The Ontogeny of the Peptide Innervation of the Human Pylorus with special reference to understanding the Etiology and Pathogenesis of Infantile Hypertrophic Pyloric Stenosis

ROBIN MICHAEL ABEL

A thesis submitted for the degree of Doctor of Philosophy

University of London

The Institute of Child Health and the Royal Postgraduate Medical School ProQuest Number: U116382

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I wish to thank my parents, Dr. Helen Abel and Mr. Douglas Abel, my family, and Professors Lewis Spitz and Julia Polak for providing inspiration and the means to achieve this project by their support and encouragement. ABSTRACT

ABSTRACT

INTRODUCTION Despite many studies the aetiology of infantile hypertrophic pyloric stenosis is unknown. The ontogeny of the peptide innervation and structure of the normal pylorus have not been described. None of the published studies have been quantified to account for the dilutional reduction in neural tissue present. In none of the documented animal models have the sequence of changes been described.

AIMS A. To examine the Ontogeny of the Peptide Innervation of the pylorus. B. To quantify the Histochemical and Morphological changes in infantile hypertrophic pyloric stenosis. C. To quantify the Histochemical changes in Natural and Experimental animal models of infantile hypertrophic pyloric stenosis. A Developmental Study Conventional histology, immunohistochemitry, and carbocyanine neural tracers were used to examine the sequence of changes in the pattern of development of the normal human pylorus. The pattern of innervation was closely related to the expression was closely related to the gestational age and morphology of the pylorus.

B Quantitative Study The expression of a wide range of neurtoransmitters was immunohistochemically examined and the morphological changes in infantile hypertrophic pyloric stenosis were qantified. The longitudinal muscle layer was hypertrophied, nerves in different tissue layers had abnormal morphology, the ganglia were smaller, and there were selective changes in the expression of neurotranmitters in different tissues. The expression of Nitric Oxide Synthase was most greatly diminished.

C Animal Studies The morphological and histochemical changes within the pylorus of a natural, canine, model and experimental, hph-1 mouse, animal model of pyloric stenosis were examined. The chronological sequence of morphological and histochemical changes were documented in the hph-1 mice. The histological changes in this model were very similar to those in infants and dogs suffering pyloric stenosis.

CONCLUSION The ontogeny of the peptide innervation of the human pylorus is developmentally regulated. Selective changes in the expression of neurotransmitters occur in infantile hypertrophic pyloric stenosis. The fundamental abnormality appears to be aberrant Nitric Oxide Synthase activity. TABLE OF CONTENTS

ABSTRACT ...... 0

CHAPTER 1 INTRODUCTION ...... I

1.1 Py l o r ic S m o o th M u s c l e Hy p e r t r o p h y ...... 2 1.1.1 Pyloric Smooth Muscle Hypertrophy occurring in Infancy...... 2 /. 1.2 Pyloric Smooth Muscle Hypertrophy occurring in Adulthood...... 3 1.1.3 Pyloric Smooth Muscle Hypertrophy occurring in other ...... species 3 1.2 In f a n t il e Hypertrophic Py l o r ic St e n o s is ...... 5 1.2.1 Historical Background...... 5 1.2.2 Normal and Pathological Anatomy...... 6 1.2.3 Associated anomalies...... 8 1.2.4 Incidence...... 8 1.2.5 Presentation and Progress...... 9 1.2.6 Management...... 10 1.2 .7 Natural Progression oj Infantile Hypertrophic Pyloric Stenosis...... 13 1.2.8 Aetiology / Theories...... 16 1.3 T h e En t e r ic N e r v o u s S y s t e m ...... 20 1.3.] Neurotransmitters and Neuropeptides...... 20 1.4 N eurotransmitters a n d N europeptides ...... 22 1.4.1 History and Classification...... 22 1.4.2 Neuromodulators...... 23 1.4.3 Interstitial Cells o f Cajal...... 23 1.5 F e a t u r e s o f M a in N e u r a l M a r k e r s a n d N europeptides o f In t e r e s t ...... 25 1.5.1 The General Neural Marker: Protein Gene Product ...... 9.5 25 1.5.2 Substance P...... 25 1.5.3 Vasoactive Intestinal Polypeptide...... 27 1.5.4 Calcitonin Gene Related Peptide...... 28 1.5.5 Nitric Oxide...... 29 1.6 AIMS OF THE STUDY ...... 33 1.6.1 Hypothesis...... 33

CHAPTER 2 MATERIALS AND METHODS ...... 41

2.1 G e n e r a l Introduction ...... 42 2.2 IMMUNOCYTOCHEMISTRY...... 42 2 .2 .1 Fixation...... 42 2.2.2 Tissue preparation...... 43 2.2.3 Immunostaining...... 44 2.2.4 Antisera ...... 46 2.3 S p e c if ic it y c o n t r o l s ...... 48 2.3.1 Antiserum specificity...... 48 2.3.2 Methodological specificity...... 48 2.4 E xperimental p r o c e d u r e s ...... 49 2.4.1 Selective division o f the fetal vagus upon the lesser curve o f the stomach...... 49 2.4.2 Adjuvant induced deficiency o f tetrahydrobiopterin in...... mice 49 2.4.3 Carbon dioxide laser induced ablation o f the innervation o f the rat pylorus...... 51 2.5 C onventional h is t o l o g y ...... 53 2.6 A ss e s s m e n t OF IMMUNOSTAINING ...... 53 2.7 Q uantification ...... 54 2 .7 .1 Image analysis quantification of nerve fibres...... 54 2.8 St a t is t ic a l A n a l y s is ...... 55 2.9 M ic r o s c o p y a n d P hotomicrography ...... 55 2.10 T a b l e s a n d F ig u r e s ...... 56

3. CHAPTER 3 THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS ...... 60 3.1 S u m m a r y ...... 61 3.2 Introduction ...... 62 3.3 E xperimental D e s ig n ...... 64 3.3.1 Tissues...... 64 3.3.2 Antibodies (For detail see methods chapter, 2.2.4)...... 65 3.3.3 Conventional Histology...... 66 3.3.4 Immunostaining...... 66 3.3.5 Neural tracing...... 66 3.4 R e s u l t s ...... 67 3.4.1 Immunohistochemistry...... 67 3.4.2 Neuronal Tracing...... 69 3.5 D is c u s s io n ...... 70 3.6 T a b l e s a n d F ig u r e s (o v e r l e a f ) ...... 74

CHAPTER 4 A QUANTITATIVE STUDY OF THE MORPHOLOGICAL AND HISTOCHEMICAL CHANGES WITHIN THE NERVES AND MUSCLE IN INFANTILE H Y P E R T R O P H IC P Y L O R IC S T E N O S IS ...... 82

4.1 S u m m a r y ...... 83 4.2 Introduction ...... 84 4.3 E xperimental DESIGN ...... 86 4.3.1 Tissues...... 86 4.3.2 Antibodies...... 86 4.3.3 Imunostaining...... 86 4.3.4 Statistical analysis...... 87 4.4 R e s u l t s ...... 88 4.4.1 Conventional Histology...... 88 4.4.2 Immunocytochemistry...... 88 4.4.3 Morphological changes in the Dimensions of the Nerves and Longitudinal Muscle Layer88 4.4.4 The Changes in Immunoreactivity within the Circular muscle layer...... 89 4.4.5 The Changes in Immunoreactivity within the Longitudinal muscle...... layer 89 4.4.6 The Changes in Immunoreactiviy within the Myenteric Plexus...... 89 4.4.7 The Changes in Morphology and Immunoreactivity within the Ganglia...... 90 4.5 D is c u s s io n ...... 91 4.6 T a b l e s a n d F ig u r e s ...... 96

CHAPTER 5 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND PHENYLKETONURIA ...... 104

5.1 S u m m a r y ...... 105 5.2 Introduction ...... 106 5.3 E xperimental DESIGN ...... 108 5.3.1 Tissues...... 108 5.3.2 Antibodies...... 108 5.3.3 Immunocytochemistry...... 109 5.3.4 Statistical analysis...... 109 5.4 R e s u l t s ...... l lo 5.5 D is c u s s io n ...... 1 12 5.6 T a b l e s a n d F ig u r e s (o v e r l e a f ) ...... 114

CHAPTER 6 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC STENOSIS IN DOGS ...... 124

6.1 S u m m a r y ...... 125 6.2 Introduction ...... 126 6.3 E xperimental DESIGN ...... 128 6.3.1 Tissues...... 128 6.3.2 Antibodies...... 128 6.3.3 Immunostaining...... 128 6.3.4 Statistical analysis...... 129 6.4 R e s u l t s ...... 130 6.4.1 Conventional Histology...... 130 6.4.2 Immunocytochem is try...... ISO 6.4.3 The Changes in Immunoreactivity within the Circular muscle layer...... 130 6.5 D i s c u s s i o n ...... 132 6.6 Tables AND F ig u r e s ...... 137 CHAPTER 7 A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE PYLORUS...... 141

7.1 S u m m a r y ...... 142 7.2 Introduction ...... 143 7.3 Experim ental DESIGN ...... 144 7.3.1 Surgical Procedure...... 144 7.3.2 Tissues...... 144 7.3.3 Antibodies...... 144 7.3.4 Immunocytochemistry...... 145 7.4 R e s u l t s ...... 146 7.4.1 Conventional Histolog)>...... 146 7.4.2 Immunocytochemistiy...... 146 7.5 D is c u s s io n ...... 147 7.6 Figures (OVERLEAF)...... 147 CONCLUDING REMARKS...... 150

APPENDICES...... 156 9.1 APPENDIX I;...... 156 9.1.1 BUFFERS...... 156 9.2 APPENDIX II...... 157 9.2.1 FIXATIVES...... 157 9.3 APPENDIX III...... 158 9.3.1 CRYOPROCESSING OF TISSUES...... 158 9.4 APPENDIX IV...... 159 9.4.1 SLIDE CO A TING...... 159 9.5 APPENDIX V...... 160 9.5.1 CONVENTIONAL HISTOLOGY...... 160 9.5.2 A VIDIN-BIOTIN-PEROXIDASE COMPLEX (ABC) METHOD...... 160 9.6 APPENDIX VI...... 162 9.6.1 GLUCOSE OXIDASE-DAB-NICKEL-ENHANCEMENT...... 162 9.7 APPENDIX V II ...... 163 ABBREVIATIONS...... 172

REFERENCES...... 173 TABLE OF FIGURES

F ig u r e 1-1Illustration o f g a s t r ic d is t e n s io n a n d v is ib l e peristalsis in HYPERTROPHICPYLORIC STENOSIS...... 35 F ig u r e 1-2 P la in a b d o m in a l r a d io g r a p h s h o w in g a d il a t e d g a s fil l e d s t o m a c h w it h LITTLE AIR PASSING THROUGH INTO THE DISTAL PORTION OF THE INTESTINE...... 36 F ig u r e 1-3 B a r iu m m e a l examination sh o w in g t h e n a r r o w e l o n g a t e d py l o r ic c a n a l (STRING SIGN) PASSING CONVEXLY UPWARDS TO THE DUODENAL CAP...... 37 F ig u r e 1-4 U l t r a s o u n d sc a n in p y l o r ic st e n o s is s h o w in g t h e t h ic k e n e d p y l o r ic MUSCULATURE WITH A CENTRAL SONOLUSCENT AREA REPRESENTING THE LUMEN OF THE PYLORIC CANAL...... 38 F ig u r e 1-5 Illustration o f t h e p y l o r u s d u r in g pyloromyotomy ...... 39 F ig u r e 2-1 D ia g r a m o f St e p s in A B C R e a c t io n ...... 57 F ig u r e 2-2 F l o w D ia g r a m o f St e p s in Im a g e A n a l y s is a n d Q uantification ...... 58 F ig u r e 3-1- St o m a c h a n d D u o d e n u m , 8 W e e k s G e s t a t io n ...... 76 F ig u r e 3-2 CGRP I mmunoreactivity in g a n g l ia a n d n e r v e s a t 23 W ee k s G e s t a t io n 76 Fig u r e 3-3 n N O S immunoreactivity in t h e M y e n t e r ic P l ex u s a n d S u b m u c o s a a t 10 WEEKS g e s t a t io n ...... 77 F ig u r e 3-4 n N O S immunoreactivity in t h e M y e n t e r ic P lex u s a n d S u b m u c o s a a t 12 WEEKS GESTATION...... 77 F ig u r e 3-5 V IP immunoreactivity in t h e M y e n t e r ic Ple x u s a n d S u b m u c o s a a t 12 w eek s GESTATION...... 78 F ig u r e 3-6 V IP immunoreactivity in t h e M y e n t e r ic P l ex u s a n d S u b m u c o s a a t 23 w eek s GESTATION...... 78 F ig u r e 3-7 SP immunoreactivity in t h e M y e n t e r ic P le x u s a n d S u b m u c o s a a t 11 w eek s GESTATION...... 79 F ig u r e 3-8 fN C A M immunoreactivity in t h e M y e n t e r ic P lex u s a n d S u b m u c o s a a t 12 WEEKS GESTATION...... 79 Fig u r e 3-9 D il A utofluorescence w it h in g a n g l ia a t 12 w e e k s o f g e s t a t io n ...... 80 F ig u r e 4-1 He u r is t ic M o d e l o f In h ib it o r y In t e s t in a l M o t o r In n e r v a t io n ...... 94 Fig u r e 4 -2 Immunoreactivity t o PG P 9.5 in C o n t r o l a n d D ise a s e d S p e c im e n s o f Py l o r u s demonstrating t h e g r o s s m u s c l e hypertrophy a n d d is t o r t e d m o r p h o l o g y in INFANTILE HYPERTROPHIC PYLORIC STENOSIS...... 99 F ig u r e 4-3 CGRP I mmunoreactivity , p r im a r il y l o c a t e d in t h e n e r v e s a n d g a n g l ia o f THE My e n t e r ic P l e x u s ...... 100 F ig u r e 4 -4 n N O S immunoreactivity w it h in t h e d is e a s e d p y l o r u s ...... 100 F ig u r e 4-5 V IP immunoreactivity w it h in t h e d is e a s e d p y l o r u s ...... 101 F ig u r e 4 -6 SP immunoreactivity w it h in t h e d is e a s e d p y l o r u s ...... 10 1 F ig u r e 4-7 G r a p h ic a l presentation o f t h e p r o p o r t io n o f N e r v e s e x p r e s s in g N e u r o a c t iv e A g e n t s w it h in t h e M y e n t e r ic P l e x u s ...... 102 F ig u r e 5-1 D ia g r a m Illustrating T h e Relationship b e t w e e n T etrahydrobiopterin in f a n t il e hypertrophic py l o r ic st e n o s is a n d phenylketonuria ...... 117 F ig u r e 5-2 W e ig h t , L ongitudinal M u s c l e T r e n d ...... 118 F ig u r e 5-3 W e ig h t , C ir c u l a r M u s c l e T r e n d ...... 118 F ig u r e 5-4 W e ig h t , M u s c u l a r is m u c o s a e T r e n d ...... 1 19 F ig u r e 5-5 F e t a l , o n e d a y o l d M o u s e M u s c l e Hy p e r t r o p h y ...... 119 F ig u r e 5-6 40 d a y o l d C o n t r o l M o u s e ...... 120 F ig u r e 5-7 40 d a y o l d Hp h -1 M o u s e ...... 120 F ig u r e 5-8 St o m a c h s o f 40 d a y C o n t r o l M o u s e ...... 121 F ig u r e 5-9 St o m a c h s o f 40 d a y o l d Hp h -1 M o u s e ...... 121 F ig u r e 5 -10 L o w P o w e r photomicrograph o f T/S o f Py l o r u s o f 40 d a y o l d C o n t r o l M o u s e ...... 122 F ig u r e 5-11 L o w Po w e r photomicrograph o f T/S o f Py l o r u s o f 40 d a y o l d Hph - l M o u s e 122 F ig u r e 6-1 M e d ia n P r o p o r t io n o f N e r v e s E x p r e ss in g A n t ig e n w it h in t h e C ir c u l a r M u s c l e LAYER ...... 137 F ig u r e 6-2 Immunostaining f o r n N O S in a n o r m a l C a n in e p y l o r u s ...... 138 Figure 6-3 Immunostaining for n NOS in a Canine pylorus of congenital pyloric STENOSIS...... 139 Figure 7-1 PGP 9.5 Immunostaining within control and laser irradiated w istar ra t PYLORUS...... 148 TABLE OF TABLES

T a b l e 2-1 T a b l e o f p r im a r y a n t ib o d ie s ...... 56 T a b l e 3-1 A g e a n d N u m b e r o f T issu e s c o l l e c t e d ...... 75 T a b l e 3-2 T a b l e O f T h e T e m p o r a l P a t t e r n O f E x p r e s s io n O f A n t ig e n s W ith in T he Hu m a n F e t a l Py l o r u s ...... 75 T a b l e 4-1 L ongitudinal M u s c l e W id th & N er v e F ib r e l e n g t h a n d w id t h ( I O’^'m m ) ...... 96 T a b le 4-2 A r ea o f Immunoreactive N er v es in t h e C ir c u l a r M u s c l e (UNITS: MM^XlO''*) ...... 96 T a b l e 4-3 A r e a o f Immunoreactive N e r v e s in t h e L ongitudinal M u s c l e (UNITS: MM^xlO"*)...... 97 T a b l e 4-4 A r e a o f Immunoreactive N e r v e s in t h e M y e n t e r ic P l e x u s (UNITS: MM^XlO"^)...... 97 T a b l e 4-5 A r e a o f G a n g l ia ( u n it s : MM^xlO ' )...... 98 T a b l e 4-6 N u m b e r o f G a n g l ia ...... 98 T a b l e 5 - 1 T h e w id th o f t is s u e l a y e r s in c o n t r o l a n d d is e a s e d m ic e 10 t o 180 d a y s o f AGE(XlO'"M)...... 115 T a b l e 5-2 T h e w id th o f t is s u e l a y e r s ex p r e s s e d a s a p r o p o r t io n o f t h e t o t a l d ia m e t e r OF THE PYLORUS...... 115 T a b l e 5-3 T h e w id t h o f t h e c ir c u l a r m u s c l e o f o n e d a y o l d a n d 14 d a y o l d f e t a l MICE(XlO"^M)...... 116 T a b l e 6-1 M ed ia n P r o p o r t io n o f N e r v e s E x p r e s s in g A n t ig e n in t h e C ir c u l a r M u sc le LAYER...... 137 T a b l e 9 -1 S Sa m p l e o f F iv e C o n t r o l C ir c u l a r M u s c l e S p e c im e n s fo r n N O S A s s a y 163 T a b l e 9 -2 S T a b l e o f A v e r a g e PGP 9.5 & NNOS ex p r e s s io n in D is e a s e d a n d C o n t r o l C ir c u l a r M u s c l e S p e c im e n s ...... 167 T a b l e 9 -3 S T a b l e o f C a l c u l a t e d P r o p o r t io n / Ra t io o f N e u r a l t is s u e e x p r e s s in g NNOS IN C o n t r o l a n d D is e a s e d C ir c u l a r M u s c l e ...... 170 CHAPTER 1- INTRODUCTION

1. CHAPTER 1 INTRODUCTION 1.1 Pyloric smooth muscle hypertrophy 1.1.1 Pyloric smooth muscle hypertrophy, in infancy 1.1.2 Pyloric smooth muscle hypertrophy, in adulthood 1.1.3 Pyloric smooth muscle hypertrophy, in other species

1.2 Infantile Hypertrophic Pyloric Stenosis 1.2.1 Historical Background 1.2.2 Pathological Anatomy 1.2.3 Associated anomalies 1.2.4 Incidence 1.2.5 Presentation and Progress 1.2.6 Management 1.2.7 Natural Progression 1.2.8 Aetiology/Theories

1.3 The Enteric Nervous System 1.3.1 Neurotransmitters and Neuropeptides

1.4 Neurotransmitters and Neuropeptides 1.4.1 History and Classification 1.4.2 Neuromodulators 1.4.3 Interstitial Cells of Cajal

1.5 Features of the Main Neural Markers of Interest 1.4.1 Protein Gene Product 9.5 1.4.2 Substance P 1.4.3 Vasoactive Intestinal Polypeptide 1.4.4 Calcitonin Gene Related Peptide 1.4.5 Nitric Oxide

1.6 Aims of the Study CHAPTER 1- INTRODUCTION

1.1. Pyloric Smooth Muscle Hypertrophy By far the most common cause of gastric outlet obstruction to affect the neonate is infantile hypertrophic pyloric stenosis. Until relatively recently the prognosis was almost universally fatal . At the Hospital for Sick Children Great Ormond Street, London, 54 cases were treated between the years 1915-1917; vrith a mortality of 80.5% . The principle factor determining outcome of these patients was the duration of symptoms prior to a definitive diagnosis being made, and thus the condition of the child if it went on to undergo surgery (Choice CC , 1932).

In infantile hypertrophic pyloric stenosis there is a presumed primary smooth muscle hypertrophy affecting the pylorus. Other discrete causes of smooth muscle hypertrophy to affect the pylorus have been described, each having a different natural progression.

1.1.1. Pyloric Smooth Muscle Hypertrophy occurring in Infancy

In addition to the common form of primary pyloric smooth muscle hypertrophy that occurs in infantile hypertrophic pyloric stenosis two other forms of infantile pyloric smooth muscle hypertrophy have been described. A reactive secondary hypertrophy of the stomach and pylorus secondary to duodenal atresia was documented by Calder in 1733. This reactive hypertrophy resolved following correction of the distal duodenal obstruction. Secondly, a familial syndrome comprising hypertrophic pyloric stenosis associated with intestinal malrotation and short small bowel was described by Tanner, which was complicated by dysfunctional intestinal obstruction probably due to a deficiency of argyrophilic neurons (Tanner MS., 1976). Infants suffering fi-om this syndrome have a poor prognosis after pyloromyotomy. CHAPTER 1- INTRODUCTION

1.1.2. Pyloric Smooth Muscle Hypertrophy occurring in Adulthood

Hypertrophic pyloric stenosis is uncommon in adulthood accounting for only 3-5% of all cases of pyloric obstruction in adulthood (Keynes WM., 1965). du Plessis has described three forms of pyloric smooth muscle hypertrophy in adulthood: (i) iate stage’ of infantile hypertrophic pyloric stenosis in which the hypertrophy persists into adulthood from infancy, (ii) secondary to other conditions affecting the upper gastrointestinal tract, and (iii) a primary hypertrophy of the pylorus commencing in adulthood which may be complicated by gastritis, du Plessis described the pathological changes in five adults with primary adult hypertrophic pyloric stenosis ( du Plessis, DJ. 1966). He concluded the underlying abnormality to be a deficiency of the longitudinal muscle of the pyloric canal. This resulted in a functional obstruction and later work hypertrophy of the circular muscle layer, du Plessis found no abnormality in the ganglia. However, Wastell has described the underlying abnormality in this condition to be fibrosis of the nerves of the myenteric plexus. The treatment of choice is pyloroplasty, gastroenterotomy or Bilroth I gastrectomy (Wastell C , 1984). Morgagni identified a close familial tendency amongst individuals affected by adult type of hypertrophic pyloric stenosis (Morgagni GB., 1971)

1.1.3. Pyloric Smooth Muscle Hypertrophy occurring in other species.

Pyloric smooth muscle hypertrophy is also known to affect animal species. Intrinsic gastric conditions causing gastric outlet obstruction in dogs are relatively uncommon. Antral pyloric hypertrophy and neoplasia are the commonest causes of chronic pyloric dysfunction. Two forms of antral pyloric hypertrophy occur: congenital and acquired . Congenital pyloric stenosis occurs most commonly in young brachycephalic dogs, for example the Boston terrier, boxer, and bull dog. Siamese cats and horses have been described to be affected by a similar condition. Acquired antral pyloric hypertrophy CHAPTER 1- INTRODUCTION tends to affect small, middle aged dogs, for example the Lhaso apso, shih tzu, and poodle. The male to female ratio is two to one (Strombeck DR., 1990) . CHAPTER 1- INTRODUCTION

1.2. Infantile Hypertrophic Pyloric Stenosis

1.2.1. Historical Background The German physician Fabricius Hildanus ( F. Hildanus 1646 Opera Omnia. Joh. Beyerns, Frankfurt) is credited with the first description of Infantile Hypertrophic Pyloric Stenosis when he described ‘spastic vomiting’ in an infant. The infant survived having been given thickened feeds. In 1717 the botanist and surgeon Blair published the post-mortem findings of a baby with the typical features of infantile hypertrophic pyloric stenosis ( Blair 1717). Further descriptions of the condition appeared in 1758 by Weber, in 1777 by Armstrong, and 1788 by Beardsly. Until 1887 little was written of the condition until the Danish paediatrician , Harold Hirschsprung gave an account of three cases and postulated the condition was due to a primary muscle hypertrophy. He gave no indication of the form of treatment for the condition (Hirschsprung H., 1888). The treatment of choice at this time was medical, using a combination of gastric lavage, antispasmodic drugs, dietary manipulations, and the application of local heat in an attempt to overcome the ‘pyloric spasm. ’ The medical treatment of infantile hypertrophic pyloric stenosis was popular because in the majority of cases the hypertrophy would resolve provided the child could be kept alive. Treatment consisted of graduated 2-3 hourly feeds only. In 1869 Kussmaul recommended gastric lavage with normal saline. In 1904 Struempel proposed the use of atropine as an antispasmodic drug. Methyl atropine was subsequently found to be more effective. Scopolamine methylnitrate was later preferred for its enhanced antispasmodic effect on the pylorus ( Tallerman KH., 1951). The earliest attempt at surgery for the treatment of infantile hypertrophic pyloric stenosis was made by Schwyzer in 1896 (Schwyzer 1896). As a result of this communication Meyer on the 23rd of June 1898 performed an anterior gastroenterostomy upon a child with infantile hypertrophic pyloric stenosis. The child died within 48hrs . Lobker in 1898 performed the first successful surgical procedure, an anterior gastroenterostomy . By 1910 Weber had a series of 49 gastroenterostomies with a mortality rate of 61% . In 1887 Loreta described 3 adult cases in which the pylorus was divulsed a gastrostomy performed and digital dilatation of the pylorus CHAPTER 1- INTRODUCTION performed . Dent in 1902 performed the first successful Heineke Mikulicz pyloroplasty.

In 1906 Nicoll, who had previously been an advocate of Loreta’s pyloric divulsion and dilatation , performed a VY submucous pyloroplasty . The technical difficulties in suturing the thickened pylorus in Heineke Mikulicz pyloroplasty led to it being abandoned. Fredet in 1908 performed a longitudinal submucous division and then sutured the thickened muscle transversely. In 1912 Ramstedt reported one such operation he had performed the previous year. While attempting transverse suture of the muscle the sutures cut through. Ramstedt abandoned the procedure, having applied an omental patch over the pylorus. A year later the child had completely recovered. Thus Ramstedt’s pyloromyotomy evolved as probably one of the most significant advances in paediatric surgery .

With the advent of safe paediatric anaesthesia Ramstedt’s pyloromyotomy is the treatment of choice for infantile hypertrophic pyloric stenosis. However, the medical management of this condition still persists amongst some senior paediatricians particularly in infants that tend to present later with less vomiting, dehydration and weight loss (Swift PGF., 1991).

1.2.2. Normal and Pathological Anatomy Although there have been numerous descriptions of the anatomy of the human stomach, upto the 1920’s, few studies specifically examined the pylorus. In 1928 Bayard Horton published the results of a histological project specifically examining the relationship of the musculature of the pylorus with that of the duodenum. The specialised nature of the arrangement of the muscle fibres of the pylorus was not recognised until relatively recently. Wemstedt and Portal (1803) were of the opinion that a morphologically well defined sphincter at the pylorus did not exist. Cruveiller described the pylorus as a true sphincter (1844). Forssell (1913) dismissed suggestions that the pylorus could not be a specialised sphincter as it was of variable thickness and therefore not always distinguishable from the rest of the stomach. He equated the arrangement of the circular and longitudinal muscle layers to that of the iris. CHAPTER 1- INTRODUCTION

The normal pylorus as described by Horton comprised a highly specialised circular muscle positioned like a fan as an inverted V, with two sphincteric loops enclosing interpositioned circular fibres. The distal loop is the gastric part of the pyloric sphincter and the proximal embraces the canal obliquely. The fibres of these loops converge on the lesser curve to form a prominence of about 2cm in length, but fanning out to form a triangular-shaped muscle extending for upto 4cm along the lesser curvature. Torgensen described the longitudinal muscle on the greater curvature of the pyloric canal as being particularly thickened along the length of the fan shaped muscle (Torgensen J., 1942). Approximately half of these fibres extend onto the duodenum (Horton BT., 1928) the remainder dip into the underlying circular muscle in the distal part of the canal to form what some have described as a dilator muscle. The first description of the ‘dilator’ muscle is attributed to Rudinger in 1879. In infantile hypertrophic pyloric stenosis there is a marked hypertrophy of the circular muscle of the pylorus ( Belding HH., 1953) so that the canal is lengthened as well as the whole pylorus becoming thickened. The pylorus becomes olive shaped, firm when relaxed and hard when the muscle is in spasm. It may measure upto 2cm in transverse diameter . In general it would appear that the larger tumours are found in larger and older infants, while the size of the tumour is not related to the length or duration of symptoms (Dodge JA , 1975). The junction between the gastric antrum and the pylorus is indistinct, there being a gradual thickening of the wall and narrowing of the canal. Distally the hypertrophy and stenosis stop abruptly as the hypertrophied pylorus projects into a normal duodenum. The muscle is greyish - white and firm to gritty in consistency. The mucosa is oedematous and thickened. In places the mucosa may become eroded and even ulcerated (Dodge JA., 1975). Oesophagitis may also cause haematemeses (Spitz L., 1979 .) . Proximally, in the well established case, the stomach becomes hypertrophied and dilated. Gastric peristalsis becomes increased in vigour and irregular in nature. CHAPTER 1- INTRODUCTION

1.2.3. Associated anomalies A number of associated morphological and biochemical anomalies have been described as being associated with infantile hypertrophic pyloric stenosis. Associated malformations include oesophageal atresia, malrotation, diaphragmatic hernia, Meckel’s diverticulum, Hirschsprung’s disease, anorectal agenesis, renal anomalies, undescended testes, and hypospadias (Croitoru, D. 1970, Pullock, W.F. 1957, Scharli, A.F. 1968, Atwell, J.D. 1977,) . Biochemical anomalies have been associated with infantile hypertrophic pyloric stenosis. Amongst which is the commonest inborn error of metabolism to affect humans: phenylketonuria. Children with untreated phenylketonuria have a higher incidence of infantile hypertrophic pyloric stenosis ( Johnson CF., 1978).

1.2.4. Incidence The incidence of infantile hypertrophic pyloric stenosis varies with geographical location, time, and race. In the United Kingdom the incidence appears to be increasing. In the 1940’s the incidence was 1.1 per 1000 births (Lawson, D., 1951) . In recent years it has increased to 2.2 per 1000 births in the 1980’s ( Knox EG., 1983, Tam PKH , 1991, Webb AR., 1983 ) . The incidence of infantile hypertrophic pyloric stenosis is recognised to be lower in Negro and Asians than in Caucasian infants (Cremin B J, 1968, Mandell G A , 1978, Swan TT., 1961) . The incidence of infantile hypertrophic pyloric stenosis is increasing amongst Negro infants in Barbados (Shim WKT., 1970) .

The male to female ratio is 4:1. There is said to be an increased incidence among first bom infants. A strong familial pattern of inheritance has been established (Carter CO., 1969, Mitchell LE., 1993) . Sons of male index patients have a 5.5% chance of developing pyloric stenosis, whereas for daughters the incidence is 2.4% . The chance that the son of a female index patient developing pyloric stenosis is 18.9% and 7% for the daughters of a female index patient. CHAPTER 1- INTRODUCTION

Infantile hypertrophic pyloric stenosis has been described to affect premature infants (Beasley SW., 1992, Cosman B C , 1988, Tack ED., 1991) The age of onset is not related to gestational age (Muayed R., 1984).

1.2.5. Presentation and Progress

i. 2. 5. l.Symptomatology Typically the condition presents with painless, projectile, non bilious vomiting between the first and fourth weeks of age. As indicated by Hirschsprung, vomiting is the predominant symptom, occurring within ten to twenty minutes of a feed. After vomiting the infant will readily feed again. If the condition remains undiagnosed the vomiting becomes regurgitant in nature occurring once or twice a day. 10-15% of children will develop haematemesis due to ulcerative oesophagitis (Spitz L , 1979, Takeuchi S., 1993). In the premature baby, projectile vomiting is not a prominent feature rather the child seems anorexic and not hungry as is the typical baby (Henderson JL., 1952).

1.2.5.2. Physical Examination Approximately 10-15% of children will present with the clinical features of at least 5% dehydration: a sunken anterior fontanelle, sunken eyes, reduced skin turgor, and dry mucous membranes.

Between 2-3% of infants will be jaundiced at presentation (Dodge JA., 1975). An absolute reduction in Glucuronyl transferase activity has been demonstrated in infantile hypertrophic pyloric stenosis (Woolley MM., 1974) . The cause of glucuronyl transferase deficiency unknown. It has been associated with dehydration and alkalosis which inhibit the activity of the enzyme. Calorie deprivation is another possibility as this parallels the jaundice of Gilberts syndrome (Cochrane WD., 1975). Once the pyloric obstruction is relieved the jaundice rapidly fades (Chaves-Carballo E., 1968).

Visible gastric peristalsis, passing from left to right across the upper abdomen, is best seen shortly after a feed. This is not a pathognemonic sign of infantile hypertrophic pyloric stenosis. The palpation of a pyloric tumour is the cardinal sign. The tumour lies in the right upper quadrant beneath the liver edge. It is most easily palpated during the CHAPTER 1-______INTRODUCTION______^ early stages of a test feed or immediately after an episode of vomiting, when the abdomen is relaxed, the pylorus in spasm, and the stomach relatively empty. (Figure 1).

1.2.6. Management The diagnosis of infantile hypertrophic pyloric stenosis is not always a simple one. In the vast majority of cases the diagnosis can be established on clinical skills alone. The adage that one should only operate upon a palpable pyloric tumour is probably correct. If a pyloric tumour is felt, further investigations to establish the diagnosis are unnecessary. In skilled hands a tumour is palpable in 90% of cases (Shaw A., 1990, Breaux JB., 1988, ZeidanB., 1988).

Radiological investigations are only necessary when a pyloric tumour cannot be palpated. A plain abdominal x-ray may show a distended gas filled stomach containing an air fluid level and little gas beyond the pylorus (Figure 2) . Often it may fail to demonstrate any such signs. Surprisingly therefore Solowiejzyk, in 1980, described more than seventy per cent of the children in his series as having undergone a plain abdominal x-ray. Upper gastrointesinal contrast studies may demonstrate a variety of radiological signs. These include a ‘string sign’ and ‘double tracking’ of contrast along the elongated, narrow pylorus and between the enfolded mucosa (Figure 3).

In recent years ultrasonography has superceded contrast studies as the diagnostic investigation of choice. The advantages of ultrasound namely accuracy, ease, speed, repeatability, low risk of aspiration and absence of ionising radiation, have been widely reported (Rollins MD., 1991, Lamki N., 1993) . A pyloric muscle wall of 3-4 mm thickness has been reported as being strongly suggestive of infantile hypertrophic pyloric stenosis. Ultrasound examination has thus been described as an ‘extension of the examining hand’ (Figure 4). Failure to demonstrate a wall thickness of 3-4mm does not exclude the diagnosis of infantile hypertrophic pyloric stenosis, and in the infant in whom clinical suspicion is high a repeated ultrasound examination or contrast study may be appropriate.

The value of radiological imaging has in recent years been increasingly questioned. Macdessi in 1993 concluded an increasing use of diagnostic imaging did not result in CHAPTER 1-______INTRODUCTION______U_ earlier diagnosis or in better management. While valuable as an aid to diagnosis in difficult cases, increasing reliance on imaging may reduce a doctor’s skills in diagnosing pyloric stenosis clinically. Misra in 1996 went further in identifying an increased risk of unnecessary laparotomies due to false positive radiological examinations ( Misra D., 1996).

1.2.6.1.Differential Diagnosis In the absence of a palpable tumour the differential diagnosis of persistent non bilious vomiting in an infant includes feeding problems, gastroesophageal reflux, urinary tract infections , pneumonia and adrenal hyperplasia.

1.2.6.2.Preoperative Preparation Pyloromyotomy for infantile hypertrophic pyloric stenosis is not an emergency procedure. Rehydration and correction of electrolyte and acid base balance is required urgently. The biochemical disturbance in infantile hypertrophic pyloric stenosis is typically a hypochloraemic alkalaemia associated with dehydration. This is the result of persistent vomiting resulting in the loss of gastric secretions, mainly hydrochloric acid and smaller amounts of sodium and potassium as chloride salts. When potassium depletion coexists with alkalaemia, sodium reabsorption across the distal renal tubules in exchange for hydrogen ions occurs in preference to potassium ions resulting in a paradoxical aciduria in the presence of an alkalaemia.

Rehydration is essential as hypokalaemia may cause cardiac arrhythmias, and the alkalosis delay extubation. Conn reported a postoperative apnoea incidence of 0.9% while Kumar and Bailey an incidence of 2.7% (Conn AW., 1963, Kumar V., 1975) .

Rehydration with a saline solution is the mainstay of therapy. Assessment of the degree of dehydration has been the subject of much interest ( Goh DW., 1990, Dawson KP., 1991). The serum chloride is widely accepted as an accurate indicator of the degree of dehydration. CHAPTER 1-______INTRODUCTION______

i. 2 6,3. Operative Technique The pyloromyotomy described by Fredet and Ramstedt remains essentially unchanged to date. Modifications such as the double V-shaped pyloric incision have been described and subsequently proven to be of no benefit (Scorpio RJ., 1993). A variety of different skin incisions have been described, including a right transverse muscle cutting or muscle splitting (Robertson DE., 1940), circumumbilical (Fitzgerald PG.,1990, Ali Gharaibeh KI. 1992) and a laparoscopic approach (Tan HL., 1993). The right muscle cutting incision allows good access, a secure closure and a cosmetically acceptable scar. Alternative procedures include balloon dilatation of the pylorus (Hayashi AH., 1990). Essentially this is a less invasive modification of the procedure first described by Loreta in 1887 for pyloric stenosis and subsequently by Jaboulay for peptic ulcer induced pyloric and duodenal stenoses. The efficacy of this technique has not yet been fully assessed in infantile hypertrophic pyloric stenosis and is not widely accepted in the United Kingdom. Curiously a similar technique was independently described to ensure complete pyloromyotomy (Shaw RB , 1994).

A potential technical failure of this technique involves tearing the mucosal lining of the pylorus. This may occur most commonly at the junction of the pylorus with the duodenum. The likelihood of this complication may be reduced by invaginating the duodenum into the pylorus over an index finger during pyloromyotomy. (Figure 5)

The mortality associated with pyloromyotomy for infantile hypertrophic pyloric stenosis is negligible if the child is appropriately rehydrated preoperatively. The postoperative morbidity is principally related to wound failure, either infection or dehiscence. The predisposing factors to both include defective operative technique and the child’s malnourished condition (Goh DW., 1991, Rao N., 1989 Spicer R , 1989). CHAPTER 1-______INTRODUCTION______U.

1.2.7. Natural Progression of Infantile Hypertrophic Pyloric Stenosis

Infantile hypertrophic pyloric stenosis has been documented to occur as early as the first week of life in full term as well as in premature babies . Thomson proposed that the condition was due to a prenatal incoordination of the pyloric muscle (Thomson J., 1897). Pyloric smooth muscle hypertrophy has been described in a seven month old foetus (Choyce CC., 1932) and premature babies (Beasley SW., 1992, Cosman BC., 1988, Tack ED., 1991). Wallgren investigated 1000 apparently normal infants at birth by barium meal radiology, five of whom subsequently developed infantile hypertrophic pyloric stenosis ( Wallgren A., 1948). Several studies have investigated gastric function following conservative, medical treatment or pyloromyotomy for infantile hypertrophic pyloric stenosis. Okorie 1988, examined the pylorus using ultrasound upto 12 weeks following pyloromyotomy in 25 children. Two processes were identified to occur simultaneously, a regression of the muscle hypertrophy and growth of the child: the pylorus was found to have returned to a normal size by twelve weeks postoperatively. Steinicke studied the function and morphology of the pylorus after medical or surgical management of infantile hypertrophic pyloric stenosis using barium studies. He described normal gastric emptying within days of surgery with the appearance of the pylorus returning to normal within three to ten months of surgery.(Steinicke 0 , 1959, 1960).Vilman, in 1986, studied 72 children between 20-42 years following pyloromyotomy using double contrast barium examination. Three of the children had been re-operated upon for persistent vomiting due to an ‘inadequate pyloromyotomy’. There was no significant functional or morphological abnormality of the stomach in any of the patients studied. Solowiejczyk in 1980 studied 41 individuals 15-30 years following pyloromyotomy. The majority of patients in this group were troubled by symptoms related to the upper gastrointestinal tract. Contrast studies performed in 31 cases showed no thickening or abnormality in morphology or function of the stomach or pylorus. However in almost 50% there were radiological features consistent with peptic ulceration. Solowiejczyk concluded the symptoms of ‘dyspepsia’ after pyloromyotomy are due to increased speed of emptying of gastric acid into the duodenum. However, less than fifty per cent CHAPTER 1-______INTRODUCTION______

(41 cases) of the total cohort of 228 children operated upon in this study were followed up. The method of selecting the 41 analysed is unclear. Furthermore Solowiejczyk’s conclusion that abnormal pyloric emptying is the cause for the peptic ulceration is at odds with his description of normal gastric emptying and a normal pylorus upon examination with contrast studies.

Wanscher in 1971 followed up 14 of a total of 24 children who had undergone pyloromyotomy for infantile hypertrophic pyloric stenosis up to 21 years previously. In only two were there symptoms suggestive of peptic ulceration. All had normal upper gastrointestinal contrast studies, in four of which there was delayed gastric emptying. Acid secretion was examined for the first time, Wanscher identified an increased basal and pentapeptide stimulated acid secretion. Ludtke, in 1994, published a study of gastric emptying 16-26 years following either conservative or surgical treatment for infantile hypertrophic pyloric stenosis in 56 patients . Technetium scintigraphy was used rather than Barium meal examination as the authors believed it to be a more precise, quantitative analysis. He also examined ultrasongraphically the dimensions of the pylorus in 48 of these patients. In neither study was there any significant morphological or functional difference between either the medically and surgically treated groups, or between the infantile hypertrophic pyloric stenosis group and control groups. Berglund found upper gastrointestinal symptoms were no more common among children that had had infantile hypertrophic pyloric stenosis than normal control children (Berglund G , 1973). Rasmussen in 1988 specifically studied the relationship between infantile hypertrophic pyloric stenosis and subsequent ulcer dyspepsia in 284 of a total of 324 children managed either medically or surgically up to 58 years previously. While this is the largest follow up study of infantile hypertrophic pyloric stenosis published, the medically and surgically treated groups were not strictly comparable as the medically treated children were managed chronologically earlier, before surgery was the accepted treatment of infantile hypertrophic pyloric stenosis. Rasmussen concluded that statistically there was no significant difference in the incidence of ‘dyspeptic’ symptoms between the medically and surgically treated groups, and that therefore any transient changes in gastric emptying following pyloromyotomy are probably insignificant. CHAPTER 1- INTRODUCTION 15

The morphology of the pylorus following pyloromyotomy has intrigued many surgeons. The pylorus, at laparotomy performed for unrelated conditions, appears to be normal within a few months of pyloromyotomy. The only abnormality that may indicate previous surgery being the presence of a fine, white line along the length of the healed myotomy. Wollstein in 1922 first described these macroscopic findings. The only histological abnormality he identified was that the muscle fibres were in places separated by scar tissue.( Wollstein M., 1922) In 1995 Vanderwinden described the histological and histochemical characteristics of the pylorus after pyloromyotomy for infantile hypertrophic pyloric stenosis. He found that the arrangement of the muscle fibres had regained a normal organised pattern and that the expression of nitric oxide synthase had returned to normal.

One may conclude that the pyloric smooth muscle hypertrophy becomes clinically apparent shortly after birth. The muscle hypertrophy is a transient phenomenon in the vast majority of children with infantile hypertrophic pyloric stenosis whether they are treated surgically or conservatively. The long-term prognosis of children that survive the condition is excellent.

As du Plessis indicated in 1966 a small proportion of children with ‘Infantile Hypertrophic Pyloric Stenosis’ will have a pyloric muscle hypertrophy that persists into adulthood CHAPTER 1- INTRODUCTION 16

1.2.8. Aetiology / Theories

Despite the dramatic improvement in the management of infantile hypertrophic pyloric stenosis, little is known of the aetiology of the condition . The possible causes may be divided into four groups, 1) genetic and environmental factors. 2) work hypertrophy, 3) neuronal and neuropeptide activity abnormalities, 4) elevated gastrin level.

1.2.8.1.Genetic and environmental factors A seasonal trend in the prevalence of infantile hypertrophic pyloric stenosis has been described, with a preponderance of cases in autumn and spring ( Walpole IR., 1981, Dodge JA. 1975). Associations with high birth weight (Lammel EJ., 1987, Adelstein P., 1976) , ABO Blood group, (Rasmussen L., 1989Dodge JA., 1967) and transpyloric tube feeding ( Raine PAM. 1982, Tack ED. 1988) are well recognised.

The familial aggregation pattern demonstrated by infantile hypertrophic pyloric stenosis has led to the proposal that it is inherited by a multifactorial threshold model of inheritance. This model assumes that the ‘liability’ to develop infantile hypertrophic pyloric stenosis is determined by the additive effects of numerous genetic and environmental factors. The condition is expressed when a certain critical threshold is exceeded . Mitchell’s study, 1993, supported the multifactorial threshold mode of inheritance or multiple interacting loci inheritance. The study excluded a maternal factor that might increase the risk to female offspring. However studies of familial recurrence patterns are possibly biased by increased reporting of second-degree relatives . More contemporary studies will be relatively unaffected by the previous high mortality associated with infantile hypertrophic pyloric stenosis. CHAPTER 1- INTRODUCTION 17

1.2.8.2. Work hypertrophy

Lynn in 1960 proposed that milk curds propelled by the gastric musculature against a pylorus in spasm induced oedema of the pyloric mucosa and thus further narrowing of the pylorus, which in turn resulted in work hypertrophy of the pylorus and stomach (LynnHB., 1960). The first experimental model of hypertrophic pyloric stenosis induced in rabbits was produced by Heinisch in 1967 (Heinisch HM., 1967). This was achieved by attaching glass beads in rubber bags to the gastric fundus.

1.2.8.3.Neuronal and Neuropeptide Activity Abnormalities

Several anomalies have been attributed to the nerves and ganglia of the pylorus. Belding in 1953 described a reduction in the number of ganglion cells and nerve fibres which was ascribed to degenerative changes. Spitz and Kaufman concluded that degenerative changes occur within the myenteric plexus upon examining 25 cases of infantile hypertrophic pyloric stenosis histologically using haematoxylin and eosin staining (Spitz L. et al. 1975). Friesen proposed in 1956 these changes may be due to immaturity of the ganglion cells, on comparing the normal pylorus of the human fetus and infant with that of 19 cases with infantile hypertrophic pyloric stenosis, as evidenced by a reduced number of mature cells. Friesen and Pearce 1963 lent further support to this hypothesis of arrested development using cholinesterase and mitochondrial enzymatic assays. Tam 1986 demonstrated intense neuron specific enolase activity within the ganglia and diminished Substance P activity by the nerves and ganglia of children affected by infantile hypertrophic pyloric stenosis. He concluded the nerves were neither degenerate nor immature. Kobayashi 1994 identified a gross reduction in activity for markers to the neural supporting cells as well as acetylcholine . Others have identified a reduction in Vasoactive Intestinal Polypeptide, met Enkephalin, and Neuropeptide Y (Malmfor G , 1988, Tam PKH , 1986) CHAPTER 1-______INTRODUCTION______1 ^

Diminished nitric oxide synthase activity has been identified in infantile hypertrophic pyloric stenosis ( Vanderwinden JM., 1992) congenital aganglionosis (Vanderwinden JM., 1993) , and achalasia of the cardia (Moncada S., 1994). The authors proposed that the abnormal expression of neural Nitric Oxide Synthase (NOS) underlies these three motility disorders affecting infants . Vanderwinden suggested the diminished NOS activity was restricted to the circular muscle layer in infantile hypertrophic pyloric stenosis. This resulted in pylorospasm and thus hypertrophy of the circular muscle layer . However the assay of NOS activity using NADPH reactivity is non-specific for neuronal NOS, and may be influenced by the presence or absence of inhibitors, such as haemoglobin, in the diseased or control specimens. The reduced NOS activity may be the result rather than the cause of the hypertrophy. The results of this study are therefore non-specific and inconclusive.

1.2.8.4.Elevated Gastrin Level.

In 1976 Dodge and Karim successfully induced infantile hypertrophic pyloric stenosis in beagle puppies following the prolonged perinatal administration of pentagastrin . However none of the animals were described to have vomited or lost weight, as occurs in dogs that develop the condition naturally. The histological changes were not quantified and the model has never been successfully reproduced .

Gastrin levels in infantile hypertrophic pyloric stenosis have variously been described as elevated (Bleicher MA., 1978, Spitz L , 1976) or normal (Rogers IM., 1975, Hambourg MA., 1979) . Werlin in 1978 measured cord serum gastrin levels prospectively in children who subsequently developed infantile hypertrophic pyloric stenosis and found no difference compared with control specimens.

Serum gastrin levels, somatostatin immunoreactivity and receptor binding sites have been measured 4-15 years following pyloromyotomy for infantile hypertrophic pyloric stenosis (Barrios 1994). The fasting serum gastrin levels and the serum gastrin response to a standard breakfast were significantly elevated in children that had undergone pyloromyotomy. Somatostatin immunoreactivity was diminished within the gastric fundus and antrum. There was found to be an increase in the number of CHAPTER 1-______INTRODUCTION______somatostatin receptor binding sites . The authors proposed that somatostatin inhibits basal and stimulated gastric acid secretion as well as gastrin release, the decreased gastric somatostatin concentration may be one mechanism by which hypergastrinaemia occurs in children who had undergone pyloromyotomy for infantile hypertrophic pyloric stenosis. Somatostatin and nitric oxide synthase have been co-localised to the same myenteric neurons ( Vincent SR., 1992). CHAPTER 1-______INTRODUCTION______^

1.3. The Enteric Nervous System

1.3.1. Neurotransmitters and Neuropeptides The human nervous system has many subdivisions: the brain and spinal cord form the central nervous system and the nerves extending from it the peripheral nervous system. The peripheral nervous system can be further subdivided into autonomic, somatomotor, and sensory systems . The autonomic nervous system controls and preserves the homeostasis of the visceral functions and is subdivided into three functionally and anatomically different systems: a) postganglionic sympathetic neurons which innervate the peripheral target organs, b) postganglionic parasympathetic neurons which innervate locally their target organs and c) preganglionic autonomic neurons which innervate the sympathetic, parasympathetic and enteric ganglia. The enteric nervous system consists of two ganglionated plexuses, the myenteric and submucosal plexuses . The myenteric plexus lies between the longitudinal and circular muscle layers . The submucosal plexus is located in the submuscosa . Neurons of this plexus synapse with neurons in both plexuses and project fibres to the mucosa and in the human to the innermost layer of the circular muscle (Wattchow D.A., 1988). A reciprocal innervation exists between enteric neurons and adrenergic and noradrenergic neurons located in paravertabral ganglia, which relay input from the central nervous system . Neurons of the enteric nervous system may be characterised according to their size and shape, electrophysiological properties, and neurotransmitter content. By neurotransmitter content, the nerves of the myenteric plexus may be subdivided into two major and distinct groups : neurons that contain vasoactive intestinal polypeptide (VIP) and nitric oxide synthase( NOS) , and neurons that contain the tachykinins, substance P(SP) and substance K, and probably also acetylcholine . Many of these neurons are motor neurons, innervating smooth muscle cells directly; the remainder are intemeurons . The opioid peptides, dynorphin and met Enkephalin, are present in both VTP/NOS and tachykinin/acetylcholine (Furness JB., 1992, Llewellyn-Smith IT, 1988). Some neurons contain somatostatin, gamma-aminobutyric acid (GABA), or serotonin . Such neurons do not innervate smooth muscle directly, but via other myenteric neurons. CHAPTER 1-______INTRODUCTION______^

The enteric smooth muscle sphincters differ from other regions of the gastrointestinal tract in that they have myogenic tone that is inhibited by neural stimulation . Acetylcholine and noradrenalin are established neurotransmitters in the autonomic nervous system. Following blockade of these classical neurotransmitters, inhibitory responses persist within the gastrointestinal tract and especially the enteric smooth muscle sphincters . This autonomy of the enteric nervous system has aroused much interest in the nonadrenergic and noncholinergic innervation of the bowel . However despite many histological and pharmacological studies, much remains to be understood of the innervation of the bowel and in particular the pylorus (Grider JR., 1992, 1993, DesaiKM., 1995). CHAPTER 1-______INTRODUCTION______^

1.4. Neurotransmitters and Neuropeptides

1.4.1. History and Classification In 1904 , Elliot proposed the concept that the nervous system utilises chemical transmission. He suggested the stimulation of peripheral nerves may cause the release of substances from nerve endings, which may affect the innervated organ. This suggestion was later confirmed by the separate studies of Loe^vi (1921) and of Cannon and Uridil (1921), and led subsequently to the discoveries of acetyl and norepinephrine, the first neurotransmitters identified . The nervous system produces a large number of substances possessing a wide range of actions on both neural and non-neural tissue but these substances have to meet the following criteria to be classified as transmitters (Orrego F., 1979). The substance along with its precursor and synthetic enzymes must be present in the presynaptic neuron. The substance should be released upon physiological stimulation of the presynaptic neuron. Exogenously applied transmitter candidates and the release of the endogenous substance should have the same effect upon the presynaptic neuron, and both should have the same effect on the postsynaptic neuron, and both should be antagonised by the same pharmacological agents . Specific receptors should be present and the interaction of the substance with its receptor should induce changes in the postsynaptic membrane permeability leading to excitatory or inhibitory postsynaptic potentials . There should exist an inactivation or removal mechanism by an enzyme or an uptake process for the transmitter candidate to conform .

Neurotransmitters are divided into four main groups: aromatic amino acids and their derivatives, aliphatic amino acids, purine derivatives and peptides ( Osborne NN., 1983). The aromatic and aliphatic amino acid groups function as classical neurotransmitters while purine derivatives and peptides most probably function as neurotransmitters and neuromodulators . The aromatic amino derivatives include adrenaline, nordrenaline, dopamine, serotonin, histamine, and tyramine while the CHAPTER 1-______INTRODUCTION______^

aliphatic amino acids include acetylcholine, aspartate, y-amino butyric acid (GABA), glutamate and glycine . The purine nucleotides adenosine, adenosine monophosphate (AMP) and adenosine triphosphate (ATP) are considered as neurotransmitters (BumstockG., 1972).

1.4.2. Neuromodulators Neuromodulators are compounds which are essential for the maintenance and communication between the neurons but do not have a trans-synaptic inhibitory or excitatory action as such on post-synaptic receptors . Neuromodulators act rather in altering the neuronal activity or acting together with neurotransmitters by altering their effect. Neuromodulators therefore function in neurotransmitter synthesis, in neurotransmitter release, receptor expression and uptake . The categorisation of neuroactive compounds into neurotransmitters and modulators may not always be justified as there are increasing numbers of examples which have multiple effects in the nervous system and the peripheral tissues . They clearly have potent effects upon many body functions, and are often synthesised and co-released with classical neurotransmitters from the nerve terminals ( Hockfelt 1987). In addition some neuropeptides can be synthesised and released by non-neuronal cells as well. Attempts to classify them according to their function in the synapse are therefore premature and it may be that at present the action of any given neurochemical exocytosis is more relevant.

1.4.3. Interstitial Cells of Cajal

In recent years increasing interest has been shown in the Interstitial Cells of Cajal (ICC). They were first described by Antiago Ramon y Cajal, a Spanish Neuroanatomist in 1893. These cells have large, oval indented nuclei with little perinuclear cytoplasm and branching cell processes giving them a stellate appearance. The cytoplasm contains abundant smooth endoplasmic reticulum and intermediate filaments. They do not contain myosin. In humans a few gap junctions have been described while in dogs there are none. CHAPTER 1-______INTRODUCTION______24 antibodies to c-kit receptors on the cells surface. Thus these cells have been seen to be arranged in bundles and to be innervated by nerve fibres from Auerbach’s Plexus. The ICC then relay onto muscle layers by specialised muscle fibres arranged into groups. Several functions have been attributed to the ICC. These include acting as an intermediaries between nerves and muscle and as pacemakers which generate rhythmic slow wave activity. The evidence for these proposed roles is the ultrastructure and location of the ICC and the results of ablation studies using methylene blue, monoclonal antibody to c-kit and W AW mice bred to be deficient of c-kit (Ward SM., 1993,HaggerR., 1997).

Clearly this group of cells has an important role in the function of the gastrointestinal tract, one which is as yet to be fully understood. CHAPTER 1-______INTRODUCTION______^

1.5. Features of Main Neural Markers and Neuropeptides of interest A review of the literature will be given here of the peptide substances of main focus of this project: Vasoactive Intestinal Polypeptide(VIP), Calcitonin Gene Related Peptide (CGRP), Substance P (SP), and Nitric Oxide (NO).

1.5.1. The General Neural Marker: Protein Gene Product 9.5 Protein Gene Product 9.5 (PGP 9.5) is a major component of the neuronal axolemma . This protein was originally detected by using two dimensional electrophoresis of crude homogenates of human organs (Jackson P., 1981) and was later isolated and purified from human cadaver brain tissue . It was shown that PGP 9.5 has a molecular weight of 27 kDa and constitutes approximately 1-2% of the total brain soluble proteins (Doran 1983). Immunocytochemical localisation showed that PGP 9.5 is widely distributed in both the central and peripheral nervous system (Thompson RJ., 1983) and is accepted now that antibodies to PGP 9.5 are perhaps the best markers of neural elements since even the smallest nerve fibres can be delineated imunocytochemically (Gulbenkian S., 1987) .

1.5.2. Substance P Substance P was the first neuropeptide to be discovered. It was originally extracted from the central nervous system in 1931 by von Euler and Gaddum (von Euler and Gaddum, 1931) . It is an 11 amino acid peptide belonging to the tachykinin family, a group of peptides which share the sequence similarities among the six aminos at their carboxyl terminal end . The other members of the tachkykinin family identified to date are neurokinin A (substance K, neurokinin a, neuromedin L,) and neurokinin b (neuromedin, neurokinin P) which are found in mammalian tissues, and eleidosin, kassinin and physalemin which have been detected only in the lower vertebrates (Kimura S., 1983; Tatemoto M., 1985). In rats, it has been demonstrated that two genes encode the precursor molecules for the tachykinins: preprotachykinin A (PPT-A) and B genes (PPT-B) The rat PPT-A gene encodes a, p, y- preprotachykinins, which contain the sequence for substance P, neurokinin A and neurokinin K, while the PPT-B gene encodes neurokinin B (Kraiuse CHAPTER 1-______INTRODUCTION______^

YT., 1987) . Three different receptor subtypes (NKl, NK2, NK3 ) have been identified for the tachykinins, each receptor type having its preferential binding affinity to substance P, neurokinin A and neurokinin B, respectively ( Lee CM., 1986) . The distribution of substance P is widespread in the central and peripheral nervous system . A study by Ljundgahl demonstrated substance P-immunoreactivity in more than 30 cells groups of the central nervous system, indicating it may have diverse functional effects in the body (Ljungdahl A I, 1978) . While immunoreactivity to VIP is found in both the gastric epithelium and smooth muscle, SP is found primarily in the myenteric plexus and to a lesser degree in the submucosal plexus (Costa M., 1987) . The injection of SP analogues into the pylorus results in an immediate increase in intraluminal pressure (Allescher HD., 1989). There is an initial tonic contraction followed by an increase in phasic activity which may be reduced by atropine or TTX. SP slows gastric emptying by a time dependent, atropine sensitive mechanism (Holzer P., 1986) . SP both in vitro and in vivo has a TTX independent stimulation of the sphincter, indicating a direct myogenic activity (Parodi JE., 1990). SP receptors have been localised mainly to isolated smooth muscle cells. A greater concentration was located in the distal stomach and pylorus. In the ileocaecal sphincter a greater proportion of SP receptors were isolated upon the circular than the longitudinal muscle layer (Souquet JC., 1985) The neurokinin receptor most specific for SP (NK-1) was found on the smooth muscle of the pylorus (Rothstein RD., 1989). The distribution of SP immunoreactivity, actions, and receptor localisation suggest a role for SP in both afferent and efferent pathways effecting sphincteric function in several regions of the gut. CHAPTER 1- INTRODUCTION 27

1.5.3. Vasoactive Intestinal Polypeptide Vasoactive Intestinal Polypeptide (VIP), a 28 amino acid peptide, was originally discovered from extracts of porcine duodenum by Said and Mutt in 1970. It is derived from a precursor preproVCP molecule whose cleavage generates the other peptides of this family: a 27 amino peptide histidine isoleucine (PHI) and peptide histidine valine (PHV-42). In human tissues a peptide closely related to PHI was found to be derived from the same prepromolecule as VIP and was subsequently named as ‘peptide histidine methionine’ ,(PHM),which is the human equivalent to PHI (Itoh N, et al 1983). VIP is both structurally and biologically related to secretin and glucagon (Said SI., 1982) These findings were supported by the finding that VIP and PHI/PHM are co­ stored in the same neurones. (Anand P., 1984, McGreegor GP , 1984). VIP has been localised in all regions of the gastrointestinal tract. It appears to be present primarily in gastrointestinal neurons innervating circular muscle and mucosa. (Larsson LT., 1979, Reinecke M., 1981) VTP has been suggested to be in greater concentrations in gastrointestinal sphincters (Alumets J., 1979). The majority of VIP- containing neurons in the gastrointestinal tract are intrinsic in origin. Approximately 45% of neurons in the myenteric plexus are immunoreactive for VIP. The terminals of these neurons project onto either other neurons or smooth muscle cells of the circular and to lesser degree longitudinal muscle layers.(Costa M., 1983, Furness JB., 1981). Antegrade neurotracer studies have identified efferent vagal fibres, especially to the proximal gastrointestinal tract, to contain VIP and not enkephalin and galanin. Some vagal fibres were seen to surround VIP containing myenteric neurons. (Kirschgessner AL, et al. 1989) This study demonstrated that at least a portion of the VIP immunoreactive fibres are of preganglionic vagal origin, although some VIP immunoreactive postganglionic myenteric neurons were noted to be present. VIP release, resulting in lower oesophageal relaxation, has been documented after vagal stimulation. (For further discussion please see subsequent chapters.) PHI and pituitary adenylate cyclase activating peptide, a recently identified VIP-like compound, have been localised to the lower oesophagus. (Biancani P., et al. 1989, Uddman R., 1991). PACAP (Pituitary Adenylate Cyclase Activating Peptide) a newer CHAPTER 1-______INTRODUCTION______28

VIP-like substance has been colocalized with VTP to the lower rat oesophagus. (Uddman R., 1991) VIP is often co localised with other classical nonpeptide neurotransmitters and neuropeptide substances including acetylcholine, enkephalin, dynorphin, galanin, PHI, neuropeptide Y, and CGRP. (Lundberg JM., 1981, Furness IB., 1986, Ekblat ER., 1984). However VTP is never co-localised with the excitatory neuropeptide Substance P.(Bartho L ., 1985, Costa M., 1985)

1.5.4. Calcitonin Gene Related Peptide Calcitonin gene related peptide (CGRP) is a 37 amino acid neurotransmitter molecule. It is an important neurotransmitter present in the central and peripheral nervous system. Its precursor is encoded by alternatively spliced messenger RNA from the primary RNA transcript of the calcitonin gene. CGRP is colocalized with substance P and tachykinins in peripheral sensory neurons (Yamano M , 1988) in primary afferent fibres in the dorsal column of the spinal cord (Tamatani M., 1989 ) , and in the limbic system of the brain (Skofttsch G , 1985 ). CGRP is distributed widely within the gastrointestinal tract, including the lower oesophageal sphincter and the internal anal sphincter (Rodrigo J., 1985) . Fibres immunoreactive to CGRP are mainly located in the myenteric plexus. CGRP is generally accepted as having a sensory function within the stomach as more than 85% of spinal afferents to the stomach contain CGRP (Dockray G J, 1991) .

CGRP is a potent relaxant of smooth muscle in general. It has been demonstrated to cause relaxation of both the lower oesophageal sphincter and the internal anal sphincter. Two types of CGRP (I and II ) have been recognised, with the possibility of different receptors and actions within the same tissue.(Baurenfeind PR., 1989). Relaxation of the opossum lower oesophageal sphincter is dose dependent to CGRP. As this action is partially sensitive to TTX it was thought to be a mixed direct smooth muscle and nerve effect ( Rattan S., 1988) . CHAPTER 1- INTRODUCTION 29

1.5.5. Nitric Oxide In recent years a great deal of interest and study has been invested in the relationship between Nitric Oxide (NO) and the pathogenesis of infantile hypertrophic pyloric stenosis, since Vanderwinden in 1993 proposed the aetiology is due to grossly diminished Nitric Oxide Synthase (NOS) expression by the circular muscle resulting in pylorospasm and muscle hypertrophy. Three of the five published animal models of pyloric stenosis are based upon aberrant NO expression (Huang PL., 1993, Bredt D , 1993, Abel RM , 1995, Voelker CA , 1995) .

NO has been attributed a wide spectrum of physiological functions. NO is a free radical, which by definition contains at least one unpaired electron in its atomic radical configuration, making it a highly reactive molecule. Thus NO has a half life of only a few seconds in physiological solutions. For these reasons until relatively recently NO was recognised as only an atmospheric pollutant.

As a biological entity NO has been studied for only a few years. In this time the study of its synthesis and functions has generated a massive number of publications. So great and sudden has the impact of NO been upon medical and scientific research that in 1992 it was named the ‘Molecule of the year’ by the well respected journal Science. ( Korshland 1992; 258: (Editorial) 1861).

While the biological functions of NO are a new scientific discovery, NO is phylogenetically an ancient biological entity. The horseshoe crab ( linulus polyphenus) by archaeological studies has remained unchanged for over 500 million years, produces NO to prevent aggregation of its circulating haemocytes (Radomski MW., 1991). To date NO has been implicated in several physiological functions: in vascular signalling contributing to the maintenance of vascular homeostasis, as a neurotransmitter and neuromodulator in the central and peripheral nervous system and as an agent of cellular killing as part of the immune system. Aberrant NO function has been implicated in several disease processes including atherogenesis (Moncada S., 1991) pulmonary CHAPTER 1-______INTRODUCTION______30 hypertension (Frostell 1991) Hirschsprung’s disease and infantile hypertrophic pyloric stenosis (Vanderwinden JM., 1992) .

That mammalian cells may generate endogenous nitrites and nitrates was first suspected when it was demonstrated that germ free rats continued to excrete similar amounts of nitrates as control animals. This dispelled the proposition that enteric flora were responsible for the greater excretion of nitrates than the dietary intake (Tannenbaum 1987). L-Arginine was then identified as the essential substrate for the synthesis of nitrites and nitrates (lyenger R., 1987). The biosynthesis of nitrites and nitrates also yielded the amino acid L-citrulline and could be inhibited by NG - monomethyl-L-arginine (LNMMA), a close structural analogue of L-arginine (Hibbs JB., 1987).

Separate though almost concurrent work resulted in the identification of a substance, subsequently termed ‘Endothelium Derived Relaxing Factor’ (EDRF) , released by endothelial cells that was essential for the vasodilator activity of acetylcholine. Similarities between the properties of EDRF and nitrovascular compounds used in the treatment of ischaemic heart disease soon became apparent. In 1987 pharmacological and biochemical evidence was published that EDRF was NO ( Palmer RMJ., 1987).

The biochemical characterisation of Nitric Oxide Synthase proved to be difficult as the synthesis of nitric oxide is closely controlled. The first isoform to be isolated was that derived from rat brain, now commonly referred to as neural NO synthase (nNOS) (Bredt D., 1990). It was found to be sensitive to the presence of Ca^^ ions and calmodulin . Tetrahydrobiopterin ( BH 4 )and NADPH were cofactors to the enzyme (Mayer BJ., 1990). Electrophoresis using sodium dodecyl polyacrylamide gel (SDS/PAGE) identified the enzyme to migrate to a single band at ~150kDa. The molecular weight of NOS was later calculated as ~280kDa, leading Scmidt in 1991 to propose the neural brain derived isoform to be a dimer. Activity of nNOS is almost entirely dependent upon the ambient Ca^^ concentration and the presence of calmodulin. The activity is further regulated by phopshorylation, demonstrating consensus sequence for phosphorylation by cyclic adenine monophosphate (cAMP)- CHAPTER 1-______INTRODUCTION______3j_ dependent-protein kinase II, cGMP-dependent protein kinase C and it appears that phosphorylation decreases activity of the enzyme. Using electrophoresis the endothelial form of NO synthase (eNOS) was characterised from bovine aortic endothelial cells (Pollock 1991). SDS-PAGE demonstrated a single band at ~135kDa. This required Ca^^ and calmodulin. BHj and NADPH were cofactors just as for the brain NOS. Unlike the brain isoform, the endothelial derived NO synthase retained almost all enzymatic activity within the particulate, rather than the cytosolic, fraction of the homogenate ( Pollock 1991). Recently Sessa (1995) reported eNOS to be a golgi-associated protein. Unlike nNOS and the macrophage derived isoform eNOS is unique amongst NO synthases in that it contains an N- terminal myristolation consensus site which appears to be instrumental in localising eNOS to the plasma membrane (Busconi 1993). Phophorylation of eNOS determines its subcellular localisation resulting in its translocation from the particulate to the cytosolic fraction and this is also marked by a decrease in its activity (Michel 1993). Macrophage NO synthase was demonstrated by electrophoresis at ~125-135kDa, whilst the active enzyme was demonstrated to be a dimer of ~250kDa (Stuehr 1991). The majority of activity of this isoform resided in the cytosolic fraction. The macrophage derived isoform did not require Ca^^ and was dependent upon BH4 , NADPH as well as flavin mononucleotide (FMN), flavine-adenine-dinucleotide (FAD) and calmodulin. Calmodulin binds so tightly to this isoform that some consider it an integral part of the molecule. Unlike the other isoforms, that derived from the macrophage is not constitutively expressed but requires transcriptional activation and de novo protein synthesis which may be induced by lipopoysaccaride from gram negative bacteria and/or cytokines such as IFN-1. Thus this isoform of NO synthase is not normally present in the resting cell and has acquired the term inducible NO synthase (iNOS). Most smooth muscle tissues of the periphery are supplied by a sub-set of nerves whose activity is not regulated by classical adrenergic or cholinergic neurotransmitters. These so called non-adrenergic non-cholinergic (NANG) nerves evoke, in response to electrical field stimulation, relaxation of the smooth muscles they are supplying. Much of the early work in attempting to identify the nature of this unknown neurotransmitter was performed on the rat anococcygeus and bovine retractor penis muscles which were found to be particularly rich in NANG nerves. Studies on these tissues demonstrated CHAPTER 1-______INTRODUCTION______3 ^ that the response to NANC stimulation could be blocked by haemoglobin and that cGMP was a probable mediator of the relaxant effect. (Gillespie JS., 1989). It was subsequently shown that the characteristics of this unknown neurotransmitter were similar to EDRF and that its activity could be abolished by NO synthesis inhibitors (Rand 1995). It is now evident that NO modulates (at least in part) all NANC responses, controlling smooth muscle tone in various tissues including the gastrointestinal tract, the respiratory tract, the urogenitary tract and some blood vessels. A possible role for NO as a neural agent in the intestine was demonstrated in studies by Bredt in 1990. NOS immunoreactivity was demonstrated in the myenteric neurons of rat intestine. Subsequently the presence of nNOS containing nerves has been demonstrated throughout the gastrointestinal tract from the oesophagus to the gastric sphincters and from the small intestine to the colon (Vanderwinden JM., 1993). From this distribution it is evident that NO synthesis contributes to gastric motility and compartmentalisation (Desai JR., 1991, Anggard E.,1994) . Interestingly, the neurally-mediated VTP induced fall in resting internal anal sphincter tone and relaxation was partially suppressed by L-NMMA (Rattan S., 1992) raising the possibility of a link between VTP neurons and NO producing cells. Although the exact nature of NOS containing neurons is not known, one may ask, are VIP and NOS co­ present and co-released? The possibility of co-existence of VTP and NO in the Non Adrenergic Non Cholinergic (NANC) inhibitory neurons may raise important issues in the NANC nerve mediated relaxation of sphincteric and non sphincteric smooth muscles of the gastrointestinal tract and other organ systems. In this regard it is remarkable that 25% of Dogiel type 1 neurons in the guinea pig small intestine were NOS containing. These NOS positive neurons co-expressed VTP (Costa MJ., 1983). Three other Non cholinergic intemeurons are now known to be involved in the pathways leading to cephalad intestinal relaxation: the neurons contain variously somatostatin, GABA, and opioid peptides. The inter-relationship between these agents and VIP and NO has been the subject intense research (Please see discussion Chapter 4) . However to date little is known of the development, distribution and character of the nerves containing NOS in the human stomach. (Please see section 4.5 for further discussion) CHAPTER 1-______INTRODUCTION______33^

1,6, AIMS OF THE STUDY

1.6.1. Hypothesis The pathogenesis of infantile hypertrophic pyloric stenosis is unknown. Changes in the muscle innervation and neurotransmitters are significant factors. Little is known of the of the innervation of the normal pylorus or how this develops. Quantified morphological and immunohistochemical changes in the muscles and nerves in infantile hypertrophic pyloric stenosis have not previously been documented.

It is postulated that the morphological changes in the nerves and muscles are due to a selective loss of neuroactive agents. In order to investigate this hypothesis a number of defined tissue processes were studied. These studies can be grouped into three major areas: I. Developmental studies of the normal human pylorus. (Ontogeny) II. Quantitative studies of the morphological and histochemical changes in the muscles and nerves in infantile hypertrophic pyloric stenosis to define precisely the underlying lesion. III.The identification of naturally occurring and experimentally induced models of pyloric smooth muscle hypertrophy, to then quantify and compare the histological changes between species.

1.6.1.1. The main aims o f the present thesis were as follows: 1. To test the hypothesis that within the fetal pylorus the expression of peptides by the muscle and nerves is developmentally regulated. 2. To trace the vagal innervation of the human fetal pylorus. 3. To study the relationship between the developing muscle and nerves. 4. To quantify the morphological changes in the dimensions of the nerves and muscle, that occur in infantile hypertrophic pyloric stenosis. 5. To characterise the changes in neuropeptide and nitric oxide expression in infantile hypertrophic pyloric stenosis.

6 . To quantify the chronological sequence of changes within the pylorus of an experimental animal model of diminished NO synthase activity. CHAPTER 1-______INTRODUCTION______^

7. To quantify the morphological and histochemical changes in the pylorus of dogs suffering from congenital pyloric stenosis.

8 . To study the comparative anatomy of the two animal models of pyloric stenosis

described in 6 & 7 above. 9. To study the effects ablation of the myenteric plexus on the innervation and muscle morphology of the rat pylorus. CHAPTER 1- INTRODUCTION 35

Figure 1-lIllustration of gastric distension and visible peristalsis in hypertrophic pyloric stenosis. CHAPTER 1- INTRODUCTION 36

Figure 1-2 Plain abdominal radiograph showing a dilated gas filled stomach with little air passing through into the distal portion of the intestine CHAPTER 1- INTRODUCTION 37

Figure 1-3 Barium meal examination showing the narrow elongated pyloric canal (string sign) passing convexly upwards to the duodenal cap.

• f e f = C •■’-■ ÿ f e CHAPTER 1- INTRODUCTION 38

Figure 1-4 Ultrasound scan in pyloric stenosis showing the thickened pyloric musculature with a central sonoluscent area representing the lumen of the pyloric canal. CHAPTER 1- INTRODUCTION 39

Figure 1-5 Illustration of the pylorus during pyloromyotomy. CHAPTER 1- INTRODUCTION 40 CHAPTER 2______MATERIALS AND METHODS______41

2. Chapter 2 MATERIALS AND METHODS 2.1 General Introduction 2.2 Immunocytochemistry 2.2.1 Fixation 2.2.2 Tissue Preparation 2.2.3 Immunostaining 2.2.4 Antisera

2.3 Specificity Controls 2.3.1 Antiserum specificity 2.3.2 Methodological specificity

2.4 Experimental procedures 2.4.1 Selective division of the fetal vagus upon the lesser curve of the stomach 2.4.2 Adjuvant induced deficiency of tetrahydrobiopterin 2.4.3 Selective vagotomy and carbon dioxide laser induced ablation of the rat pylorus

2.5 Conventional histology

2.6 Assessment of immunostaining

2.7 Quantification

2.8 Statistical analysis

2.9 Microscopy and Photomicrography CHAPTER 2______MATERIALS AND METHODS______42

2.1. General Introduction The present study has mainly been concerned with the localisation of intraneuronal proteins and peptides at light microscopical level in the pylorus. The ability to identify neural substances in tissue sections by immunohistochemistry has resulted in rapid advances in the neurosciences. Immunocytochemistry, originally introduced by Coons in 1941, is now in wide use and several modifications of the method have been developed since the original work. The avidin-biotin-peroxidase complex method (Hsu SM., 1981) was applied in most studies of different experimental models and human tissues. In some instances the possible origin and nature of the nerve fibres was determined by surgical or neuropharmacological manipulations.

During the present study it became necessary to modify and develop specific software and applications of image analysis to obtain quantitative data of the different parameters of neural changes seen in the models studied. This methodology, which is based upon the separation of different grey scales captured from immunostained sections, presents now an additional powerful methodology in morphological techniques. The rapid advance in computer sciences and in image manipulation thus provide a semi-automatic technique for objective evaluation of the structures revealed by immunocytochemistry.

2.2. Immunocytochemistry

2.2.1. Fixation

Tissue fixation serves two main purposes in immunocytochemistry i) preservation of tissue structure and ii) preservation of the antigen of interest in situ without significant loss of antigenicity. Treatment of fresh tissues with a suitable fixative is therefore required to prevent autolysis and to trap soluble antigens within the cells. Peptides, as small molecules can be degraded rapidly especially in tissues rich in proteinases such as are found in the intestine. The fixative may either coagulate proteins (alcohols and CHAPTER 2______MATERIALS AND METHODS______43 acetone) or cause inter or intra-molecular cross linkages of the cellular components tightly to each other (paraformaldehyde based fixatives). Inadequate fixation therefore may result in loss of antigens and tissue destruction whereas overfixation may lead to excessive cross-linking and masking of antigens of interest.

For optimal fixation, tissues are preferentially perfusd with a choice of a fixative which will produce rapid penetration via the vascular tree and immobilisation of the proteins and peptides throughout the tissues (Palay 1962). However, this method can only be used when the subject of study is either a whole organ with arteries relatively easily accessible or as an animal killed for tissue collection. In most cases, therefore, immersion fixation is used. This method can provide very satisfactory results but dissected tissue has to be small enough for rapid immobilisation of proteins and peptides to occur. One method which delays tissue changes before optimal fixation is to immerse the tissue in cold fixative immediately after removal.

In the present study, human and animal tissues were immersion fixed immediately after removal. The fixative of choice was Zamboni’s fluid as this fixative could be stored for longer periods. (Appendix II). This was significant particularly when human or animal tissues were obtained during surgical procedures.

2.2.2. Tissue preparation

After fixation, tissues were washed thoroughly with 1 .OmM phopshate-buffered 0.15 saline (PBS, ph7.4) containing 15% sucrose as tissue preservative and 0.01% sodium azide to inhibit microbial contamination (Appendix I). In some cases tissues were stored upto three months in this buffer at 4°C without any apparent loss of morphological detail or antigenicity. CHAPTER 2______MATERIALS AND METHODS______44

Cryostat blocks were prepared by snap-freezing (Appendix III). Cryostat sections 10pm sections were cut and mounted on slides coated with poly-L-lysine or Vectabond. Poly-L-lysine provided better adhesion and was preferred in most studies. In some cases (in particular the human and mouse fetal immunostaining) whole mount preparations were used. Tissues were dissected under a dissecting microscope and processed as a free floating preparations through the different steps of the immunostaining procedure.

2.2.3. Immunostaining

Immunocytochemistry is a technique employed for the visualisation of antigens in situ in tissue sections using polyclonal or monoclonal antibodies. The method is based upon the conjugation of chromogen molecules with antibodies so that the binding site can be detected either by the use of direct methods (primary antibody conjugated with chromogen molecule) (Coons 1941) or by indirect methods (antibodies raised to the immunoglobulins of the donor species of the primary antisera) (Coons 1951). The chromogens may be either directly emitting light of specific wavelength (fluorochromes) or they can be enzymes chosen to visualise by chemical reaction. Indirect methods are more sensitive than direct ones since multiple secondary chromogen labelled antibodies can attach to the primary antibody revealing the antigenic sites (Stemberger, 1979). This increased sensitivity allows lower concentrations of primary antiserum to be used and also has the advantage that any possible interference of labelling process and loss of primary antibody during this process can be avoided.

The introduction of enzyme labelled antibodies produced a further increase in the sensitivity of immunostaining and provided stable end products with electron dense properties so that they could be employed in application of electron microscopy for ultrastructural localisation of binding sites. The peroxidase-antiperoxidase (PAP) method (Stemberger 1970) has been widely used as it provides simple light microscopic means of detecting antigens with the added advantage of conventional CHAPTER 2______MATERIALS AND METHODS______45 histochemical counterstaining which is particularly important in histopathology. This method was first introduced as a four step technique and was later modified to a three step method with the increased sensitivity resulting in a further dilution of the primary antiserum. The binding sites were detected by using the DAB/hydrogen peroxide reaction to visualise the horseradish peroxidase which was used as a chromogen molecule. The avidin-biotin-complex (ABC) method, which was utilised in most studies of the present thesis, is quite similar to the PAP method, with the exception that it relies upon the binding properties of avidin and biotin, which have an extremely high affinity to each other (Appendix VI; Hsu SM., 1981). In this method, the primary antibody is unlabelled, and the secondary antibody is covalently biotinylated. The third step involves the application of the ABC complex, which binds to the secondary antibody by virtue of the avidin-biotin high affinity reaction. As in the PAP method, the antigen-antibody binding sites are visualised by the use of chromogens such as DAB. Several small biotin molecules can be conjugated in the same antibody molecule, allowing networks of ABC complexes to bind to the antibody. This is a significant advantage as it helps to detect very low tissue concentrations of antigens. Supra- optimal dilutions of the primary antibody can then be used to provide comparisons of the relative density of antigens between tissues of interest. There are disadvantages in the ABC method due to endogenous biotin present in the tissues. For example, mast cells are rich in biotin and therefore a false positive result may occur. Negative controls without the primary antiserum are therefore necessary in all studies. Fig 1 shows the basic principles of the ABC method with the various possibilities to reduce undesirable non-specific reactions.

The ABC complex is usually visualised by the use of DAB and hydrogen peroxide to give a brown permanent end-product. The principle is based upon the use of the enzyme peroxidase in the ABC complex. Peroxidase donates electrons to its substrates hydrogen peroxide and the oxidised enzyme is then stabilised by the donation of electrons from the DAB molecule. The oxidised DAB then polymerises to produce a highly insoluble and amorphous brown deposit clearly visible under the light microscope (Seligman 1968). This end product reaction can also be used for study with transmission electron microscopy since the deposit is reasonably electron-dense. CHAPTER 2______MATERIALS AND METHODS______46

This method generally provides perfectly satisfactory resolution in most studies, especially when larger structures like cells are studied. Morphological tissue alterations and nerve fibre destruction in a given tissue are usually reported in a descriptive manner and photomicrographic documentation is then highly important. An ideal preparation should have an end product as dark as is possible with minimal background staining. The use of heavy metal ions in immunohistochemistry for intensification can provide the means to increase density and resolution significantly. During the studies in this thesis one such intensification method was used. This procedure produced a more intense end product permitting more sensitive microscopic detection. By this technique an incubation medium prepared from DAB, ammonium nickel sulphate, ammonium chloride, g-D-glucose oxidase in acetate buffer (Appendix VII; Shu Y., 1988). Ammonium nickel sulphate provides the heavy metal ions to produce a bluish black end product deposit. The peroxidase substrate, hydrogen peroxide, is produced by continuous oxidation of the glucose oxidase substrate, glucose.

Additional enhancement of the staining density was achieved by prolonged incubation times, and repetition of incubation with secondary antisera. Pretreatment with detergents, like Tween and Triton X-100, were not found to improve the staining quality and there was frequent poor adherence of the sections on the microscope slides despite the prior slide coating.

2.2.4. Antisera Most of the antisera to the neuropeptide epitopes used in these studies were raised at the Hammersmith hospital. Polyclonal antisera were preferred as immunocytochemical studies of neuropeptides involve the use of cross linking fixatives which may often alter the primary antigen structure so that more specific monoclonal antibodies cannot recognise the epitope under investigation. The neuropeptides selected for immunisation were first coupled to larger carrier proteins (keyhole limpet haemocyanin (KLH) or bovine serum albumin (BSA)) to evoke a better immune response. Bis-diazobenzidine (BDB), gluteraldehyde and carbodiimide (GDI) were used as a coupling agents. BDB has preferential coupling with tyrosine groups, gluteraldehyde preferentially couples CHAPTER 2______MATERIALS AND METHODS______47 amino groups and CDI with amino and carboxyl groups. Thus the choice of coupling agents determines to some extent which regions of the peptide are to be exposed to the immune system. New Zealand white rabbits were used for immunisation. Typically Img of respective antigen in 200|il of vehicle was used for subcutaneous multiple injections. Booster injections were given four and eight weeks after the first injection and thereafter, injections were given at monthly intervals. Maximal titres of antibodies were usually obtained after the third or fourth booster immunisation and the titres usually rapidly declined after the seventh or eighth booster. All antisera were tested by ELISA, RIA and immunocytochemistry using known positive controls. Antiserum cross reactivity was tested by absorption of a range of dilutions of various synthetic peptides. Significant cross-reactivity was reported if reactivity of the antiserum was decreased or quenched.

The general characteristics of all of the antibodies used in this project are presented in table 2.1. CHAPTER 2______MATERIALS AND METHODS______48

2,3 . Specificity controls

2.3.1. Antiserum specificity

Neuropeptides often belong to families of structurally related peptides which have a region of common amino acid sequence. Therefore, antibodies raised against a peptide often recognise other members of a given peptide family. This, and the fact that specificity tests provide only indirect and partial evidence of specificity, means that immunostaining should be referred to ‘antigen like immunoreactivity.’ The generally accepted means to test antiserum specificity is to carry out liquid phase absorption of the antiserum prior to immunostaining.

2.3.2. Methodological specificity

Methodological specificity includes all other reactions except that of the primary antiserum with its corresponding antigen. Methodological specificity includes background staining, false positive and false negative immunostaining and other possible causes of interference. Several steps were taken in the studies to improve method specificity. Primary antisera were used at their optimal concentrations so that unfavourable serum proteins were diluted out. Hydrogen peroxide in PBS was used to inactivate the endogenous peroxidase in tissue sections. Sites which could possibly bind secondary antiserum were saturated with the use of normal non immune serum from the same species. Carrier protein (BSA) is added to the primary and secondary antiserum dilutions in order to remove antiserum reactivity with albumin. Primary and subsequent immunoreagents were omitted to serve as negative controls and to monitor the methodological specificity. CHAPTER 2______MATERIALS AND METHODS______49

2.4. Experimental procedures

2.4.1. Selective division of the fetal vagus upon the lesser curve of the stomach

Experimental procedures followed the guidelines of the Polkinghom report upon the use of embryos in research. Especial care was taken not to cause unnecessary suffering to the mice or rats used in these experiments.

To trace the vagal innervation of the pylorus the carbocyanine compound 1,1’- dioctatdecyl-3,3,3 ’,3 ’-tetramethylindocarbocyanine percholate (Dil) was used. Dil is a lipophilc crystal that passively diffuses and autofluoresces in aldehyde-fixed tissue, thus allowing selective neuronal tracing post mortem. This technique has been used extensively in both the central and peripheral nervous system to trace neural pathways, though never in human embryology (Gotz M., 1992) . Thus a crystal of Dil was placed upon the severed surface of the vagus nerve as it traverses the lesser curvature of the stomach. The fetal stomachs were then incubated at room temperature for six months.

2.4.2. Adjuvant induced deficiency of tetrahydrobiopterin in mice.

The hph-1 mouse model was originally created as an animal model of hyperphenylalanaemia. Phenylketonuria is the commonest inborn error of metabolism to affect humans. As the laboratory mouse has been extremely well characterised genetically and physiologically, a mouse model of phenylalanine catabolism was a very attractive prospect. In humans, the normal catabolism of phenylalanine is initiated by phenylalanine hydroxylase (McDonald JD., 1988). The cofactor to this reaction is the pteridine tetrahydrobiopterin. Tetrahydrobiopterin is synthesised from GTP (Brand MP., 1995) and maintained in the required reduced state by quinonoid- dihydropteridine reductase (QDHPR). Thus the forms of hyperphenylalanaemia may be CHAPTER 2______MATERIALS AND METHODS______50 classified as mutations that reduce phenylalanine hydroxylase activity ( Types I and II), mutations that reduce QDHPR activity (Type IV), and mutations that disrupt synthesis of tetrahydrobiopterin (Type V). Tetrahydrobiopterin is a cofactor to all the isoforms of phenylalanine hydroxylase and NOS.

A colony of hph-1 mice deficient of tetrahydrobiopterin has been developed as a model of phenylketonuria (McDonald 1988 a & b). The hph-1 mouse was created using the sperm mutagen N-ethyl-N’-nitrosourea (Russell W. 1979). The underlying biochemical defect is a deficiency of GTP-cyclohydrolase activity (McDonald JD., 1988), which catalysed the initial rate limiting step in the synthesis of tetrahydrobiopterin (Nichol C. 1978).

Untreated Phenylketonuria, is associated with infantile hypertrophic pyloric stenosis (Johnson CF., 1978), further implicating NOS in the pathogenesis of the condition.

Experimental procedures followed the guidelines of the Home Office for the care and use of laboratory animals and the procedures used were approved by the local ethical committee. All of the animals were killed by a schedule three procedure. Details of the experiments are given in the relevant chapters of this thesis.

Pyloric specimens were gathered from control (C57BlxCBA) and diseased hph-1 mice at two weeks of gestation, one, ten, twenty, forty, ninety, and one hundred and eighty days after birth. Six control and six diseased specimens were collected at each age from established breeding colonies. The age of the animals was calculated from the date of timed mating. The pups were weaned to a solid diet of a predominantly grain laboratory Chow, at approximately twenty days of age. The animals were weighed immediately prior to being killed by cervical dislocation. The stomach and pylorus were dissected and fixed, fresh, in Zamboni’s fixative for six hours at 4°C and rinsed three times in 15% (wt/vol) sucrose in 0.1 mol/L phosphate-buffered saline (PBS; pH 7.2) with 0.01% (wt/vol) sodium azide. Sections of 10pm thickness were cut perpendicular to the long axis of the gut from snap-frozen blocks in a cryostat at - 25°C. CHAPTER 2 MATERIALS AND METHODS 51

2.4.3. Carbon dioxide laser induced ablation of the innervation of the rat pylorus

The functional role of the myenteric plexus is important to consider in infantile hypertrophic pyloric stenosis. Chemical or surgical ablation of the myenteric plexus has been demonstrated to produce enteric smooth muscle hypertrophy (Hadzijahic N., 1993). Changes in innervation similar to those in infantile hypertrophic pyloric stenosis have been induced in the rat pylorus (Bumstock G., 1993). Electromagnetic irradiation with a carbon dioxide laser has been demonstrated to selectively ablate neural tissue (Kadota T., 1992) . The rat has been used to study the physiological effects of carbon dioxide laser induced irradiation of the stomach (Kadota T., 1992) .

All of the animals were examined, to ensure they were fit for surgery, and weighed. The operating theatre was appropriately lit, ventilated and heated. The doors were closed during surgery, and bore signs notifying staff the carbon dioxide laser was operational. Both the assistants and surgeon were appropriately scrubbed, gowned and gloved. While the carbon dioxide laser was operational all staff wore protective equipment.

The animals were placed supine on a warming blanket on the operating table. An inhalation general anaesthetic, methoxyfluramine, was used. This was administered by a Baines circuit.

Peroperatively the following parameters were monitored; the skin colour, respiratory pattern, and body temperature.

The anaesthetised, adult rats’ abdominal fur was clipped from around the site of incision the skin then prepared vrith an antisceptic solution such as povidone. The stomach was exposed through a vertical midline incision through the linea alba. The leA lobe of the liver was gently reflected cranially to expose the lesser curve of the stomach and the pylorus. Prior to laser irradiation a saline soaked, fenestrated disk of CHAPTER 2______MATERIALS AND METHODS______52 filter paper was placed over the abdomen so that only the pylorus was exposed to irradiation by the carbon dioxide laser.

Following CLIP the abdomen was closed using a standard, layered, interrupted technique using polydioxanone (PDS) sutures.

Following the procedure the animals’ condition was monitored as they recovered in a warm, quiet incubator containing appropriate bedding, such as ‘vet-therm.’

2.4.3.1. Postoperative analgesia.

During recovery and especially the first 24hrs postoperatively, the rats were closely monitored for signs of distress or pain: abnormal behaviour, (eg. huddling and sedantry, or not feeding.) dishevelled fur, lack-lustre/glazed eyes.

As the bowel was minimally handled, the period of postoperative ileus was short. Thus the rats could suckle or drink within a few hours of recovery, and temgesic could be administered as analgesia in blackcurrant jelly.

2.4.3.2. Postoperative care.

The animals were clinically examined and weighed on a daily basis. The adult rats were examined twice a day for the first five days and then once a day. CHAPTER 2 MATERIALS AND METHODS 53

2.5. Conventional histology

In order to preserve high contrast between immunostained structure and background, counterstaining was not usually performed for sections used for image analysis. Instead some sections were cut from each case and studied using haematoxylin and eosin staining to assess the morphological features of the tissues and their structures (Appendix V) .

2.6. Assessment of immunostaining

All immunostained sections were carefully examined with low and high power objectives. In each case, tissue morphology was assessed from conventionally stained sections and immunoreactive structures were related to the general morphology. Care was taken that at least five non-consecutive sections from each tissue block were immunostained for each antiserum in order to avoid over estimation of nerve fibre distribution and their subjective density. A microscopic scoring system was used in the developmental and rat studies. This system is based upon an arbitrary grading of results (density of innervation ) from zero to four pluses. Otherwise subjective quantitative assessment was avoided as this method can provide false-non informative results. However image analysis quantification was used, as is described below, when it appeared likely that quantitative assessment could provide useful information on the physiological or pathophysiological changes involved in infantile hypertrophic pyloric stenosis. CHAPTER 2______MATERIALS AND METHODS______54

2. 7. Quantification

Numerical presentation of immunostaining results has received attention in recent years. In most cases, such results can be assessed by simple cell counting but quantification of changes in complex structures presents a difficult task. Image analysis methods provide critical structural information and offer a possibility to assess a magnitude of differences between experimental groups with good reproducibility. In this thesis the results were quantified wherever possible. During the last three to four years there has been considerable improvement in image analysis methodology and computer software has been modified and developed in the department for particular projects allowing quantitative presentation of results in some instances (Abrams D , 1994).

2.7.1. Image analysis quantification of nerve fibres

Once the optimal conditions for tissue antigen preservation and antisera properties were established, the control and experimental tissues were processed in parallel and image analysis quantification was carried out either in a randomised manner or using control/experimental tissues as pairs in the process. A low light charge-screen coupled CCTV camera (HV-720K, Hitachi, Denshi Ltd., Japan) was mounted on an Olympus BH-2 light microscope linked to a VIDAS image analyser (Kontron, Munich, Germany). The captured image was automatically digitised to a 512 x 512 pixel array with each pixel having one of the total 256 grey levels. Image segmentation was achieved by setting maximal and minimal thresholds so that the darkest areas, i.e. immunoreactive structures, were recognised and counted as positive nerve fibres. Pixels which did not fall in this grey level range were considered to be background. Several field parameters were used depending on the application. These included area percentage, intercepts per mm^ and total immunoreactive area. Preparations were orientated so that the image analysis quantification was carried out in the same way for each preparation. Thus, for example, the tissue blocks of control and diseased human pylorus were sectioned perpendicular to the longitudinal axis of the gut. CHAPTER 2______MATERIALS AND METHODS______55

2.8. Statistical Analysis

The statistical analysis of the results was carried out using the Mann Whitney U test and 95% confidence intervals to compare the median values between the diseased and control groups. A P value of less than 0.05 was taken as evidence of a significant difference.

2.9. Microscopy and Photomicrography

Transmitted light preparations were examined with an Olympus BH 2 microscope and photographed on a Polyvar microscope, fitted with Nomarski interference contrast optics (Reichert-Jung, U.K). Black and white photomicrographs were taken with Kodak Technical Pan 2415 film and developed in Kodak HC 110 developer (1:30) for 8.5 minutes at +20°C. Black and white paper ilfbspeed Grade III or Kodabrome Grade 5 paper was developed in Suprol developer (1:5) (May and Baker, U.K). CHAPTER 2 MATERIALS AND METHODS 56

2. 10. Tables and Figures

Table 2-iTable of primary antibodies Antigen Donor Species Dilution Souree PGP 9.5 Rabbit 1/5000 Ultraclone CGRP Rabbit 1/2000 RPMS SP Rabbit 1/5000 RPMS VIP Rabbit 1/10,000 RPMS nNOS Rabbit 1/5000 V. Riveros- Moreno fNCAM Mouse 1/5000 G. Rougon nmNCAM Mouse 1/10,000 J.J. Hemperly nNCAM Mouse 1/5000 J.J. Hemperly aSM A Mouse 1/20,000 Sigma CHAPTER 2 MATERIALS AND METHODS 57

Figure 2-1 Diagram of Steps in ABC Reaction

UmmWdw CHAPTER 2 MATERIALS AND METHODS 58

Figure 2-2 Flow Diagram of Steps in Image Analysis and Quantification

Hanning of opennm t accordngto image analytic and statistical reqpirermts

Cptinization of tissue processing

Ajustmert o f nicrosocpe and illurrination

Capture iiTBge o f structures to be anal}5ed

processing ard cxxtnast enhanoernat

DtMnloading o f results fcr statistical analysis CHAPTER 2 MATERIALS AND METHODS 59 CHAPTER 3 :THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 60

3. CHAPTER 3 The Ontogeny of the Innervation of the Human Pylorus

3.1 Summary

3.2 Introduction

3.3 Experimental design

3.3.1 Tissues 3.3.2 Imunostaining 3.3.3 Neural tracing

3.4 Results

3.5 Discussion

3.6 Tables and Figures (overleaf) CHAPTER 3:THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS______W

3.1. Summary

There have been many studies documenting the biochemical and histological changes that occur in infantile hypertrophic pyloric stenosis. However relatively little is known of the development or anatomy of the normal pylorus. A knowledge of the development and distribution of the innervation and morphology of the pylorus is important for gaining a better understanding of the function of the enteric nervous system as well as the pathogenesis of infantile hypertrophic pyloric stenosis. In this study the intrinsic and extrinsic vagal innervation was examined. The detailed immunocytochemical localisation of PGP 9.5, nNOS, SP, VTP, and CGRP was examined in thirty four human fetal and infant pyloruses ranging from eight weeks of gestation to six months postnatally. The vagal innervation was traced using Dil. In general the primitive stomach tube developed discrete tissue structures from eight weeks of gestation. The enteric nerves colonise the pylorus from the serosal surface inwards from eight weeks of gestation. All of the antigens examined had a craniocaudal pattern of expression. These findings are consistent with the reported innervation of other parts of the intestine. Actin is first expressed within the muscularis propria at eight weeks of gestation then the muscularis mucosae at fourteen weeks of gestation. VIP and NOS are both first expressed at eleven and twelve weeks within the myenteric plexus and submucosa, where the peptides are colocalized. VIP and vagal fibres, traced with Dil, were first identified in the myenteric plexus at twelve weeks. Overall, this study has illustrated that the ontogeny of the innervation of the pylorus can be examined histologically in the developing human stomach. Each of the antigens studied could be identified using immunohistochemistry and the vagal innervation traced post-fixation using Dil. Significantly, these studies have provided a baseline from which to assess possible disease-related changes in the development of the stomach such as infantile hypertrophic pyloric stenosis and Duplication cysts of the alimentary tract. CHAPTER 3 :THB ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 62

3.2. Introduction

In 1888 Harold Hirschsprung in his treatise entitled ‘angeborener pylorusstenose’ proposed that infantile hypertrophic pyloric stenosis was a congenital anomaly which he considered to be a persistent form of the fetal pyloric musculature. More recent theories have proposed degenerative changes (Belding HH., 1953, Spitz L , 1975), immaturity of the neuropetide innervation (Friesen SR., 1956), and diminished nitric oxide synthase activity (Vanderwinden JM., 1992). Few studies have investigated the innervation of the developing human intestine ( Kelly EJ., 1993, Timmermans J., 1994, Facer P., 1992, Hitchcock R., 1992) and none have concentrated specifically on the pylorus. While some studies have described the chronological sequence of an antigen’s expression (Timmermans J., 1994), the pattern of inter-relationships, both temporal and spatial, between groups of nerves expressing different neural agents of the intrinsic and extrinsic innervation of the intestine has not been examined. Furthermore the relationship between developing muscle cells and the innervation of the pylorus remains unexamined. Muscle development is influenced by nerves by both the regulation of mitosis of myoblasts and the pattern of expression of proteins. Neural cell adhesion molecule is a key regulator of cell to cell interactions in the human gastrointestinal tract. The expression of NCAM by muscle fibres has been correlated with the development of the neuromuscular junction and innervation (Romanska HH., 1993).

The importance of studying these relationships in the development of the pylorus with reference to understanding the pathogenesis of infantile hypertrophic pyloric stenosis lies in the complex evolving roles NO has with other neural agents such as VIP, GAB A, somatostatin and the opioids (Desai KM., 1994; Grider JR., 1994) .

Immunocytochemistry is a relatively rapid, reliable and efficient method of localising proteins and by the end of 1994, an extensive number of antibodies had been raised to CHAPTER 3:THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS______« the various neural agents studied. All were well characterised, specific antibodies raised to either purified protein or to synthetic peptides selected fi’om predicted protein sequences of the cloned protein. The value of such studies has been twofold: firstly they have allowed me to determine the detailed, normal and developing anatomy of the human pylorus, and secondly they have provided a basis fi^om which I was able to assess the involvement the expression of these substances has in disease-related processes, through detailing changes in their presence or distribution. CHAPTER 3 THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS______64

3.3. Experimental Design

3.3.1. Tissues

Pyloric specimens were obtained, fresh, from twenty aborted human fetuses ranging from 8 to 23 weeks of gestation and fixed in Zamboni’s fixative for immunochistochemical study. A further fourteen specimens, ranging between 10 and 23 weeks gestation, were collected fixed in 2% formalin for the carbocyanine neuronal tracer Dil study (Table 1). The developmental age of the fetal specimens was determined by the foot length. Ethical approval for this work was obtained in accordance with the Polkinghome report. Twenty pyloric specimens were collected from children ranging in age from one week to eight months: 4 were obtained during surgery for gastric transposition, 16 were obtained at post-mortem within 4 hours of death, from causes unrelated to the gastrointestinal tract. All tissues were collected with local ethical committee approval.

Specimens for immunocytochemistry were immersed in Zamboni’s fixative for six hours at 4°C and then rinsed three times in 15% (wt/vol) sucrose (Appendix II) .

Cryostat sections of 10pm thickness were cut perpendicular to the long axis of the pylorus from snap frozen blocks in a cryostat at -25°C. To identify the craniocaudal and temporal patterns of development within the pylorus, serial sections of 10pm thickness were cut at 200pm intervals from blocks oriented as above. These were mounted on poly-L-lysine-coated slides and air dried for 2 hours prior to staining.

Three sections of each specimen were stained using haematoxylin and eosin. CHAPTER 3 ;THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 65

3.3.2. Antibodies (For detail see methods chapter, 2.2.4)

To demonstrate the total innervation of the developing pylorus the sections were immunostained using antibodies to PGP 9.5 (Gulbenkian S., 1987). Neural subtypes were identified by immunostaining for following antigens: the neural isoform of nitric oxide synthase, a potent nonadrenergic noncholinergic smooth muscle relaxant factor that has been implicated in the pathogenesis of infantile hypertrophic pyloric stenosis (Vanderwinden JM., 1992, Grider JR., 1994, Desai KM., 1994 ); vasoactive intestinal polypeptide a potent nonadrenergic noncholinergic smooth muscle relaxant factor known to be highly expressed in gastrointestinal sphincters (Alumets J., 1979, Grider JR., 1994, Desai KM., 1994); calcitonin gene-related peptide which is involved in the afferent innervation of the stomach (Dockray GJ. 1991); substance P, a tachykinin with excitatory, motor function (Kimura S., 1983); in addition antibodies to the fetal (fNCAM), neural (nNCAM) and neuromuscular (nmNCAM) isoforms of neural cell adhesion molecule, and to aSMA were used to investigate the relationship between the expression of NCAM and smooth development. (Table 2.1). CHAPTER 3 :THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 66

3.3.3. Conventional Histology Tissue sections were stained conventionally using haemotoxylin and eosin.

(Appendix V)

3.3.4. Immunostaining Tissue sections were stained by the ABC method (Appendix VI) and peroxidase activity enhanced by the DAB-glucose oxidase nickel enhancement technique (Appendix VII).

3.3.5. Neural tracing To correlate the extrinsic, vagal innervation of the pylorus with that of the intrinsic enteric innervation, the vagal innervation was traced using the carbocyanine compound l,r-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine percholate (Dil). Dil is a lipophilc crystal that passively diffuses and autofluoresces in aldehyde-fixed tissue, thus allowing selective neuronal tracing post mortem ( Gotz N., 1992, Kressel M., 1994) . To minimise the transport time of Dil along the vagus to the pylorus, and thus the time tissue may decompose, the vagus nerve was divided on the lesser curve of the stomach just proximal to the pylorus, upon four fetal stomachs ranging in age from 10, 12, 15, 20, weeks. A crystal of Dil was placed upon the severed surface of the nerve. Four control specimens consisted of the Dil placed directly upon the surface of the pyloruses from foetuses at 10, 12, 15, and 20 weeks of gestation. The specimens were incubated at room temperature in PBS with 0.01% sodium azide for six months. At the end of this period 15 micron thick sections were cut. The sections were cut perpendicular to the long axis of the pylorus, and examined immediately under an immunofluorescence microscope. CHAPTER 3 :THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS______

3.4. Results

3.4.1. Immunohistochemistry

3.4.1.1 .Adventitia. The adventitia consisted of loose amorphous sheets of cells that contained blood vessels. Nerve fibres immunoreactive for PGP 9.5 and expressing SP were identified by 8 weeks of gestation. VIP was expressed from eleven weeks, while nNOS was not expressed until thirteen weeks. Immunoreactivity for fNCAM, and nNCAM, was identified from twelve weeks. Immunoreactivity for nmNCAM, aSMA, and CGRP was not detected.

3.4.1.2.Muscularis Propria. The longitudinal and circular muscle layers were present as discrete and circumferentially continuous structures from eleven weeks. Smooth muscle actin was expressed by the circular muscle layer from eight weeks. Ganglia were immunoreactive for PGP 9.5 from eight weeks, following which stage progressively more nerves fibres were found to lie between muscle fibres. The number of nerves and ganglia was found to increase craniocaudally (Figure 3.1). a smooth muscle actin (aSMA) was expressed by the muscle fibres from 8 weeks The nerves and ganglia expressed fNCAM, nNCAM, nmNCAM and SP from eight weeks. Both VTP and nNOS were expressed by these structures by eleven weeks. The density of immunoreactivity for all the antigens reflected that of the underlying neural tissue, craniocaudally. CGRP was expressed by the nerve fibres fi’om twelve weeks, while the ganglion cells were immunoreactive from twenty-three weeks (Figure 3.2) .

3.4.1.3.Submucosa Up to eleven weeks of gestation, the submucosa consisted of loosely packed, amorphous, eosinophilic cells. At eleven weeks more basophilic and more densely aggregated cells appeared that were radially oriented. These may be rudimentary lymphatic or blood vessels. PGP 9.5-immunoreactive nerves were first apparent at eight weeks as triangular cell bodies and nerve processes within the outer third of the CHAPTER 3:THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS______^ submucosa (Figure 3.3) . By ten weeks these processes reached the middle third and by thirteen weeks the mucosa. Fewer cell bodies, but more nerve processes were present toward the duodenum.

Nerve fibres immunoreactive for fNCAM, that were radially and craniocaudally oriented , were first apparent at eight weeks. Both nNCAM and nmNCAM were first expressed within the submucosa at twelve weeks. No reactivity to aSMA was seen in the submucosa.

By twelve weeks nNOS immunoreactivity was demonstrated in the outer third of the submucosa within cell bodies and nerve processes (Figure 3.3) . At twelve weeks the immunoreactive processes extended to the inner third of the submucosa (Figure 3.4) .

VIP was first expressed at twelve weeks in cell bodies and nerve processes extending to the inner third of the submucosa (Figure 3.5) . By twenty-three weeks more transversely sectioned, craniocaudally oriented nerve fibres were present (Figure 3.6).

From eleven weeks, primarily, radially oriented nerve fibres were found to express SP (Figure 3.7). However, CGRP immunoreactivity was not expressed until twenty-three weeks, when it was found primarily in nerve fibres extending to the inner third of the submucosa.

3.4.1.4 Mucosa Up to eleven weeks of gestation the mucosa was seen to consist of tall columnar cells, following which they became more cuboidal and folded to form rugae. The rugae were more prominent toward the duodenum. The submucosal, PGP-immunoreactive nerve fibres were first demonstrated at eleven weeks, the submucosal plexus was demonstrated at fourteen weeks, and fine mucosal fibres were not apparent until CHAPTER 3:THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 69

fifteen weeks. The highest concentration of submucosal fibres was in the pyloric cushions.

VIP was first detected in fine nerve fibres running up into mucosal folds at twelve weeks, while nNOS was first detected at fifteen weeks. fNCAM, nNCAM and nmNCAM were all expressed in intrarugal nerves and submucosal plexus from twelve weeks (Figure 3.8). CGRP and SP were not expressed in the mucosa until twenty- three weeks, primarily as fine fibres running in the rugae. aSMA was expressed by the muscle fibres of the muscularis mucosa from 14 weeks.

The temporal pattern of expression of the antigens is summarised in table 2.

All of the antigens examined had a craniocaudal pattern of expression within the muscularis propria with the highest concentration of immunoreactivity toward the duodenum. Only PGP 9.5-, VIP- and nNCAM- immunoreactive nerves show a similar craniocaudal pattern of expression in the submucosa.

The morphology of each of the tissues described and the pattern of expression of each of the antigens examined immunohistochemically within the 23 week gestation pylorus did not change postnatally up to 223 days of age.

3.4.2 Neuronal Tracing Dil autofluorescence was first seen in ganglia of twelve week fetuses. All the controls were negative, with no Dil fluorescence seen within the pylorus (Figure 3.9). CHAPTER 3:THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS______70

3.5. Discussion

The aim of this project was to study the relationship between the pattern of expression of neuropeptides, nNOS and NCAM with the morphological changes within the developing human, fetal pylorus. The results confirm our original hypothesis that the expression of antigens examined within the fetal pylorus is developmentally regulated. In discussing the findings of this study, the results will be correlated with the known overall macroscopic and microscopic development of the stomach. Where possible associations between the development of the stomach and conditions affecting the stomach, that require paediatric surgery, will be identified.

The stomach develops from the cranial part of the foregut, caudal to the primitive lung buds and initially behind the heart. The stomach first becomes apparent in the fifth to sixth week as the foregut just distal to the septum transversum expands slightly. From day 26 the distal foregut elongates rapidly. In the fifth week the dorsal wall of the stomach grows faster than the ventral, resulting in the formation of the greater and lesser curvatures of the stomach (Patten BM., 1946) . The developing stomach then turns through 90° around a craniocaudal axis so that its greater curvature lies to the left and the dorsal mesogastrium thins to form the bursa omentalis (His 1895). The left and right vagal trunks thus rotate as well to form the posterior and anterior vagal trunks respectively. Fibres from the left and right vagal plexuses, which originally ran with the vagal trunks in the mesoderm on either side of the stomach, are mixed to some extent especially in the more caudal parts of the posterior and anterior vagal trunks. The fundus of the stomach grows by elongation and differential growth of the greater curvature. The more common form of gastric volvulus, along the long axis of the stomach, occurs in the same direction as this initial rotation. Gastric volvulus in the older child and adult is associated with eventration of the diaphragm, anorexia bulimia and the ingestion of sudza a maize based cereal meal in the Southern African states. A high incidence occurs in the ‘deep chested’ breeds of dogs such as the Irish Setter. CHAPTER 3:THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS______T\_

Salenius studied 134 human fetal stomachs using conventional histology and histochemistry. His findings were published on ‘The Ontogenesis of the Human Gastric Epithelial Cells’ in 1962. Glandular pits were first identified on the lesser curvature from 6 weeks, in the corpus from 8 weeks, and in the pylorus from 10 weeks. Histochemically, succinate dehydrogenase, and thus parietal cells, was demonstrated from 9 weeks. The parietal cell was thus the first glandular cell type identified although it was not morphologically fully differentiated until 11 weeks. Chief cells appeared in the gastric pits from 12 weeks and goblet cells from 11-12 weeks. The pyloric glands developed from 11 and were fully developed by 13 weeks. The description of the early development of primitive gastric epithelium has been proposed as an explanation of why gastric epithelium is so often found within duplication cysts ( Bajpai M., 1994). However subsequent immunohistochemical studies (Kelly EJ., 1993) have identified a more dynamic pattern of development of the gastric epithelium, the distribution of parietal cells not being fully developed until the third trimester. Kelly identifed parietal cells by immunoreactivity for intrinsic factor and hydrogen-potassium ATP-ase from 13 weeks of gestation and in 20% of cases these were found to vanish from the pylorus and antrum in the third trimester.

Our observation that the enteric nerves colonise the fetal pylorus from the serosal surface inwards is consistent with studies of other parts of the enteric nervous system (Facer P., 1992, Hitchcock RJ., 1992). Nerve cells appear to colonise the pylorus during the five to eight weeks, initially in the intermuscular margin to then spread transmurally. By twenty to twenty-three weeks, a full term infant’s pattern of innervation was noted.

The pattern of expression of actin by muscle fibres is similar in the pylorus to that described in the large bowel (Kressel M., 1994): actin first appears in the muscularis propria at eight weeks, while its expression in the muscularis mucosae occurs at fourteen weeks. Gianelli L., 1915, did not identify well developed muscle fibres until 20 weeks of gestation. Upto this stage he described ‘fibrocells’ predominantly in the circular muscle layer, external to which were longitudinally orientated ‘fibrocells’. The CHAPTER 3:THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS______72

‘dilator’ longitudinal muscle fibres were first identified at 24 weeks of gestation. Later studies described the ‘dilator’ muscle fibres as being present from 9.5 to 12 weeks (Welch EH., 1921) . The results of this study are consistent with these subsequent studies. The expression of NCAM by colonic muscle fibres has been correlated with the development of the neuromuscular junction and innervation. Muscle development is influenced by nerves by both the regulation of mitosis of myoblasts and the pattern of expression of proteins. aSMA is known to be expressed by 8 weeks of gestation by human colonic muscle fibres. Thus colonic muscle is differentiated by 8 weeks of gestation (Romanska H., 1993) .

The temporal and craniocaudal pattern of expression of VIP is similar to that described elsewhere in the gastrointestinal tract (Facer P., 1992). That the vagal innervation and VIP expression within the pylorus were both first demonstrated at 12 weeks within the muscularis propria, using Dil and immunohistochemistry respectively, is significant as VIP has been localised to vagal fibres.

Furthermore, it is remarkable that within the pylorus VIP and nNOS are both expressed at the same time within the muscularis propria and the submucosa where these antigens are colocalized within the same nerves ( Furness IB., 1992) . In the adventitia nNOS is expressed two weeks after VTP, at thirteen weeks. The relationship between VTP and nNOS as inhibitory motor neuroactive compounds is complicated and is not fully established. Pharmacological studies have recently demonstrated a role for exogenous NO-mediated VTP release from isolated ganglia (Grider JR., 1993) and VTP-mediated NO release and muscle relaxation from innervated, target muscle cells ( Grider JR., 1992). More recent studies have questioned these conclusions and proposed NO as being the main neurotransmitter of vagally induced gastric relaxation (Desai KM., 1994). Little is known of the interaction between the expression of NO and other neurotransmitters in the developing pylorus. Please see subsequent chapters (Section4.5) for further discussion. CHAPTER 3:THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS______7^

The expression of SP and CGRP in the developing human pylorus has not previously been described. The pattern of expression of these antigens reflects that of the other antigens examined in the pylorus by this study.

That the pylorus has not developed fully until twenty-three weeks is consistent with the results of previous studies examining the development of the gastric mucosa ( Kelly EJ., 1993) and small intestine (Facer P., 1992).

The fetus has been known to swallow in utero since the time of Hippocrates. Fetal swallowing contributes to the regulation of amniotic fluid volume. Mechanical obstruction such as duodenal atresia is associated with polyhydramnios. Isotope studies indicate effective swallowing from 16-17 weeks’ gestation (Prichard J., 1966, Abramovitch DR., 1970) . Radiographic studies plot a progressive increase in gastrointestinal motility by a decrease in gastrocolic transit time ( McClain C R , 1963). The age of onset of pyloric muscle activity is unknown. Furthermore, little is known of the physiological functions of the pylorus in the neonate or infant. The neural complex of ganglia, nerves, synaptic vesicles and immunoreactivity for neuroactive agents continues to mature in the pylorus up to 23 weeks gestation relative to nerve area, as demonstrated by PGP 9.5 immunoreactivity. This is consistent with the observation that fetal upper gastrointestinal motility increases with gestational age.

In conclusion these results provide the first detailed description of the development of the human pylorus in terms of its morphology and phenotypic expression of both nerves and muscle. This study has fulfilled its primary objectives: it has demonstrated the expression of neural and muscle antigens is developmentally regulated. The pattern of expression of these substances, both craniocaudally and radially, is similar to that described in other parts of the intestine as well as the gastric mucosa throughout gestation. The gastric mucosa has not developed the infant distribution of parietal cells until after the third trimester. In addition the localisation of the antigens examined correlates well with the functional actions of these agents recorded in postnatal studies. CHAPTER 3 :THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS______74

Thus this study has established a baseline from which to investigate, understand and manage diseases, specifically infantile hypertrophic pyloric stenosis.

3.6. Tables and Figures (overleaf) CHAPTER 3 THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 75

Table 3-1 Age and Number of Tissues collected FETUSES FETUSES CHILDREN IMMUNOHISTOCHEMICAL NEURONAL TRACING STUDY STUDY AGE No. AGE No. AGE No. SPECIMENS SPECIMENS SPECIMENS (Weeks) (Weeks) (Days) 8 1 8 0 I I 10 2 10 2 2 3 II I 11 I 6 3 12 5 12 3 14 I 13 3 13 0 21 2 14 2 14 0 35 2 15.5 2 15 2 56 3 16 1 16 0 70 3 22 I 20 3 224 2 23 2 23 3

Table 3-2 Table Of The Temporal Pattern Of Expression Of Antigens Within The Human Fetal Pylorus

Adventitia Muscularis Submucosa Mucosa propria (weeks) (weeks) (weeks) (weeks) PGP 9.5 8 8 8 11 SP 8 8 11 23 fNCAM 12 8 8 12 nNCAM 12 8 12 12 nmNCAM - 8 12 12 Actin - 8 - 14 VIP 11 11 12 12 nNOS 13 11 12 15 CGRP - 12 23 23 CHAPTER 3 THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 76

Figure 3-1- Stomach and Duodenum, 8 Weeks Gestation

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Figure 3-2 CGRP Immunoreactivit} in ganglia and nerves at 23 Weeks Gestation

à CH.AfTER 3 THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 77

Figure 3-3 nNOS immunoreactivitj in the Myenteric Plexus and Submucosa at 10 weeks gestation

i

Figure 3-4 nNOS immunoreactivity in the Myenteric Plexus and Submucosa at 12 weeks gestation

« * % CHAPTER 3 THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 78

Figure 3-5 VIP immunoreactivity in the Myenteric Plexus and Submucosa at 12 weeks gestation

' %

Figure 3-6 VIP immunoreactivity in the Myenteric Plexus and Submucosa at 23 weeks gestation

i CHAPTER 3 THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 79

Figure 3-7 SP immunoreactivity in the Myenteric Plexus and Submucosa at 11 weeks gestation

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Figure 3-8 fNCAM immunoreactivity in the Myenteric Plexus and Submucosa at 12 weeks gestation

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I

✓ CHAPTER 3 THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 80

Figure 3-9 Dil Autofluorescence within ganglia at 12 weeks of gestation. CHAPTER 3 : THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 81 CHAPTER 4: 82 A QUANTITATrVB STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

4. CHAPTER 4 A Quantitative Study of the Morphoiogicai and Histochemicai Changes within the Nerves and Muscie in infantile hypertrophic pyloric stenosis.

4.1 Summary

4.2 Introduction

4.3 Experimental design

4.3.1 Tissues 4.3.2 Imunostaining 4.3.3 Statistical analysis

4.4 Results

4.5 Discussion

4.6 Tables and Figures CHAPTER 4: 83 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

4.1. Summary A number of histological changes have been described in infantile hypertrophic pyloric stenosis. However, none of these findings have been quantified and statistically analysed to account for the apparent dilutional reduction of neural tissue due to the muscle hypertrophy. In order to better understand the morphological changes it is important to quantify the changes in dimensions of nerves and muscle, and the proportionate expression of neural antigens in infantile hypertrophic pyloric stenosis . Twenty specimens of pylorus from children with infantile hypertrophic pyloric stenosis and age/ sex-matched controls were examined using conventional histology and immunohistochemistry for a range of nerve and muscle antigens. The changes in the proportion of nerves expressing each antigen were quantified and statistically analysed. The longitudinal muscle was found to be hypertrophic (median control muscle width = 55, diseased =114 x lO'^^m, p = 0.01) and PGP 9.5 stained nerves appeared longer and thicker in the myenteric plexus (median myenteric plexus nerve control length = 4.31 diseased length = 4.84, p =0.02; control width = 3.43, diseased width = 4.01x lO'^^mm, p =0.02) and shorter in the longitudinal muscle layer (median longitudinal muscle control nerve length = 3.76 diseased length = 3.34x lO'^^mm, p= 0.06) in infantile hypertrophic pyloric stenosis. The proportion of nerves that expressed nNOS was found to be diminished in all the infantile hypertrophic pyloric stenosis tissues examined. In the circular muscle and myenteric plexus, the proportion of nerves that expressed VTP and nNOS was almost identically diminished (Difference in medians 34, p = 0.06 and 40, p = 0.001 respectively). The expression of CGRP and SP was proportionately reduced in the myenteric plexus (Difference in Medians 23, p = 0.001 and 39, p = 0.001 respectively). The results of this study represent the first quantitative analysis of nerves and muscle in infantile hypertrophic pyloric stenosis. The muscle hypertrophy is not restricted to circular muscle layer. The changes in nerve morphology cannot be attributed to a dilutional effect of the muscle hypertrophy. The selective changes in nerve and ganglion morphology varies between tissue layers and neural antigen expressed. The findings of reduced proportions of nerves expressing, in particular, nNOS may shed some light on the aetiology of this condition. CHAPTER 4: 84 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

4.2, Introduction upon comparison of a histological preparation of a normal pylorus with that from a child suffering from infantile hypertrophic pyloric stenosis there is an obvious gross distortion of the overall morphology in the diseased pylorus due to the massive smooth muscle hypertrophy (Figure 4.1). This makes accurate, subjective assessment of the changes almost impossible. Simple examination by eye reveals a reduction in the amount of neural tissue seen relative to the increased amount of hypertrophic muscle present. It is unknown if there is a dilution in the total amount neural tissue apparent due to the muscle hypertrophy that characterises this condition or if there is an actual reduction, either generalised or selective, in the nerves and ganglia present.

A number of histological changes have been ascribed to the nerves and ganglia including degenerative (Belding HH,. 1953, Spitz L.,1975 ), regenerative (Friesen SR., 1953) and artefactual changes secondary to compression by the hypertrophied muscle (Stringer M., 1990). All of the published histochemicai studies have reported diminished reactivity to the substances examined ( Friesen SR., 1963, TamPKH., 1986, Malmfors G,. 1986, WatchowDA., 1987, Dieler R , 1980 & 1990, Vanderwinden JM., 1992, Kobayashi H., 1994). (Please see Chapter 1 and Section 4.5 for further discussion ).

However, none of the studies have been quantified to account for the apparent dilutional reduction in neural tissue present and in none have the histological changes been described in the various tissue layers. It remains unclear whether the muscle hypertrophy is restricted to the circular muscle layer alone. Thus the fundamental underlying histological changes in infantile hypertrophic pyloric stenosis remain unknown.

In this study a range of antibodies was selected for the following reasons: Protein gene Product 9.5 is a sensitive pan neuronal marker, reactivity to it provides a CHAPTER 4: 85 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS measure of total neural tissue present (Gulbenkian S., 1987), VTP and nNOS are both potent nonadrenergic-noncholinergic smooth muscle relaxants the function of which are closely inter-related (Desai KM., 1994) , SP is a tachykinin (Kimura S, 1983), and CGRP is involved with the afferent innervation to the stomach (Dockray GJ. 1991) .

Thus this study tested the hypothesis that in infantile hypertrophic pyloric stenosis there is a selective loss of neuroactive agents.

The aims of this project were to quantify a) the changes in the dimensions of the nerves and muscle that occur in infantile hypertrophic pyloric stenosis and b) the proportion of the total neural tissue expressing neural antigen. Thus the possible dilutional reduction in total neural tissue due to muscle hypertrophy was assessed. CHAPTER 4; 86 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

4.3 Experimental design

4.3.1 Tissues Twenty pyloric muscle biopsies were collected fresh from infants undergoing pyloromyotomy for infantile hypertrophic pyloric stenosis. Twenty age-and-sex matched control pyloric specimens were collected. Four were obtained during surgery for gastric transposition, 16 were obtained at post-mortem^ within 4 hours of death, secondary to causes unrelated to the gastrointestinal tract. The specimens were then immersed in Zamboni’s fixative for six hours at 4^C. (Appendix II) .

4.3.2 Antibodies Antibodies to the following range of antigens was selected: PGP 9.5, VIP, nNOS, CGRP, and SP. (Please see Section 4.1).

4.3.3 Imunostaining Five randomly selected sections of 10pm thickness were cut, for each of the antigens examined, perpendicular to the long axis of the bowel from snap-frozen blocks in a cryostat at -25°C. The slides were processed for DAB-nickel enhancement method. (Appendix VI).

The developed slides were counterstained with eosin and examined under a transmitted light microscope. A computer-assisted image analysis system (Seescan) was used to analyse the following parameters in the control and diseased tissues: thickness of the longitudinal muscle layer; thickness and length of the nerves as measured in a transverse, 10pm thick section of the pylorus, in the myenteric plexus, longitudinal and circular muscles; number and size of ganglion cells expressing each of the antigens examined; density of immunostaining by the nerves within the myenteric plexus, longitudinal and circular muscle layers. For each parameter quantified, five images in each of the tissue areas studied were analysed for each section. There were 20 individuals in each group. For each individual, five randomly selected sections CHAPTER 4; 87 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS were stained for each antigen and the mean of these five sections was used in the statistical analysis. In total 49,350 images were examined. The principle steps in data management are illustrated in Appendix VII tables IS, 28, 38. The data derived from these tables was used to create the row detailing immunoreactivity to nNOS in the circular muscle layer of table 4.2.

4.3.4. Statistical analysis

Data were analysed by calculating the median and 95 % confidence interval for the immunostaining by antibodies to each antigen in the control and infantile hypertrophic pyloric stenosis groups. The median and 95% confidence interval for antigen expression was calculated as the percentage of total nerves as shown by PGP immunoreactivity in each group, the difference (control - infantile hypertrophic pyloric stenosis) in the median percentage stained and its 95% confidence interval. The P value was derived from a Mann Whitney U test comparing the median percentage immunostained in the two groups. CHAPTER 4: 88 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

4.4 Results

4.4.1 Conventional Histology Haematoxylin and eosin staining revealed that the longitudinal and circular muscle layers were thickened in infantile hypertrophic pyloric stenosis samples compared with the controls. The mucosa and submucosa were not included in the specimens. The muscle fibres were arranged in abnormally disorganised whorls. The muscle fibres appeared to be normal. Interspersed between these fibres were occasional nerves and ganglia. In the diseased specimens, the density of these neural structures was much lower. Some of the nerves appeared thicker than in the controls.

4.4.2 Immunocytochemistry Subjective examination, by eye, revealed an apparent reduction in the total amount of neural tissue expressing PGP in all the tissues of the diseased specimens (Figure 4.2 a,b). Subjective comparison between control and diseased specimens was complicated by the muscle hypertrophy. CGRP expression was restricted primarily to the nerves near ganglia (Figure 4.3), while nNOS (Figure 4.4), VIP (Figure 4.5), and SP (Figure 4.6) were more widely expressed in nerves of the longitudinal muscle, circular muscle, and myenteric plexus. In the diseased specimens subjective assessment revealed an apparent lack of nerves expressing these antigens. The relative changes in each of the tissues and the changes in antigen expression by the ganglia was impossible to assess by only simple examination.

4.4.3 Morphological changes in the Dimensions of the Nerves and Longitudinal Muscle Layer

The longitudinal and circular muscle layers were found to be hypertrophied in the diseased samples (longitudinal muscle thickness 114 xlO’^m) as compared with normal controls (longitudinal muscle thickness 55 xlO’^^m). (Table 1 ) The only CHAPTER 4; 89 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS significant differences in the size of the nerves was in the myenteric plexus, where the nerves were longer (control 4.31, diseased 4.84 x 10"^ mm) and thicker ( control 3.43, diseased 4.01 x 10"^ mm) in infantile hypertrophic pyloric stenosis than in the control tissue and in the longitudinal muscle layer where the nerves were shorter (control 3.76, diseased 3.34 x 10"'^ mm).

4.4.4. The Changes in Immunoreactivity within the Circular muscle layer The area of nerves expressing each of the antigens examined was reduced in infantile hypertrophic pyloric stenosis within the circular muscle layer. When calculated as a proportion of the total innervation (PGP-immunoreactive) nerves expressing nNOS were found to be reduced significantly in the circular muscle layer (difference in medians = 34, P =0.001) . The reduction in VIP immunoreactive nerves approached statistical significance (difference in medians = 40, P =0.06).

4.4.5. The Changes in Immunoreactivity within the Longitudinal muscle layer

Within the longitudinal muscle layer the proportions of the total area of PGP- immunoreactive nerves expressing CGRP, SP nNOS, and VTP were reduced. The change only reached statistical significance for nNOS (difference in medians = 37, P =0.02).and SP (difference in medians = 19, P =0.03).

4.4.6. The Changes in Immunoreactiviy within the Myenteric Plexus

Within the myenteric plexus the proportion of the total number of PGP - immunoreactive nerves expressing each of the antigens examined was reduced. The reduction in expression of all the antigens was greatest in the myenteric plexus. CHAPTER 4: 90 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

4.4.7. The Changes in Morphology and Immunoreactivity within the Ganglia

Each ganglion was taken to include the cell bodies and nerve fibres. The proportion of the total PGP immunoreactive neural tissue per ganglion expressing CGRP, SP and nNOS was reduced in infantile hypertrophic pyloric stenosis. The quantity of neural tissue expressing VTP was increased in infantile hypertrophic pyloric stenosis (median control staining 24, diseased 35 mm^ xlO'^), despite the overall size of the ganglia being smaller (median PGP immunoreactivity 2.8, diseased 1.0). Only the change in expression of nNOS reached statistical significance (difference in medians = 44, P = 0.06).

The proportions of the total number of PGP-immunoreactive ganglion cells expressing CGRP, SP, and nNOS were reduced in infantile hypertrophic pyloric stenosis. The number of ganglia expressing VIP was increased (difference in medians = -80, P = 0.01). CHAPTER 4: 91 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

4.5. Discussion

The aims of this study were to quantify the changes in the dimensions of the nerves and muscle and the proportion of the total neural tissue expressing each of a range of neural antigens in infantile hypertrophic pyloric stenosis. Significant muscle hypertrophy and selective abnormalities in the morphology of the nerves were seen. These results demonstrate that the selective changes in the nerves cannot be attributed entirely to the increase in the muscle in infantile hypertrophic pyloric stenosis. Thus the longer and wider nerves of the myenteric plexus and the shorter nerves of the longitudinal muscle cannot be attributed to compression by the muscle hypertrophy (Stringer 1990).

This study demonstrates that longitudinal muscle hypertrophy is a characteristic of infantile hypertrophic pyloric stenosis. Thus this dispels the proposition that a deficiency of the specialised ‘dilator’ longitudinal muscle, first described by Rudinger in 1879 and subsequently Horton in 1928 as a possible cause of infantile hypertrophic pyloric stenosis (Please see Chapter 1 section 1.2.2).

NOS has been implicated in the pathogenesis of infantile hypertrophic pyloric stenosis by the description that only the circular muscle is hypertrophied and the diminished activity of NADPH is localised to this tissue (Vanderwinden JM., 1992) . nNOS expression is diminished in the circular and longitudinal muscle layers as well as the myenteric plexus. That the proportionate reduction in expression of nNOS and VIP is the same in the circular muscle and myenteric plexus is biologically significant as the peptides are colocalized to the same nerves in the guinea pig (Furness JB , 1992). Only nNOS expression is diminished in all of the tissues studied within both the nerves and ganglia.

The apparent increase in number of ganglia expressing VIP may be an artefact of the increase in size of these ganglia in infantile hypertrophic pyloric stenosis: the larger ganglia may by random be ‘seen’ more often per image analysed. The ncreased area of each ganglion expressing VTP in infantile hypertrophic pyloric CHAPTER 4: 92 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS stenosis is a curious phenomenon given the total size of the ganglia is reduced by PGP staining.

The anatomical and functional relationship between nNOS and VIP within the gastrointestinal tract is the subject of much interest and study. NO and VIP are both major mediators of NANC inhibition at various sites in the gastrointestinal tract.( Desai 1991 a & b, Gibson A., 1990 , Li C G , 1991). The motor involvement of NOS immunoreactive nerve fibres has been implicated by the demonstration that such fibres in the myenteric plexus of the guinea pig ileum innervate the circular muscle and synapse with other neurons (Llewellyn-Smith I I , 1992). Dogiel type I morphology has been attributed to NOS-immunoreactive nerves in the rat duodenum and canine colon. Axons from these neurons lead to muscle (Ward SM., 1992, Aimi HA., 1993). NOS involvement in gastric relaxation has been implicated by the demonstration of NOS immunoreactive neurons within the myenteric plexus of the pylorus (Timmermans J., 1994, Vanderwinden JM., 1993) and in vagal efferent fibres lead to the stomach ( Forster ER., 1993).

VIP has been proposed as a mediator of gastric relaxation in the guinea pig, rat, cat, and ferret (Grider JR., 1985, De Beurme JA., 1988, Grundy D., 1993) It has also been suggested that VIP and NO are co-transmitters of gastric relaxation in the guinea pig, rat and ferret (Li C G , 1990, Grider JR., 1992 & 1993, Grundy D , 1988). The regulation of enteric smooth muscle relaxation is not fully understood. The release of VIP and NO during relaxation from motor neurones is regulated by other neurones that act as modulatory interneurones. At least three other noncholinergic intemeurons, as well as possibly a cholinergic one, are known to be involve in this regulation: these neurons contain somatostatin, GAB A, and opioid peptides. Somatostatin neurons synapse with other neurons and do not project fibres to the muscle layers (Costa M., 1980). They seem to act as primary regulatory neurons. In human intestine (Grider JR., 1989) and rat colon (Grider JR., 1987) somatostatin is released concomitantly with VTP and NO during relaxation. Administration of somatostatin produces muscle relaxation, while blocking CHAPTER 4; 93 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS somatostatin with antiserum inhibits VIP release and muscle relaxation (Grider JR., 1987). The action of somatostatin appears to be indirect and through the stimulation of GABA and the inhibition of opioid containing neurones. Anatomically GABA neurons project fibres onto circular muscle directly. However there is no direct action of GABA on the muscle cells. GABA, Somatostatin, and VIP are released concomitantly during relaxation. The release of GABA like that of VTP is augmented by somatostatin and inhibited by somatostatin antiserum. GABA induces muscle relaxation and VTP release that is abolished by TTX and biculline, a GABA-A receptor antagonist; the actual muscle relaxation being abolished by VIP antagonists, implying this is mediated by VIP. (Grider JR., 1992). Muscle relaxation and VIP release induced by somatostatin can be partially blocked by biculline. (Grider JR., 1991). Taken together these findings imply Somatostatin, GABA and VIP act in series to produce muscle relaxation.

Opioid neurons inhibit VIP release and muscle relaxation. (Grider JR., 1987) The activity of opioid neurons is controlled by somatostatin neurons. Somatostatin inhibits and somatostatin antiserum augments opioid peptide release. The stimulatory effect of somatostatin on VCP release is inhibited by opioid peptides and augmented by naloxone (Grider JR., 1991). These findings imply somatostatin inhibits the release of opioid peptide, which in turn, results in the release of VIP. These relationships are summarised overleaf. The relationship between VCP and NO remains unclear. That VIP may act through the release of NO has been demonstrated in guinea pig gastric smooth muscle cell (Grider JR., 1992) . NO has recently been ascribed a more dominant role over VIP in vagally induced guinea pig gastric relaxation by some elegant pharmacological studies (Desai KM., 1994) . However the absence of nNOS- immunoreactivity demonstrated in this study within the smooth muscle cells does not exclude the presence of other isoforms of NOS and thus the possibility of interaction with VTP. CHAPTER 4: 94 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

Figure 4-1 Heuristic Model of Inhibitory Intestinal Motor Innervation

SOMATOSTATIN NEURONS

STIMATE IBIT

GABA OPIOID neurons neurons

STIMULATE

VIP/NO NEURONS

The increase in size and number of VIP expressing ganglia is therefore functionally consistent with that of a reduction in metEnkephalin expression in infantile hypertrophic pyloric stenosis (Wattchow DA., 1987): endogenous endorphins tonicly inhibit VIP release ( Hoyle CH., 1990, Grider JR., 1987). The increase in VIP expression by the ganglia may represent a compensatory mechanism by which the condition naturally regresses: VIP release results in the release of nitric oxide from smooth muscle cells (Grider JR., 1992).

SP expression is not diminished in the circular muscle while it is in the myenteric plexus and longitudinal muscle layers (Tam PKH., 1986). Within the normal intestine the expression of SP is primarily located to the myenteric plexus. SP has a potent contractile effect upon enteric smooth muscle. CGRP expression is only reduced in the myenteric plexus. The significance of the reduced somatic afferent transmitter, CGRP (Kressel M., 1994), expression demonstrated by this study is unclear. Within the myenteric plexus the nerves are longer and thicker and the same proportion express all of the antigens examined. (Figure 4.6) The CHAPTER 4: 95 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS significance of these changes restricted to the myenteric plexus is yet to be established.

This study characterises the only significant morphological abnormalities in infantile hypertrophic pyloric stenosis to be shorter nerves in the longitudinal muscle and longer, thicker nerves within the myenteric plexus. The variations in the abnormal morphology of the nerves in the tissue layers cannot be attributed to ‘compression’ by the hypertrophied muscle (Stringer M., 1990). Furthermore the changes in expression of the antigens examined cannot be accounted for by the morphological changes of the nerves alone. The altered expression of peptides and nNOS by the nerves may represent a secondary phenomenon, reflecting a more fiindamental abnormality in the development of the nerves. Notably, despite the ganglia being of a smaller cross sectional area, as determined by PGP immunoreactivity, in infantile hypertrophic pyloric stenosis theproportionate area of the ganglia expressing VIP is increased.

Thus for the first time the histological changes underlying infantile hypertrophic pyloric stenosis have been quantified and statistically analysed. The changes described here represent a ‘snap -shot’ in the natural progression of the condition, that has been taken when the gastric outlet obstruction was so severe as to be clinically significant to warrant pyloromyotomy. A natural evolution and regression of the condition has been described (Please see Chapter 1) That the greatest reduction in expression was in that of nNOS further implicates an abnormality in the production of the inhibitory factor nitric oxide in the aetiology of this condition. CHAPTER 4; 96 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

4.6. Tables and Figures

Table 4-1 Longitudinal Muscle Width & Nerve Fibre length and width (lO’^^mm) Muscle type Dimension Group Median Difference P (95% Cl) in medians value (95% CD Circular Nerve Fibre Control 4.21 (3.29 to 4.75) 2.51 (-0.56 to 1.12) 0.6 Length Diseased 3.68 (3.05 to 4.81) Nerve Fibre Control 3.28 (2.76 to 3.92) 0.08 (-0.45 to 0.80) 0.8 Width Diseased 2.96 (2.47 to 4.20) Longitudinal Muscle Control 55 (43 to 64) -64 (-134 to -14) 0.01 Width Diseased 114 (37 to 197) Nerve Fibre Control 3.76 (3.24 to 4.42) 0.57 (-0.05 to 1.43) 0.06 length Diseased 3.34 (2.81 to 3.77) Nerve Fibre Control 3.11 (2.66 to 3.48) 0.24 (-0.33 to 0.82) 0.4 Width Diseased 2.78 (2.47 to 3.48) Myenteric Nerve Fibre Control 4.31 (3.41 to 4.84) -0.81 (-1.51 to -0.11) 0.02 plexus Length Diseased 4.84 (4.44 to 5.42) Nerve Fibre Control 3.43 (2.56 to 3.87) -0.86 (-1.51 to -0.13) 0.02 Width Diseased 4.01 (3.46 to 5.29)

Table 4-2 Area of Immunoreactive Nerves in Circular Muscle (units: mm'^xlO '*) Antigen Group Median staining Median % Difference in P (95% CD PGP medians valu (95% CD (95% CD CGRP Control 0.49 (0.24 to 0.90) 33 (16 to 55) 2(-20 to 25) 0.8 Diseased 0.13 (0.09 to 0.18) 28 (11 to 52) nNOS Control 0.74 (0.60 to 1.10) 53 (39 to 93) 34 (13 to 57) 0.001 Diseased 0.06 (0.05 to 0.09) 23 ( 9 to 34) SP Control 0.24 (0.11 to 0.45) 21 (9 to 48) 5 (-6 to 22) 0.3 Diseased 0.04 (0.02 to 0.16) 10 ( 4 to 29) VIP Control 0.72 (0.43 to 1.87) 96 (29 to 109) 40 (-2 to 85) 0.06 Diseased 0.12 (0.06 to 0.30) 27 (10 to 76) PGP Control 1.17 (0.97 to 2.44) Diseased 0.55 (0.27 to 0.69) CHAPTER 4; 97 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

Table 4-3 Area of Immunoreactive Nerves in the Longitudinal Muscle (units: mm^xlO"^) Antigen Group Median staining Median % Difference P (95% Cl) PGP in medians value (95% Cl) (95% Cl) CGRP Control 0.47 (0.15 to 0.59) 60 (44 to 61) 32 (-6 to 62) 0.10 Diseased 0.12(0.09 to 0.17) 38 (12 to 64) nNOS Control 0.42 (0.30 to 0.71) 82 (61 to 82) 37 (5 to 65) 0.02 Diseased 0.07 (0.04 to 0.17) 22 (16 to 73) SP Control 0.20 (0.08 to 0.34) 43 (22 to 43) 19 (7 to 32) 0.03 Diseased 0.06 (0.03 to 0.11) 25 (12 to 25) VIP Control 0.38 (0.13 to 0.66) 49 (34 to 62) 9 (-6 to 25) 0.19 Diseased 0.13 (0.05 to 0.20) 40 (28 to 45) PGP Control 0.78 (0.34 to 0.98) Diseased 0.33 (0.21 to 0.66)

Table 4-4 Area of Immunoreactive Nerves Myenteric Plexus (units: mm^xlO'"^) Antigen Group Median staining Median % Difference P (95% Cl) PGP in medians value (95% Cl) (95% Cl) CGRP Control 1.21 (0.48 to 1.55) 47 (27 to 83) 39 (22 to 66) <0.001 Diseased 0.25 (0.15 to 0.42) 10 (7 to 12) nNOS Control 1.72(1.13 to 2.04) 71 (51 to 131) 89 (47 to 131) <0.001 Diseased 0.25 (0.14 to 0.42) 9 (4 to 25) SP Control 0.53 (0.34 to 1.08) 32 (18 to 51) 23 (10 to 41) <0.001 Diseased 0.25 (0.15 to 0.42) 10 (7 to 12) VIP Control 0.99 (0.44 to 2.24) 67 (25 to 74) 53 (19 to 66) <0.001 Diseased 0.25 (0.15 to 0.42) 9 ( 7 to 12) PGP Control 2.20(1.53 to 3.04) Diseased 3.02 (2.25 to 3.57) CHAPTER 4: 98 A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

Table 4-5 Area of Ganglia (units: mm^xlO'*) Antigen Group Median staining Median % PGP Difference in P (95% Cl) (95% Cl) medians value (95% Cl) CGRP Control 1.2 (0.4 to 3.42) 26 (8 to 52) 15 (-5 to 48) 0.2 Diseased 0.65 (0.27 to 3.27) 7 (4 to 53) nNOS Control 3.45 (2.21 to 4.15) 69 (36 to 77) 44 (-1 to 59) 0.06 Diseased 1.3 (0.66 to 2.84) 21 (10 to 66) SP Control 1.65 (0.83 to 2.27) 34 (18 to 63) 27 (-7 to 73) 0.3 Diseased 0.85(0.2 to 2.00) 7 (4 to 39) VIP Control 1.0 (0.62 to 2.68) 24 (9 to 43) -14 (-59 to 14) 0.2 Diseased 2.2 (0.60 to 6.02) 35 (18 to 97) PGP Control 2.8 (2.25 to 3.20) Diseased 1.0 (0 to 1.35)

Table 4-6 Number of Ganglia Antigen Group Median staining Median % PGP Difference in P (95% Cl) (95% Cl) medians value (95% Cl) CGRP Control 2.55(0.42 to 2.6) 69(16 to 88) 36(0 to 71) 0.17 Disease 0(0 to 1) 0( 0 to 76) d nNOS Control 2.6(1.02 to 2.98) 72(39 to 99) 36( 0 to 67) 0.11 Disease 0.1(0 to 0.58) 25(0 to 67) d SP Control 2(1.05 to 2.78) 65(47 to 99) 59(33 to 93) <0.001 Disease 0(0 to 0) 0(0 to 23) d VIP Control 0.83(0 to 2.28) 29(0 to 64) -80(-127 to -14) 0.01 Disease 1.2(0 to 1.8) 100(23 to 183) d PGP Control 4.05(3.43 to 5.35) Disease 5.1(4.38 to 8.18) d CHAPTER 4: 99 A QUANTITATIVE STUDY OF THE MORPHOLOGICAL AND HISTOCHEMICAL CHANGES WITHIN THE NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

Fit];ures 4-2 a Immunoreactivity to PGP 9.5 in Control Specimen of Pylorus

•:N

Fig 4-2 b Immunoreactivity to PGP 9.5 in Diseased Specimen of Pylorus demonstrating gross muscle hypertrophy and distortion of nerves CHAPTER 4; 100 A QUANTITATIVE STUDY OF THE MORPHOLOGICAL AND HISTOCHEMICAL CHANGES WITHIN THE NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

F ig u re 4-3 CGRP Immunoreactivity within the diseased pylorus

X *

%

% ' ^ & u - . # %

3 V

F ig u re 4-4 nNOS immunoreactivity within the diseased pylorus CHAPTER 4: 101 A QUANTITATIVE STUDY OF THE MORPHOLOGICAL AND HISTOCHEMICAL CHANGES WITHIN THE NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

Figure 4-5 \TP immunoreactivity within the diseased pylorus

?

s

y

Figure 4-6 SP immunoreactivity within the diseased pylorus CHAPTER 4: 102 A QUANTITATIVE STUDY OF THE MORPHOLOGICAL AND HISTOCHEMICAL CHANGES WITHIN THE NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

Figure 4-7 Graphical presentation of the proportion of Nervesexpressing Neuroactive Agents within the Myenteric Plexus.

Antigen Expression by the Nerves of the Myenteric Plexus

M 0.7 control

□ diseased

a. 0.2

CGRP VIP Antigen CHAPTER 4: 103 A QUANTITATIVE STUDY OF THE MORPHOLOGICAL AND HISTOCHEMICAL CHANGES WITHIN THE NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS CHAPTER 5; 104 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND ______PHENYLKETONURIA______

5. CHAPTER 5 A Mouse Model of Infantile Hypertrophic Pyloric Stenosis and Phenylketonuria.

5.1 Summary

5.2 Introduction

5.3 Experimental design

5.3.1 Tissues 5.3.2 Imunostaining 5.3.3 Statistical analysis

5.4 Results

5.5 Discussion

5.6 Tables and Figures CHAPTER 5: 105 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND ______PHENYLKETONURIA______

5.1. Summary Several experimental animal models of infantile hypertrophic pyloric stenosis have been described in none of which have the morphological changes been quantified and statistically verified, and in none have chronological sequence of events been described.

The aim of this study was to quantify the chronological sequence of changes in the morphology and to examine the expression of neuroactive agents in the pylorus of an animal model of infantile hypertrophic pyloric stenosis and phenylketonuria.

Thirty specimens of pylorus from hph-I mice and age/ sex matched controls (age range: 10-180 days) were examined using conventional histology and immunohistochemistry. The changes in the morphology of the muscle layers was quantified and statistically analysed in each age group.

A transient hypertrophy occurred in muscle layers of the hph-I mouse pylorus aged from 10 to 90 days (10 day, hph-I longitudinal muscle mean diameter = 3.4, control = 1.8, p = <0.001; hph-I circular muscle width = 11.5, control = 4.7, p =<0.001) during which period they weighed significantly less than the controls (40, day hph-I weight = lOgms, control = 25 gms). Upon subjective examination there was no change in the pattern of expression of the antigens studied ( Protein Gene Product 9.5, Vasoactive Intestinal Polypeptide, Substance P, Calcitonin Gene Related Peptide, and neuronal Nitric Oxide Synthase) within the hph-I mice compared with the controls. hph-I mice develop a transient smooth muscle hypertrophy of all the muscle layers of the pylorus associated with gastric distension and transient weight loss. The only known anomaly throughout this period is diminished nitric oxide synthase activity due to a deficiency of tetrahydrobiopterin. The pyloric muscle hypertrophy may be secondary to the diminished activity of nitric oxide synthase, despite the expression of neuronal nitric oxide synthase being unchanged within the hph-1 mouse’s pylorus. CHAPTERS: 106 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND ______PHENYLKETONURIA______5-2. Introduction Several histological abnormalities have been attributed to infantile hypertrophic pyloric stenosis. The morphological changes in the muscle, nerves and ganglia as well as the altered expression of a wide range of neural agents in the different tissue layers has been quantified and statistically verified (Abel RM., 1995) . However these features represent only a ‘snapshot’ in the natural progression of the condition to evolve a transient smooth muscle hypertrophy, that in the majority of infantile cases in humans, will resolve. The advantages of identifying an experimental animal model of infantile hypertrophic pyloric stenosis include: • The underlying lesion in the hph-1 mouse is well defined and understood. Studying this experimental animal as a model of pyloric stenosis may thus shed light upon the aetiology and pathogenesis of infantile hypertrophic pyloric stenosis. • The chronological sequence of morphological changes may be quantified and statistically identified. • Conditions (PKU) naturally associated with infantile hypertrophic pyloric stenosis may be studied in this experimental model. Thus the mechanism by which these naturally associated conditions occur may be examined. • The results of these studies may shed light upon the mechanism by which pyloromyotomy ‘cures’ infantile hypertrophic pyloric stenosis and ultimately provide information for the development of a nonsurgical cure for the condition.

While several animal models of infantile hypertrophic pyloric stenosis have been described ( Bredt D., 1993, Dodge JA., 1976, Huang PL., 1993, Voelker C A , 1995), in none have the morphological changes in the pylorus been quantified and statistically verified or the sequence of events in the evolution of the condition been analysed.

Phenylketonuria (PKU) is associated with an increased incidence of infantile hypertrophic pyloric stenosis (Johnson CF. 1978). PKU is due to aberrant activity of phenylalanine hydroxylase. Tetrahydrobiopterin is a cofactor to phenylalanine hydroxylase as well as the enzyme nitric oxide synthase (NOS) and thus the formation of nitric oxide (NO). NO has been implicated in the pathogenesis of infantile CHAPTERS: 107 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND ______PHENYLKETONURIA______hypertrophic pyloric stenosis (Bredt D., 1993, Huang PL., 1993, Vanderwinden JM., 1992).

The hph-I mouse was created as an animal model of PKU (McDonald JD., 1988 a & b. Brand MP., 1995) . It is deficient of tetrahydrobiopterin, and has recently been described as having diminished NOS activity (Brand MP., 1995).

The aims of this study were to test the hypothesis that the hph-1 mouse develops pyloric muscle hypertrophy, to quantify the chronological sequence of morphological changes, and to assess the pattern of expression of neuroactive agents within the experimental mouse’s pylorus. CHAPTERS: 108 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND ______PHENYLKETONURIA______

5.3 . Experimental design

5.3.1. Tissues Pyloric specimens were gathered from control (C57BlxCBA) and experimental hph-1 mice at two week of gestation, ten, twenty, forty, ninety, and one hundred and eighty days after birth. Six control and six experimental specimens were collected at each age from established breeding colonies. The age of the animals was calculated from the date of timed mating. The pups were weaned to a solid diet of a predominantly grain laboratory Chow, at approximately twenty days of age. The animals were weighed immediately prior to being killed by cervical dislocation. The stomach and pylorus were dissected and fixed, fresh, in Zamboni’s fixative for six hours at 4°C (Appendix II) and rinsed three times in 15% (wt/vol) sucrose in 0.1 mol/L phosphate-buffered saline (PBS; pH 7.2) with 0.01% (wt/vol) sodium azide. Sections of 10pm thickness were cut perpendicular to the long axis of the gut from snap-frozen blocks in a cryostat at -25°C.

Sections from each specimen were conventionally stained using haematoxylin and eosin (Appendix V) .

5.3.2. Antibodies PGP: a highly sensitive pan neuronal marker (Gulbenkian S., 1987) VTP: a mediator of the parasympathetic innervation to the stomach nNOS: both NO and VIP are potent nonadrenergic-noncholinergic smooth muscle relaxants (Desai KM., 1994) SP: an excitatory motor innervation (Kimura S., 1983) CGRP: the sensory innervation to the stomach (Dockray GJ., 1991) CHAPTERS: 109 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND PHENYLKETONURIA

5.3.3. Immunocytochemistry Tissue sections were stained by the avidin-biotinylated-peroxidase complex (ABC) method with nickel enhancement (Appendix VI & VII). The developed slides were counterstained with eosin and examined under a transmitted light microscope. The width of the muscle layers was measured using a graticule. Photographs were taken using Technical PAN film (ASA 50; Kodak Ltd., Hemel Hempstead, UK.).

5.3.4. Statistical analysis

The results were analysed statistically using Bartlett’s analysis of variance.

For each muscle diameter, a two way analysis of variance was performed with factors group (control, diseased) and age .

As the diseased animals were smaller than the controls, for each animal the diameters of each tissue were expressed as a proportion of the diameter of the pylorus, to ascertain whether there were differences in the relative proportions between the groups. CHAPTERS; 110 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND ______PHENYLKETONURIA______

5,4. Results

The diameter of the hph-1 mouse’s pylorus was smaller than that of the control mouse. Haematoxylin and eosin staining revealed that although the pylorus of the experimental mouse was smaller, the circular and longitudinal muscle layers appeared to be disproportionately thicker than those of the control specimens. Subjective comparison of the diseased and control specimens was complicated by the disparity in size of the tissues relative to the size of the pylorus in each group. The morphology and arrangement of the muscle fibres was unchanged in the hph-1 mice. The nerves and ganglia appeared to be similar in the experimental and control specimens. Qualitative assessment revealed no apparent change in immunoreactivity for any of the antigens examined at any age, between the control and experimental animals, in any of the tissues examined (Figures 5.10, 5.11).

The mean values of each muscle layer examined for each group at each age, and the pooled estimate of the standard deviation from the analysis of variance is presented in table 5.1. The p value was used to examine whether there was a significant interaction between group and age, that is whether the difference between the control and experimental groups varies with age.

Table 5.2 presents the mean diameters expressed as a proportion of the pylorus diameter, standard errors of the differences between any pair of means (sed) and the P value testing for a significant interaction between group and age. All of the muscle layers in the experimental mouse pylorus are disproportionately thicker than those of the control mice. The hypertrophy of all the muscle layers is transient and resolves as the hph-1 mice approach 90 days of age (Figures 5.1, 5.2, 5.3).

The stomachs of fourteen day old fetal and one day old hph-1 and control mice were examined. Due to technical difficulties precipitated by the small size of the specimens, only 3 stomachs were examined per group at each age. The circular muscle layer alone was examined, as the muscularis mucosae and longitudinal muscle layer could not be differentiated as discrete structures at these ages. A two way analysis of variance with CHAPTERS: 111 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND ______PHENYLKETONURIA______factors group (control, diseased) and age (14 day fetus, I day) was performed. Table 5.3 presents the mean diameter for the control and hph-I mice at each age, the pooled estimate of the standard error of the difference between any pair of means (sed) and the P value testing whether there is a significant interaction between group and age.

There is a highly significant (P=O.OOI) relationship between group and age for pylorus diameter. For the absolute circular muscle diameter there was no significant interaction between group and age (P=0.2), but there was a highly significant interaction (P<0.001) for the relative diameter of the circular muscle expressed as a percentage of the pylorus diameter.

The hph-I mice are smaller and have a smaller pylorus than the control mice of the same age. However, the pylorus of these mice has disproportionately thicker longitudinal, circular and muscularis mucosae muscle layers than those of the control mice. These changes are transient: the hypertrophy of the muscle layers begins in utero and resolves by 180 days (Fig 1-4) in all the muscle layers. CHAPTERS: 112 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND PHENYLKETONURIA

5.5. Discussion

Nitric oxide has been implicated in the pathogenesis of infantile hypertrophic pyloric stenosis since diminished NADPH activity in the circular muscle was documented, a NOS-gene deleted ‘knockout’ mouse described and more recently an experimental model in which perinatal inhibition of NOS resulted in pyloric smooth muscle hypertrophy. (Bredt D., 1993, Huang PL., 1993 Voelker CA., 1995). In the ‘knockout’ mouse the only abnormality identified was pyloric muscle hypertrophy.

The hph-I mouse was studied as it has a defect of GXP cyclohydrolase activity (McDonald JD., 1988) thus a deficiency of tetrahydrobiopterin and diminished NOS activity (Brand MP., 1995). Tetrahydrobiopterin is a cofactor to nitric oxide synthesis. Nitric oxide is formed from two monoxygenation steps of arginine. The first step produces N hydroxyarginine as an intermediate species using one oxygen molecule and one NADPH in the presence of tetrahydrobiopterin. The second step involves the oxidation of N-hydroxyarginine to form citrulline and nitric oxide.

The hph-I mouse was originally created as a model of PKU. Phenylketonuria is the commonest inborn error of metabolism to affect humans. It is characterised biochemically by hyperphenylalaninaemia. Untreated phenylketonuria is associated with an increased incidence of infantile hypertrophic pyloric stenosis (Johnson CF., 1978) . The hph-I mouse serum phenylalanine levels rise from day four to peak at day six at nearly ten times normal, following which the levels gradually decline to normal by the time the mice are being weaned at twenty days. Rarely is the hph-I mouse hyperphenylalaninaemic after weaning. (McDonald JD., 1988). The relationship between tetrahydrobiopterin, phenyalanine hydroxylase, NOS, phenylketonuria and infantile hypertrophic pyloric stenosis are illustrated in Figure 5.1.

Unlike the ‘knockout’ mouse, the hph-I mouse is transiently smaller than its control. That the hph-I mice are smaller until ninety days of age cannot be attributed simply to a period of hyperphenylalanaemia, as the size difference persisted long after the period CHAPTERS: 113 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND ______PHENYLKETONURIA______of ketosis had resolved. The difference in body weight between the control and diseased mice resolved as the hypertrophy of each of the muscle layers within the hph- 1 mouse’s pylorus resolved. The changes in body size are consistent with the hph-1 mouse developing gastric outlet obstruction due to a transient hypertrophy of the pyloric musculature. The onset of the muscle hypertrophy was demonstrated to be in utero in the hph-1 mouse. This is consistent with reports of premature babies developing infantile hypertrophic pyloric stenosis (Henderson XL., 1952) . The transient nature of the pyloric muscle hypertrophy in the hph-1 mice is consistent with clinical reports of the successful conservative management of infantile hypertrophic pyloric stenosis (Tallerman KH., 1951, Swift PGF., 1991). That both the longitudinal and circular muscle layers hypertrophy within the hph-1 mouse’s pylorus is consistent with our quantified findings that both these layers are hypertrophied in infantile hypertrophic pyloric stenosis (Abel RM., 1995).

As in all rodents, except ferrets, the hph-1 mice could not vomit despite the gross gastric distension. This is probably related to the relatively long intrabdominal segment of the rodent oesophagus.

Pyloric smooth muscle hypertrophy was found to occur in an experimental mouse model treated with NOS inhibitors (Voelker CA., 1995) . It is unclear whether in this model the muscle hypertrophy was restricted to only the circular muscle layer, as the morphological changes of experimental mouse’s pylorus were not quantified nor were the chronological sequence of changes described. The prenatal mice, that were administered L-NAME in utero, were noted to be smaller than control mice. However the morphology of the pylorus was not described or quantified. The animals administered L-NAME postnatally were found to suffer a selective weight loss, with relative sparing of cerebral mass. In this group pyloric muscle hypertrophy was identified. However, these morphological changes were not quantified. In neither the prenatally or postnatally treated groups was the morphology of the nerves described. Furthermore, while NOS activity was described to be inhibited, it was not measured in this animal model. CHAPTERS: 114 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND ______PHENYLKETONURIA______Unlike other NO deficient mouse models (Bredt D. 1993, Huang PL. 1993, Voelker CA. 1995) the hph-1 mice behave abnormally, are less fertile, and lose weight after birth to regain it by forty days as the pyloric smooth muscle hypertrophy resolves Figs 1-3.

Despite the hph-1 mice developing pyloric muscle hypertrophy there was no change, upon qualitative visual examination, in the expression of the neuroactive agents studied immunohistochemically. Reduced NOS activity due to a deficiency of tetrahydrobiopterin has been demonstrated in the hph-1 mouse ( Brand MP., 1995). Thus although the enzyme NOS may be demonstrated by immunohistochemistry within the pylorus, the underlying defect may be reduced activity of the enzyme and so diminished nitric oxide production. The relationship between nitric oxide and the other neuroactive agents within the pylorus is not established.

This is the first study to describe the chronological sequence of changes that occur in an experimental model of pyloric stenosis and the first to identify an in utero onset of the hypertrophy. Furthermore it is the only animal model of infantile hypertrophic pyloric stenosis that was originally created and verified as a model of another naturally occurring human condition known to be associated with infantile hypertrophic pyloric stenosis.

5.6. Tables and Figures (overleaf CHAPTERS: 115 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND PHENYLKETONURIA

Table 5-1 The width of tissue layers in control and diseased mice 10 to 180 days of age (xlO'^^m)

Diameter Group Age, days from birth sed P value

10 20 40 90 180

Pylorus Control 43.1 42.8 54.6 58.8 60.4 1.69 <0.001 Diseased 24.8 37.8 46.3 45.6 53.1 Longitudinal Control 0.76 0.61 0.55 0.58 0.66 0.14 <0.001 Muscle Diseased 0.85 0.99 1.49 0.51 0.26 Circular Control 2.01 1.98 0.92 3.94 5.22 0.49 <0.001 Muscle Diseased 2.85 8.17 5.87 5.42 4.18 Muscularis Control 0.37 0.28 0.68 0.36 0.42 0.05 <0.001 Mucosa Diseased 0.41 0.60 0.33 0.32 0.32

Table 5-2 The width of tissue layers expressed as a proportion of the total diameter of the pylorus

Diameter Group Age, days from birth sed P value

10 20 40 90 180 Longitudinal Control 1.8 1.4 1.0 1.0 1.1 0.35 <0.001 Muscle Diseased 3.4 2.6 3.2 1.1 0.5 Circular Muscle Control 4.7 4.7 1.7 6.7 8.6 1.3 <0.001 Diseased 11.5 21.8 12.7 11.9 7.7 Muscularis Control 0.85 0.65 1.26 0.61 0.70 0.12 <0.001 Mucosa Diseased 1.65 1.59 0.71 0.71 0.60 CHAPTERS: 116 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND PHENYLKETONURIA

Table 5-3 The width of the circular muscle of one day old and 14 day old fetal mice(xlO'^m)

Diameter Group Age sed P value

14 day fetus 1 day Pylorus Control 23.8 50.7 0.56 0.001 Diseased 9.2 40.0 Circular Muscle Control 1.33 2.78 0.27 0.2 Diseased 1.94 2.86 Circular Muscle % Control 5.6 5.5 1.9 <0.001 Pylorus Diseased 21.1 7.1 CHAPTERS: 117 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND ______PHENYLKETONURIA______Figure 5-1 Diagram Illustrating The Relationship between Tetrahydrobiopterin infantile hypertrophic pyloric stenosis and phenylketonuria.

Phenylalanine

Phen^lanine Nitric Oxide Hydroxylase Synthase Tetrahydrobiopterin

Tyrosine 4-Tetrahydrobiopterin

Phenylketonuria

Untreated

Pyloric Stenosis CHAPTERS: 118 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND PHENYLKETONURIA

Figure 5-2 Weight, Longitudinal Muscle Trend

W d ^ Longitudinal Misde Trend

35- - 0.035 fe ■CcrtidW. 30- - 0.03

2 5 - -0.025 I ■HHIW. 2 0 - - 0.02 I 15- -0.015 a ■Ccitrci,LMDP 1 0 - ? - 0.005 k -D-HFH1,IMCP

10 20 40 90 180 ^ (D a js)

Figure 5-3 Weight, Circular Muscle Trend

Weight,Circular Muscle Trend

T 0.16 ■Control W t 35 -- - 0.14

30 - - 0.12 •HPHl Wt. I - 0.1 20 -

I ■Control 15 - - 0.06 I CM/DP 10 -- - 0.04

- 0.02 k -HPH-1 CM/DP

10 20 40 90 180 Age (Days) CHAPTERS: 119 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND PHENYLKETONURIA

Figure 5-4 Weight, Muscularis mucosae Trend

Weight, Muscularis Mucosae Trend

40 j 0.018

-- 0.016 ■Control Wt. 35 k -- 0.014 I 30 -- -- 0.012 I 25 -- ■HPHlWt. Ü -0.01 S 2 r 20 ■S s - 0.008 I ^ I 15 + ■Control - 0.006 g MM/DP 10 -- - 0.004 2 5 - - 0 . 0 0 2 Oh ■HPH MM/DP

0 10 20 40 90 180

Age (Days)

Figure 5-5 Fetal, one day old Mouse Muscle Hypertrophy

Estimated Difference in Means of Proportionate Circular Muscle Width to Diameter Pylorus.

-50 100 200

O (/) Ë § O— *o := ■a

Age (Days)

DP: Diameter Pylorus, CM: diameter of Circular Muscle, LM: diameter of Longitudinal Muscle MM: diameter of Muscularis Mucosae CHAPTER 5: 120 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND ______PHENYLKETONURIA______

Figure 5-6 40 day old Control Mouse

CONTROL, 40 DAY OLD MOUSE

T 7 r ..... ■...... ■’0 30 40 50 60 70 80 90 100 110 120 130 140 ISO 160 170 180 190

Figure 5-7 40 day old Hph-1 M ouse

DISEASED, 40 DAY OLD MOUSE

'n 2 0 30 40 50 s o 7 0 8 0 9 0 100 110 120 730 140 ISO 160 170 CHAPTER 5: 121 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND PHENYLKETONURIA

Figure 5-8 Stomachs of 40 day Control Mouse

liiitiiininiininiitii

Figure 5-9 Stomachs of 40 day old Hph-1 Mouse

il il tin II in CHAPTERS: 122 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND PHENYLKETONURIA

Figure 5-10 Low Power photomicrograph of T/S of Pylorus of 40 day old Control Mouse

■V

» *KS

Figure 5-11 Low Power photomicrograph of T/S of Pylorus of 40 day old Hph-1 Mouse

s!" ^--v- » # # # 4 ^ %

■^. 4 • y,'4> * , 1 -./// Î c . . . y^~.^ / » # :-W ' ' : * . * / r' 1 r - -f \ /J . : * • f'~- J * CHAPTERS: 123 A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND PHENYLKETONURIA CHAPTER 6 124 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______

6. CHAPTER 6 A Quantitative Study of the Histochemical Changes underlying Pyloric Stenosis in Dogs.

6.1 Summary

6.2 Introduction

6.3 Experimental design

6.3.1 Tissues 6.3.2 Imunostaining 6.3.3 Statistical analysis

6.4 Results

6.5 Discussion

6.6 Tables and Figures CHAPTER 6 125 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______

6.1. Summary

A number of studies have described the histological changes that occur in infantile hypertrophic pyloric stenosis in humans and a number of animal models of the condition have been created. The histological features of naturally occurring congenital pyloric stenosis in dogs have never been described. In order to better understand the pathogenesis of the infantile hypertrophic pyloric stenosis and canine congenital pyloric stenosis conditions it is important to compare the morphological changes between the species in both conditions. A precise comparison of the histological changes is best achieved by quantifying these changes and thus studying the comparative anatomy.

Eight specimens of pylorus from dogs with pyloric stenosis and six control specimens were examined using conventional histology and immunohistochemistry for a wide range of antigens. The changes in the proportion of nerves expressing each antigen were quantified and statistically analysed.

Conventional histology revealed gross hypertrophy of the diseased animal’s circular muscle layer similar to that in the human condition. The nerves appeared to be unchanged. Subjective assessment of the immunostaining suggested there was no change in the expression of any of the antigens examined. These findings were confirmed upon statistical analysis of the results.

The results of this study are the first quantitative analysis of the histological changes of pyloric stenosis occurring in canine pylorus. This allowed the changes in the canine pylorus to be compared with those in infantile hypertrophic pyloric stenosis and other experimental animal models of pyloric stenosis. The findings that the proportion of nerves expressing all of the antigens examined ( PGP, nNOS, CGRP, SP and VIP) is unchanged is at variance with our findings in infantile hypertrophic pyloric stenosis. However similar features were described in the hph-1 mouse model and LNAME mouse models of this condition. The result of this study thus further validate these as animal models of naturally occurring pyloric smooth muscle hypertrophy. CHAPTER 6 126 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______

6.2. Introduction ‘Pyloric stenosis’ is a term that describes a group of conditions in dogs in which there is an intrinsic abnormality of gastric emptying. In ‘Antral pyloric hypertrophy’ the pyloric canal is characterised by pyloric smooth muscle hypertrophy and or mucosal hyperplasia. Two forms have been described congenital and acquired pyloric hypertrophy. In congenital pyloric hypertrophy there is pyloric smooth muscle hypertrophy resulting in luminal obstruction. This condition tends to occur in young brachycephalic breeds of dogs such as the Boston terrier, boxer, and bulldog. Acquired antral pyloric hypertrophy is usually encountered in middle aged to older dogs, between one and fifteen years of age. The Lhapso apso, Shih Tzu, Pekingese and poodle are most commonly affected breeds. The aetiology of these conditions is ill understood. Little has been written of congenital pyloric hypertrophy in dogs. A similar and more widely described dichotomy of conditions resulting in primary pyloric smooth muscle hypertrophy has been described in humans. The presentation and management of infantile hypertrophic pyloric stenosis and congenital pyloric stenosis is very similar. The fundamental histological changes underlying canine pyloric stenosis remain unknown. Thus it remains unclear whether the two conditions, infantile hypertrophic pyloric stenosis and canine pyloric stenosis share the same underlying morphological changes.

In this study a range of antibodies was selected for the following reasons: Protein gene Product 9.5 is a sensitive pan neuronal marker, reactivity to it provides a measure of total neural tissue present (Gulbenkian S 1987), Vasoactive Intestinal Polypeptide and neural Nitric Oxide Synthase are both potent nonadrenergic-noncholinergic smooth muscle relaxants the function of which are closely inter-related (Desai KM., 1994) , and substance P is a tachykinin (Kimura S, 1983) .

This study tested the hypothesis that in canine congenital pyloric stenosis, as in infantile hypertrophic pyloric stenosis, there is a selective loss of neuroactive agents. CHAPTER 6 127 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______The aims of this study were to quantify and compare the histological changes that occur in dogs and humans as well as in other experimental animal models of infantile hypertrophic pyloric stenosis. CHAPTER 6 128 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______

6.3. Experimental design

6.3.1. Tissues Eight pyloric muscle biopsies were collected fresh from dogs undergoing pyloroplasty for Congenital pyloric stenosis. Six control pyloric specimens were collected. Four from dogs undergoing post-mortem within six hours of death, and two at surgery for unrelated conditions. The stomach and pylorus were dissected and immersed in Zamboni’s fixative for six hours at 4°C (Appendix II)and rinsed three times in 15% (wt/vol) sucrose in O.I mol/L phosphate-buffered saline (PBS; pH 7.2) with 0.01% (wt/vol) sodium azide.

6.3.2. Antibodies Antibodies to the following range of antigens was selected: PGP 9.5, VIP, nNOS, and SP. (Please see Section 6.1 for discussion) .

6.3.3. Immunostaining Five randomly selected sections of lOjim thickness were cut, for each of the antigens examined, perpendicular to the long axis of the bowel from snap-frozen blocks in a cryostat at -25°C. The slides were processed for DAB-nickel enhancement method. (Appendices VI & VII).

The developed slides were counterstained with eosin and examined under a transmitted light microscope.

The developed slides were counterstained with eosin and examined under a transmitted light microscope.

A computer-assisted image analysis system (Seescan) was used to analyse the following parameters in the control and diseased tissues: width of the longitudinal muscle layer; width and length of the nerves as measured in a transverse, 10pm thick section of the pylorus, in the myenteric plexus, longitudinal and circular muscles; CHAPTER 6 129 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______number and size of ganglion cells expressing each of the antigens examined; density of immunostaining by the nerves within the myenteric plexus, longitudinal and circular muscle layers. For each parameter quantified, five images in each of the tissue areas studied were analysed for each section.

6.3.4. Statistical analysis

Data were analysed by calculating the median and 95 % confidence interval for the immunostaining by antibodies to each antigen in the control and diseased groups. The median and 95% confidence interval for antigen expression was calculated as the percentage of total nerves as shown by PGP immunoreactivity in each group, the difference (control - diseased) in the median percentage stained and its 95% confidence interval. The P value was derived from a Mann Whitney U test comparing the median percentage immunostained in the two groups. CHAPTER 6 130 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______

6.4. Results

6.4.1. Conventional Histology Haematoxylin and eosin staining revealed that the morphological changes in the muscle of the canine pylorus affected by congenital pyloric stenosis were very similar to those found in infantile hypertrophic pyloric stenosis: longitudinal and circular muscle layers were thickened in diseased samples compared with the controls. The mucosa and submucosa were not included in the specimens. The muscle fibres were arranged in abnormally disorganised whorls. The muscle fibres appeared to be normal. Interspersed between the muscle fibres were nerves and ganglia that appeared to be unchanged in morphology. In the diseased specimens, the density of these neural structures was slightly lower.

6.4.2. Immunocytochemistry Subjective examination, by eye, revealed an apparent reduction in the total amount of neural tissue expressing PGP in all the tissues of the diseased specimens. Subjective comparison between control and diseased specimens was complicated by the muscle hypertrophy. nNOS, VIP, and SP were widely expressed in nerves of the longitudinal muscle, circular muscle, and myenteric plexus. In the diseased specimens subjective assessment revealed no apparent difference in nerves expressing these antigens. The relative changes in each of the tissues and the changes in antigen expression by the ganglia was impossible to assess by simple examination alone.

Due to technical difficulties in recovering specimens, the circular muscle layer was the predominant tissue received. Statistical analysis of all the parameters measured proved to be valid in only this tissue.

6.4.3. The Changes in Immunoreactivity within the Circular muscle layer The area of nerves expressing each of the antigens examined was found to be unchanged in congenital pyloric stenosis within the circular muscle layer. When calculated as a proportion of the total innervation (PGP-immunoreactive) nerves CHAPTER 6 131 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______expressing nNOS were found to be reduced insignificantly in the circular muscle layer. The reduction in VIP immunoreactive nerves approached statistical significance (Table 1, Figure I). CHAPTER 6 132 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______

6.5. Discussion Two distinct groups of dogs are affected by ‘pyloric stenosis.’ In each group there is a distinct clinical condition: Acquired Antral Pyloric Hypertrophy (AAPH) and Congenital Pyloric Stenosis (CPS). AAPH is usually diagnosed in middle-aged or older dogs (range 1-15 years). Published case studies include about twice as many males and castrated males as females and spayed female dogs. The majority are smaller breeds of dogs (less than 10kg) such as Lhapso Apso, Shih Tzu, Pekingese and miniature poodle. AAPH is known to occur in a few of the larger breeds of dogs such as the Doberman Pinscher, Collie and German Shepherd. A similar syndrome has been identified in cats and horses. In those dogs whose sole underlying pathology is AAPH there are no consistent features either upon physical examination, haematological or biochemical analysis. A hypochloraemic, hypokalaemic, metabolic alkalosis associated with severe gastric outlet obstruction may occur as can a metabolic acidosis associated with a moderate dehydration. However the majority of dogs have no significant acid base or electrolyte imbalance (Matthiesen DT., 1986, Sikes RI., 1986, Walter MC,. 1985) Contrast examination may show gastric distension, delayed gastric emptying, pyloric intraluminal filling defects, and thickening of the pyloric wall. The pylorus may be narrowed and tip into the duodenal cap to produce the ‘beak’ sign as in infantile hypertrophic pyloric stenosis. Fluoroscopy may demonstrate normal or vigorous gastric contractions. Endoscopy, and biopsy, is a useful investigation to exclude other rarer causes of gastric outlet obstruction in the older dog: neoplasia, foreign bodies, eosinophilic granulomas, and fungal disease. The endoscopic appearance in AAPH is of occlusion of the pyloric canal by bulging hyperplastic mucosal folds. Histology of mucosal biopsy specimens in AAPH demonstrate a pronounced papillary and branching pattern of the surface foveolae, hyperchromasia, and increased numbers of mitotic figures in the foveolar epithelial cells (Leib MS., 1993).

Congenital pyloric hypertrophy, in dogs, has not been widely described in the scientific literature. It tends to affect younger brachycephalic breeds dog: the bull dog, the boxer, and the Boston terrier. The cardinal morphological change is pyloric muscle hypertrophy. This has been described as being identical to that produced in the pentagastrin induced dog model of infantile hypertrophic pyloric stenosis (Strombeck CHAPTER 6 133 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______DR., 1990) .The degree of hypertrophy is variable. The relationship between the degree of hypertrophy and breed of dog or duration and severity of symptoms is unclear. In humans there is known to be no such association (Dodge J., 1990) . The predominant sign is of vomiting. Occasionally individual dogs may present with weight loss anorexia and abdominal distension. The vomiting tends to occur at a relatively constant interval after feeding. The vomitus consists of unchanged food and gastric juice. Amongst breeds that significantly swallow air, such as the bulldog, the vomitus is frothy. Affected animals are usually ravenously hungry immediately after vomiting. The symptoms usually start shortly after weaning. The natural progression of the condition tends to be determined by the age at onset of the symptoms. Older dogs may maintain their body weight while smaller puppies rapidly become weak, lose weight and growth may be virtually arrested. Despite this the dogs generally appear to be remarkably well. Hypochloraemic, hypokalaemic metabolic alkalosis may occur. The hypochloraemic, hypokalaemic metabolic alkalosis is corrected with isotonic saline as in humans. A diagnosis may be made upon the history and clinical signs, however radiological examination is useful. Plain abdominal radiographs may demonstrate the size and contents of the fasted dog’s stomach as well as exclude foreign bodies. Contrast radiographs are of variable benefit, false positives and negatives may occur. Gastric emptying time may be delayed either by an obstructive lesion of the pylorus or simply by the dog being anxious. More reliable features of pyloric outlet obstruction would be antral dilatation or the ‘beak, string and tit’ signs, similar to those in Infantile Hypertrophic Pyloric Stenosis. Spasmolytic agents may relieve pylorospasm. However the definitive treatment of congenital pyloric stenosis today is surgery. The simplest and safest procedure is Ramstedt’s pyloromyotomy. Pyloroplasty such as Mickulicz’ or Finney’s have been performed for congenital pyloric stenosis. These are becoming less popular. The long term prognosis for congenital pyloric stenosis, after surgery, is excellent (Srombeck DR., 1990) . Thus CPS closely resembles infantile hypertrophic pyloric stenosis as a clinical entity. Diseased specimens from dogs suffering from CPS were only collected in this study. The smooth muscle hypertrophy in this condition was found to be very similar to that in infantile hypertrophic pyloric stenosis. These results are remarkable as the quantified, massive reduction in the proportion of nerves that expressed all the antigens studied in infantile hypertrophic pyloric stenosis ( Abel RM., CHAPTER 6 134 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______1995) are not reflected in dogs suffering from congenital pyloric stenosis. nNOS was the only antigen found to diminished in expression in all of the tissues examined in the diseased human pylorus. In the diseased canine pylorus there was found to be no significant reduction in expression of nNOS.

The pylorus of the pentagastrin induced canine model of infantile hypertrophic pyloric stenosis said to identical with that of naturally occurring canine CPS (Dodge J.,1976, Strombeck DR., 1990). However the histological changes in either of these conditions has not been quantified to date. The hypergastrinaemia described by some in infantile hypertrophic pyloric stenosis (Bleicher MA., 1978, Spitz L., 1976) may be secondary to diminished somatostatin immunoreactivity (Barrios V., 1994). Somatostatin, VTP, and NO are major mediators of NANG inhibitory innervation to the intestine. Somatostatin normally inhibits the release of gastrin. The proportion of nerves expressing VTP and nNOS in the circular muscle layer of the diseased human pylorus is diminished by an almost identical amount (Abel RM., 1995) . Somatostatin and NOS are co-localised to the same neurons in the myenteric plexus (Vincent SR., 1992).

Pyloric smooth muscle hypertrophy has been demonstrated histologically in mice treated postnatally with LNAME, a NOS inhibitor (Voelkner C A , 1995) . Prenatally treated mice were described as being smaller. It is unclear, from this study, if the muscle hypertrophy was restricted to the circular muscle layer alone or if it occurred in the prenatally treated mice. The histological features of the pre or postnatally treated mice were not quantified. The morphology and histochemical changes in the nerves were not described.

A nitric oxide gene-deleted mouse has been described (Bredt D., 1993, Huang P., 1993) in which the only abnormality demonstrated was pyloric smooth muscle hypertrophy. It was not described to be impotent and did not behave abnormally as one might expect in the presence of NO deficiency. Furthermore these animals did not lose weight. As these animals did not lose weight and as the histological changes in the nerves and muscle were not quantified it is unclear how closely this model reflects the changes in infantile hypertrophic pyloric stenosis. CHAPTER 6 135 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC STENOSIS IN DOGS.

In none of the animal models discussed, was the chronological sequence of events in the development of the muscle hypertrophy described.

The changes identified in this study of the canine pylorus affected by CPS are consistent with those in the hph-1 mouse (Chapter 5) . The underlying lesion in the experimental animal model is a deficiency of tetrahydrobiopterin and diminished NOS activity (Brand MP., 1995). A transient smooth muscle hypertrophy of the pylorus causing gastric outlet obstruction and weight loss, upto 40 days of age, has been demonstrated in these mice (Abel RM., 1995). The muscle hypertrophy was found to occur in utero in the hph-1 mouse. This is consistent with reports of premature infants developing infantile hypertrophic pyloric stenosis (Beasley SW., 1989) . The clinical progression of infantile hypertrophic pyloric stenosis, CPS, and the hph-1 mouse is very similar.

The quantified results of this study, those of the histological changes in infantile hypertrophic pyloric stenosis and those of the hph-1 mouse suggest that diminished NOS activity may be the principle lesion in the pathogenesis of infantile hypertrophic pyloric stenosis in humans and the hph-1 mouse, and perhaps in dogs. The reduced expression of the other neuropeptides examined may be secondary changes reflecting a more fundamental aberration in the expression of NO in the developing, diseased pylorus.

In humans such a deficiency of NO synthesis is unlikely to be due to a relative lack of dietary substrate as a result of vomiting, as infantile hypertrophic pyloric stenosis is known to be associated with oesophageal atresia and has been identified on the first day of life at the time of laparotomy and insertion of gastrostomy (Spitz L., personal communication). A deficiency of substrate would not account for the localised loss of nNOS in the pylorus.

Thus this study has achieved its objectives of describing the histological features of canine CPS Thus allowing these changes to be compared with those in infantile CHAPTER 6 136 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______hypertrophic pyloric stenosis and experimental animal models of this condition. In so doing some fascinating insights into the pathogenesis of this common condition affecting dogs and men have been revealed. CHAPTER 6 137 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC STENOSIS IN DOGS.

6.6. Tables and Figures

Figure 6-1 Median Proportion of Nerves Expressing Antigen within the Circular Muscle layer.

Dogs: Median Proportion of Nerves Expressing Antigen within the Circular Muscle.

Ç 0 .6 - I CONTROL

0.4 -- □ DISEASED

VIP Antigen

Table 6-1 Median Proportion of Nerves Expressing Antigen in the Circular Muscle layer.

ANTIGEN CONTROL DISEASED STANDARD ERROR P VALUE NOS 0.602 0.435 0.6 0.14 0.8 SP 0.415 0.387 0.82 0.22 0.76 VIP 0.57 0.17 0.09 0.11 0.065 CH.AfTER6 138 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC ______STENOSIS IN DOGS.______

Figure 6-2 Immunostaining for nNOS in a Normal Canine pylorus CHAPTER 6 139 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC STENOSIS IN DOGS.

Figure 6-3 Immunostaining for nNOS in a Canine pylorus of congenital pyloric stenosis CHAPTER 6 140 A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC STENOSIS IN DOGS. CHAPTER? 141 A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE ______PYLORUS.______

7. CHAPTER 7 A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE PYLORUS,

7.1 Summary

7.2 Introduction

7.3 Experimental design

7.3.1 Surgical procedure 7.3.2 Tissues 7.3.3 Imunostaining 7.3.4 Statistical analysis

7.4 Results

7.5 Discussion

7.6 Figures CHAPTER 7 142 A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE ______PYLORUS.______7A. Summary

A number of studies have reported ablation or degeneration of the enteric innervation to be associated with smooth muscle hypertrophy of the affected region of intestine. The aims of this study were to examine the effects of carbon dioxide laser irradiation upon the innervation and morphology of the pylorus The carbon dioxide laser has been reported to selectively ablate both the intrinsic and extrinsic innervation of the pylorus.

24 adult wistar rats underwent carbon dioxide laser irradiation of the pylorus. The dose of irradiation employed was limited by delayed ischaemic necrosis of the pylorus. The pylorus was examined at 3, 7, 14, and 21 days following the procedure.

The enteric innervation was found not to be ablated by laser irradiation at the doses employed. Following irradiation there was a significant increase in serum gastrin levels. Subjective histological examination revealed there to be no change in the innervation or the morphology of the muscles of the pylorus.

The apparent lack of disturbance to the innervation and morphology of the pylorus, following carbon dioxide laser irradiation, may reflect two possible causes: the changes were too subtle to be identified by simple, subjective assessment of such restricted number of cases, and secondly the intrinsic and extrinsic innervation of the pylorus are involved in complex anatomical and functional inter-relationships that are not easily distinguishable by simple ablation studies. CHAPTER 7 143 A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE PYLORUS.

7.2. Introduction

A gross reduction in the total amount of neural tissue present associated with degenerative changes in the nerves and ganglia have been described in infantile hypertrophic pyloric stenosis by several authors (Belding HH.,1953, Spitz L.,1975) . Hadzijahic has reported producing smooth muscle hypertrophy of the small intestine after chemical ablation of the myenteric innervation (Hadzijahic N., 1995) . Muscle development is influenced by nerves by both the regulation of mitosis of myoblasts and the pattern of expression of proteins. aSMA is known to be expressed by 8 weeks of gestation by human colonic muscle fibres. The functional relationship between nerves and enteric smooth muscle remains poorly understood (Romanska H , 1993) . The use of lasers has become increasingly popular in abdominal surgery. Recent reports have described the use of these instruments to selectively ablate neural tissue (Kadota T., 1992 Queresh A., 1992). The aim of this study was to investigate the relationship between the extrinsic and intrinsic innervation and muscle by ablating both the extrinsic and intrinsic innervation by carbon dioxide laser irradiation. CHAPTER? 144 A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE ______PYLORUS.______

7.3 . Experimental design

7.3.1. Surgical Procedure All operations were performed at the Institute of Child Health, London by Mr. RM. Abel under Home Office and local ethical committee guidelines for the care of laboratory animals and the use of operating lasers. Under clean conditions and under general anaesthesia (methoxyfluoramine administered by a Baines circuit) a limited laparotomy was performed to deliver the stomach from a vertical, upper abdominal midline incision. To restrict the area of irradiation a fenestrated, saline-soaked swab was placed over the pylorus. Thus the heat energy was absorbed and tissue other than the pylorus was protected. A dose of O.SmWatts of irradiation was pulsed three times over 0.1 secs at a distance of 1 meter from the pylorus. The dose was calibrated initially upon fresh cadaveric rat pylorus. A ‘sham’ laparotomy was performed upon litter-mates (n=24). These animals were housed under similar conditions and tissues were processed in parallel with tissues from the experimental groups. The experimental and control animals were killed by a schedule one procedure at 3,7,14, and 21 days postoperatively. If any of the animals lost 10% of their preoperative body weight they were killed by a schedule one procedure. (Please see section 2.4.3)

7.3.2. Tissues The stomach and pylorus were dissected from each animal and fixed, fresh, in Zamboni’s fixative for six hours at 4°C (Appendix II) then rinsed three times in 15% (wt/vol) sucrose in 0.1 mol/L phosphate-buffered saline (PBS; pH 7.2) with 0.01% (wt/vol) sodium azide. Sections of 10pm thickness were cut parallel to the long axis of the gut from snap-frozen blocks in a cryostat at -25°C.

7.3.3. Antibodies PGP: this is a highly sensitive pan neuronal marker (Gulbenkian S. 1987) VTP: a mediator of the parasympathetic innervation to the stomach nNOS: both and VIP are potent nonadrenergic-noncholinergic smooth muscle relaxants (Desai KM. 1994) SP: provides the excitatory motor innervation (Kimura S, 1983) CHAPTER 7 145 A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE ______PYLORUS.______CGRP: the sensory innervation to the stomach (Dockray GJ.I99I)

7.3.4. Immunocytochemistry Tissue sections were stained by the avidin-biotinylated-peroxidase complex (ABC) method with nickel enhancement (Appendix VI & VII) . The developed slides were counterstained with eosin and examined under a transmitted light microscope. The width of the muscle layers was measured using a graticule. Photographs were taken using Technical PAN film (ASA 50; Kodak Ltd., Hemel Hempstead, UK.). CHAPTER 7 146 A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE ______PYLORUS.______

7.4. Results

7.4.1. Conventional Histology Haematoxylin and eosin staining revealed evidence of the previous surgery in the experimental animals: the adventitia was disrupted in some areas. The morphology of the longitudinal and circular muscle layers in the experimental group was unchanged compared with the control specimens. This relationship was unaffected by the length of the postoperative period prior to sacrifice. The muscle fibres appeared to be normal. Interspersed between these fibres were nerves and ganglia. In the experimental specimens, the density and morphology of these neural structures was unchanged.

7.4.2. Immunocytochemistry Subjective examination, by eye, revealed no apparent change in the total amount of neural tissue that expressed PGP in all of the tissues. CGRP expression was restricted primarily to the nerves and ganglia of the myenteric plexus, while nNOS, VIP, and SP were more widely expressed in nerves of the longitudinal muscle, circular muscle, and myenteric plexus. CHAPTER 7 147 A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE ______PYLORUS.______

7.5. Discussion The results of this study showed that carbon dioxide laser irradiation, at a maximal dose limited by late ischaemic necrosis, fails to selectively ablate neural tissue within the pylorus. That serum gastrin levels were significantly elevated in both the experimental groups suggests that some injury to the vagus nerve had been produced. Serum gastrin levels are frequently elevated following vagotomy (Koop H.,I993, Uvnas-Moberg K., 1992) .

The carbon dioxide laser has a spectral emission in the middle of the infrared portion of the spectrum at 1,600 nm (10.6 \im) . The active medium is the carbon dioxide molecule. To obtain a population inversion, proper energy transfer from the electric discharge from pump source is necessary. To obtain adequate energy transfer an intermediary nitrogen atom is first excited. Its energy is then transferred to the carbon dioxide molecule. After the carbon dioxide molecule decays with the emission of infrared energy, the molecule is brought down to ground state through collisions with the helium atoms. Thus a gas mixture of carbon dioxide, nitrogen and helium is used in the laser cavity. The carbon dioxide laser emission wavelength is absorbed by water. The absorption coefficient of water is 230 cm'^ at 10.6 |im. As tissues are composed of 70-90% water and this acts as the primary absorbing medium in the tissue. The absorption coefficient is so high that 98% of the incident energy is absorbed in 0.17mm of tissue. The depth of penetration is referred to as the extinction length. Despite such a short extinction length it proved impossible to selectively ablate the neural tissue within the pylorus without causing fatal, late ischaemic necrosis.

In conclusion the use of a carbon dioxide laser to selectively ablate neural tissue in a clinical setting, such as vagotomy (Kadota H., 1993 Quereshi A., 1992), must therefore be considered with some caution.

7.6. Figures (overleaf CHAPTER 7 148 A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE ______PYLORUS.______

Figure 7-1 PGP 9.5 Immunostaining within control and laser irradiated wistar rat

pylorus.

V.

* ^ \

Jt / JL. X / y CHAPTER 7 149 A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE PYLORUS. CHAPTERS______CONCLUDING REMARKS______150

CONCLUDING REMARKS

In the introductory chapter of this thesis a detailed dissertation was presented on the anatomy and morphology of the normal human pylorus, and the pathophysiology, natural history and treatment of infantile hypertrophic pyloric stenosis. This detailed literature review illustrated that little is known of either the development or the innervation and morphology of the normal pylorus.

An understanding of the ontogeny and anatomy of the normal pylorus and the precise changes in infantile hypertrophic pyloric stenosis is essential to understanding the aetiology and pathogenesis of this condition. By acquiring this information this study tested the following hypothesis.

The expression of neuro transmitters and the morphology of the normal pylorus is developmentally regulated. The morphological changes within the nerves and muscles in infantile hypertrophic pyloric stenosis are due to a selective loss of neuroactive agents.

There has been a profusion of reports discussing the changes in morphology and innervation of the pylorus in infantile hypertrophic pyloric stenosis. The detailed morphology and ontogeny of the peptide innervation of the normal pylorus has never been examined. None of the published studies has been quantified to accommodate the distortion in appearance due to the muscle hypertrophy in either infantile hypertrophic pyloric stenosis or related naturally occurring or experimentally induced animal models of pyloric stenosis. The results and conclusions of these studies have not been statistically verified and none have been as extensive as in this study. Thus the observations made in this study are entirely original.

The principal technique used in these studies has been immunohistochemistry. It is widely accepted as a reliable method of identifying specific proteins in tissues. Some of the proteins may share epitopes with unrelated compounds resulting in CHAPTERS______CONCLUDING REMARKS______151 the possibility of false positive cross reactivity. Thus the specificity of each of the antibodies used in these investigations was validated using Western blotting. By this technique the immunoprecipitate of the antibody and protein of the correct molecular weight is ensured.

In addition to such well recognised methods other techniques were modified in new ways to be used for specific aspects of this study. For example the carbocyanine compound Dil was used to trace the vagal innervation of the human fetal pylorus.

Some of the most significant observations made in the course of this thesis were in the studies of the ontogeny of the peptide innervation of the pylorus. The fundamental principle that the expression of neuroactive agents is developmentally regulated was confirmed to occur within the pylorus as in other regions of the gut such as the small intestine. The temporal and spatial patterns of expression, both radial and craniocaudal, of a wider range of substances was examined than has ever previously been examined in any region of the intestine. The relationship between the patterns of expression of these substances and the development of the overall morphology of the pylorus have thus been documented. The inhibitory neurotransmitters NO and VIP were found to be expressed at the same time. The extrinsic vagal innervation of the human fetal pylorus has never previously been examined. By unrelated techniques of carbocyanine tracing and immunohistochemistry the vagal, VTP, containing fibres were found to be first present at the same time as intrinsic VIP containing nerves and ganglia. These studies provide a baseline from wliich further investigations of infantile hypertrophic pyloric stenosis may be evaluated.

The quantitative study of the histological changes in infantile hypertrophic pyloric stenosis revealed several previously unrecognised features of this condition. The most fundamental observations were that the longitudinal muscle layer is hypertrophied as well as the circular muscle, the nerves of the myenteric plexus are longer and thicker and that ganglia expressing VTP are larger and increased in CHAPTERS______CONCLUDING REMARKS______152 number. NOS and VIP expression was almost identically diminished in the circular muscle and myenteric plexus. Of all the neurotransmitters studied, NOS was the most greatly diminished in all of the tissues examined. The expression of CGRP was found to be diminished in selective tissues such as the myenteric plexus. These findings confirmed the hypothesis that selective changes in neurotransmitter expression underlie infantile hypertrophic pyloric stenosis and that these changes are not artefacts due to compression or dilution secondary to the muscle hypertrophy.

The fundamental lesion underlying infantile hypertrophic pyloric stenosis may be a selective aberration of the neural population expressing NOS and VIP as the expression of both of these neurotransmitters was almost identically diminished and both have been colocalized to the same nerve population. The age of onset of this lesion may be at, or shortly after, both NOS and VTP are expressed within the fetal pylorus. NOS and VEP were found to be first expressed concurrently at 11 weeks of gestation. The abnormal morphology of nerves and muscle may be developmental changes secondary to the aberrant expression of these neurotransmitters. The increased expression of VIP containing ganglia may be a mechanism by which infantile hypertrophic pyloric stenosis naturally resolves. The complex relationship between the neural expression of neurotransmitters, the expression of NO by muscle fibres and the activity of the interstitial cells of Cajal is yet to be fully assessed. The study of the chronological sequence of changes in the hph-1 mouse may help to clarify these relationships and so the aetiology of pyloric stenosis.

The hph-1 mouse model is a unique animal model of infantile hypertrophic pyloric stenosis in several ways: it is the first experimental model of infantile hypertrophic pyloric stenosis that was originally described and verified of a condition naturally associated with infantile hypertrophic pyloric stenosis (PKU), the fundamental biochemical lesion of reduced NOS activity and NO production has been identified in the hph-1 mouse, the chronological sequence of histological changes within the experimental mouse pylorus have been quantified and statistically CHAPTERS______CONCLUDING REMARKS______153 verified. The hph-1 mouse behaved clinically as do children with infantile hypertrophic pyloric stenosis. The mice developed a transient weight loss, associated with gastric distension, that resolved as the pyloric muscle hypertrophy resolved. The muscle hypertrophy affected the longitudinal and circular muscle layers as described by this study in infantile hypertrophic pyloric stenosis.

The histological features of the naturally occurring condition congenital pyloric stenosis, in dogs, were described for the first time in this study. The muscle hypertrophy was found to be very similar to that in the human condition: disorganised whorls of circular and longitudinal muscle fibres between relatively sparse numbers of nerves and ganglia. The proportion of nerves that expressed NOS was found to be statistically unchanged. Similar immunohistochemical features were found in the hph-1 mouse. There was no apparent change in the expression of nNOS by the nerves of the hypertrophied hph-1 mouse pylorus.

Thus the histological changes in a rodent model, originally described for a condition naturally associated with infantile hypertrophic pyloric stenosis, were found to occur in a related naturally occurring form of infantile hypertrophic pyloric stenosis known to affect another species, dogs.

In conclusion the studies presented in this thesis have described the development of morphology and the ontogeny of the neuropeptide innervation of the normal infant pylorus and quantified the changes in these parameters in infantile hypertrophic pyloric stenosis. A selective loss of nNOS was identified in all of the diseased tissues studied. The hph-1 mouse has been verified as an experimental animal model of a condition (PKU) naturally associated with infantile hypertrophic pyloric stenosis. This study quantified and verified a transient muscle hypertrophy of the mouse pylorus associated with gastric distension and weight loss which resolved as the muscle hypertrophy resolved. Clinically the hph-1 mice behaved as do children with infantile hypertrophic pyloric stenosis. The underlying biochemical abnormality is a selective reduction of tetrahydrobiopterin and thus its cofactor activity to NOS in the production of NO. As the production of NOS is CHAPTERS______CONCLUDING REMARKS______154 unchanged, the neural expression of this enzyme within the hph-1 mouse pylorus was found to be unaltered as assessed by imunohistochemistry. These histochemical findings were reflected in dogs suffering from naturally occurring congenital pyloric stenosis.

By studying the comparative anatomy in three different species suffering from pyloric stenosis the underlying lesion was found to be muscle hypertrophy of both longitudinal and circular muscle layers, abnormal morphology of the nerves and ganglia in different tissues, and a selective reduction in the production of Nitric Oxide. A mechanism by which the condition naturally resolves in infants may be by the increased expression of VIP within ganglia. The primary pathology may be the abnormal Nitric Oxide and VTP innervation and the muscle hypertrophy the mechanism by which the lesion often naturally resolves in infantile hypertrophic pyloric stenosis.

Thus these studies have quantified and characterised the changes underlying infantile hypertrophic pyloric stenosis and pyloric stenosis in two other animals. In so doing these studies provide a base line from which further investigations into infantile hypertrophic pyloric stenosis may proceed. CHAPTERS CONCLUDING REMARKS 155 APPENDIX 156

APPENDICES

APPENDIX I:

BUFFERS mMol phosphate buffered saline (PBS)

1. Dissolve 87.9 g NaCl, 2.72g KH 2 PO4 anhydrous to 9 litres of deionised water. 2. Adjust to pH 7.2-7.4 with concentrated HCl. 3. Dilute xlO with deionised water before use.

PBS-sucrose (1 litre)

Dissolve 150g of sucrose in 1 litre of 10 niM PBS and add 10 mg of sodium azide (NaNs) to inhibit microbial/ fungal contamination.

Diluents for antibodies

Primary antibodies and normal sera PBS; 0.05% bovine serum albumin (BSA): 0.1% sodium azide (NaNsMPBS BSA: NaNs) In 100ml of PBS dissolve 0.05g BSA and O.lg NaNs (stored at 4®C)

Secondary antibodies PBSBSA In 100ml of PBS dissolve 0.05g BSA. NaN3 is not added as it can interfere with the precipitation of chromogens such as diaminobenzidine. APPENDIX 157

APPENDIX II

FIXATIVES

Zamboni’s solution (1 litre)

1. Dissolve 17g of paraformaldehyde in 850 ml of O.IM phosphate buffer (PBS, pH7.4). 2. Heat to 60°C and stir until solution is clear. 3. Cool and add 150ml of saturated picric acid. Store at 4®C until used. Shelf life approximately 2-months.

Immersion Fixation In this study all tissues were fixed by immersion of the tissue in at least 20 times its volume of Zamboni’s fixative. The size of the tissue should not exceed 2 x 0.5 cm^ since penetration rates of fixatives is usually relatively slow. Fix for 6 hours at room temperature or overnight.

Post Fixation tissue washes and storage

Rinse tissues in several changes of PBS-sucrose at 4 °C. Tissues may be stored in PBS- sucrose at 4°C for upto 6 months. Storage in tissue blocks at -40®C is advisable for longer periods. APPENDIX 158

APPENDIX III

CRYOPROCESSING OF TISSUES

Preparation o f tissue blocks

1. Blot tissue with filter paper to remove excess PBS-sucrose. 2. Mount tissue at optimum orientation in embedding medium (OCT compound, Miles Scientific) onto a piece of cork. Very small tissue can be held in place with forceps in a well of OCT or within larger supporting tissue such as liver. 3. Cover the entire tissue with embedding medium and plunge into melting isopentane (iso-pentane 2-methylbutane, BDH ltd.) pre-cooled in liquid nitrogen. 4. Store tissue blocks in -400C. 5. Let blocks warm up in cryostat cabinet before sectioning. APPENDIX 159

APPENDIX IV

SLIDE COATING

Poly-L-lysine coated slides

1. Dissolve 1 mg of poly-L-lysine (PLL; MW 150,000-300,000; Sigma) in 1ml of distilled water 2. Apply lOpl of PLL solution onto each slide, and spread PLL so that it forms a thin film covering the slide using a similar technique to that for blood smear preparations. 3. Use the slide immediately afterwards.

Vectabond™ -treated slides 1. Add 1 bottle of vectabond reagent (Vector Laboratories ltd. Peterborough,UK) to 400ml of Acetone. 2. Immerse glass slides in acetone for 10 seconds. 3. Transfer slides to Vectabond - acetone solution for 5 minutes. 4. Allow to dry. 5. Slides may be stored at room temperature for several months. APPENDIX 160

APPENDIX V

CONVENTIONAL HISTOLOGY

Haematoxylin and Eosin (H&E) 1. Thaw-mount frozen sections onto coated glass slides and allow them to air dry for ~1 hour at room temperature. 2. Immerse sections in Harris’s haematoxylin (undiluted) for ~ 1 minute. 3. Wash under tap water for -10 minutes or until a deep blue colour develops. 4. Differentiate in acid-alcohol (70% (v/v) industrial methylated spirit : 1% (v/v) concentrated HCl) for 5 seconds so that only cell nuclei retain the stain (check under the microscope) 5. Wash in tap water and counterstain in 4% eosin for -30 seconds. 6. Wash in tap water for 5 minutes. 7. Dehydrate through graded alcohols. 8. Clear in inhibisol and mount in Pertex.

AVIDIN-BIOTIN-PEROXIDASE COMPLEX (ABC) METHOD

1. Thaw-mount frozen sections onto coated glass slides and allow them to air dry for - 1 hour at room temperature. 2. Dehydrate the sections through graded alcohols to inhibisol 3. Block the endogenous peroxidase by immersion in 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature. 4. Rehydrate the sections to distilled water. 5. Block non-specific binding sites by incubating sections for 20 minutes with a 3% solution of normal serum (from the same species in which the secondary antibody was raised) in lOmM PBS containing 01% bovine serum albumin (BSA; Sigma). 0.01% sodium azide (Sigma) and 3% normal serum. 6. Rinse the slides in PBS three times for 5 minutes. 7. Blot excess PBS from around sections. 8. Incubate the sections Avith biotinylated secondary antisera (usually goat anti-rabbit IgG for polyclonal primary antisera and horse anti-mouse IgG (IgM) for APPENDIX 161

monoclonal antisera - obtained from Vector Laboratories, Peterborough, UK.) diluted 1:100 in PBS, containing 0.1% BSA and 3% normal serum (No NaN3)ovemight at room temperature. 9. Rinse the slides in PBS three times for 5 minutes. 10.Incubate sections in ABC complex ( combine reagents A and B at a final dilution of 1:200 ) for one hour at room temperature. 11 .Rinse the slides in PBS three times for 5 minutes. 12.Prepare a solution containing 0.025% (w/v) 3.3’ diaminobenzidine (DAB; Sigma) with 0.03% (v/v) hydrogen peroxide in PBS. 13.Develop the slides by immersing in the DAB solution for 5 minutes at room temperature. Alternatively, use the modified method described in Appendix VII. H.Rinse the slides in PBS three times for five minutes. 15.Rinse the slides in tap water, dehydrate through graded alcohol, clear in inhibisol and mount in pertex. APPENDIX 162

APPENDIX VI

GLUCOSE OXIDASE-DAB-NICKEL-ENHANCEMENT

Follow the protocol shown in Appendix VI up to step 11, then proceed as follows;

1. Rinse the slides three times for 5 minutes in 0.1 M acetate buffer (I3.6Ig sodium acetate in one litre of distilled, adjusted to pH 6 with acetic acid). 2. Immerse the slides in glucose oxidase-DAB-nickel solution (see below) for 5 min. 3. Rinse the slides thrice for five minutes in 0.1 M acetate buffer. 4. Rinse the slides in tap water, dehydrate through graded alcohol, clear in inhibisol and mount in pertex.

Preparation of glucose oxidase-DAB-nickel solution: 1. Dissolve 5.25g nickel ammonium sulphate (BDH, UK.) in I25ml of 0.2M sodium acetate buffer (pH6.0). Dissolve 500mg (3-D-glucose (BDH) and lOOmg ammonium chloride (BDH) in the mixture. 2. Dissolve 50mg DAB (or take 4ml aliquot) in 125ml distilled water. 3. Combine solutions 1 and 2 and add 5mg glucose oxidase to the mixture just before use to start the reaction. 4. Immerse the slides to develop for 5 minutes. 5. Upon completion, rinse and mount as for appendix VI APPENDIX 163

APPENDIX VII

Supplementary tables of quantification of histological changes in infantile hypertrophic pyloric stenosis.

Table 0-lS Sample of Five Control Circular Muscle Specimens for nNOS Assay

FrameArea RedlnPrame %Red Count C1 NOS 1 01

0 01 0.06922 0.0001738 0.2511 21 02 0.06922 0.00009469 0.1368 33 03 0.06922 0.0009785 1.414 96 AvOi 0.06922 0.000415663 0.600633333 50 01 NOS II 01

0 01 0.06922 0.0003157 0.4561 54 02 0.06922 0.0002859 0.4131 67 03 0.06922 0.0004174 0.603 89 AvOi 0.06922 0.000339667 0.490733333 70

01 NOS III 01

0 01 0.06922 0.0008347 1.206 73 02 0.06922 0.001172 1.694 123 03 0.06922 0.0002095 0.3027 65 AvOili 0.06922 0.000738733 1.067566667 87

01 NOS lU 01

0 01 0.06922 0.000588 0.8495 84 02 0.06922 0.0005308 0.7669 61 03 0.06922 0.0001499 0.2166 42 AvOiv 0.06922 0.0004229 0.611 62.33333333

01 NOS V 01

0 01 0.06922 0.0003203 0.4627 71 02 0.06922 0.0003444 0.4976 55 03 0.06922 0.0002492 0.36 62 AvOv 0.06922 0.000304633 0.4401 62.66666667 02 NOS 1 01

0 APPENDIX 164

Cl 0.06922 0.0001794 0.2592 56 02 0.06922 0.0001623 0.2344 60 03 0.06922 0.0002535 0.3662 61 AvOi 0.06922 0.0001964 0.2866 59

02 NOS II 01

0 01 0.06922 0.00008315 0.1201 30 02 0.06922 0.0001151 0.1662 33 03 0.06922 0.0001151 0.1662 33 AvOi 0.06922 0.00010445 0.150833333 32

02 NOS III 01

0 01 0.06922 0.0001644 0.2375 67 02 0.06922 0.00009334 0.1349 43 03 0.06922 0.0002299 0.3321 40 AvOiii 0.06922 0.000162547 0.234833333 50

02 NOS IV 01

0 01 0.06922 0.0007143 1.032 220 02 0.06922 0.0001379 0.1992 67 03 0.06922 0.0001065 0.1538 62 AvOiv 0.06922 0.000319567 0.461666667 116.3333333

02 NOS V 01

0 01 0.06922 0.0007156 1.034 236 02 0.06922 0.0004056 0.5859 152 03 0.06922 0.000009388 0.01356 10 AvOv 0.06922 0.000376863 0.544486667 132.6666667

FrameArea Redin Frame %Red Count 03 NOS 1 01

0 01 0.06922 0.0001419 0.205 33 02 0.06922 0.0001255 0.1814 34 03 0.06922 0.0001674 0.2418 45 AvOI 0.06922 0.000144933 0.2094 37.33333333

03 NOS II 01

0 01 0.06922 0.0001974 0.2852 65 02 0.06922 0.0007843 1.133 78 03 0.06922 0.0006518 0.9417 44 APPENDIX 165

AvCi 0.06922 0.0005445 0.766633333 62.33333333 C3NNSIIIC1

C C1 0.06922 0.0007232 1.045 50 C2 0.06922 0.000452 0.653 91 C3 0.06922 0.0001711 0.2472 43 AvCiii 0.06922 0.000448767 0.6484 61.33333333

C3 NOS III C1

C C1 0.06922 0.0003162 0.4569 82 C2 0.06922 0.0003074 0.4441 89 C3 0.06922 0.001514 2.187 197 AvCIv 0.06922 0.000712533 1.029333333 122.6666667

C3 NOS IV 01

C 01 0.06922 0.0001033 0.1492 40 02 0.06922 0.0003069 0.4433 87 03 0.06922 0.0003026 0.4371 93 AvOv 0.06922 0.0002376 0.3432 73.33333333

04 NOS 1 01

0 01 0.06922 0.000519 0.7498 90 02 0.06922 0.0012 1.733 151 03 0.06922 0.0002055 0.2968 58 AvOI 0.06922 0.0006415 0.926533333 99.66666667

04 NOS II 01

0 01 0.06922 0.0007645 1.104 72 02 0.06922 0.0002473 0.3573 59 03 0.06922 0.0003543 0.5119 98 AvOI 0.06922 0.000455367 0.657733333 76.33333333

04 NOS III

0 01 0.06922 0.0005931 0.8568 71 02 0.06922 0.0002165 0.3127 76 03 0.06922 0.0007427 1.073 85 AvOiii 0.06922 0.000517433 0.7475 77.33333333

04 NOS IV 01

0 APPENDIX 166

C1 0.06922 0.000794 1.147 84 C2 0.06922 0.0006601 0.9537 163 C3 0.06922 0.0008372 1.209 115 AvCiv 0.06922 0.000763767 1.103233333 120.6666667

C4 NOS V C1

C C1 0.06922 0.0002374 0.343 61 C2 0.06922 0.0002787 0.4026 46 C3 0.06922 0.0006982 1.009 84 AvCv 0.06922 0.000404767 0.584866667 63.66666667

C5 NOS 1 C1

C Cl 0.06922 0.0002739 0.3957 81 02 0.06922 0.0005493 0.7936 175 03 0.06922 0.0003704 0.5352 153 AvOi 0.06922 0.000397867 0.574833333 136.3333333

05 NOS II 01

0 01 0.06922 0.002081 3.007 355 02 0.06922 0.001401 2.024 285 03 0.06922 0.0004356 0.6293 169 AvOi 0.06922 0.001305867 1.886766667 269.6666667 05 NOS III 01

0 01 0.06922 0.0004029 0.5821 133 02 0.06922 0.001043 1.506 205 03 0.06922 0.001052 1.519 235 AvOiil 0.06922 0.000832633 1.202366667 191

05 NOS IV 01

0 01 0.06922 0.001597 2.307 284 02 0.06922 0.000441 0.6371 110 03 0.06922 0.0005842 0.844 140 AvOlv 0.06922 0.000874067 1.2627 178

05 NOS V 01

0 01 0.06922 0.0004632 0.6693 135 02 0.06922 0.001458 2.106 262 03 0.06922 0.0005818 0.8405 121 AvOv 0.06922 0.000834333 1.205266667 172.6666667 APPENDIX 167

Table 0-2STable of Average PGP 9.5 & nNOS expression in Diseased and Control Circular Muscle Specimens

NOS CONTROL CONTROL DISEASED DISEASED PGP CONTROL CONTROL DISEASED DISEASED CM CM %Red Count %Red Count %Red Count %Red Count 1i 0.600633333 50 11 0.627266667 63 1i 0.662533333 142.3333333 0.8023 71 1ii 0.490733333 70 lii 0.222966667 21 lii 1.500766667 130 0.5325 55.33333333 1iii 1.067566667 87 liii 0.333666667 62.33333333 liii 1.068666667 85.33333333 0.316833333 43.33333333 1iv 0.611 62.33333333 liv 0.3348 47 liv 0.938766667 76 0.531266667 59.66666667 1v 0.4401 62.66666667 1v 0.6106 93.33333333 1v 0.672466667 76 0.9186 73 1 0.642006667 66.4 0.42586 57.33333333 0.96864 101.9333333 0.6203 60.46666667

21 0.2866 59 2i 0.467233333 51.66666667 2i 0.583333333 82.66666667 0.018168067 5.666666667 2ii 0.150833333 32 2ii 0.304616667 28 2ii 0.532233333 42.33333333 0.006667367 2.666666667 2iii 0.234833333 50 2iii 0.027899333 6.666666667 2iii 0.418266667 40 0.032806667 8.333333333 2iv 0.461666667 116.3333333 2iv 0.110826667 22.33333333 2iv 0.349233333 31.66666667 0.002614 2 2v 0.544486667 132.6666667 2v 0.131373333 26 2v 0.264433333 46 0.001568667 1 2 0.335684 78 0.208389867 26.93333333 0.4295 48.53333333 0.012364953 3.933333333

3i 0.2094 37.33333333 31 0.069367 11 3i 1.8001 122.3333333 0.160866667 16.33333333 3ii 0.786633333 62.33333333 3ii 0.054266667 23 3ii 1.1401 109.3333333 0.155266667 21.66666667 3iii 0.6484 61.33333333 3iii 0.033586667 15.33333333 3iii 0.740433333 75 0.283093333 12 3iv 1.029333333 122.6666667 3iv 0.051153333 8.666666667 3iv 1.1401 109.3333333 0.011763667 6 3v 0.3432 73.33333333 3v 0.155513333 21 3v 0.740433333 75 0.02954 5.333333333 3 0.603393333 71.4 0.0727774 15.8 1.112233333 98.2 0.128106067 12.26666667

4i 0.926533333 99.66666667 4i 0.186543333 11.66666667 4i 0 0 0.420633333 30 4ii 0.657733333 76.33333333 4ii 0.031776667 12.66666667 4ii 3.334666667 126 0.116212333 7.666666667 4iii 0.7475 77.33333333 4iii 0.063426667 12.66666667 4iii 1.741666667 88.66666667 0.1277 19 4iv 1.103233333 120.6666667 4iv 0.072596667 17 4iv 1.5472 64 0.386233333 33.33333333 4v 0.584866667 63.66666667 4v 0.035653333 9 4v 0.794033333 43.66666667 0.652966667 49.66666667 4 0.803973333 87.53333333 0.077999333 12.6 1.483513333 64.46666667 0.340749133 27.93333333

5i 0.574833333 136.3333333 5i 0.063813333 3.333333333 5i 0.583333333 82.66666667 0.331033333 19.33333333 5ii 1.886766667 269.6666667 5ii 0.036696 1.333333333 5ii 0.532233333 42.33333333 0.428976667 30.66666667 5iii 1.202366667 191 5iii 0.031776667 6.666666667 5iii 0.418266667 40 0.120119333 10 5iv 1.2627 178 5iv 0.01886 7.666666667 5iv 0.349233333 31.66666667 0.07738 18 5v 1.205266667 172.6666667 5v 0.02984 8 5v 0.264433333 46 5 1.226386667 189.5333333 0.0361972 5.4 0.4295 48.53333333 0.239377333 19.5

61 0.1181 185.3333333 6i 0.416596667 14.33333333 6i 0.079743333 9.666666667 0.543466667 57 6ii 0.4316 160 6ii 0.234967667 12.33333333 6ii 0.1481 18.33333333 0.610133333 39 6iii 0.9983 150 6iii 0.06561 10.33333333 6iii 0.1379 30.66666667 0.5218 40.66666667 6iv 0.948333333 148.3333333 6iv 0.117026667 11.33333333 6iv 1.3238 78 0.7041 40 6v 0.927 189.3333333 6v 0.13021 14.33333333 6v 0.16925 22 0.544 49.66666667 6 0.684666667 166.6 0.1928822 12.53333333 0.371758667 31.73333333 0.5847 45.26666667

7i 0.4742 85.66666667 7i 0.044306667 14.33333333 7i 1.016433333 49 0.19996 45.66666667 7ii 0.5996 110.6666667 7ii 0.041596667 14.66666667 7ii 0.6522 84.33333333 0.159233333 56.66666667 7iii 0.546766667 134.3333333 7iii 0.05528 15.33333333 7iii 0.974033333 96 0.250166667 17.33333333 7iv 0.399533333 107.3333333 7iv 0 014856667 3 7iv 1.493066667 156 0.2465 32.33333333 7v 0.572533333 97 7v 0.109533333 14.33333333 7v 0.8671 96.66666667 0.296566667 32.33333333 7 0.518526667 107 0.053114667 12.33333333 1.000566667 96.4 0.230485333 36.86666667

8i 1.281666667 173 8i 0.1302 21 8i 2.565333333 270.6666667 0.159733333 21 8ii 1.099466667 166 8ii 0.031777667 5.333333333 8ii 0.783333333 66.66666667 0.131873333 18 8iii 1.172966667 124.6666667 8iii 0.003358667 2 8iii 2.795 187.3333333 0.096843333 14 8iv 1.601666667 193.6666667 8iv 0.03126 12 8iv 2.814333333 161 0.2545 43 8v 0.872966667 112 8v 0.003486667 0.333333333 8v 3.327 186 0.2307 19.33333333 8 1.205746667 153.8666667 0.0400166 8.133333333 2.457 174.3333333 0.17473 23.06666667

9i 0.8648 97 9i 0.04702 10.66666667 9i 0.9034 129.6666667 0.197756 8 9ii 0.854333333 144 9ii 0.043531667 13.33333333 9ii 0.9034 129.6666667 0.107566667 21.33333333 9iii 0.8467 101 9iii 0.129846667 22.66666667 9iii 1.0966 103 0.25697 30 APPENDIX 168

9iv 1.091466667 119.6666667 9lv 0.0996 21.33333333 9iv 0 0 0.418233333 37.66666667 9v 1.442666667 198.6666667 9v 0.05787 14.66666667 9v 0 0 0.427 33.66666667 9 1.019993333 132.0666667 0.075573667 16.53333333 0.9678 120.7777778 0.2815052 26.13333333

10i 0.296933333 58.66666667 101 0.05619 14.33333333 lOi 0 0 2.355333333 58.66666667 10ii 1.058666667 144 1011 0.047796667 15.66666667 lOii 0 0 3.264666667 67.33333333 10iii 0.810866667 110 lOiii 0.076216667 17.33333333 lOiii 0 0 3.584 86.66666667 10iv 1.168933333 140 lOiv 0.036556667 12 lOiv 0 0 3.697333333 101.3333333 lOv 0.745233333 98.66666667 lOv lOv 4.608333333 235 3.222 100 10 0.816126667 110.2666667 0.05419 14.83333333 4.608333333 235 3.224666667 82.8

Hi 0.6747 122.6666667 111 0.4608 102 Hi 2.632666667 190.6666667 0.3235 77 1111 0.4307 115.6666667 liii 0.2193 48 liii 3.231333333 214.3333333 0.347 21 11111 1.009733333 131 11 Hi 0.2348 47 Hiii 6.101 287 0.2819 11 lllv 0.9756 113 Iliv 0.1562 47 Iliv 4.958666667 239 0.2772 0 11v 0.8244 139 liv 0.2782 19 liv 1.653666667 146 0.227 13 11 0.783026667 124.2666667 0.26986 52.6 3.715466667 215.4 0.29132 30.5

121 0.232366667 39.66666667 12i 0.044306667 14.33333333 12i 2.997 194 0.369633333 29 1211 0.361033333 60.66666667 12ii 0.041596667 14.66666667 12ii 4.67 252.3333333 1.294666667 84.66666667 12111 0.7403 115.6666667 12iii 0.05528 15.33333333 12iii 4.918 192.6666667 0.7735 22.66666667 12iv 0.502233333 92.66666667 12iv 0.014856667 3 12iv 4.154666667 201.6666667 0.601633333 45.66666667 12v 0.472533333 103.6666667 12v 0.109533333 14.33333333 12v 5.129333333 218 0.756266667 26.33333333 12 0.461693333 82.46666667 0 053114667 12.33333333 4.3738 211.7333333 0.75914 41.66666667

131 1.219 164 13i 0.0279 15 13i 5.128 184 0.3853 29 131! 1.952666667 213.6666667 13ii 0.053476667 10.33333333 13ii 4.291 208.6666667 0.525433333 49 13111 0.956366667 141.3333333 13iii 0.040043333 11.66666667 13iii 3.792666667 194.3333333 0.4372 50.66666667 13iv 1.833 171.3333333 13iv 0.030223333 9.666666667 13iv 2.205333333 219 0.514333333 58 13v 3.352 318.6666667 13v 0.043916667 10.66666667 13v 8.6737 232.3352412 0.387666667 51.33333333 13 1.862606667 201.8 0.039112 11.46666667 4.81814 207.6670482 0.449986667 47.6

141 0.495533333 111 14i 0.082016667 18.33333333 14i 0.155526667 27.33333333 0.095946667 14 141! 0.2563 62.33333333 14ii 0.095576667 13.5 14ii 0.640066667 30 0.277633333 42.66666667 14111 0.610466667 118.3333333 14iii 0.024673333 6.666666667 14iii 0.951133333 107.6666667 1.893 105.3333333 14iv 0.296333333 86.66666667 14iv 0.075566667 11.66666667 14iv 1.426133333 114 1.406 60.33333333 14v 1.827266667 170.3333333 14v 0.02299 6 14v 0.513 40.33333333 1.259866667 45.33333333 14 0.69718 109.7333333 0.060164667 11.23333333 0.737172 63.86666667 0.986489333 53.53333333

151 0.268566667 86.66666667 15i 0.029323333 7.333333333 15i 0.952766667 79.33333333 1.181333333 99.66666667 1511 0.33666 62 15ii 0.081526667 15.33333333 15ii 0.652233333 73.33333333 0.384 60 15111 0.134466667 38.33333333 15iii 0.167033333 16.33333333 15iii 0.369266667 35 0.888366667 113.6666667 15iv 0.089283333 30.33333333 15iv 15iv 1.395666667 114.3333333 1.1852 108.6666667 15v 0.11224 20.33333333 15v 0.206533333 30.33333333 15v 2.428 147.6666667 1.223833333 126 15 0.188243333 47.53333333 0.121104167 17.33333333 1.159586667 89.93333333 0.972546667 101.6

161 0.916066667 78.66666667 16i 0.088473333 14.33333333 16i 2.626333333 131.3333333 0.8285 35.33333333 16il 1.611 209.6666667 16ii 0.06615 7 333333333 16ii 1.479666667 72.33333333 0.7535 53 16111 3.906666667 388 16iii 0.067566667 15.33333333 16iii 1.19 84.66666667 1.065933333 43.66666667 16iv 2.000666667 281 16iv 0.062533333 14.66666667 16iv 0.850933333 65.33333333 0.8907 32 16v 2.673333333 290.6666667 16v 0.079306667 17 16v 0.7698 53.66666667 1.1027 85.33333333 16 2.221546667 249.6 0.072806 13.73333333 1.383346667 81.46666667 0.928266667 49.86666667

171 0.32719 55 17i 0.04573 14.33333333 17i 4.182666667 237.3333333 0.099903333 21 1711 0.370066667 107 17ii 0.03759 14.66666667 17ii 3.727 175.6666667 0.07661 35.33333333 17111 0.198033333 66 17iii 0.03436 14.66666667 17iii 1.058533333 153.6666667 0.063926667 27.33333333 17lv 0.490566667 80 33333333 17iv 0.032553333 12.33333333 17iv 1.117333333 142 0.133963333 53 17v 1.673 249.6666667 17v 0.033067667 18.66666667 17v 1.502233333 178 0.1111 42.33333333 17 0.611771333 111.6 0.0366602 14.93333333 2.317553333 177.3333333 0.097100667 35.8

181 2.962666667 125 18i 0.070656667 17 66666667 18i 3.979666667 186.6666667 0.126576667 10.66666667 1811 0.548 232.6666667 18ii 0.07944 22.33333333 18ii 3.884333333 167.3333333 0.3488 24 18111 1.246333333 202.3333333 18iii 0.458566667 77.33333333 18iii 1.0357 110 0.2726 15.33333333 18lv 1.121666667 214.6666667 18iv 0.153466667 34.33333333 18iv 1.802733333 138.6666667 1.423433333 48 18v 0.681333333 157.6666667 18v 0.1983 39.66666667 18v 3.765 213.6666667 0.559966667 39.33333333 18 1.312 186.4666667 0.192086 38.26666667 2.893486667 163.2666667 0.546275333 27.46666667 APPENDIX 169

19i 0.268333333 172.6666667 191 0.096246667 30.33333333 191 2.388 132.3333333 0.823766667 45.33333333 19ii 0.429 188 19ii 0.04883 15 19ii 0.636 85 0.551466667 33 19iii 0.017 157.6666667 19iii 0.07067 9.666666667 19iii 0.842233333 97.33333333 0.842333333 39.33333333 19iv 1.888666667 212 19iv 0.04508 9.666666667 19iv 0.714566667 83.33333333 0.41 38 19v 0.307833333 155 19v 0.052313333 21 19v 1.284266667 119.6666667 0.649466667 21 19 0.582166667 177.0666667 0.062628 17.13333333 1.173013333 103.5333333 0.655406667 35.33333333

20i 1.369666667 152.6666667 20i 0.050634333 11.33333333 20i 1.049933333 132 0.638133333 44.66666667 20ii 1.671666667 194 20ii 0.06058 10 20ii 0.843733333 98.66666667 1.351333333 76 20iii 0.652866667 89.66666667 20iii 0.048443333 6.666666667 20iii 0.866333333 93.33333333 0.981533333 69.66666667 20iv 0.832233333 105 20iv 0.050376667 5 20iv 1.8274 145 0.000677733 0.979133333 20v 1.027033333 109.3333333 20v 0.06135 5 20v 0.8354 84 0.000845133 1.221166667 20 1.110693333 130.1333333 0.054276867 7.6 1.08456 110.6 0.594504573 38.50672667 APPENDIX 170

Table 0-3S Table of Calculated Proportion/ Ratio of Neural tissue expressing nNOS in Control and Diseased Circular Muscle

no subj antig pgpc group ratio no. subj antig pgpc group ratio 1. 1 .642007 .96864 control .6627921 21. 1 .42586 .6203 diseased .6865388 2. 2 .335684 .4295 control .7815692 22. 2 .20839 .012365 diseased 16.85321 3. 3 .603393 1.112233 control .5425059 23. 3 .072777 .128106 diseased .5680999 4. 4 .803973 1.483513 control .5419387 24. 4 .077999 .340749 diseased .2289046 5. 5 1.226387 .4295 control 2.855383 25. 5 .036197 .239377 diseased .1512133 6. 6 .684667 .371759 control 1.841696 26. 6 .192882 .5847 diseased .329882 7. 7 .518527 1.000567 control .5182332 27. 7 .053115 .230485 diseased .2304488 8. 8 1.205747 2.457 control .4907395 28. 8 .040017 .17473 diseased .2290219 9. 9 1.019993 .9678 control 1.053929 29. 9 .075574 .281505 diseased .2684642 10. 10 .816127 4.608333 control .1770981 30. 10 .05419 3.224667 diseased .0168048 11. 11 .783027 3.715467 control .2107479 31. 11 .26986 .29132 diseased .9263353 12. 12 .461693 4.3738 control .1055588 32. 12 .053115 .75914 diseased .0699673 13. 13 1.862607 4.81814 control .3865822 33. 13 .039112 .449987 diseased .0869181 14. 14 .69718 .737172 control .9457494 34. 14 .060165 .986489 diseased .060989 15. 15 .188243 1.159587 control .1623362 35. 15 .121104 .972547 diseased .1245225 16. 16 2.221547 1.383347 control 1.605922 36. 16 .072806 .928267 diseased .0784322 17. 17 .611771 2.317553 control .2639728 37. 17 .03666 .097101 diseased .377545 18. 18 1.312 2.893487 control .4534321 38. 18 .192086 .546275 diseased .3516288 19. 19 .582167 1.173013 control .4963006 39. 19 .062628 .655407 diseased .0955559 20. 20 1.110693 1.08456 control 1.024095 40. 20 .054277 .594505 diseased .0912978 APPENDIX 171 ABBREVIATIONS 172

ABBREVIATIONS

AAPH Acquired Antral Pyloric Hypertrophy ABC Avidin Biotin Complex BOB Bis-diazobenzidine

BH4 T etrahydrobiopterin BSA Bovine Serum Albumin cAMP cyclic adenine monophosphate GDI Carbodiimide cGMP cyclic guanyl monophosphate CGRP Calcitonin Gene Related Product CPS Congenital Pyloric Stenosis Dil I, I’-dioctatidecyl-3,3,3 ’,3 ’-tetramethylindocarbocyanine perchlorate EDRF Endothelial Derived Relaxing Factor eNOS Endothelial Nitric Oxide Synthase ICC Interstital Cell of Cajal iNOS Inducible Nitric Oxide Synthase kDA Kilo Dalton LNMMA NG-monomethyl—L-arginine NADPH Nicotinamide Adenine Dinucleotide Phosphate Diaphorase NANC Non Adrenergic Non Cholinergic nNOS Neural Nitric Oxide Synthase NO Nitric Oxide NOS Nitric Oxide Synthase PACAP Pituitary Adenylate Cyclase Activating Peptide PAP Peroxidase-antiperoxidase PBS Phosphate Buffered Solution PGP 9.5 Proten Gene Product 9.5 PHI Peptide Histidine Isoleucine PHM Peptide Histidine Methionine PHV Peptide Histidine Valine PPT-A Preprotachykinin A PPT-B Preprotachykinin B QDHPR Quinonoid-dihydropteridine Reductase SDS/PAGE Sodium Dodecyl Polyamide Gel TTX Tetrodotoxin VIP Vasoactive Intestinal Polypeptide REFERENCES 173

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By Robin Michael Abel London, England

# Pyoric stenosis (PS) is a common condition in infancy, none of the reports has described the chronological which is associated with smooth muscle hypertrophy that changes underlying pyloric hypertrophy. results in pyloric outlet obstruction. The author examines the The objectives of this project were threefold: (A) to ontogeny of the peptide innervation of the pylorus in fetal tissues and an experimental model in mice and evaluates the examine the ontogeny of the peptide innervation of histochemical and morphological changes in the pylorus. The the pylorus; (B) to quantify the histochemical and data suggest that PS is an intrauterine lesion that occurs by morphological changes in PS; and (C) to identify an 12 weeks' gestation. This is associated with diminished nitric experimental model of PS. oxide in human tissues and reduced enzyme activity (result­ ing from a deficiency in an enzyme cofactor) in mice. In­ A. THE DEVELOPMENTAL STUDY creased vasoactive intestinal polypeptide expression in py­ loric myenteric ganglia may be an intrinsic mechanism for A im s resolving this condition. 1. To study the ontogeny of the intrinsic and Copyright © 1996by W.B. Saunders Company extrinsic innervation of the pylorus. INDEX WORDS: Hypertrophic pyloric stenosis, nitric oxide 2. To identify the developmental stage at which PS synthase, fetal pylorus, vasoactive intestinal polypeptide. develops. Tissue was collected from 35 human fetuses (age NFANTILE HYPERTROPHIC pyloric stenosis range, 8 to 23 weeks’ gestation) and 20 children (age I (PS) was first described in 1646 by Fabricius range, 1 week to 8 months). To trace the vagal Hildanus. It is the most common cause of emergency innervation of the pylorus, the carbocyanine com­ abdominal surgery in infancy.^ A related condition pound Dil was used.This is a lipophilic crystal occurs in brachycephalic dogs. The aetiology is un­ that is absorbed by nerves. Dil passively diffuses and known. Before the development of Ramstedt’s pyloro­ autofluoresces in aldehyde-fixed tissue, which allows myotomy in 1911 and the development of safe paedi­ selective neuronal tracing postmortem. atric anaesthesia, some cases were treated successfully To minimise the transport time of Dil along the by conservative management. vagus to the pylorus, and thus the tissue decomposi­ Many investigators have described the histological tion time, a selective vagotomy was performed on four changes underlying PS,^'^ but the ontogeny and struc­ foetal stomachs (age range, 10 to 23 weeks). A crystal ture of the normal infant pylorus has not been of Dil was placed on the severed surface of the nerve. described previously. To date, none of the biochemi­ Control specimens had Dil placed directly on the cal or histochemical studies of PS have accounted for serosa of the pylorus. The specimens were incubated the apparent dilutional reduction in neural tissue that for 6 months. occurs after muscle hypertrophy. To identify the craniocaudal and temporal patterns Although animal models of PS have been re- of development, serial sections were cut at 200-jxm ported,^-^^ none has been verified or quantified, and intervals through the pylorus. The sections were stained with haematoxylin and eosin and immuno- stained using antisera to the general neural marker Mr Robin Abel was the Research Fellow at the Institute of Child protein gene product 9.5 (PGP 9.5), vasoactive intes­ Health, London, England. tinal polypeptide (VIP), the neural isoform of nitric Presented at the 42nd Annual International Congress of the British oxide synthase (NOS), calcitonin gene-related pep­ Association of Paediatric Surgeons, Sheffield, England, July 25-28, tide (CGRP), substance P (SP), the foetal isoform of 1995. This essay was the prize-winning award for the BAPS Trainees Prize. neural cell adhesion molecule (fNCAM), the neural Mr Abel’s supervisors were Professor L. Spitz (Institute of Child isoform of neural cell adhesion molecule (nNCAM), Health, London) and Professor J.M. Polak (The Royal Postgraduate the neuromuscular isoform of neural cell adhesion Medical School, London). molecule (nmNCAM), and actin. Address reprint requests to Mr Robin Michael Abel, Department of Surgery, Birmingham Children’s Hospital, NHS Trust, Ladywood R e su lts Middleway, Birmingham B16 SET, England. Copyright © 1996 by W.B. Saunders Company The temporal pattern of expression of the antigens 0022-3468l96l3104-0006$03.00l0 is summarised in Table 1. All the antigens examined

490 Journal of Pediatric Surgery, Vol 31, No 4 (April), 1996; pp 490-497 INFANTILE HYPERTROPHIC PYLORIC STENOSIS 491

Table 1. Temporal Pattern of Antigen Expression (no. of weeks) proportionate numbers of ganglia expressing SP, Muscularis CGRP, and NOS were reduced significantly. The size Mucosa Submucosa Propria Adventitia of these ganglia was unchanged. However, ganglia PGP 9.5 11 8 8 8 expressing VIP increased in number and size. VIP 12 12 11 11 The longitudinal muscle layer was hypertrophied, NOS 15 12 11 13 SP 23 11 8 8 as was the circular muscle layer (Fig 6).

CGRP 23 23 12 — fNCAM 12 8 8 12 C. EXPERIMENTAL ANIMAL MODEL nNCAM 12 12 8 12 A im s nmNCAM 12 12 8 — Actin 14 12 8 — 1. To identify an animal model of PS. 2. To quantify the temporal pattern of morphologi­ cal changes underlying PS. had a craniocaudal pattern of expression within the A colony of hph-1 mice deficient in tetrahydrobiop- muscularis propria. The highest concentration of terin has been developed as a model of phenylketon- immunoreactivity was toward the duodenum. Only uria.15,16 Untreated phenylketonuria is associated with PGP 9.5, VIP, and nNCAM had a craniocaudal PS.'^ Tetrahydrobiopterin is a cofactor to NOS and pattern of expression in the submucosa. The cranio­ phenylalanine hydroxylase. caudal changes in VIP expression in the pylorus are Six specimens were gathered from control and consistent with those described in the small intes­ diseased animals (age range, 2-week-old foetuses to tine. 180-day-old animals). The pylorus was examined The fetal pylorus had developed the morphology immunohistochemically. The width of the pylorus, and histochemical structure of the infant pylorus by and its muscle layers, was measured using a graticule. 23 weeks of gestation. R e su lts Extrinsic Innervation The circular, longitudinal, and muscularis mucosae The vagal innervation of the pylorus was first layers had significant hypertrophy up to 40 days of demonstrated by Dil autofluorescence and was con­ age. Qualitatively, there was no change in immunore­ firmed by VIP immunoreactivity at 11 weeks. (VIP is activity to PGP 9.5, VIP, NOS, SP, CGRP, or nNCAM. expressed by vagal flbres.^^) The relationship between body weight and the width B. THE QUANTITATIVE STUDY of each muscle layer is illustrated in Figs 7-10.

A im s DISCUSSION 1. To quantify the morphological changes underly­ NOS has been implicated in the pathogenesis of PS ing PS. since the description that only the circular muscle is 2. To characterise the histochemical changes under­ hypertrophied and the diminished expression of NOS lying PS in humans, by quantifying the p r o p o r ­ is localised to this tissue.^^ The present study shows tio n of the total neural tissue expressing antigen, that longitudinal muscle hypertrophy is a characteris­ thus accommodating the dilutional reduction in tic of PS, and that NOS expression is diminished in total neural tissue owing to muscle hypertrophy. the circular and longitudinal muscle layers as well as Twenty pyloric muscle biopsy specimens from chil­ in the myenteric plexus. That the proportionate dren who underwent pyloromyotomy for PS were reduction in expression of NOS and VIP is the same studied, along with 20 age- and sex-matched control in the circular muscle and myenteric plexus is biologi­ specimens. The tissues were immunostained for PGP cally significant, because the peptides are colocalised 9.5, VIP, NOS, SP, and CGRP. Five nonserial sec­ to the same nerves in these structures.Only NOS tions were examined for each antigen, A computer- expression was diminished in all the tissues studied. assisted image analysis system was used to quantify 25

images for each parameter measured and in each of Table 2. Relative Changes in Proportion of Nerves tissue studied. The mean of these values was used for Expressing Antigen

statistical analysis. Overall, 49,350 images were ana­ Antigen Circular Muscle Myenteric Plexus Longitudinal Muscle lysed. VIP i 1 1 1 t NOS i i f i i i i R e su lts SP ; i The relative changes in the proportion of nerves CGRP i i expressing antigens is summarised in Table 2. The NOTE. The relative changes are illustrated in Figs 1-5. Fig 1. Estim ates of differences in medians of proportion of nerves expressing antigen in the circular muscle.

Fig 2. Estim ate of differences in median and 95% confidence inter­ vals of proportion of nerves ex­ pressing antigen in the myenteric plexus Anticen

Fig 3. Estimated differences in median proportions of nerves ex­ pressing antigen in the longitudi­ nal muscle. Fig 4. Estimate of differences in median antigen staining per gan­ glion.

Fig 5. Differences in m edian numbers of ganglia expressing an­ tigen.

S 0.08

Fig 6. Width of the longitudinal muscle in control (■) and diseased (□) patients. 494 ROBIN MICHAEL ABEL

30 r 15

10

10 Fig 7. W eight and muscularis mucosae trend.

It is notew orthy that SP expression is not dim in­ nal muscle, and thus this cannot account for the ished in the circular muscle but is so in the myenteric histochemical changes. plexus and longitudinal muscle layers. SP has a potent Recently, a NOS-gene deleted “knockout mouse” contractile effect on enteric smooth muscle. CGRP model was described,*^’^* in which the only abnormal­ expression is reduced only in the myenteric plexus. ity is gasric outlet obstruction due to pyloric hypertro­ The significance of the reduced expression of CORP*-"* phy. Because nitric oxide is required by erectile demonstrated by this study is unclear. It is remark­ tissues, it is strange that the knockout mice were able that the same proportion of nerves in the described as being just as fertile as normal mice. myenteric plexus express all the antigens in PS Furthermore, these mice did not lose weight or (Fig 11). behave abnormally despite having gastric outlet ob­ Several investigators have attributed various mor­ struction and gastric distension. After a 3-year study phological changes to the nerves in PS. It has been of the genetic lesion underlying PS, no evidence was proposed that compression of the nerves may account found to suggest that an aberration of NOS gene for the histochemical changes^^; however, the present expression underlies PS in humans.^’ Therefore, the study shows that the only significant morphological significance of the knockout mouse as an animal change is a shortening of the nerves in the longitudi­ model of PS is unclear.

- O — HPHl CM/DP

Fig 8. Weight and circular 10 muscle trend. INFANTILE HYPERTROPHIC PYLORIC STENOSIS 495

Fig 9. W eight and longitudinal 20 muscle trend.

The hph-1 model identified in this study is signifi­ cular differentiation.^^ In Hirschsprung’s disease, cant for several reasons. (1) This is the first animal fNCAM is reexpressed by the muscularis mucosae^^; model of PS that was originally created and verified as however, in PS, nNCAM is expressed by muscle a model of another human condition known to be cells.25 Presently this finding is being further charac­ associated with PS. (2) hph-1 mice behave abnor­ terised by immuno-electron microscopy. mally, are less fertile, and lose weight after birth, then The increase in size and number of VIP expressing regain it by 40 days as the pyloric smooth muscle ganglia is functionally consistent with that of a reduc- hypertrophy resolves. (3) That the hypertrophy is transient is consistent with reports of successful conservative management of PS. (4) That the hyper­ trophy occurs in fetal hph-1 mice is consistent with reports of the condition being found in premature babies. Because pyloric hypertrophy develops in prema­ ture babies and hph-1 fetal mice, the lesion underly­ ing PS probably occurs in utero. That NOS and VIP are colocalised to the same nerves in the circular muscle and the myenteric plexus, that the expression of both is diminished by the same proportion in PS, C and that both are expressed at approximately 12 8 weeks within the myenteric plexus and submucosa of the human pylorus suggest that the lesion underlying PS occurs by 12 weeks. That, qualitatively, the mice did not develop the 1 histochemical changes seen in children with PS, suggests that these may be secondary changes in the Series! pathogenesis of pyloric smooth muscle hypertrophy. This study’s results suggest that a reduction in nitric oxide results in abnormal smooth muscle growth and differentiation. The mechanism for this is un­ clear. It is remarkable that the ontogeny of NCAM in the pylorus is different from that in the large bowel,^^ -20 where fNCAM is expressed by the fetal muscularis Age (Days)

mucosae, but not in the fetal pylorus. NCAM has Fig 10, Estimated differences in means of proportionate circular been implicated in cell-cell adhesion and neuromus­ muscle width to pylorus diameter. 496 ROBIN MICHAEL ABEL

Fig 11. Antigen expression by nerves of the myenteric plexus.

tion in metEnkephalin expression in PS^^: endog­ caused by a reduction in the amount of nitric oxide enous endorphins tonicly inhibit VIP release.^^’^® The synthase present in all tissue layers (humans) or by a increase in VIP expression by the ganglia may repre­ reduction in the functional activity of the enzyme sent a compensatory mechanism by which the condi­ secondary to a cofactor deficiency (tetrahydrobiop­ tion naturally regresses: VIP release results in the terin in hph-1 mice), underlies the condition. In­ release of nitric oxide from sm ooth muscle cells.^^ creased VIP expression by the ganglia may represent These data suggest that pyloric smooth muscle a mechanism by which the condition naturally re­ hypertrophy is an intrauterine lesion that becomes solves. clinically apparent as “infantile hypertrophic steno­ The present study identified two potential path­ sis” after feeding is established. The intrauterine ways for possible nonoperative treatment of PS: lesion occurs by 12 weeks of gestation. The condition enhancing nitric oxide production by the use of nitric is characterised by hypertrophy of the longitudinal oxide donors or NOS cofactors, or augmenting VIP and circular muscle layers. Diminished nitric oxide, activity (eg, by opiate antagonists).

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