathetic autonomic nerves fed by the thoracolumbar trunk, which lie posterior to the endopelvic fascia and just inferior to the bifurcation o f the abdominal aorta. The S P H splits as it passes over the pelvic  °1  promontory into the left and right inferior hypogastric nerves where they descend into the pelvis under the endopelvic fascia, and expand into a fenestrated fan-like projection called the inferior hypogastric plexus (IHP) (Fig. 7) (Maggi, 1993; Butler-Manuel et al, 2000; Maas e t a l , 1999). The sacral trunk is located on the posterior wall o f the pelvis and is largely derived from parasympathetic fibers originating from L5-S4. While a significant portion o f these nerves leave the pelvis to innervate the lower limb, our focus is on those branches which form the pudendal and pelvic splanchnic nerves. The pudendal nerve is typically derived from parasympathetic fibers o f the anterior divisions o f S3-4 with occasional input from S2 (Jang et al., 1987). Sympathetic fibers from the lumbar trunk portion o f the sympathetic chain ganglia attach to the sacral roots prior to the branching o f the pudendal nerve (deGroat et al., 1993). The pathway o f the pudendal leaves the pelvis via the greater sciatic foramen and after hooking past the ischial spine, re-enters via the lesser sciatic foramen to innervate the perineum. The pudendal is largely the somatosensory nerve o f the external genitalia, terminating as the dorsal nerve o f the penis and clitoris. A s w e l l , branches o f the pudendal feed motor to the muscles o f the perineum (Moore and Dalley, 1999). The parasympathetic pelvic splanchnic nerves, originate from sacral roots o f S2-4 and course laterally under the endopelvic fascia to j o i n the I H P (Butler-Manuel et al, 2000)(Maas et al, 1999). A t this point the I H P contains afferent and efferent sympathetic and parasympathetic autonomic nerves as well as some sensory nerves supplying the rectum, uterus, vagina, vestibular bulbs, clitoris, bladder and urethra (Maas et al, 1999).  ID  Fig. 1 Representative course o f the cavernous nerve from the pelvic plexus to the penile shaft.  This is one o f the clearest documentations o f the pathway and branching o f the  cavernous nerves. Note the pathway and branching as it comes from the posterolateral position on the prostate to the lateral walls o f the urethra as it passes through the levator ani muscle. (Modified from Paick et al.,1993).  Fig 2. Bones of the pelvis with focus on general terminology of pelvic areas. Note the pelvic promontory which is a landmark for  13  Os coxa  Symphysis False pelvis •  True pelvis  \4  Fig 3. Muscles o f the pelvic diaphragm viewed from above. It is important to understand that these muscles work as a support system for both the internal organs above them and the external genitals below. They also play an important involuntary contraction role during orgasm.  t.S  ANTERIOR  POSTERIOR 1U  Fig 4:  O r i e n t a t i o n o f the uterus, ovaries a n d associated structures w i t h i n the b r o a d ligament. N o t e that the c a r d i n a l a n d uterosacral ligaments are not w r a p p e d w i t h i n the b r o a d ligament. T h e y do h o w e v e r p l a y as e q u a l l y important r o l e as the b r o a d l i g a m e n t as a c o n d u i t for nerves a n d vessels f r o m the lateral p e l v i c w a l l to the p e l v i c organs.  The  dotted l i n e is the course o f the a u t o n o m i c nerves traced o n gross m a c r o d i s s e c t i o n d u r i n g this study.  16  Fig 5: Illustration of an inferior view of the female perineum with the musculature of the pelvic diaphragm. The inferior pelvic diaphragm is anatomically divided into two triangle which are named by the more superficial structures located within each triangle. The urogenital triangle contains many of the organs/tissues that are important for this study.  ANTERIOR  Pubic symphysis  F i g 6. Female Urogenital Diaphragm showing the orifices o f the vagina and urethra. A l o n g with the muscles o f the pelvic diaphragm, the urogenital diaphragm provides both support for the internal structures and attachment for the perineal structures which sit inferior to it. These structures have been added to show their orientation.  2d  Pubic Symphysis  Bartholins Glands  Perourethral  Diaphragm  Fig 7: Innervation o f female pelvic viscera. A generalized schematic overview o f the autonomic innervation to the pelvis. Note that the parasympathetics as the pelvic splanchnic nerves to form the pelvic plexus (also known as the inferior hypogastric plexus).  Sympathetic trunk  Superior hypogastric plexus  Inferior hypogastric nerve  Nerves  Inferior hypogastric plexus  Pudendal Nerve  Muscles Muscles Somatic Somatic  of the pelvic d i a p h a g m of the urogenital d i a p h a g m sensory to the external genitals afferents  External Genitalia  THESIS RATIONALE AND OBJECTIVE The autonomic innervation o f the erectile tissue in the human male has long been identified. In men the cavernosal nerves arise from the prostatic plexus then pierce the urogenital diaphragm to supply the cavernosal bodies and thence the penis. In contrast, the autonomic innervation o f the female (via the vaginal plexus) has been described as "supplying its mucosa and erectile tissues at its anterior parts".  Unfortunately, the  description is not fully supported by published data as there has been no description o f the actual neural pathways that supply the erectile structures o f the vulva, nor have their site(s) o f origin in the vaginal plexus been defined. Specifically, it has always been supposed, but never definitively demonstrated, that the female innervation generally parallels that o f the male ( i . e . that there are two major nerve bundles that are equivalent in form and function to the male cavernosal nerves). It is also unclear whether or not there are individual branches to the individual erectile structures, which include the right and left clitoral corpora, vestibular bulb, and periurethral tissue. Based on similarities o f anatomy, I hypothesize that the female innervation o f the erectile tissue generally parallels that o f the male. Specifically, I predict that the cavernous nerve travels from the pelvicplexus as two major bundles, then branches inferior to the urogenital diaphragm to innervate the left and right clitoral corpora, the vestibular bulb and the periurethral tissue. To test my hypothesis I macro-dissected preserved female cadavers to determine the pattern o f travel through the pelvic and vaginal plexuses. Due to the interwoven nature o f the plexuses and the density o f the connective tissue between the vagina and urethra, I used histological processing, serial sectioning and 3 D reconstruction o f both fresh and fixed female cadaver tissue to better visualize the path through the urogenital diaphragm to the external structures o f the vulva.  lb  MATERIALS AND METHODS This study involved cadaver tissue obtained from the Faculty o f Medicine at the University o f British Columbia. A total o f ten specimens were used to fully document the morphology o f the neural pathways supplying the female erectile tissue (Table 1). Techniques used ranged from an intact pelvic macro-dissection to specific histological processing. Digital reconstruction a 3D image o f the external genitalia was also employed using serial sections.  TISSUE PRESERVATION O f the ten cadavers, eight were preserved by morgue staff via standard embalming procedures upon arrival at the Department o f Anatomy and C e l l B i o l o g y ' s morgue at the University o f British Columbia. The cadavers were embalmed using the following mixture: 20% isopropanol, 20% propylene glycol, 4% phenol and 5% formalin. This solution was infused under positive pressure (20 psi) v i a the left and right brachial arteries. T o allow infusion o f the embalming fluid to the cellular level, the cadavers are left in a cooler at 7° Celsius for two months. To ensure that the embalming process did not affect data integrity through cell damage, two cadaver specimens had their reproductive organs removed directly upon their arrival at the morgue, both within 24 hours o f death.  This tissue was immediately processed  in tissue prep cassettes in a Leica Automatic 12 Bath Tissue Processor using standard fixation method o f 30% xylene/70%) alcohol m i x for 5 baths, five baths o f 100% xylene and 2 baths o f liquid paraffin. The samples were then embedded on the cassettes in paraffin and sectioned on a Leica model 1212 microtome.  GROSS ANATOMICAL DISSECTION The first portion o f this study involved the dissection o f an intact pelvis. The abdominal cavity was exposed v i a a mid-sagittal incision. The abdominal organs were removed to a i d l n the exposure o f the posterior abdominal wall and the pelvic organs. The pelvic bones were then cut approximately 3 inches lateral to the pubic symphysis, the bone  then being removed leaving the periosteum intact (Fig 8). The area under the pubic symphysis was then dissected aided by Surgitel 2.75X binocular loupes.  The intention o f  the first dissection was primarily to establish methods o f approach to the area in question. However, due to the limited number o f female cadavers with intact reproductive organs the first cadaver, along with one other were then further dissected as described below. The two pelvi were removed by transecting the body at L 4 and removing the legs at the acetabulum. L i k e the previous pelvis, the pelvic organs remained intact however the rectum was removed just above the level o f the coccyx to aid with visualization o f the posterior pelvis (Fig. 9). The entire pelvic block was then cut mid-sagittally.  Dissection, aided by Surgitel binocular  loupes, began at the superior hypogastric plexus, which was identified prior to transection at L 4 , and moved caudally following the pelvic plexus to the external genitalia. The results were documented using still photos on a Fuji Finepix A201 digital camera. HISTOLOGICAL PROCESSING Eight cadavers were prepared for histological processing. The first processing done was with an intact pelvis which had been transected at the body at L 4 and had both legs removed at the acetabulum.  The entire block was then placed in 10 % Formic acid to  decalcify the bone thereby enabling sectioning. The pelvis was then cut transversely into 10 blocks, approximately 1 to 3 cm in width. Due to the density o f the pelvic bones only one or two cuts could be made prior to the block being returned to the acid for the newly exposed bone to decalcify. The tissue sections were then further processed to ensure cellular fixation for embedding in a large glass jar with an internal mixer in the following manner: 10% Formalin for six hours, 70% Xylene/30%) alcohol for 6 hours with 5 bath changes, 100% Xylene for 6 hours with 5 bath changes and 100% Paraffin for 2 hours with 2 bath changes. The sections were then placed in containers, covered in paraffin and allowed to cool in a vacuum oven overnight. The tissue was then cut serially at thickness o f 10 to 25 microns using a Jung K motorized sliding microtome and a carbon tip blade. They were placed on specially cut  27  8x13 cm glass slides (due to the large section size) that was treated with five percent polylysine (Sigma) for tissue adherence. The slides were then stained using standard preparation of hematoxylin and eosin ( H & E ) which was chosen because o f its easy adjustability ' m i d staining' with the varying thickness o f tissue, and cover slipped using Permount and second glass slide. W e were unable to locate commercially coverslips that were large enough.  This  portion was done to obtain a preliminary understanding o f cross-sectional relationships o f not only bones and organs but also vasculature, muscles, innervation and fascia. Specimens o f five embalmed and two fresh cadavers were used to further the histological component o f this study. Each o f the seven had the reproductive organs removed enbloc from the pelvis. This was accomplished by cutting the pubic rami 8 cm lateral to the pubic symphysis maintaining the pubic arch. Laterally the ovaries were removed by cutting mid fallopian tube following the broad ligament caudally approximately 2.5cm lateral to the uterine edges. Once removed from the cadaver, each block was then further dissected to remove the compact bone and unneeded tissue. Five o f the blocks (two fresh and three embalmed) were then cut mid-sagittally, keeping one side for future reference while the other side was further cut to fit standard tissue prep cassettes. This material was then processed in a Leica Automatic Tissue Processor using 30%Xylene/70%alcohol mix and then a 100%Xylene bath. The samples were then embedded in paraffin and sectioned on a Leica model 1212 microtome. Two o f the embalmed blocks were also processed using the above procedure however these blocks were cut transversely into sections o f approximately 5mm keeping right and left together (Fig 10). These 'intact' blocks were used to ensure that we did not lose important structure that lay midline. These sections were embedded in paraffin and cut on a Jung K motorized microtome. Due to the larger format o f these sections they were placed on specially cut 8x13cm microscope slides treated with 5% poly-lysine (Sigma) for tissue adherence. A l l o f the sections were subsequently stained with H & E for morphometric analysis o f nerve size, orientation and general histological organization.  28  3D MODEL One o f the mid-sagitally cut reproductive blocks was used to develop a 3D model o f the U G D and erectile tissue. To obtain a representative specimen from each section the embedded tissue was trimmed on the microtome to get a cross-section o f the entire section. Several 5 micron samples were then obtained, placed on slides and stained using H & E . This procedure was repeated for the twenty eight sections o f the block. The resulting slides were then scanned in a Kodak slide scanner, processed in Adobe P h o t o s h o p ® 6.0 to grayscale to aid i n visualization (Fig 11). Each o f the following structures was identified within each scan: the pudendal nerve, the dorsal vein, the clitoral crura, the urogenital diaphragm and a secondary nerve bundle. Each structure was color coded and the orientation was maintained by orientation points on the scans which were obtained by using a cardiac syringe on the original section prior to cutting. This left a small spherical hole that was later located using the microscope. Knowledge o f anatomy was essential when generating these structures into 3D as their inherent structure had to be added. For example a nerve is flat but tubular where as muscle is solid. The scans with their labeled structures were then converted using the imaging software Cinema 4 D X L ® , by Carbon Digital M e d i a o f Vancouver, B . C . , Canada to generate a three dimensional image (Fig 12). When used in conjunction with a computer video player the image can be rotated and viewed on all sides ( F i g l 3 ) .  29  Table 1. Table showing the variety o f processing and techniques employed to determine nerve pathway.  31  Number of specimens (n)  Technique or process used Gross Anatomical Dissection Macro dissection - intact pelvis  3  Macro dissection - pelvis cut mid-sagittal  2 (both previous dissected when intact)  Total specimens  3  Histologic Processing Embalmed  1  - whole pelvis - enbloc reproductive organs - whole  2  - cut mid-sagital  2  - U G D and gentials only  1 (used for 4 D X L )  Fresh  2  - enbloc reprod. organs cut mid-sagittal  Total specimens  32  8  F i g 8: Schematic o f cuts made to cadaver to expose underlying organs A) Illustration o f cuts made to outer abdominal wall to access pelvis. Procedure the same for access to reproductive organs for harvest 'enbloc' with removal o f pubic symphysis being done as part o f the block, not as a separate step as illustrated for the procedure below. B) Illustration o f exposure o f the pelvic organs with the abdominal contents removed. Note the removal o f the pubic symphysis but retention o f underlying structures.  33  F i g 9: V i e w o f transected pelvis that has been cut mid-sagitally prior to nerves to rectal area being removed. Most the dissected material is being lifted up to aid in the visualization o f the rectal branches.  35  F i g 10: Representative block o f tissue from the external genitalia. This section was further cut into 7 - 5 m m sections, processed for fixation, embedded in paraffin and sliced into  approximately 5 micron sections and plated on glass slides. A ) Inferior and superior views o f a tissue block showing the external genitals B) A 10 micron slice o f tissue from one o f the seven sections that has been stained with H and E and mounted on a glass slide.  37  F i g 11:  Serial section scan used in 3 D reconstruction. Note each type o f structure was identified via microscope and circled accordingly.  39  I  1  2mm  40  12:  Schematic o f how Cinema 4 D X L develops the 3D image. A representative slice o f tissue from each section was scanned into the computer and then the major structures were identified and outlined using 4 D X L . The computer then rendered the 3D image maintaining structural orientation. The resulting image can be shown as static images as seen in figure 16 or used in video software to create a movable 3D image.  41  4Z  Fig 13.  Static images o f external genitalia and urogenital diaphragm from Cinema 4 D X L imaging software. A + B Medial and lateral view o f the generated 3 D reconstuction. Both o f these view give a great view o f the orientation o f the different layers o f the uro-genital diaphragm and external genitals. C + D. V i e w s from inferior and superior. Note on D the nerve bundle associated only with the urogenital diaphragm. This reconstruction along with other serial sections helped determine the path o f the autonomic nerves through the U G D and was identified as the cavernous nerve.  43  RESULTS Gross anatomical dissections o f cadaver material has been carried out for centuries. Human neuroanatomy has been heavily studied in male cadavers and much o f the information can be carried over to the female. However, in some areas, such as the pelvis, differences in structure and function requires a direct approach. Cadaver studies have traced autonomic nerves throughout the female pelvis i n some detail. They have demonstrated that sympathetic nerves flow into the pelvis v i a the bilateral inferior hypogastric plexus (IHP) and j o i n with the parasympathetic nerves o f S2-4 via the pelvic splanchnic nerves at the pelvic plexus (Campbell, 1950; Maas et al.,1999). These plexuses sit on the lateral walls o f the pelvis, and move medially to the reproductive organs via ligaments to supply the bowel, bladder, urethra and entire genital system. (Maas et al., 1999; Butler-Manuel, 1999). The missing component to date is how these nerves continue their path inferiorly to the erectile tissue and the external genitalia. In the present study, dissection and histochemical evaluation o f fresh and fixed tissue definetively demonstrated the structure and pathway o f the autonomic nerves which feed the external genitals and are involved in sexual arousal. MACROSCOPIC The initial gross dissection was undertaken to primarily orientate the researcher with the general layout o f the female pelvis. W e were, however, able to superficially demonstrate previously well documented structures o f the reproductive organs, major pelvic vasculature, the pelvic promontory, the sacral nerves, the sympathetic chain ganglia, the superior hypogastric plexus (SHP), the inferior hypogastric plexus, and the pelvic splanchnic nerves (Fig 14). The only discrepancy noted was the variations o f the orientation o f the superior hypogastric plexus. Netter, 1997 illustrates the S H P as beginning at the bifurcation o f the aorta and ending at the promontory. However there is documentation that the S H P can lay as much as 1-2 cm below the promontory (Maas et al.,1999; Butler-Manuel et al., 2000). The specimen shown in figure 14 is somewhat unusual in that the S H P begins below the promontory but has a short course until it bifurcates into the inferior hypogastric plexus.  45  Dissection o f the two hemi pelvises were then undertaken to properly document in detail the above noted structures. In both cases we obtained access to the posterior abdominal wall with no difficulties. The superior hypogastric plexus (SHP), our starting point, was found attached via fascia one cm below the aortic bifurcation on the posterior abdominal wall, posterior to the endopelvic fascia. This finely fenestrated network o f neurons receives sympathetics from the left and right sympathetic chain ganglia. A s the S P H continued inferiorly it spread laterally to a width o f two to three cm. The network clung firmly to the posterior wall while it continued two and a half cm below the pelvic promontory. A t the most inferiolateral ends the S H P bifurcated into three millimeter diameter left and right hypogastric nerves (Fig 9). Each o f these nerves followed a singular course, one centimeter medial and parallel to the ureters. In both cases the inferior hypogastric nerve began 1 cm above the level o f the common iliac artery bifurcation into the internal and external iliac branches and ended one centimeter below the coccyx at the inferior hypogastric plexus (IHP) (Fig 15). Parasympathetic nerves entered the pelvis v i a the sacral nerves o f S2-4. These sacral nerves left the sacrum via the anterior foramina and joined together approximately two centimeters inferiolaterally and coursed over the muscles o f the posterior pelvic w a l l . In both pelvises, approximately one centimeter after exit from the foramina, branches arose from the roots o f S3 and S4. They ran inferolaterally to independently pierce the endopelvic fascia and join medially with the I H P four to five centimeters later (Fig 9). These nerves were identified as the pelvic splanchnic nerves. The I H P , like the S H P was a highly fenestrated web encapsulated in thick connective tissue.  The plexus was three centimeter wide and 3 centimeter long triangular shaped  structure and it lay approximately three centimeters from the mid-sagittal plane at its superior border at the level o f the rectouterine fold.  It extended laterally to encompass many o f the  lower branches o f the internal iliac artery and medially to the target organs o f the uterus, vagina and bladder (Fig. 16). A t the level o f the rectouterine fold several branches o f the  46  I H P branched off the to rectum. Once seen the branches were cut and the rectum removed to aid in visualization o f the distal area o f the I H P (Fig 9). The density o f the connective tissue made it very difficult to differentiate nerves particularly as they joined with other connective tissue support systems o f the broad, uterosacral and cardinal ligaments. It was noted however that the majority o f the plexus appeared to turn more medial at the level o f the vaginal vault (Fig 15). Despite the large amount o f branching a major part o f the plexus turns toward the midline running in an anteroinferior direction moving from the lateral wall o f the cervix and vaginal vault to a position anterior and lateral to the urethra (Fig 17). L i k e Olelrich, 1983, we noted the problem o f tissue identification, despite using 2.75 magnification binocular loupes, through the dense fibrous area between the urethra and vagina at the level o f the U G D . Several very small branches o f nerves appeared to pierce the U G D and carried on in the same orientation to the external genitals. However due to the density and apparent atrophy o f the tissue o f the U G D we were unable to confirm unequivocally that these branches were in fact the cavernous nerve feeding the erectile tissue.' To attempt to confirm the pathway o f the cavernous nerve microscopic processing was employed. MICROSCOPIC In general, the hematoxylin and eosin ( H & E ) staining allowed for easy identification o f a structures such as skeletal muscle, erectile tissue and nerves. O f course, the one disadvantage was that it did not allow us to differentiate nerve types. For this study however our main goal was to determine the pathway o f major nerves piercing the U G D . This was done not only to aid in further the understanding o f general gynecological anatomy but also to give a focus for further immuno and neurochemical studies. The histological sections o f the eight cadavers were evaluated starting at the external genitals and moving superiorly to the internal structures at the rectouterine pouch. This allowed the nerves to be followed directly from their target organ to their source. W e found two large neurovascular bundles running into the external genitals: one which was found  47  only on the inferior side o f the U G D which we identified as the pudendal nerve and the accompanying vasculature (Paick et al., 1993; Benoit, 1999;Lundberg, 2001) the second came from the lateral wall o f the vagina (Fig 18), moved anterior as it coursed inferiorly and pierced the U G D anteriolateral to the urethra to its target erectile tissue. This key feature, that is the well documented course o f the pudendal nerve running along the inferior wall o f the U G D versus the cavernous nerve which pierces the U G D allowed us to identify this second nerve as the cavernous nerve and vasculature (Fig 19). Second to pathway, size difference is significant between the two with the pudendal being much larger (Fig. 20). The putative cavernous nerve followed a similar course in each o f the cadavers. The cavernous nerve appeared on the lateral wall o f the vagina at the level of the vesicouterine pouch. A t this point it coursed inferiorly along with the vagina and urethra towards the U G D . A s the nerve coursed inferior it moved into the dense connective tissue between the urethra and vagina and ended up 5 m m lateral to the urethra at the level o f the U G D . O'Connell et al, 1999 identified the cavernous nerve as a single, large, easily dissected nerve within the erectile tissue which surrounds the periurethral sponge. B y histological analysis we found that the cavernous nerve was most commonly a single nerve bundle until it pierced the U G D where it branched.  The largest o f these branches remained medial and continued  in it's orientation to the urethra moving 2-3mm anterior just prior to piercing the vestibular bulbs(Fig 20) After leaving the U G D the cavernous nerve comes within 1mm o f the pudendal nerve as it courses anteriorly to become the dorsal nerve o f the clitoris . Despite only being separated by deep perineal connective tissue we were unable to confirm any communicating nerves. W e were able to easily see a significant size difference with the pudendal being much larger (Fig. 20 ). Given the whole mount nature o f some o f our specimens we compared the results to current anatomical literature. In particular the orientation o f the ligaments and pelvic plexus was similar to that o f Butler-Manuel et al., 2000. L i k e O'Connell et al., 1999 we found a significant variation from the description o f the vestibular bulbs. W e were unable in our ten  48  specimens to find any evidence o f the tear drop shaped, bisected vestibular bulbs. W e found all our specimens to have much more erectile tissue o f the bulbs continue around the anterior wall o f the urethra in the shape o f a inverted u (Fig 25). A s well the bulbs had significant fullness o f depth than previously documented (Fig 17).  3D Model The 3D model o f the U G D and external genitals provided further confirmation for several anatomical points discussed in this thesis. The first o f which is that two large bundles o f nerves could easily be identified. Their orientation to the U G D allowed us to confirm them as the pudendal and cavernous nerves.  Specifically the superior view o f the  model allowed for visualization o f the course o f the cavernous nerve from within the pelvic cavity. The inferior view confirms the pathway o f the pudendal nerve on the inferior surface o f the U G D (Fig 13). These differences as noted before were further confirmation that we did indeed have two separate nerves with two separate pathways. A s well the model confirmed the inverted u-shape o f the erectile tissues o f the vestibular bulbs. The post-sectioning shape more like a boomerang than a tear drop. A similar shape was also noted on the macrodissected cadavers however the depth o f the tissues(inferior to superior) was as much as 1" at the widest area. (Fig. 17)  49  Fig 14: Pictures o f a partially dissected pelvis which has not yet been cut mid-sagittal. A.  Lateral view o f the pelvis allowing visualization o f the uterus and bladder  in orientation to other structures o f the abdominal area such as the kidney and muscles. B.  V i e w looking inferior to superior showing the superior hypogastric  plexus.  50  F i g 15:  Macro-dissection o f the lateral pelvic wall. Note the lines added for orientation to the pelvic promontory, interal iliac artery bifurcation and the hypogastric nerve.  52  b3  F i g 16:  Lateral view of pelvic bowl showing orientation of IHP to the pelvic contents  54  F i g 18.  Histological section o f area superior to the U G D . The neurovascular bundle is close to the wall o f the vagina and is quite large. This bundle moves anterior as it approaches the U G D as seen in F i g 18 and 19.  58  Posterior  Neurovascular Bundle  Anterior *note this section is superior to the U G D  9fl  F i g 19:  Histological sections o f the U G D . The urethra and external sphincter are easily identified as well as their orientation to the anterior vaginal wall. The neurovascular bundle o f the cavernous nerve can be seen in the inset.  60  Fig 20:  Histological sections o f external genitals just inferior to the U G D . The neurovascular bundle o f the dorsal nerve and vein are very easily visualized do to the large size o f the bundle. A cross-section o f the bundle can be seen on the top left hand side were a small piece o f the crua can be seen just beginning to enter the sections.  62  SUPERIOR  i  INFERIOR  1  2mm  F i g 21: shown  Histological sections o f U G D and surrounding area which repeat the work  in F i g 18-20. Note that a U G D representative block has been added to aid in visual orientation. A. B. C.  Area superior to the U G D Within the U G D Inferior to the U G D  64  Urethra  Vagina  UGD  DoraaT^ri Vein  Ischiocavernous Muscle  A.  Pudendal^ Nerve v  Posterior  Anterior Levator Ani Muscle Cavernous  ''^HP  B.  Bulbospongiosus Muscle Ischiocavernous Muscle Crua Levator Ani Muscle 9m % Vestibular Bulb  Cavernous Nerve  5mm Pudendal Nerve  Crua  F i g 22:  Histological sections o f U G D and surrounding area which repeat the work shown in F i g 18-20. Note that a U G D representative block has been added to aid in visual orientation. A . Area superior to the U G D B . Within the U G D C. Inferior to the U G D  66  VAGINA  Levator Ani Muscle  Levator Ani Muscle  5Wv\  Fig 23:  Histological sections o f U G D and surrounding area which repeat the work shown i n F i g 18-20. Note that a U G D representative block has been added to aid in visual orientation. A . Area superior to the U G D B . Within the U G D C. Inferior to the U G D  68  VAGINA  LATERAL  Fig 24: Histological sections o f U G D and surrounding area which repeat the work shown i n F i g 18-20. Note that a U G D representative block has been added to aid in visual orientation. A . Area superior to the U G D B . Within the U G D C. Inferior to the U G D  70  VAGINA  UGD  Posterior i  ~~^~ —ijL_Cavernous Nerve  <  J  Urethra  Medial  B  Vaginal Wall  Lateral  1 _i Urethra' Cavernous Nerve Vaginal Wall  Urethra Cavernous Nerve  Anterior 71  5mm  Fig 25:Histological section o f U G D . Note this section has been cut and mounted whole to give a better understanding o f the structural relations o f the U G D .  72  ANTERIOR Pubic Symphysis  1 cm POSTERIOR  11  Fig 26:  Whole mount tissue cross-section o f the external genitals showing the structures.  Note how the erectile tissue o f the vestibular bulbs continues  around the anterior wall o f the urethra and it is not bifurcated like the crua.  74  Neurovascular bundles  DISCUSSION To our knowledge this is the first study to trace the complete pathway o f the cavernous nerve in the human female pelvis.  The autonomic innervation o f the erectile  tissue in the human male has long been identified. In men the cavernosal nerves arise from the prostatic plexus then pierce the U G diaphragm to supply the cavernosal bodies and thence the penis.  There has been no description o f the actual neural pathways that supply the  erectile structures o f the vulva, nor have their site(s) o f origin in the vaginal plexus been defined. The morphology o f these pathways has therefore also remained unknown. Specifically it has always been supposed, but never proven, that the female innervation generally parallels that o f the male (ie. that there are two major nerve bundles that are equivalent in form and function to the male cavernosal nerves). It has also been unclear whether or not there are individual branches to the individual erectile structures, which include the right and left clitoral corpora, bisected vestibular bulb, and periurethral tissue. Sympathetic fibres to the pelvis arise from the thoracolumbar spinal cord (Pick and Sheehan, 1946) and then reach the pelvic plexus v i a two outflows, namely the prevertebral hypogastric nerve (Barber et al 2002, Latarjet and Bonnet 1913, Learmonth 1931, K u o et al 1984) and the paravertebral sacral sympathetic chain. In general parasypmpathetic innervation arises from the sacral spinal cord (Kokotas et al 1978) and proceeds to the pelvic plexus via the pelvic nerve. However there is some histochemical evidence (Benoit et al 1991) that the pelvic nerve contains both adrenergic and cholinergic (ie. sympathetic and parasympathetic, respectively) fibres in the pelvic roots o f the male pelvic plexus (Huselboch and Coggeshall 1982, Downie et al 1984, K u o et al 1984, Lepor et al 1985). Research in male humans and animals has demonstrated that efferent and afferent sympathetic and parasympathetic neurotransmission occurs via the cavernous nerves (de Groat et al 1993, Vernet 1964) and also terminal branches o f the pudendal nerves.  We  found no evidence to support autonomic branches o f the cavernous feeding the pudendal nerve. The role o f the sympathetic fibres o f the cavernous nerve remains unclear as the  76  erectile response elicited in animals by stimulation o f the sympathetic chain was still present after unilateral or bilateral transections o f the cavernous nerves near their origin (Giuliano et al 1995). The pudendal nerves arise from the sacral roots (typically S2, S3, and S4) and contain motor fibres (for the perineal muscles), sensory fibres, and likely more sympathetic fibres (Katagiri et al 1986, M c K e n n a and Nadelhaft 1986, Lavoisier et al 1988). M u c h o f the research to date in human females has concerned itself with either the internal structures o f the pelvis or the external structures o f the genitalia. There has been little work on the neuropathways between these two areas. There are several reasons behind our ignorance o f female genital neurophysiology and its anatomic basis. First is a tendency for investigators to be preoccupied with vaginal lubrication, which is but one component o f arousal. Second, the majority o f women do not have an accurate appreciation o f erectile tissue engorgement in any direct sense. Third, the often used measurement o f genital congestion does not correlate well with subjective arousal. Finally, information about the effects o f surgical procedures on vaginal or vulval innervation is incomplete at best. Consider the issue o f vaginal lubrication and the autonomic nerve supply allowing vasodilation o f the submucosal capillary plexus with subsequent increase in transudation. The common plexus that supplies both the vaginal mucosa and the proximal vulval erectile structures, w i l l be affected differently by various surgical procedures. Surgeries which potentially damage cavernous nerves and vaginal branches o f the utero-autonomic plexus include Birch procedures (controlling o f involuntary urination by elevating the urethra and bladder), anterior vaginal repairs, vaginal hysterectomies (primarily in estrogen-deficient women where there is shrinkage o f tissues such that the urethra becomes approximated to the anterior vaginal fornix), radical hysterectomies for vulval cancer (where there is deep dissection o f the vesico-vaginal plexus) and direct vulva procedures for various benign and malignant conditions (Ziessen, et al.,2002). It is also noteworthy that the neurotransmitters involved are likely to be different i n the two areas. It seems that vasoactive intestinal peptide (VIP) (Ottesen et al., 1987) and another as yet unrecognized neurotransmitter primarily  77  mediate increased transudation whereas nitric oxide (NO) is a major neurotransmitter o f the "erectile"or congestive response o f the vulva (Burnett et al.,1997). It w i l l not come as a surprise to most persons that women generally do not have an appreciation o f their degree o f erectile tissue engorgement. It is equally unsuprising that most men are exquisitely aware o f their degree o f erectile tissue engorgement. A woman generally cannot tell you how engorged her vestibular bulb is, assuming she knows that she even has one. Nevertheless the simple fact is that vasocongestion o f her erectile structures is critical for her sexual enjoyment for several reasons: Her source o f sexual stimulation and subsequent arousal may well be genital touch. If the structures do not engorge, the touch is not sexually effective. Her further stimulation to orgasm usually requires direct massaging o f the congested structures. This may occur through the labia, the mons, direct clitoral stimulation, or through the anterior vaginal wall to the erectile tissue around the urethra. Lack of engorgement o f these structures tends to cause their stimulation to unpleasantly sensitive, irritating, and even uncomfortable and painful. Arousal is obviously dampened and orgasm is not reached. The issue o f genital congestion raises the issue o f its use as an objective measure o f arousal. Typically vaginal plethysmography is employed for these studies, and this procedure measures increases i n vaginal blood volume and/or pulse amplitude. These measurements are typically taken when the subject is exposed to an erotic stimulus (usually an erotic video). Unfortunately, the data is really representative only o f objective arousal and Brotto et al., 2002 have shown that there is poor correlation between objective and subjective arousal. The last major reason that female genitalia neurophysiology/neuroanatomy is so poorly understood is that the effects o f surgical interference on autonomic innervation o f the vagina and vulva have only been minimally studied. This knowledge gap has opened the door to conjecture about these effects and there is no shortage o f opinions. Not only are there  78  markedly different views concerning the disruption o f the autonomic nerve supply to the vulva and vagina but the possible repercussions on sexual function are rarely delved into. Loss o f sexual arousal and pleasure from stimulation o f vulval erectile tissue is not considered i n detail. Lack o f vaginal lubrication is sometimes raised as a concern but the confounding effects o f estrogen status are often not clarified. Typically the focus o f research has been centred on "intercourse frequency" and "intercourse satisfaction". In fact most studies o f sexual consequences of hysterectomy refer to orgasm. Using criteria such as intercourse frequency and/or satisfaction to assess the effect of a given surgical procedure on a patient's sex life is fraught with problems. This is because it may be completely impossible to differentiate between the negative effects o f nerve damage and the positive effects o f vaginal repair (or whatever). For example, the patient that has had a cyst the size o f a grapefruit removed may say that intercourse is more frequent/pleasureable because it doesn't hurt anymore. However, it also may not be as complete as it could have been had nerve-sparing surgical techniques been employed. A review o f the literature regarding hysterectomy's, the most common gynecological surgery performed on women in the U K and U S A (Farquhar et al., 2002), is anything but clear. In 1983 K i l k k u et al. reported that the frequency o f orgasm was significantly reduced one year post operatively in women who underwent total hysterectomy versus those patients who kept their cervixes (ie. subtotal hysterectomy). K i l k k u also found that dyspareunia decreased from 3 1 % preoperatively to 16% postoperatively with total hysterectomy, while with subtotal hysterectomy a more spectacular decrease occurred, ie. from 29% pre-op to 6% post-op. A n important finding o f this study was the decrease in frequency o f orgasm only with those patients who had undergone total hysterectomy. Ten years later, Virtanen (1993) (working in the same institution as K i l k k u ) concluded that the anatomic basis for his observed lack o f problems with orgasm after a total hysterectomy is due to the fact that damage to pelvic nerves and the plexus should be minimal since the nerves are posterior and the main plexus lies below the cardinal ligaments,  79  which are preserved in total hysterectomy.  "Because the pelvic plexus remains intact, the  vaginal walls and the erectile tissue o f the vestibule and clitoris remain sensitive and, consequently, no symptoms occur". Virtanen did, however, find a decreace in dyspareunia (pain in the pelvic area during or after intercourse) after total hysterectomy. Similarly, Nathorst-Boost (1992) comparing total abdominal hysterectomy with supravaginal hysterectomy in 576 women less than 55 years o f age, found no differences in sexual outcome between the two groups with over 80% in both groups reporting improvement or no change. Thakar et al., 2002, in study involving 279 women determined that there was little no statistical difference in either type o f hysterectomy when related to sexual function. Their results however did show a significant decrease in dypareunia from 46.2%) to 6% in subtotals and 39.3% to 14.3 % in total hysterectomies. Interestingly 10% o f the subtotal group had an increase in what was termed "good sexual relationship" however there was no change in the total hysterectomy group. In contrast, Hasson wrote in 1993 that the loss o f the major portion o f the uterovaginal plexus through excision o f the cervic "is bound to have an adverse effect on sexual arousal and orgasm in women who previously experienced internal orgasm". He based this statement on the concept o f stimulation to the cervix leading to a different type of orgasm than one arising from stimulation o f the vestibule and clitoris or other parts o f the external genitalia - forgetting, perhaps, that orgasm is a reflex and the efferent component, ie. the contraction o f internal and external genitalia is a constant entity even i f its intensity varies with the type and degree o f stimulation. Zekam et al., 2003 studied gynecologists attitudes regarding hysterectomy's. Interestingly, advocates for total hysterectomies state that there has been no demonstration that the cervix has any role in sexual, urinary or bowel function. Advocates for subtotals focus on surgical times, decreased morbidity and fewer complications. Ultimately the authors concluded that most gynecologists surveyed favor total abdominal hysterectomy over  80  subtotal. Few counsel women regarding the options o f total and subtotal hysterectomy or offer a choice between the procedures. These studies emphasize the need to discriminate between various dimensions o f surgical outcome. Patients may perceive the benefits to be more important that the problems. For example, patients with significant pelvic pathology may be gratified by relief o f their symptoms but may become discontented with other results such as loss of sexual function and vault prolapse over time. One possible way to determine patient perceived differences in outcome would be to run a longnitudinal studies involving both groups.  The variations in  outcome post total hysterectomy for benign disease is likely based in anatomy not patient psychology. The pelvic organs, including the uterus and cervix are supported by the endopelvic fascia which attaches to the pelvic sidewalls. Sympathetic and parasympathetic nerves travel from the pelvic plexus inferiorly along the posterio-lateral wall o f the pelvis, move medial along the uterosacral and cardinal ligaments (Kato et al., 2002) and pierces the U G D lateral and slightly posterior to the urethra to innervate the external genitals.  Cervical  removal, and cutting o f the cardinal and/or utero-sacral ligaments may result in the loss o f innervation which relays with the U G D and external genitals . The pundendal nerve, which has a more exterior route, as previously described may explain why sexual function may only be minimally effected. If there is an anastomosis between pudendal and cavernous, this connection may help replace function that could be lost through lack of surgical skill or anatomical anomalies.  Despite our continued lack o f knowledge i n the area o f womens sexual arousal and gynecological anatomy, there have been significant strides made in the last 10 years to finding better answers than the simple application o f male anatomy. With the knowledge o f the pathway o f the automonics through the U G D , educators can ensure dissemination at all levels o f health care from developing more precise surgical techniques to giving women a better understanding o f the balance o f options with outcomes. A s women become more  81  educated on their options for sexual intimacy they may feel more control and with that comes a great enjoyment and comfort level.  Future directions Ultimately while the information obtained from this study may have clarified some points regarding neuroanatomy o f the pelvis, it has also raised may more. Certainly the nerve content o f the neurovascular bundles o f both the pudendal and cavernous nerves need to further clarified. Does the pudendal receive sympathetics from the pelvic sympathetic trunk or are there communicating branches between the pudendal and cavernous nerves. If these nerves do have communicating branches is there an approximate point that they consistently occur. A l o n g with that comes the need for clarification o f the neurotransmitters involved in erectile tissue o f the genitals. One o f the drawbacks o f the cadavers we used was their age with the oldest being over 100. O'Connell, 1998 raised the question o f a link between age and atrophy o f vasculature and genital tissue. W i t h this in mind it would be prudent to repeat the work, using fresh tissue from a variety o f ages. In particular, looking at fetal, pre and post puberty, pre, peri and postmenopausal women. One interesting aspect may be to look at the changes in vasculature and how it correlates to the changes in tissue.  82  REFERENCES Barber M . , Bremer R., Thor K . , Dolber P., Kuehl., Coates K . 2002. 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