The_ontogeny_of_the_peptide_in.pdf - UCL Discovery

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 o f Doctor o f Philosophy

University o f London

The Institute o f Child Health and the Royal PostgraduateMedical School

ProQuest Number: U116382

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ACKNOWLEDGEMENTS

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

INTRODUCTIONDespite 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.

AIMSA. 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 StudyConventional 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 StudyThe 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 StudiesThe 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.

CONCLUSIONThe 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

A B S T R A C T ......................................................................................................................................................................0

C H A P T E R 1 IN T R O D U C T IO N ............................................................................................................................ I

1.1 Py l o r ic S m o o th M u s c l e H y p e r t r o p h y ................................................................................................... 21.1.1 Pyloric Smooth Muscle Hypertrophy occurring in Infancy ........................................................ 2/. 1.2 Pyloric Smooth Muscle Hypertrophy occurring in Adulthood...................................................31.1.3 Pyloric Smooth Muscle Hypertrophy occurring in other species.............................................. 3

1.2 In f a n t il e H y p e r t r o p h ic Py l o r ic St e n o s is ............................................................................................. 51.2.1 Historical Background ................................................................................................................................... 51.2.2 Normal and Pathological Anatomy ........................................................................................................... 61.2.3 Associated anomalies......................................................................................................................................81.2.4 Incidence..............................................................................................................................................................81.2.5 Presentation and Progress............................................................................................................................91.2.6 Management................................................................................................................................................... 101.2.7 Natural Progression oj Infantile Hypertrophic Pyloric Stenosis.............................................. 131.2.8 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 ...................................................................................................................... 201.3.] Neurotransmitters and Neuropeptides.................................................................................................. 20

1.4 N e u r o t r a n s m it t e r s a n d N e u r o p e p t id e s .............................................................................................. 221.4.1 History and Classification .........................................................................................................................221.4.2 Neuromodulators...........................................................................................................................................231.4.3 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 e u r o p e p t id e s o f In t e r e s t ................................. 251.5.1 The General Neural Marker: Protein Gene Product 9 .5 ...............................................................251.5.2 Substance P ...................................................................................................................................................... 251.5.3 Vasoactive Intestinal Polypeptide........................................................................................................... 271.5.4 Calcitonin Gene Related Peptide ............................................................................................................ 281.5.5 Nitric Oxide...................................................................................................................................................... 29

1.6 A IM S O F T H E S T U D Y ..................................................................................................................................... 331.6.1 Hypothesis.........................................................................................................................................................33

C H A P T E R 2 M A T E R IA L S A N D M E T H O D S ............................................................................................41

2.1 G e n e r a l In t r o d u c t io n ....................................................................................................................................422.2 IMMUNOCYTOCHEMISTRY...................................................................................................................................42

2 .2 . 1 Fixation ..............................................................................................................................................................422.2.2 Tissue preparation ........................................................................................................................................ 432.2.3 Immunostaining ............................................................................................................................................. 442.2.4 Antisera ..............................................................................................................................................................46

2.3 S p e c if ic it y c o n t r o l s ........................................................................................................................................482.3.1 Antiserum specificity .................................................................................................................................... 482.3.2 Methodological specificity .........................................................................................................................48

2.4 E x p e r im e n t a l p r o c e d u r e s ............................................................................................................................ 492.4.1 Selective division o f the fe ta l vagus upon the lesser curve o f the stom ach .............................492.4.2 Adjuvant induced deficiency o f tetrahydrobiopterin in mice........................................................492.4.3 Carbon dioxide laser induced ablation o f the innervation o f the rat pylorus ....................... 51

2.5 C o n v e n t io n a l h is t o l o g y ...............................................................................................................................532.6 A ss e s s m e n t OF IMMUNOSTAINING................................................................................................................. 532.7 Q u a n t if ic a t io n .....................................................................................................................................................54

2 .7 . 1 Image analysis quantification o f nerve fib re s .................................................................................542.8 St a t is t ic a l A n a l y s is ........................................................................................................................................552.9 M ic r o s c o p y a n d P h o t o m ic r o g r a p h y .......................... 552.10 T a b l e s a n d F ig u r e s ......................................................................................................................................... 56

3. C H A P T E R 3 T H E O N T O G E N Y O F T H E IN N E R V A T IO N O F T H E H U M A N P Y L O R U S ...................................................................................................................................................................... 60

3.1 S u m m a r y ................................................................................................................................................................. 613.2 In t r o d u c t io n ........................................................................................................................................................ 623.3 E x p e r im e n t a l D e s i g n .......................................................................................................................................64

3.3.1 Tissues........................................................................................................................................ 643.3.2 Antibodies (For detail see methods chapter, 2.2.4).............................................................. 653.3.3 Conventional Histology........................................................................................................... 663.3.4 Immunostaining........................................................................................................................ 663.3.5 Neural tracing........................................................................................................................... 66

3.4 R e s u l t s ..................................................................................................................................................................... 673.4.1 Immunohistochemistry............................................................................................................. 673.4.2 Neuronal Tracing......................................................................................................................69

3.5 D is c u s s io n ...............................................................................................................................................................703.6 T a b l e s a n d F ig u r e s (o v e r l e a f ) .................................................................................................................. 74

C H A P T E R 4 A Q U A N T IT A T IV E S T U D Y O F T H E M O R P H O L O G IC A L A N D H IS T O C H E M IC A L C H A N G E S W IT H IN T H E N E R V E S A N D M U S C L E IN IN F A N T IL E 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 ................................................................................................................................................................. 834.2 In t r o d u c t io n ........................................................................................................................................................ 844.3 E x p e r im e n t a l DESIGN........................................................................................................................................86

4.3.1 Tissues........................................................................................................................................864.3.2 Antibodies..................................................................................................................................864.3.3 Imunostaining........................................................................................................................... 864.3.4 Statistical analysis....................................................................................................................87

4.4 R e s u l t s .....................................................................................................................................................................884.4.1 Conventional Histology...........................................................................................................884.4.2 Immunocytochemistry.............................................................................................................. 884.4.3 Morphological changes in the Dimensions o f the Nerves and Longitudinal Muscle Layer884.4.4 The Changes in Immunoreactivity within the Circular muscle layer..................................894.4.5 The Changes in Immunoreactivity within the Longitudinal muscle layer.......................... 894.4.6 The Changes in Immunoreactiviy within the Myenteric Plexus.......................................... 894.4.7 The Changes in Morphology and Immunoreactivity within the Ganglia........................... 90

4.5 D is c u s s io n .............................................................................................................................................................. 914 .6 T a b l e s a n d F ig u r e s ..................................................................... 96

C H A P T E R 5 A M O U S E M O D E L O F IN F A N T IL E H Y P E R T R O P H IC P Y L O R IC S T E N O S IS A N D P H E N Y L K E T O N U R IA ................................................................................................... 104

5.1 S u m m a r y ............................................................................................................................................................... 1055.2 In t r o d u c t io n ...................................................................................................................................................... 1065.3 E x p e r im e n t a l DESIGN......................................................................................................................................108

5.3.1 Tissues.................................................................................................................................... 1085.3.2 Antibodies.............................................................................................................................. 1085.3.3 Immunocytochemistry.......................................................................................................... 1095.3.4 Statistical analysis..................................................................................................................109

5.4 R e s u l t s ...................................................................................................................................................................l lo5.5 D is c u s s io n .......................................................................................................................................................... 1 125.6 T a b l e s a n d F ig u r e s (o v e r l e a f ) ..............................................................................................................114

C H A P T E R 6 A Q U A N T IT A T IV E S T U D Y O F T H E H IS T O C H E M IC A L C H A N G E S U N D E R L Y IN G P Y L O R IC S T E N O S IS IN D O G S .................................................................................. 124

6.1 S u m m a r y ...............................................................................................................................................................1256.2 In t r o d u c t io n ......................................................................................................................................................1266.3 E x p e r im e n t a l DESIGN......................................................................................................................................128

6.3.1 Tissues.................................................................................................................................... 1286.3.2 Antibodies.............................................................................................................................. 1286.3.3 Immunostaining.................................................................................................................... 1286.3.4 Statistical analysis................................................................................................................ 129

6.4 R e s u l t s ...................................................................................................................................................................130

6.4.1 Conventional Histology....................................................................................................... 1306.4.2 Immunocytochem is try ........................................................................................................... ISO6.4.3 The Changes in Immunoreactivity within the Circular muscle layer................................ 130

6.5 D i s c u s s i o n ..................................................................................................................................... 1326.6 T a b l e s 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 ........................................................................................................................................1427.2 I n t r o d u c t i o n ................................................................................................................................1437.3 E x p e r im e n ta l D ESIG N ......................................................................................................................................144

7.3.1 Surgical Procedure............................................................................................................... 1447.3.2 Tissues.................................................................................................................................... 1447.3.3 Antibodies.............................................................................................................................. 1447.3.4 Immunocytochemistry........................................................................................................... 145

7.4 R e s u l t s ...........................................................................................................................................1467.4.1 Conventional Histolog)>....................................................................................................... 1467.4.2 Immunocytochemistiy........................................................................................................... 146

7.5 D is c u s s io n .............................................................................................................................................................1477.6 F ig u r e s (O V ER LEA F)........................................................................................................................................... 147

CONCLUDING REMARKS............................................................................................................ 150

APPENDICES..................................................................................................................................... 156

9.1 APPENDIX I ; ............................................................................................................................... 1569.1.1 BUFFERS.............................................................................................................................. 156

9.2 APPENDIX II............................................................................................................................... 1579.2.1 FIXATIVES..............................................................................................................................157

9.3 APPENDIX III................................................................................................................................1589.3.1 CRYOPROCESSING OF TISSUES..................................................................................... 158

9.4 APPENDIX IV.............................................................................................................................. 1599.4.1 SLIDE CO A TING.................................................................................................................. 159

9.5 APPENDIX V .................................................................................................................................1609.5.1 CONVENTIONAL HISTOLOGY......................................................................................... 1609.5.2 A VIDIN-BIOTIN-PEROXIDASE COMPLEX (ABC) METHOD ..................................... 160

9.6 APPENDIX VI................................................................................................................................1629.6.1 GLUCOSE OXIDASE-DAB-NICKEL-ENHANCEMENT................................................162

9.7 APPENDIX V II............................................................................................................................ 163

ABBREVIATIONS.............................................................................................................................172

REFERENCES.................................................................................................................................... 173

TABLE OF FIGURES

F ig u r e 1-1Il l u s t r a t io n o f g a s t r ic d is t e n s io n a n d v is ib l e p e r is t a l s is in

HYPERTROPHICPYLORIC STENOSIS..................................................................................................................35F 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...............................36F ig u r e 1-3 B a r iu m m e a l ex a m in a t io n 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............................................37F 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 THEPYLORIC CANAL.....................................................................................................................................................38

F ig u r e 1-5 Il l u s t r a t io n o f t h e p y l o r u s d u r in g p y l o r o m y o t o m y ................................................ 39F ig u r e 2-1 D ia g r a m o f St e p s in A B C Re a c t io n ......................................................................................... 57F 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 u a n t if ic a t io n ........................... 58F 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 ................................................................76F ig u r e 3-2 C G R P Im m u n o r e a c t iv it y 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 76F ig u r e 3-3 n N O S im m u n o r e a c t iv it y 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 ............................................................................................................................................... 77F ig u r e 3 -4 n N O S im m u n o r e a c t iv it y 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............................................................................................................................................... 77F ig u r e 3-5 V IP im m u n o r e a c t iv it y 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...............................................................................................................................................................78F ig u r e 3 -6 V IP im m u n o r e a c t iv it y 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...............................................................................................................................................................78F ig u r e 3 -7 SP im m u n o r e a c t iv it y 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.............................................................................................................................................................. 79F ig u r e 3 -8 fN C A M im m u n o r e a c t iv it y 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............................................................................................................................................... 79F ig u r e 3 -9 D il A u t o f l u o r e s c e n c e 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 ........................80F ig u r e 4-1 H e 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 ...........................94F ig u r e 4 -2 Im m u n o r e a c t iv it y 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

d e m o n s t r a t in g t h e g r o s s m u s c l e h y p e r t r o p h y a n d d is t o r t e d m o r p h o l o g y inINFANTILE HYPERTROPHIC PYLORIC STENOSIS...........................................................................................99

F ig u r e 4-3 C G R P Im m u n o r e a c t iv it y , 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 M y e n t e r ic P l e x u s .................................................................................................................................100F ig u r e 4 -4 n N O S im m u n o r e a c t iv it y w it h in t h e d is e a s e d p y l o r u s ............................................ 100F ig u r e 4-5 V IP im m u n o r e a c t iv it y w it h in t h e d is e a s e d p y l o r u s ................................................. 101F ig u r e 4 -6 SP im m u n o r e a c t iv it y w it h in t h e d is e a s e d p y l o r u s ..................................................10 1F ig u r e 4 -7 G r a p h ic a l pr e s e n t a t io n 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 ..................................................................102F ig u r e 5-1 D ia g r a m Il l u s t r a t in g T h e Re l a t io n s h ip b e t w e e n T e t r a h y d r o b io p t e r in

in f a n t il e h y p e r t r o p h ic py l o r ic st e n o s is a n d p h e n y l k e t o n u r ia .......................................117F ig u r e 5-2 W e ig h t , L o n g it u d in a l M u s c l e T r e n d ................................................................................118F 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 ...........................................................................................118F 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 19F 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 H y p e r t r o p h y ....................................................... 119F 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 .................................... 120F ig u r e 5 -7 40 d a y o l d H p h -1 M o u s e ..............................................................................................................120F 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 ................................................................................... 121F ig u r e 5 -9 St o m a c h s o f 40 d a y o l d H p h -1 M o u s e .................................................................................121F ig u r e 5 -10 L o w Po w e r ph o t o m ic r o g r a p h 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 .................................................................................................................................................................... 122F ig u r e 5-11 L o w Po w e r p h o t o m ic r o g r a p h o f T /S o f Py l o r u s o f 40 d a y o l d H ph - 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................................................................................................................................................... 137F ig u r e 6 -2 Im m u n o s t a in in g 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

F ig u r e 6-3 I m m u n o s ta in in g f o r nNOS in a C a n in e p y l o r u s o f c o n g e n i t a l p y l o r i cSTENOSIS ............................................................................................................................................................... 139

F ig u r e 7-1 PGP 9.5 I m m u n o s ta in in g w i th in c o n t r o l a n d l a s e r i r r a d i a t e d w i s t a r r a 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 ..........................................................................................................56T 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 ..................................................................................75T 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

H u m a n F e t a l Py l o r u s ................................................................................................................................... 75T a b l e 4-1 L o n g it u d in a l 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 ) ......... 96T a b le 4 -2 A r ea o f Im m u n o r e a c t iv e N er v es in t h e C ir c u l a r M u s c l e

(UNITS: MM^XlO''*)....................................................................................................................................................... 96T a b l e 4-3 A r e a o f Im m u n o r e a c t iv e N e r v e s in t h e L o n g it u d in a l M u s c l e

(UNITS: MM^xlO"*)..........................................................................................................................................................97T a b l e 4 -4 A r e a o f Im m u n o r e a c t iv e 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"^)..........................................................................................................................................................97T a b l e 4-5 A r e a o f G a n g l ia

( u n it s : MM^xlO ' ) ........................................................................................................................................................ 98T a b l e 4 -6 N u m b e r o f G a n g l i a ........................................................................................................................... 98T 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

A G E(X lO '"M )..................................................................................................................................................... 115T 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............................................................................................................................................... 115T 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).................................................................................................................................................... 116T 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.................................................................................................................................................................... 137T 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 163T a b l e 9 -2 S T a b l e o f A v e r a g e PG P 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 .................................................................................................................... 167T 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 INTRODUCTION1.1 Pyloric smooth muscle hypertrophy1.1.1 Pyloric smooth muscle hypertrophy, in infancy1.1.2 Pyloric smooth muscle hypertrophy, in adulthood1.1.3 Pyloric smooth muscle hypertrophy, in other species

1.2 Infantile Hypertrophic Pyloric Stenosis1.2.1 Historical Background1.2.2 Pathological Anatomy1.2.3 Associated anomalies1.2.4 Incidence1.2.5 Presentation and Progress1.2.6 Management1.2.7 Natural Progression1.2.8 Aetiology/Theories

1.3 The Enteric Nervous System1.3.1 Neurotransmitters and Neuropeptides

1.4 Neurotransmitters and Neuropeptides1.4.1 History and Classification1.4.2 Neuromodulators1.4.3 Interstitial Cells of Cajal

1.5 Features of the Main Neural Markers o f Interest1.4.1 Protein Gene Product 9.51.4.2 Substance P1.4.3 Vasoactive Intestinal Polypeptide1.4.4 Calcitonin Gene Related Peptide1.4.5 Nitric Oxide

1.6 Aims o f the Study


1.1. Pyloric Smooth Muscle HypertrophyBy 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.


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


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) .


1.2. Infantile Hypertrophic Pyloric Stenosis

1.2.1. Historical BackgroundThe 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


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 AnatomyAlthough 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.


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.


1.2.3. Associated anomaliesA 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. IncidenceThe 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.


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. ManagementThe 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 DiagnosisIn 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 PreparationPyloromyotomy 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


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.


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 NeuropeptidesThe 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.


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).


1.4. Neurotransmitters and Neuropeptides

1.4.1. History and ClassificationIn 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


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. NeuromodulatorsNeuromodulators 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.


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 PSubstance 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


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.


1.5.3. Vasoactive Intestinal PolypeptideVasoactive 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


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 PeptideCalcitonin 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) .


1.5.5. Nitric OxideIn 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


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 ( BH4 )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. HypothesisThe 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.


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.


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


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


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


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.


Figure 1-5 Illustration of the pylorus during pyloromyotomy.


CHAPTER 2_________________ MATERIALS AND METHODS_________________________ 41

2. Chapter 2 MATERIALS AND METHODS2.1 General Introduction2.2 Immunocytochemistry2.2.1 Fixation2.2.2 Tissue Preparation2.2.3 Immunostaining2.2.4 Antisera

2.3 Specificity Controls2.3.1 Antiserum specificity2.3.2 Methodological specificity

2.4 Experimental procedures2.4 . 1 Selective division of the fetal vagus upon the lesser curve of the stomach2.4.2 Adjuvant induced deficiency of tetrahydrobiopterin2.4.3 Selective vagotomy and carbon dioxide laser induced ablation of the rat pylorus

2.5 Conventional histology

2.6 Assessment o f immunostaining

2.7 Quantification

2.8 Statistical analysis

2.9 Microscopy and Photomicrography


2.1. General IntroductionThe 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


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.


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


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.


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. AntiseraMost 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


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.


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


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


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.


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.


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.


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).


2.10. Tables and Figures

Table 2-iTable o f primary antibodies

Antigen Donor Species Dilution SoureePGP 9.5 Rabbit 1/5000 UltracloneCGRP Rabbit 1/2000 RPMSSP Rabbit 1/5000 RPMSVIP Rabbit 1/10,000 RPMSnNOS Rabbit 1/5000 V. Riveros-

MorenofNCAM Mouse 1/5000 G. RougonnmNCAM Mouse 1/10,000 J.J. HemperlynNCAM Mouse 1/5000 J.J. HemperlyaSM A Mouse 1/20,000 Sigma


Figure 2-1 Diagram of Steps in ABC Reaction

UmmWdw


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

DtMnloading of results fcr

statistical analysis

processing ard cxxtnast enhanoernat

Capture iiTBge of structures to be anal}5ed

Cptinization of tissue processing

Ajustmert of nicrosocpe and illurrination

Hanning of opennm t accordngto image analytic and statistical reqpirermts


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 Tissues3.3.2 Imunostaining3.3.3 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 HistologyTissue sections were stained conventionally using haemotoxylin and eosin.

(Appendix V)

3.3.4. ImmunostainingTissue 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 tracingTo 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.SubmucosaUp 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 MucosaUp 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 TracingDil 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 collectedFETUSES FETUSES CHILDREN

IMMUNOHISTOCHEMICAL NEURONAL TRACING

STUDY STUDY

AGE No. AGE No. AGE No.

(Weeks)SPECIMENS (Weeks) SPECIMENS (Days) SPECIMENS

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

(weeks)

Muscularispropria

(weeks)

Submucosa

(weeks)

Mucosa

(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


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

2 i . ' . . : - A ' :

% %V,

Ï -

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

à

CH.Af TER 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

« * %


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


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

‘A

%

T

//

/’ I

J

Figure 3-8 fNCAM immunoreactivity in the Myenteric Plexus and Submucosa at12 weeks gestation

/ >

I

✓


Figure 3-9 Dil Autofluorescence within ganglia at 12 weeks of gestation.

CHAPTER 3 : THE ONTOGENY OF THE INNERVATION OF THE HUMAN PYLORUS 8 1

CHAPTER 4: 82A 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


4.1 Summary

4.2 Introduction


4.3.1 Tissues4.3.2 Imunostaining4.3.3 Statistical analysis

4.4 Results

4.5 Discussion

4.6 Tables and Figures

CHAPTER 4: 83A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES


4.1. SummaryA 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.



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


WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSISmeasure 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; 86A QUANTITATIVE STUDY OF MORPHOLOGICAL & HISTOCHEMICAL CHANGES



4.3.1 TissuesTwenty 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 AntibodiesAntibodies to the following range of antigens was selected: PGP 9.5, VIP, nNOS,

CGRP, and SP. (Please see Section 4.1).

4.3.3 ImunostainingFive 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


WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSISwere 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.



4.4 Results

4.4.1 Conventional HistologyHaematoxylin 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 ImmunocytochemistrySubjective 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



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.



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).



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


WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSISstenosis 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


WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSISsomatostatin 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.



Figure 4-1 H euristic M odel o f Inhibitory Intestinal M otor Innervation

S O M A T O S T A T I N N E U R O N S

A T ES T I M I B I T

G A B An e u r o n s

S T I M U L A T E

O P I O I Dn e u r o n s

V I P / N O N E U R O N S

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


WITHIN NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSISsignificance 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 the proportionate 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.



4.6. Tables and FiguresTable 4-1 Longitudinal Muscle Width & Nerve Fibre length and width (lO’ ^mm)

Muscle type Dimension Group Median (95% Cl)

Difference in medians (95% CD

Pvalue

Circular Nerve Fibre Length

Control 4.21 (3.29 to 4.75) 2.51 (-0.56 to 1.12) 0.6

Diseased 3.68 (3.05 to 4.81)

Nerve Fibre Width

Control 3.28 (2.76 to 3.92) 0.08 (-0.45 to 0.80) 0.8

Diseased 2.96 (2.47 to 4.20)

Longitudinal MuscleWidth

Control 55 (43 to 64) -64 (-134 to -14) 0.01

Diseased 114 (37 to 197)

Nerve Fibre length

Control 3.76 (3.24 to 4.42) 0.57 (-0.05 to 1.43) 0.06

Diseased 3.34 (2.81 to 3.77)

Nerve Fibre Width

Control 3.11 (2.66 to 3.48) 0.24 (-0.33 to 0.82) 0.4

Diseased 2.78 (2.47 to 3.48)

Myentericplexus

Nerve Fibre Length

Control 4.31 (3.41 to 4.84) -0.81 (-1.51 to -0.11) 0.02

Diseased 4.84 (4.44 to 5.42)

Nerve Fibre Width

Control 3.43 (2.56 to 3.87) -0.86 (-1.51 t o -0.13) 0.02

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 (95% CD

Median % PGP

(95% CD

Difference in medians

(95% CD

Pvalu

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)



Table 4-3 Area of Immunoreactive Nerves in the Longitudinal Muscle

(units: mm^xlO"^)Antigen Group Median staining

(95% Cl)Median %

PGP(95% Cl)

Difference in medians (95% Cl)

Pvalue

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 (95% Cl)

Median % PGP

(95% Cl)


Pvalue

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)



Table 4-5 Area of Ganglia (units: mm^xlO'*)


Median % PGP (95% Cl)

Difference in medians

(95% Cl)

Pvalue

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


Median % PGP (95% Cl)


Pvalue

CGRP Control 2.55(0.42 to 2.6) 69(16 to 88) 36(0 to 71) 0.17

Diseased

0(0 to 1) 0( 0 to 76)

nNOS Control 2.6(1.02 to 2.98) 72(39 to 99) 36( 0 to 67) 0.11

Diseased

0.1(0 to 0.58) 25(0 to 67)

SP Control 2(1.05 to 2.78) 65(47 to 99) 59(33 to 93) <0.001

Diseased

0(0 to 0) 0(0 to 23)

VIP Control 0.83(0 to 2.28) 29(0 to 64) -80(-127 to -14) 0.01

Diseased

1.2(0 to 1.8) 100(23 to 183)

PGP Control 4.05(3.43 to 5.35)

Diseased

5.1(4.38 to 8.18)

CHAPTER 4: 99A QUANTITATIVE STUDY OF THE MORPHOLOGICAL AND HISTOCHEMICAL CHANGESWITHIN THE NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

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

•:N

Fig 4-2 b Immunoreactivity to PGP 9.5 in Diseased Specimen o f Pylorus demonstrating gross m uscle hypertrophy and distortion o f nerves

CHAPTER 4; 100A QUANTITATIVE STUDY OF THE MORPHOLOGICAL AND HISTOCHEMICAL CHANGESWITHIN 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


Figure 4-5 \TP immunoreactivity within the diseased pylorus

?

s

y

Figure 4-6 SP immunoreactivity within the diseased pylorus


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

expressing Neuroactive

Antigen Expression by the Nerves of the Myenteric Plexus

M 0.7

a. 0.2

CGRP

control

□ diseased

AntigenVIP

CHAPTER 4: 103A QUANTITATIVE STUDY OF THE MORPHOLOGICAL AND HISTOCHEMICAL CHANGES WITHIN THE NERVES AND MUSCLE IN INFANTILE HYPERTROPHIC PYLORIC STENOSIS

CHAPTER 5; 104A 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



5.4 Results

5.5 Discussion


CHAPTER 5: 105A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND

________________________________PHENYLKETONURIA________________________________

5.1. SummarySeveral 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: 106A MOUSE MODEL OF INFANTILE HYPERTROPHIC PYLORIC STENOSIS AND

_________________________________ PHENYLKETONURIA__________________________________

5-2. IntroductionSeveral 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


_________________________________ 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.


________________________________ PHENYLKETONURIA________________________________

5.3. Experimental design

5.3.1. TissuesPyloric 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. AntibodiesPGP: 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)


PHENYLKETONURIA

5.3.3. ImmunocytochemistryTissue 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.).


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; 110A 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


_________________________________ 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.


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


_________________________________ 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.


_________________________________ 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


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

LongitudinalMuscle

Control 0.76 0.61 0.55 0.58 0.66 0.14 <0.001

Diseased 0.85 0.99 1.49 0.51 0.26

CircularMuscle

Control 2.01 1.98 0.92 3.94 5.22 0.49 <0.001

Diseased 2.85 8.17 5.87 5.42 4.18

MuscularisMucosa

Control 0.37 0.28 0.68 0.36 0.42 0.05 <0.001

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 Pvalue

10 20 40 90 180

LongitudinalMuscle

Control 1.8 1.4 1.0 1.0 1.1 0.35 <0.001

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

MuscularisMucosa

Control 0.85 0.65 1.26 0.61 0.70 0.12 <0.001

Diseased 1.65 1.59 0.71 0.71 0.60


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 Pvalue

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 % Pylorus

Control 5.6 5.5 1.9 <0.001

Diseased 21.1 7.1


_______________________________ PHENYLKETONURIA_______________________________

Figure 5-1 Diagram Illustrating The Relationship between Tetrahydrobiopterin infantile hypertrophic pyloric stenosis and phenylketonuria.

Phenylalanine

Phen^lanine Hydroxylase

Tyrosine

Tetrahydrobiopterin

Nitric Oxide Synthase

4-Tetrahydrobiopterin

Phenylketonuria

Untreated

Pyloric S tenosis


PHENYLKETONURIA

Figure 5-2 Weight, Longitudinal Muscle Trend

W d ^ Longitudinal M isde Trend

35- - 0.035 fe

30- - 0.03

2 5 - -0.025II 2 0 - - 0.02

15- -0.015 a

10 -

?- 0.005 k

10 20 40 90 180

■CcrtidW.

■HHIW.

■Ccitrci,LMDP

-D-HFH1,IMCP

^(D ajs)

Figure 5-3 Weight, Circular Muscle Trend

Weight,Circular Muscle Trend

T 0.16

35 -- - 0.14

30 - - 0.12

- 0.1II 20 -

15 - - 0.06

10 - - - 0.04

- 0 .02 k

10 20 40 90 180

I

■Control W t

•HPHl Wt.

■ControlCM/DP

-HPH-1CM/DP

Age (Days)


PHENYLKETONURIA

Figure 5-4 Weight, Muscularis mucosae Trend

Weight, Muscularis Mucosae Trend

40

35

30 --

25 --Ür 20I 15 +

10 - -

5 -

010 20 40

Age (Days)

90 180

j 0.018

-- 0.016k

-- 0.014 I

-- 0.012 I

- 0 . 0 1 S 2 ■S s

- 0.008 I ^

- 0.006 g

- 0.0042

- 0 . 0 0 2 Oh

■Control Wt.

■HPHlWt.

■ControlMM/DP

■HPH MM/DP

Figure 5-5 F e ta l, one day old Mouse Muscle Hypertrophy

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

-50 200100

O (/)Ë §O :=— *o ■a <D « 2

Age (Days)

DP: Diameter Pylorus,CM: diameter of Circular Muscle,LM: diameter of Longitudinal Muscle MM: diameter of Muscularis Mucosae


___________________________________PHENYLKETONURIA___________________________________

Figure 5-6 40 day old Control Mouse

C O N T R O L , 40 D A Y O L D M O U S E

T 7 r ... .. ■............ .................... .... .■’ 0 3 0 40 50 6 0 70 8 0 9 0 100 110 120 130 140 ISO 160 170 180 190

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

D IS E A S E D , 40 D A Y O L D M O U S E

'n 2 0 3 0 40 50 s o 7 0 8 0 9 0 100 110 120 730 140 ISO 160 170


PHENYLKETONURIA

Figure 5-8 Stomachs o f 40 day Control Mouse

liiitiiininiininiitiiFigure 5-9 Stomachs o f 40 day old Hph-1 Mouse

il il tin II in


PHENYLKETONURIA

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

■V

» *KS

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

s!"^ - - v - » # # # 4 ^ %

/ r 'r - -f \* • f'~-

■ . 4 •

* , y , ' 4 >1 - . / / /

Î c . . .y^~.^ / » # :-W ' ' : *

. *1

/ J . :J *


PHENYLKETONURIA

CHAPTER 6 124A 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



6.4 Results

6.5 Discussion



________________________________ 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.


________________________________ 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.


__________________________________ 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



________________________________ STENOSIS IN DOGS.________________________________


6.3.1. TissuesEight 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. AntibodiesAntibodies 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. ImmunostainingFive 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;


__________________________________ 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.


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.


________________________________ STENOSIS IN DOGS.________________________________

6.4. Results

6.4.1. Conventional HistologyHaematoxylin 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. ImmunocytochemistrySubjective 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


__________________________________ 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).


________________________________ STENOSIS IN DOGS._________________________________

6.5. DiscussionTwo 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


__________________________________ 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.,


__________________________________ 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.


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


__________________________________ 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.


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.

I CONTROL

□ DISEASED

Ç 0 .6 -

0.4 --

VIP

Antigen

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

ANTIGEN CONTROL DISEASED STANDARD ERROR PVALUE

NOS 0.602 0.435 0.6 0.14 0.8SP 0.415 0.387 0.82 0.22 0.76

VIP 0.57 0.17 0.09 0.11 0.065

CH.AfTER6 138A QUANTITATIVE STUDY OF THE HISTOCHEMICAL CHANGES UNDERLYING PYLORIC

________________________________ STENOSIS IN DOGS.________________________________

Figure 6-2 Immunostaining for nNOS in a Normal Canine pylorus


STENOSIS IN DOGS.

Figure 6-3 Immunostaining for nNOS in a Canine pylorus o f congenital pyloric stenosis


STENOSIS IN DOGS.

CHAPTER? 141A 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


7.3.1 Surgical procedure7.3.2 Tissues7.3.3 Imunostaining7.3.4 Statistical analysis

7.4 Results

7.5 Discussion

7.6 Figures

CHAPTER 7 142A 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.


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? 144A STUDY OF THE HISTOLOGICAL CHANGES FOLLOWING LASER IRRADIATION OF THE

_____________________________________ PYLORUS._____________________________________


7.3.1. Surgical ProcedureAll 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. TissuesThe 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. AntibodiesPGP: 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)


_____________________________________PYLORUS._____________________________________CGRP: the sensory innervation to the stomach (Dockray GJ.I99I)

7.3.4. ImmunocytochemistryTissue 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.).


_____________________________________ PYLORUS._____________________________________

7.4. Results

7.4.1. Conventional HistologyHaematoxylin 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. ImmunocytochemistrySubjective 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.


_____________________________________ PYLORUS._____________________________________

7.5. DiscussionThe 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


________________________________________ PYLORUS.________________________________________

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

pylorus.

V .

* ^\

Jt / JL. X

y /


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 o f neuro transmitters and the morphology o f 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 o f

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


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


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


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


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 KH2PO4 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 fo r antibodies

Primary antibodies and normal seraPBS; 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 FixationIn 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 slides1. 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

001 0.06922 0.0001738 0.2511 2102 0.06922 0.00009469 0.1368 3303 0.06922 0.0009785 1.414 96

AvOi 0.06922 0.000415663 0.600633333 5001 NOS II 01

001 0.06922 0.0003157 0.4561 5402 0.06922 0.0002859 0.4131 6703 0.06922 0.0004174 0.603 89

AvOi 0.06922 0.000339667 0.490733333 70

01 NOS III 01

001 0.06922 0.0008347 1.206 7302 0.06922 0.001172 1.694 12303 0.06922 0.0002095 0.3027 65

AvOili 0.06922 0.000738733 1.067566667 87

01 NOS lU 01

001 0.06922 0.000588 0.8495 8402 0.06922 0.0005308 0.7669 6103 0.06922 0.0001499 0.2166 42

AvOiv 0.06922 0.0004229 0.611 62.33333333

01 NOS V 01

001 0.06922 0.0003203 0.4627 7102 0.06922 0.0003444 0.4976 5503 0.06922 0.0002492 0.36 62

AvOv 0.06922 0.000304633 0.4401 62.6666666702 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

001 0.06922 0.00008315 0.1201 3002 0.06922 0.0001151 0.1662 3303 0.06922 0.0001151 0.1662 33

AvOi 0.06922 0.00010445 0.150833333 32

02 NOS III 01

001 0.06922 0.0001644 0.2375 6702 0.06922 0.00009334 0.1349 4303 0.06922 0.0002299 0.3321 40

AvOiii 0.06922 0.000162547 0.234833333 50

02 NOS IV 01

001 0.06922 0.0007143 1.032 22002 0.06922 0.0001379 0.1992 6703 0.06922 0.0001065 0.1538 62

AvOiv 0.06922 0.000319567 0.461666667 116.3333333

02 NOS V 01

001 0.06922 0.0007156 1.034 23602 0.06922 0.0004056 0.5859 15203 0.06922 0.000009388 0.01356 10

AvOv 0.06922 0.000376863 0.544486667 132.6666667

FrameArea Redin Frame %Red Count03 NOS 1 01

001 0.06922 0.0001419 0.205 3302 0.06922 0.0001255 0.1814 3403 0.06922 0.0001674 0.2418 45

AvOI 0.06922 0.000144933 0.2094 37.33333333

03 NOS II 01

001 0.06922 0.0001974 0.2852 6502 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

CC1 0.06922 0.0007232 1.045 50C2 0.06922 0.000452 0.653 91C3 0.06922 0.0001711 0.2472 43

AvCiii 0.06922 0.000448767 0.6484 61.33333333

C3 NOS III C1

CC1 0.06922 0.0003162 0.4569 82C2 0.06922 0.0003074 0.4441 89C3 0.06922 0.001514 2.187 197

AvCIv 0.06922 0.000712533 1.029333333 122.6666667

C3 NOS IV 01

C01 0.06922 0.0001033 0.1492 4002 0.06922 0.0003069 0.4433 8703 0.06922 0.0003026 0.4371 93

AvOv 0.06922 0.0002376 0.3432 73.33333333

04 NOS 1 01

001 0.06922 0.000519 0.7498 9002 0.06922 0.0012 1.733 15103 0.06922 0.0002055 0.2968 58

AvOI 0.06922 0.0006415 0.926533333 99.66666667

04 NOS II 01

001 0.06922 0.0007645 1.104 7202 0.06922 0.0002473 0.3573 5903 0.06922 0.0003543 0.5119 98

AvOI 0.06922 0.000455367 0.657733333 76.33333333

04 NOS III

001 0.06922 0.0005931 0.8568 7102 0.06922 0.0002165 0.3127 7603 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 84C2 0.06922 0.0006601 0.9537 163C3 0.06922 0.0008372 1.209 115

AvCiv 0.06922 0.000763767 1.103233333 120.6666667

C4 NOS V C1

CC1 0.06922 0.0002374 0.343 61C2 0.06922 0.0002787 0.4026 46C3 0.06922 0.0006982 1.009 84

AvCv 0.06922 0.000404767 0.584866667 63.66666667

C5 NOS 1 C1

CCl 0.06922 0.0002739 0.3957 8102 0.06922 0.0005493 0.7936 17503 0.06922 0.0003704 0.5352 153

AvOi 0.06922 0.000397867 0.574833333 136.3333333

05 NOS II 01

001 0.06922 0.002081 3.007 35502 0.06922 0.001401 2.024 28503 0.06922 0.0004356 0.6293 169

AvOi 0.06922 0.001305867 1.886766667 269.666666705 NOS III 01

001 0.06922 0.0004029 0.5821 13302 0.06922 0.001043 1.506 20503 0.06922 0.001052 1.519 235

AvOiil 0.06922 0.000832633 1.202366667 191

05 NOS IV 01

001 0.06922 0.001597 2.307 28402 0.06922 0.000441 0.6371 11003 0.06922 0.0005842 0.844 140

AvOlv 0.06922 0.000874067 1.2627 178

05 NOS V 01

001 0.06922 0.0004632 0.6693 13502 0.06922 0.001458 2.106 26203 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

NOSCM

CONTROL CONTROL DISEASED DISEASED PGPCM

CONTROL CONTROL DISEASED DISEASED

%Red Count %Red Count %Red Count %Red Count1i 0.600633333 50 11 0.627266667 63 1i 0.662533333 142.3333333 0.8023 711ii 0.490733333 70 lii 0.222966667 21 lii 1.500766667 130 0.5325 55.333333331iii 1.067566667 87 liii 0.333666667 62.33333333 liii 1.068666667 85.33333333 0.316833333 43.333333331iv 0.611 62.33333333 liv 0.3348 47 liv 0.938766667 76 0.531266667 59.666666671v 0.4401 62.66666667 1v 0.6106 93.33333333 1v 0.672466667 76 0.9186 731 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 5.6666666672ii 0.150833333 32 2ii 0.304616667 28 2ii 0.532233333 42.33333333 0.006667367 2.6666666672iii 0.234833333 50 2iii 0.027899333 6.666666667 2iii 0.418266667 40 0.032806667 8.3333333332iv 0.461666667 116.3333333 2iv 0.110826667 22.33333333 2iv 0.349233333 31.66666667 0.002614 22v 0.544486667 132.6666667 2v 0.131373333 26 2v 0.264433333 46 0.001568667 12 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 16.333333333ii 0.786633333 62.33333333 3ii 0.054266667 23 3ii 1.1401 109.3333333 0.155266667 21.666666673iii 0.6484 61.33333333 3iii 0.033586667 15.33333333 3iii 0.740433333 75 0.283093333 123iv 1.029333333 122.6666667 3iv 0.051153333 8.666666667 3iv 1.1401 109.3333333 0.011763667 63v 0.3432 73.33333333 3v 0.155513333 21 3v 0.740433333 75 0.02954 5.3333333333 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 304ii 0.657733333 76.33333333 4ii 0.031776667 12.66666667 4ii 3.334666667 126 0.116212333 7.6666666674iii 0.7475 77.33333333 4iii 0.063426667 12.66666667 4iii 1.741666667 88.66666667 0.1277 194iv 1.103233333 120.6666667 4iv 0.072596667 17 4iv 1.5472 64 0.386233333 33.333333334v 0.584866667 63.66666667 4v 0.035653333 9 4v 0.794033333 43.66666667 0.652966667 49.666666674 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 19.333333335ii 1.886766667 269.6666667 5ii 0.036696 1.333333333 5ii 0.532233333 42.33333333 0.428976667 30.666666675iii 1.202366667 191 5iii 0.031776667 6.666666667 5iii 0.418266667 40 0.120119333 105iv 1.2627 178 5iv 0.01886 7.666666667 5iv 0.349233333 31.66666667 0.07738 185v 1.205266667 172.6666667 5v 0.02984 8 5v 0.264433333 465 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 576ii 0.4316 160 6ii 0.234967667 12.33333333 6ii 0.1481 18.33333333 0.610133333 396iii 0.9983 150 6iii 0.06561 10.33333333 6iii 0.1379 30.66666667 0.5218 40.666666676iv 0.948333333 148.3333333 6iv 0.117026667 11.33333333 6iv 1.3238 78 0.7041 406v 0.927 189.3333333 6v 0.13021 14.33333333 6v 0.16925 22 0.544 49.666666676 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 45.666666677ii 0.5996 110.6666667 7ii 0.041596667 14.66666667 7ii 0.6522 84.33333333 0.159233333 56.666666677iii 0.546766667 134.3333333 7iii 0.05528 15.33333333 7iii 0.974033333 96 0.250166667 17.333333337iv 0.399533333 107.3333333 7iv 0 014856667 3 7iv 1.493066667 156 0.2465 32.333333337v 0.572533333 97 7v 0.109533333 14.33333333 7v 0.8671 96.66666667 0.296566667 32.333333337 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 218ii 1.099466667 166 8ii 0.031777667 5.333333333 8ii 0.783333333 66.66666667 0.131873333 188iii 1.172966667 124.6666667 8iii 0.003358667 2 8iii 2.795 187.3333333 0.096843333 148iv 1.601666667 193.6666667 8iv 0.03126 12 8iv 2.814333333 161 0.2545 438v 0.872966667 112 8v 0.003486667 0.333333333 8v 3.327 186 0.2307 19.333333338 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 89ii 0.854333333 144 9ii 0.043531667 13.33333333 9ii 0.9034 129.6666667 0.107566667 21.333333339iii 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 37.666666679v 1.442666667 198.6666667 9v 0.05787 14.66666667 9v 0 0 0.427 33.666666679 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 58.6666666710ii 1.058666667 144 1011 0.047796667 15.66666667 lOii 0 0 3.264666667 67.3333333310iii 0.810866667 110 lOiii 0.076216667 17.33333333 lOiii 0 0 3.584 86.6666666710iv 1.168933333 140 lOiv 0.036556667 12 lOiv 0 0 3.697333333 101.3333333lOv 0.745233333 98.66666667 lOv lOv 4.608333333 235 3.222 10010 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 771111 0.4307 115.6666667 liii 0.2193 48 liii 3.231333333 214.3333333 0.347 2111111 1.009733333 131 11 Hi 0.2348 47 Hiii 6.101 287 0.2819 11lllv 0.9756 113 Iliv 0.1562 47 Iliv 4.958666667 239 0.2772 011v 0.8244 139 liv 0.2782 19 liv 1.653666667 146 0.227 1311 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 291211 0.361033333 60.66666667 12ii 0.041596667 14.66666667 12ii 4.67 252.3333333 1.294666667 84.6666666712111 0.7403 115.6666667 12iii 0.05528 15.33333333 12iii 4.918 192.6666667 0.7735 22.6666666712iv 0.502233333 92.66666667 12iv 0.014856667 3 12iv 4.154666667 201.6666667 0.601633333 45.6666666712v 0.472533333 103.6666667 12v 0.109533333 14.33333333 12v 5.129333333 218 0.756266667 26.3333333312 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 29131! 1.952666667 213.6666667 13ii 0.053476667 10.33333333 13ii 4.291 208.6666667 0.525433333 4913111 0.956366667 141.3333333 13iii 0.040043333 11.66666667 13iii 3.792666667 194.3333333 0.4372 50.6666666713iv 1.833 171.3333333 13iv 0.030223333 9.666666667 13iv 2.205333333 219 0.514333333 5813v 3.352 318.6666667 13v 0.043916667 10.66666667 13v 8.6737 232.3352412 0.387666667 51.3333333313 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 14141! 0.2563 62.33333333 14ii 0.095576667 13.5 14ii 0.640066667 30 0.277633333 42.6666666714111 0.610466667 118.3333333 14iii 0.024673333 6.666666667 14iii 0.951133333 107.6666667 1.893 105.333333314iv 0.296333333 86.66666667 14iv 0.075566667 11.66666667 14iv 1.426133333 114 1.406 60.3333333314v 1.827266667 170.3333333 14v 0.02299 6 14v 0.513 40.33333333 1.259866667 45.3333333314 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 99.666666671511 0.33666 62 15ii 0.081526667 15.33333333 15ii 0.652233333 73.33333333 0.384 6015111 0.134466667 38.33333333 15iii 0.167033333 16.33333333 15iii 0.369266667 35 0.888366667 113.666666715iv 0.089283333 30.33333333 15iv 15iv 1.395666667 114.3333333 1.1852 108.666666715v 0.11224 20.33333333 15v 0.206533333 30.33333333 15v 2.428 147.6666667 1.223833333 12615 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 35.3333333316il 1.611 209.6666667 16ii 0.06615 7 333333333 16ii 1.479666667 72.33333333 0.7535 5316111 3.906666667 388 16iii 0.067566667 15.33333333 16iii 1.19 84.66666667 1.065933333 43.6666666716iv 2.000666667 281 16iv 0.062533333 14.66666667 16iv 0.850933333 65.33333333 0.8907 3216v 2.673333333 290.6666667 16v 0.079306667 17 16v 0.7698 53.66666667 1.1027 85.3333333316 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 211711 0.370066667 107 17ii 0.03759 14.66666667 17ii 3.727 175.6666667 0.07661 35.3333333317111 0.198033333 66 17iii 0.03436 14.66666667 17iii 1.058533333 153.6666667 0.063926667 27.3333333317lv 0.490566667 80 33333333 17iv 0.032553333 12.33333333 17iv 1.117333333 142 0.133963333 5317v 1.673 249.6666667 17v 0.033067667 18.66666667 17v 1.502233333 178 0.1111 42.3333333317 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 10.666666671811 0.548 232.6666667 18ii 0.07944 22.33333333 18ii 3.884333333 167.3333333 0.3488 2418111 1.246333333 202.3333333 18iii 0.458566667 77.33333333 18iii 1.0357 110 0.2726 15.3333333318lv 1.121666667 214.6666667 18iv 0.153466667 34.33333333 18iv 1.802733333 138.6666667 1.423433333 4818v 0.681333333 157.6666667 18v 0.1983 39.66666667 18v 3.765 213.6666667 0.559966667 39.3333333318 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 45.3333333319ii 0.429 188 19ii 0.04883 15 19ii 0.636 85 0.551466667 3319iii 0.017 157.6666667 19iii 0.07067 9.666666667 19iii 0.842233333 97.33333333 0.842333333 39.3333333319iv 1.888666667 212 19iv 0.04508 9.666666667 19iv 0.714566667 83.33333333 0.41 3819v 0.307833333 155 19v 0.052313333 21 19v 1.284266667 119.6666667 0.649466667 2119 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 44.6666666720ii 1.671666667 194 20ii 0.06058 10 20ii 0.843733333 98.66666667 1.351333333 7620iii 0.652866667 89.66666667 20iii 0.048443333 6.666666667 20iii 0.866333333 93.33333333 0.981533333 69.6666666720iv 0.832233333 105 20iv 0.050376667 5 20iv 1.8274 145 0.000677733 0.97913333320v 1.027033333 109.3333333 20v 0.06135 5 20v 0.8354 84 0.000845133 1.22116666720 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 ratio1. 1 .642007 .96864 control .6627921 21. 1 .42586 .6203 diseased .68653882. 2 .335684 .4295 control .7815692 22. 2 .20839 .012365 diseased 16.853213. 3 .603393 1.112233 control .5425059 23. 3 .072777 .128106 diseased .56809994. 4 .803973 1.483513 control .5419387 24. 4 .077999 .340749 diseased .22890465. 5 1.226387 .4295 control 2.855383 25. 5 .036197 .239377 diseased .15121336. 6 .684667 .371759 control 1.841696 26. 6 .192882 .5847 diseased .3298827. 7 .518527 1.000567 control .5182332 27. 7 .053115 .230485 diseased .23044888. 8 1.205747 2.457 control .4907395 28. 8 .040017 .17473 diseased .22902199. 9 1.019993 .9678 control 1.053929 29. 9 .075574 .281505 diseased .268464210. 10 .816127 4.608333 control .1770981 30. 10 .05419 3.224667 diseased .016804811. 11 .783027 3.715467 control .2107479 31. 11 .26986 .29132 diseased .926335312. 12 .461693 4.3738 control .1055588 32. 12 .053115 .75914 diseased .069967313. 13 1.862607 4.81814 control .3865822 33. 13 .039112 .449987 diseased .086918114. 14 .69718 .737172 control .9457494 34. 14 .060165 .986489 diseased .06098915. 15 .188243 1.159587 control .1623362 35. 15 .121104 .972547 diseased .124522516. 16 2.221547 1.383347 control 1.605922 36. 16 .072806 .928267 diseased .078432217. 17 .611771 2.317553 control .2639728 37. 17 .03666 .097101 diseased .37754518. 18 1.312 2.893487 control .4534321 38. 18 .192086 .546275 diseased .351628819. 19 .582167 1.173013 control .4963006 39. 19 .062628 .655407 diseased .095555920. 20 1.110693 1.08456 control 1.024095 40. 20 .054277 .594505 diseased .0912978

APPENDIX 171

ABBREVIATIONS 172

ABBREVIATIONS

AAPH Acquired Antral Pyloric HypertrophyABC Avidin Biotin ComplexBOB Bis-diazobenzidineBH4 T etrahydrobiopterinBSA Bovine Serum AlbumincAMP cyclic adenine monophosphateGDI CarbodiimidecGMP cyclic guanyl monophosphateCGRP Calcitonin Gene Related ProductCPS Congenital Pyloric StenosisDil I , I ’-dioctatidecyl-3,3,3 ’,3 ’-tetramethylindocarbocyanine perchlorateEDRF Endothelial Derived Relaxing FactoreNOS Endothelial Nitric Oxide SynthaseICC Interstital Cell of CajaliNOS Inducible Nitric Oxide SynthasekDA Kilo DaltonLNMMA NG-monomethyl—L-arginineNADPH Nicotinamide Adenine Dinucleotide Phosphate DiaphoraseNANC Non Adrenergic Non CholinergicnNOS Neural Nitric Oxide SynthaseNO Nitric OxideNOS Nitric Oxide SynthasePACAP Pituitary Adenylate Cyclase Activating PeptidePAP Peroxidase-antiperoxidasePBS Phosphate Buffered SolutionPGP 9.5 Proten Gene Product 9.5PHI Peptide Histidine IsoleucinePHM Peptide Histidine MethioninePHV Peptide Histidine ValinePPT-A Preprotachykinin APPT-B Preprotachykinin BQDHPR Quinonoid-dihydropteridine ReductaseSDS/PAGE Sodium Dodecyl Polyamide GelTTX TetrodotoxinVIP Vasoactive Intestinal Polypeptide

REFERENCES 173

REFERENCES

Abel RM., Bishop AE., Spitz L., Polak JM.

The Expression of Neural Cell Adhesion Molecule in Infantile Hypertrophic Stenosis.

Presented at B.A.P.S Symposium, Rotterdam 1994

Abel R.

The Ontogeny of the Peptide Innervation of the Human Pylorus with Special Reference to Understanding the Aetiology and Pathogenesis of Infantile Hypertrophic Stenosis.

J. Paed. Surgery 1995; 31 (4): 490-497

Abel R.M., Bishop A.E., Spitz L., Polak J.M.

The Expression of Neural Cell Adhesion Molecule in Pyloric Stenosis.

Manuscript in preparation.


The Ontogeny of the Innervation of the Human Pylorus

Accepted for publication by the JPS


A Quantitative Study of the Muscle Hypertrophy and Selective Neural Changes in Morphology and Neurochemical Expression in Infantile Hypertrophic Pyloric Stenosis.

Accepted for publication by the JPS


Decreased Nitric Oxide Synthase Activity in an Animal Model of Hypertrophic Pyloric Stenosis and Phenylketonuria

To be submitted to the JPS

Abramovitch DR.

Fetal factors influencing amniotic fluid volume and composition of liquor amnii.

J. Obstet Gynecol Br Commonw 1970; 77: 865-877

Abrams D., Facer P., Bishop AEB.

Computer Assisted Stereological Quantification Program; Opem Stereo.

Microscopy Res. Technique 1994; 29 (3) : 24-247

Adelstein P., Fredrick J.,

Pyloric Stenosis in the Oxford Record Linkage Study area

J.Med Genet. 1976; 13: 439-448

Ahmed, S.

Infantile Hypertrophic Pyloric Stenosis associated with major anomalies of the alimentary tract.

J. Paediatric Surgery., 1970; 5: 660

Aimi Y., Kinoshita T., Fujimura M.

Histochemical localisation of nitric oxide synthase in the rat enteric nervous system.

Neuroscience 1993; 53 :287-297

Akeson RA., Wujek JR., Roe S., Warren SL., Small SJ .

Smooth muscle cells transiently express NCAM .

Molecular Brain Research 1988 ;4 : 107 - 120

Alain JL., Grousseau D., Terrier G.

Extramucosal Pyloromyotomy by laparoscopy.

J. of Paediatric Surgery 1991 ; 26: 1191-1192

Allescher HD, Kostolanska F, Tougas G, Fox JE, Regoli D, Drapeau G, Daniel EE.

The actions of neurokinins and Substance P upon the canine pylorus antrum and duodeum.

Peptides 1989; 10: 671-679

Allescher HD., Kurkak M., Huber A., Trudrung P., Schuszdiarra V.

Regulation of VIP release from rat enteric nerve terminals: evidence for a stimulatory effect of NO

Am J. Phsiol 1996; G569-G574

Altdorf K., Feher E., Donath T., Feher J.

Nitric oxide synthyase-containing nerve elements in the pylorus of the cat.

NeurosciLett 1996; 212: 195-198

Alumets J., Fahrenkmg J., Hakanson R., SchafFalitzky O., Sundler F., Uddman R.

A rich VIP nerve supply is characteristic of sphincters

Nature 1979; 280 :155 - 156

Aly H., Sahni R., Wung J.

Weaning strategy with inhaled Nitric Oxide treatment in persistent pulmonary hypertension of the newborn

Arch Dis Child 1997; 76: F118-F122

Anand P, Gibson SJ, Yiangou Y, Christofides ND, Polak JM, Bloom SR.

PHI -like immunoreactivity colocates with with VIP-containing system in the human lumbosacral cord.

Neurosci Lett 1984 46: 191-196

Anggard E.

Nitric Oxide: mediator, muderer, and medicine.

Lancet 1994; 343: 1199-1206

Asaoka Y., Nakamura S-I, and Nishizuka Y.

Intracellular Signalling by Phosholipid Hydrolysis and Protein Kinase C Activation

Biomedical Research 1993; 14: 3, 93-98

Atwell, J.D., Cook, P.L., Strong, L., Hyde, I.,

The relationship between vesico-ureteric reflux, trigonal abnormalities and a bifid pelvi- calyceal collecting system: a family study.

Br. J. Urol 1977: 49, 97-107,

Bailey PV., Tracey TF., Connors Jr. RH., Mooney DP., Lewis JE., Weber TR.

Congenital Duodenal Obstruction : a 32 year experience .

J. of Paediatric Surgery 1993 ; 28 : 92 - 95

Bajpai M., Mathur M.

Duplications of the Alimentary Tract; Clues to the missing links.

J. Paediatric Surgery 1994, 29; 10: 1361-1365

Bandyopadhyay A., Chakder S., Lynn R.B.,Rattan S.

Vasoactive Intestinal Polypeptide Gene Expression is Characteristically Higher in Opossum Gastrointestinal Sphincters

Gastroenterology 1994; 106: 1467-1476

Barrios V ., Urrutia M.J.M. , Lama R., Garcia-Novo M.D., Hemanz A., Arilla E.

Serum Gastrin Level and Gastric Somatostatin content and binding in Long-Term Pyloromyotomized Children

Life Sciences 1994; 55: (4): 317-325

Bartheld C.S., Cunningham D.E., Rubel E.W.

Neuronal Tracing with Dil : Décalcification, Cryosectioning, and Photoconversion for light and electron microscopic analysis.

J. of Histochemistry and Cytochemistry.

Bartho L, Holzer P.

Search for a physiological role of substance P in gastrointestinal motility.

Neuroscience 1985; 16: 1-32

Baurenfeind PR., Hof R., Hof A., Cucala M., Siegrist S., Von Ritter C , Fischer JA., BlumAL.,

Effects of hCGRP I and II on gastric blood flow and acid secretion in anaesthetised rabbits.

Am. J. Physiol 1989; 256: G145-149

Bayguinov O , Sanders K.

Role of nitric oxide as an inhibitory neurotransmitter in the canine pyloric sphincter

Am J Physiol 1993; G975-G986

Bealer JF., Graf J., Bruch N., Adzick S., Harrison MR.

Gastroschisis increase Small Bowel Nitric Oxide Synthase Activity

J. Paediatric Surgery 1996; 31(8); 1043-1046

Beasley S.W., Hutson J.M.

Pyloric stenosis in Sick Premature infants.

Am. J. Diseases in Childhood 1989 ; 143 : 142

Beckman J., Tsai J-H.

Reactions and Difliision of Nitric Oxide and Peroxynitrite

The Biochemist Oct/Nov 1994 8-10

Belding HH, Kernohan JW.

A Morphological Study of the Myenteric Plexus and The Musculature of the Pylorus with. Special Reference to the Changes in Hypertrophic Pyloric Stenosis

Surg Gynae Obst 1953; 97: 322-334

Benjamin N. , O'Driscoll F., Dougall H., Duncan C , VcKenzie H.

Stomach NO synthesis.

Nature 1994; 368: 502

Benson, CD.

Infantile Pyloric Stenosis Historical Aspects and Current Surgical Concepts

Prog Paediatr. Surg. 1970; 63-88

Biancani P, Beinfield MC, Hilemeier C, Behar J.

Role of peptide histidine isoleucine in the relaxation of the cat lower esophageal sphincter.

Gastroenterology 1989; 97:1083-1089

Bishop A.E., Romanska H.H., Brereton R.J., Spitz L., Polak J.M.

Abnormalities in the gut neuroendocrine system in Hirschprung's disease : implications for surgical procedures .

Advances in the innervation of the Gastrointestinal tract 1992; G.E. Hoile et at. , editors .

Bishop A.E., Polak J.M.

The gut and the autonomic nervous system.

'Autonomic Failure' A Textbook of Clinical Disorders of the Autonomic Nervous System.

Bishop A.E., Polak J.M.

Modem Histological Imaging Methods

Submitted to Surgical Endocrinology. Eds. J. Lynn, S.R. Bloom, Butterworth & Co. Ltd. Lon 1992

Bissonnette B., Sullivan P.J.

Pyloric Stenosis.

Canadian J. Anaesthesia 1991 ; 38 : 668 - 676

Blair DH.

An account of the dissection of a child.

Phil Trans 1717; 30; 631,

Blass-Kampmann S., Reinhardt-Maelicke S., Kindler-Rohrbom A , Cleeves V., Rajewsky MF.

In vitro Differentiation of E-N-CAM Expressing Rat Neural Precursor Cells Isolated by FACS During Prenatal Development.

J. of Neuroscience Research 1994; 37: 359 - 373

Bleicher MA., Shandling B.

Increase in serum immunoreactive gastrin levels in idiopathic hypertrophic pyloric stenosis .

Gut 1978; 19: 794:

Bloch R.J.

Clusters of Neural Cell Adhesion molecule at sites of cell-cell contact.

J. Cell Biology 1992 ; 116 : 449 - 463

Bola F., Hyland K..

Pterin and Neurotransmitter Amine Metabolism in the HPH-1 Mouse Mutant.

9th International Symposm. Pteridines and Folic Acid Derivatives, Program & Abstract Book D-05

Houghton N.K., Evans S.M., Hawkey C.J., Cole A.T., Balstis M., Whittle B.J.R. , Moncada S.

Nitric oxide synthase activity in ulcerative colitis and Crohn's disease.

Lancet 1993; 342 : 338 -339

Boulton C., Gartwaite J.

NO Signalling and Synaptic Plasticity


Bourchier D., Dawson KP., Kennedy JC.

Pyloric stenosis: A postoperative ultrasonic study

Austr. Paediatr. J. 1985; 21:189-190

Brady S T.

Motor Neurons and Neurofilaments in Sickness and in Health.

Cell 1993; 73: 1 -3

Brain AJL., Roberts DS.

Who should treat Pyloric Stenosis: The General Surgeon or Specialist Pediatric Surgeon?

J. Pediatric Surgery 1996; 1535-1537

Brand M.P., Heales S.J R., Land J.M., ClarkJ.B.

Tetrahydrobiopterin deficiency and brain nitric oxide synthase in the hph 1 mouse

J. Inher. Metab. Dis. 1995; 18: 33-39

Bredt DS., Hwang PM., Snyder SH.

Localisation of nitric oxide synthase indicating a neural role for nitric oxide.

Nature 1990; 347: 768-770

Bredt DS., Snyder SH.

Isolation of nitric oxide synthase , a calmodulin requring enzyme

Proc Natl Acad Soi USA 1990; 87; 682-685

Bredt D , Snyder S.H.

Nitric Oxide a Novel Neuronal Messenger.

Neuron 1992 ; 8 : 3 - 11

Bredt D., Huang P., Dawson T., Fishman M. , Snyder S.

Nitric Oxide Synthase Gene Knockout Mice : Molecular and Morphological Characterisation.

Endothelium 1993 ; 19 (supplement) s6

Brehmer A., Stach W.

Morphological classification of NADPHd-positive and -negative myenteric neurons in the porcine small intestine.

Cell Tiss Res 1997; 287: 127-134

Brereton R.J.

Letter to ed itor.

J. of Paediatric Surgery 1991 ; 27 : 796-797

Brookes S.J.H.

Neuronal Nitric oxide in the gut.

J. Gastroenterology and Hepatology 1993; 8: 590-603

Buja L.M., Eigenbrodt M.L., Eigenbrodt E.H.

Apoptosis and Necrosis

Archives Pathological Laboratory Medicine 1993; 117: 1208-1213

Bult H., Boeckxstaens G.E., Pelckmans P. A. , Jordaens F.H. , Maercke YM., Herman AG.

Nitric oxide as an inhibitory non - adrenergic non - cholinergic neurotransmitter .

Nature 1990 ; 345 : 346 - 347

Bumstock G, Dumsday B, Smythe A

Atropine-resistant excitation of the urinary bladder-the possibility of transmission via nerves releasing a purine nucleotide.

B rJ Pharmacol 1972; 44: 451-461

C alder J.

Two cases of children bom with preternatural conformations of the gut.

Med. Essays Observations Edinburgh 1733; 1: 203

Carter CO., Evans KA.,

Inheritance of congenital pyloric stenosis .

J. Med Genet 1969; 6: 233,

Cashman N.R., Covault J., Wollman R.L., Sanes J R.

Neural Cell Adhesion Molecule in Normal, Denervated, and Myopathic Human Muscle

Annals of Neurology 1987; 21: 481-489

Cestro B.

Effects of arginine, S-adenosylmethionine and polyamines on nerve regeneration

Acta Neurol Scand 1994; Supple 154 : 32-41

Chakder S., Ratten S.

Involvement of cAMP and cGMP in relaxation of internal anal anal sphincter by neural stimulation, VIP, and NO.

Am. J. Physiol. 1993; 264: G702-G707

Chaves-Carballo E., Harris LE., Lynn HB.

Jaundice associated with pyloric stenosis and neonatal small bowel obstructions.

Clin Pediat. 1968; 7: 198-202.,

Chew A.L., Friedwald J.P., Donovan C.

Diagnosis of congenital antral web by ultrasound.

Paediatric Radiology 1992 ; 22 : 342 - 343

Choyce CC., Beattie JM.

The Stomach and Duodenum

A System of Surgery Cassell & Co. 1932;,2: 214-218

Choyce C.C., Beattie J.M..

Infantile Stenosis of the Pylorus

In ' A System of Surgery,' Cassell and Company Ltd. 1932; 214-219,

Collins M.K.L., Perkins G.R., Rodriguez-Tarduchy G., Nieto M.A., Lopez- Rivas.A.

Growth Factors as Survival Factors: Regulation of Apoptosis.

Bioessays 1994; 16: 2, 133-138

Corkery J.J.

Infantile hypertrophic pyloric stenosis : where should it be treated?

Annals of the Royal College of Surgeons England 1994; 76 : 71-74

Cosman B.C., Sudekum A.E., Oakes D.D., de Vires PA.

Pyloric stenosis in a premature infan t.


Costa M, Furness JB, Pullin CO, Bornstein J.

Substance P enteric neurons mediate non-cholinergic transmission to the circular muscleof the guinea pig small intestine.

Arch Pharmacol 1985; 328: 446-453

Costa M, Furness JB.

The origins pathways and terminations of neurons with VIP like immunoreactivity in the guinea pig small intestine

Neuroscience 1983;8:665-676

Costa M, Fumess JB, Llewellyn-Smith IJ.

Histochemistry of the enteric nervous system.

In Johnson LP ed. Physiology of the gastrointestinal tract, vol 2, 2nd edition. New York: Raven Press 1987:1-40

Costa J.J.L., Averill S., Ching Y.P., Priestly JV.

Immunocytochemical localization of a growth associated protein (GAP - 43) in rat adrenal gland.

Cell Tissue Research 1994; 275 : 555 - 566

Costa M, Furness JB, Llewellyn-Smith IJ., Davies B., Oliver J.

An Immunohistchemical study of projections of somatostatin containing neurons in the guinea pig intestine.

Naunyn Schmiedebers Arch Pharmacol 1980; 5: 841-852

Courtney S.P, Spicer R.D.

Pseudo-pyloric tumours

Br. J. Clinical Practice 1990 ; 44 : 370-371

Covault J., Sanes J R.

Neural cell adhesion molecule (N-CAM) accumulates in denervated and paralyzed skeletal muscles.

Proceedings National Academy Science USA 1985; 82: 4544-4548

Covault J., Merlie J.P., Giordis C , Sanes J R.

Molecular forms of N-CAM and its RNA in Developing and Denervated Skeletal Muscle

J. Cell Biology 1986; 102: 731-739

Cremin, B.J., Klein, A.

Infantile Hypertrophic Pyloric Stenosis : a ten year survey.

S. Aff. Med J. 1968; 42: 1056-1060,

Croitoru D., Nelson I., Guttman F.M.

Pyloric stenosis assosciated with M alrotation.

J. of Paediatric Surgery . 1991 ; 26 : 1276 - 1278

Cunningham B.A..

Cell adhesion molecules and the regulation of development.

Am. J. of Obstetrics and Gynaecology 1991 ; 164 : 939 -948

Cunningham R.T., Johnston C.F., Irvine G.B., Buchanan KD.

Serum neuron specific enolase levels in patients with neuroendocrine and carcinoid tumours.

Clinica Chimica Acta 1992; 212: 123 - 131

Cynober L.

Can arginine and ornithine support gut functions?

Gut 1994; supple 1 : S42 - S45

Dahl J.L., Bloom D.D., Epstein M.L., Fox D.A., P. Bass T.

Effect of chemical ablation of myenteric neurons on neurotransmitter levels in rat jejunum .

Gastroenterology 1987 ; 92 : 338 - 344

Daniloff J.K., Levi G., Grumet M., Rieger F., Edelman G.M.

Altered Expression of Neural Cell Adhesion Molecules Induced by Nerve Injury and Repair.

J. of Cell Biology 1986; 103: 929-945

Dantzig A.H., Hoskins J., Tabas L.B., Bright S., Shepard R.L., Jenkins I.E., Duckworth D.C., Sportsman J R., Mackensen D., Rosteck Jr . P R., Skatrud P.L.

Association of Intestinal Peptide Transport with a Protein Related to the Cadherin Superfamily

Science 1994; 264: 430-433

Darley-Usmar V., Radomski M.

Free Radicals in the Vasculature: The Good, The Bad, and the Ugly


Davies A.M.

Switching neurotrophin dependence

Cell Biology 1994; 43: 273-275

Daavis J.Q., McLaughlin T., Bennett V..

Ankyrin-binding Proteins Related to Nervous System Cell Adhesion Molecules: Candidates to Provide Transmembrane and Intercellular Connections in Adult Brain.

The J. of Cell Biology 1993; 121: 121 - 133

Dawson KP., Graham D.

The assessment of dehydration in congenital pyloric stenosis.

New Zealand Medical Journal 1991; 104 : 162-163

De Beurme FA., Lefebvre RA.

Comparison of of effect of vasoactive intestinal polypeptide and non adrenergic non cholinergic neurone stimlation in the cat pylorus.

Eur J. Pharmacol. 1988; 152: 71-82

De Giorgio R., Parodi J.E., Brecha N.C., Brunicardi F.C., Becker J.M., Go V.L.W., Stemini C.

Nitric Oxide Producing Neurons in the Monkey and Human Digestive System.

J. Comparative Neurology 1994; 342: 619-627

Deluca S.A.

Hypertrophic Pyloric Stenosis.

Am. Family Physician.

Desai K.M., Sessa W.C, Vane J R.

Involvement of nitric oxide in reflex relaxation-of the stomach to accomodate food or fluid.

Nature 1991; 351-353

Desai KM., Sessa WC., Vane JR.

Involvement of Vasoactive Intestinal Polypeptide in the reflex relaxation of the stomach to accomodatefood.

Nature 1991; 351: 477-479

Desai KM., Warner TD., Bishop AEB., Polak JM., Vane JR.

Nitric Oxide, and not vasoactive intestinal peptide, as the main neurotransmitter of vagally induced realaxation of the guinea pig stomach

Br J Pharmacol 1994; 113, 1197-1202

De Vault K., Rattan S.

Phyiological Role of Neuropeptides in the Gastrointestinal Smooth Muscle Sphincters: Neuropeptide and VIP-Nitric Oxide Interactions.

Gut Peptides; Biochemistry and Physiology, Editors J.H. Walsh & G.J. Dockray, Raven Press ltd. 1994

Dickson G , Gower H.J., Barton C.H., Prentice H.M., Elsom V.L., Moore S.E., Cox R.D., Quinn C., Putt W., Walsh F.S.

Human Muscle Neural Cell Adhesion Molecule (N-CAM): Identification of a Muscle-Specific Sequence in the Extracellular Domain.

Cell 1987; 50: 1119-1130

Dieler R., Schroder J.M., Skopnik H., Steinau G.

Infantile hypertrophic pyloric stenosis : myopathic type .

Acta Neuropathologica 1990 ; 80 : 295 - 306

Dieler R., Schroder J.M.

Myenteric plexus neuropathy in infantile hypertrophic pyloric stenosis.

Acta Neuropathologica 1989 ; 78 : 649 - 661

Diket AL., Pierce MR., Munshi UK., Voelker C A , Eloby-Childress S., Greenberg SS., Zhang X-J., Clark DA., Miller MJS.

Nitric oxide inhibition causes intrauterine growth retardation and himb limb disruptions in rats

Am J Obstet Gynecol 1994; 171, 5 : 1243-1250

Dockray GJ.

Mediation and Modulation of gastric afferent functions by regulatory peptides

Brain-Gut interactions,Eds. Y. Tache, D. Windgate, CRC Press, Boca Raton, ppl23-132 1991.

Dockray GJ.

Mediation and Modulation of gastric afferent functions by regulatory peptides.

Brain-Gut interactions,Eds. Y. Tache, D. Windgate, CRC Press, Boca Raton, 1991;ppl23-132 .

Dodge JA.

ABO Blood groups and Infantile Hypertrophic Pyloric Stenosis .

BMJ. 1967; 4: 781-782

Dodge JA

Infantile Hypertrophic Pyloric Stenosis in Belfast

Arch Dis Childhood 1975; 50; 171-178

Dodge J.A., Karim A. A.

Induction of Pyloric Hypertrophy by Pentagastrin.

Gut 1976 ; 17: 280 -284

Domoto T., Oki M., Kotoh T., Nakamura . T.

Heterogenous distribution of peptide containing nerve fibres within the circular muscle layer of the human pylorus.

Clinical Autonomic Research 1992 ; 2 : 403 -407

Donellan W.L., Cobb L.M.

Intraabdominal Pyloromyotomy.

J. of Paediatric Surgery 1991 ; 26 :174 - 175

Douglas S.W.

Lesions involoving the Pyloric Region of the Canine Stomach

J. Am. Vet. Radiol. Soc 1968; 9: 89-93

Du Plessis D.J.

Primary Hypertrophic Pyloric Stenosis in the Adult

British J. Surgery 1966; 53: 6; 485-492

Ebashi S.

Phosphorylation of Myosin Light Chain is not the main activation mechanism of Smooth Muscle Contraction

Biomedical Research 1993; 14: Supplement 2, 117-119

Edelman G.M.

Morphoregulatory Molecules.

Biochemistry 1988 ; 27 : 3533 - 3543

Eedery P., Lyonnet S., Mulligan L.M.

Mutations of the RET proto-oncogene in Hirschsprung's disease

Nature 1994 367-378

Edin R., Lundberg J.M., Ahlman H., Dahlstrom A., Fahrenkmg J., Hokfelt T., Kewenter J.

On the VIP-ergic innervation of the feline pylorus .

Acta Physiol 1979 ; 107 : 185 - 187

Edin R , Lundberg J., Terenius L., Dahlstrom A., Hokfelt T., Kewenter J., Ahlman H.

Evidence of Vagal Enkephalinergic Neural control of the Feline Pylorus and Stomach


Ekblad E., Mulder H., Sundler F.

Vasoactive intestinal peptide expression in enteric neurons is upregulated by both colchicine and axotomy.

Regulatory Peptides 1996; 63: 113-121

Ekblat ER, Hakanson R, Sundler F.

VIP and PHI coexist with an NPY-like substance neurons of the cat small intestine. Regul Peptides 1984; 10; 47-55

Elberger A.J., Honig M.J.

Double-labelling of tissue containing the carbocyanine Dye Dil for Immunocytochemistry.

The J. of Histochemistry and Cytochemistry 1990; 38: 735 - 739

Eriksen C A , Anders C.J.

Audit of the results of operations for infantile pyloric stenosis in a district general hospital.

Archives of Disease in Childhood 1991; 66; 130 -133

Escrig C., Bishop AEB., Inagaki H., Moscoso G., Tkasashi K., Vamdell IM., Ghatei MA., Bloom SR., Polak JM.

Localisation of endothelin like immunoreactivity in adult developing gut

Gut 1992; 33, 212-217

Evan G.I.

Old Cells never die, they just apoptose

Trends in Cell Biology 1994; 4: 191-192

Facer P., Bishop A.E., Moscoso G., Tereghi G., Liu Y.F., Goodman R.H., Legon S., Polak J.M.

Vasoactive Intestinal Polypeptide Gene Expression in the Developing Human Gastrointestinal Tract.


Fan WQ., Smolich JJ., Wild J., Yu VYH., Walker AM.

Nitric oxide modulates regional blood flow differences in the fetal gastrointestinal tract.

Am J. Physiol 1996: G598-G604

Ferri G.L., Adrian T.E., Soimero L., Blank M., Cavalli D., Bilotti G., Polak J.M., Bloom S.R.

Intramural distribution of immunoreactive V IP , substance P , somatostatin, and mammalian bombesin in the oesophago-gastro-pyloric region of the humann g u t .

Cell and Tissue Research 1989 ;

Figarella-Branger D., Nedelec J., Pellisier J.F., Boucraut J., Bianco N., Rougon G.

Expession of various isoforms of NCAM and their highly polysialylated counterparts in diseased human m uscles.

J. Neurological Sciences 1990 ; 98 :21 -36

Figarella-Branger D., Pellissier J.F., Bianco N.

Expression of Various NCAM Isoforms in Human Embryonic Muscles: Correlation with Myosin Heavy Chain Phenotypes.

J. Neuropathology and Experimental Neurology.

Fitzgerald P.G., Lau G.Y.P., Cameron G.S.

Umbilical incision for pyloromyotomy.

J. of Paediatric Surgery 1990 ; 25 : 1117-1118

Foley L.C., Slovis T.L., Campbell IB ., Strain ID ., Harvey I.E ., Luckey D. W.

Evaluation of the vomiting in fan t.

Am J. Diseases in Childhood 1989 ; 143 ; 660 - 661

Forman H P., Leonidas J.C., Kronfeld G.D.

A rational approach to the diagnosis of pyloric stenosis : Do the results match the claims


Forster EM., Green T., Dockray GJ.

Efferent pathways in the reflex control of gastric emptying in rats

Am J Physiol 1991; G499- G504

Forster ER., Southam E.

The intrinsic and vagal extrinsic innervation of the rat stomach contains nitric oxide synthase

Neuroreport 1993 4; 275-278

Frei T., von Bohlen F., Wille W., Schachner M.

Different extracellular domains of NCAM are involved in different functions.

I Cell Biology 1992; 118 : 177 -194

French I.E ., Wohlwend A., Sappino A., Tschopp J., Schifferli J.A.

Human Clustering Gene Expression is Confined to Surviving Cells during in Vitro Programmed Cell Death.

J. Clinical Investigation 1994; 93: 877-884

Friesen SR., Boley JA., et el.

Myenteric plexus of the pylorus : Its early normal development and its changes in the hypertrophic pyloric stenosis.

Surgery 1956; 39: 21,

Friesen SR., Pearce AGE.

Pathogenesis of congenital pyloric stenosis: Histochemical analyses of pyloric ganglion cells

Surgery 1963; 53: 604,

Friesen SR., James MD., Boley JO., Miller DR.

The myenteric innervation of the pylorus: its early normal development and changes in hypertrophic pyloric stenosis

Surgery 1956; 39: 21-29

Fujisawa H.

Regulation of the Activities of Ca2+/Calmodulin-Dependent multifunctional Protein Kinases


Furness JB, Costa M.

The enteric nervous system.

Edingburgh, UK: Churchill Livingston; 1986

Furness JB, Costa M, Walsh JH.

Evidence for and significance of the projection of VIP containing neurons from the myenteric plexus to the tenia coli in the guinea pig.


Furness JB., Trussell DC., Pompolo S., Bomstein JC., Maley BE., Storm- Mathison J.

Shapes and projections of neurons with immunoreactivity gamma aminobutyric acid in the guinea pig small intestine.

Cell Tiss. Res. 1989; 256: 293-301

Furness J.B., Bomstein J.C., Murphy R., Pompolo S..

Roles of peptides in transmission in the enteric nervous system.

Trends in Neuroscience 1992; 15: 66-71

Gabella G.

Structure of Muscles and Nerves in the Gastrointestinal Tract

Physiology of the Gastrointestinal Tract, Second Edition, Ed. L.R. Johnson, Raven Press 1987

Georgeson K.E., Corbin T.J., Griffen J.W., Breaux, Jr C.W.

An Analysis of Feeding Regimens After Pyloromyotomy for Hypertrophic Pyloric Stenosis

J. Paediatric Surgery 1993; Nov; 28; 1478-1480

Ali K.I., Gharaibieh M., Ammari F., Qasaimeh G., Kasawneh B.

Pyloromyotomy through a circumumbilical incision.

J. Royal College Surgeons 1992 ; 37: 175 - 176

Gibson A., Mirzazdeh S., Hobbs AJ., Moore PK.

L-NG-monomethyl L-arginine inhibit nonadrenergic, noncholinergic relaxation of the mouse anococcygeus muscle.

BrJ. Pharmacol., 1990; 99: 602-606

Gillespie JS., Liu X., Martin W.

The effects of L-arginine and NG-monomethyl L-arginine on the response of the rat anococcygeus muscle to NANC nerve stimulation.

Br. J. Pharmacol. 1989; 98: 1080-1082

Godfraind T., Morel N., Wibo M.

Pharmacological Control of Calcium Channels and Muscle Contractility


Goh D.W., Hall S.K., Gomall P., Buick R.G., Green A., Corkery J.J..

Plasma chloride and alkalaemia in pyloric stenosis .

Br. J. Surgery 1990 ; 77 : 922 - 923

Goh D.W.

Abdominal wall dehiscence following Ramstedt's operation.

Br. J. Surgery 1991 ; 78 : 761

Gomes H., Lallemand P.

Infant apnoea and gastroesophageal reflux.

Paediatric Radiology 1992 ; 22 : 8-11

Gordon L., Wharton J., Moore S.E., Walsh F.S., Moscoso G , Penketh R., Wallwork J., Taylor K.M., Yacoub M.H., Polak J.M.

Myocardial Localization and Isoforms of Neural Cell Adhesion Molecule (N-CAM) in the Developing and Transplanted Human Heart.

J. Clinical Investigation 1990; 86: 1293-1300

Goto Y., Hollinshead J.W., Debas H.T..

A New Intraoperative Test for the Completeness of Vagotomy: The PCP-GABA Test.

The American J. of Surgery 1984; 147: 159 - 163

Gotz M., Novak N., Bastymer M.

Membrane -bound molecules in rat cerebral cortex regulate thalamic innervation.

Development 1992; 116: 507 - 519

De Graan P.N.E., Gispen W.H,

The role of B-50/Gap-43 in transmitter release: Studies with permeated synaptosomes.

Biochemical Society Transactions 1993; 21: 406 - 410

Graham DA., Mogridge RN., Abbott MD,. et al.

Pyloric Stenosis the Christchurc experience

New Zealand Medical Journal 1993 104: 57-59

Greeley Jr. G.H.

Peptide YY

Biomedical Research 1993; 14:3, 89-92

Grider JR., Cable MB., Said SI., Makhlouf M.

Vasoactive Intestinal Polypeptide as a neural mediator of gastric relaxation.

Am J. Phys. 1985; 248; G73-G78

Grider, JR., Makhlouf GM.

Suppression of inhibitory neural input to colonic muscle by opioid peptides.

J. Pharmacology Exptl Therapeutics 1987; 243: 205-210


Suppression of inhibitory neural input to colonic muscle by opioid peptides.

J. Pharmacol. Exptl. Therap. 1987; 243: 205-210

Grider JR., Arimura A., Makhlouf GM.

Role of somatostatin neurons in intestinal peristalsis: facilatory intemeurons in descending pathways.

Am J. Physiol. 1987; 253: G434-G438

Grider JR.,

Identification of neurotransmitter regulating the intestinal peristaltic reflex in humans.


Grider JR.

Somatostatin (SS) neurons regulate the descending limb of the peristaltic reflex.

Gastroenterology 1991; 100: A445


Enteric G ABA: mode of action and role in the regulation of the peristaltic reflex.

Am. J. Physiol. 1992; 262: G690-G694 -

Grider, JR., K.N. Murthy, J-G. Jin, G.M. Makhlouf

Stimulation of Nitric Oxide from muscle cells by VIP; prejunctional enhancement of VIP release.

American J. Physiology 1992;262: G774-G778

Grider JR., Jin J-G.

Vasoactive intestinal polypeptide release and L-citnilline production from isolated ganglia of the myenteric plexus: evidence for regulation of vasoactive intestinal polypeptide by nitric oxide.

Neuroscience 1993; 54: 521-526

Grider J R.

Role of Neuropeptides in Peristalsis

Gut Peptides: Biochemistry and Physiology, Editors J.H. Walsh & G.J. Dockray, Raven Press ltd. 1994

Grisoni E., De Agustin J.C., Kalhan. S.C.

Vasoactive Intestinal Polypeptide causes Relaxation of the Pyloric Sphincter in the Rabbit.

J. Paediatric Surgery 1993 ; 28 : 1117 - 1120

Gronbech J.E., Lacy E.R.

Substance P Attenuates Gastric Mucosal Hyperaemia After Stimulation of Sensory Neurons in the Rat Stomach.

Gastroenterology 1994; 106: 440 - 449

Grosser O., Lewis F.T., McMurrich J.P.

The Development of the digestive tract and organs of respiration.

Manual of Human Embryology. Eds. F. Kiebel, F.P. Hall. P. A. Philadelphia, Lippincott, 1912; Chapter 17: 291-497

Grundy D., Gharib-Naseri MK., Hutson D.

Role of nitric oxide and vasoactive intestinal polypeptide in vagally mediated relaxation of the gastric corpus in the anaesthetized ferret.

J. Auton. Nerv. Sys. 1993; 43: 241-246

Gulbenkian S, Wharton J , Polak J.

The visualization of cardiovascular innervation in the guinea pig using an antiserum to protein gene product 9.5 (PGP 9.5)

J. Auton Nerv Syst 1987; 18: 235-247

Guslandi M.

Nitric Oxide: An Ubiquitous Actor in the Gastrointestinal Tract.

Digestive Disease 1994; 12-36

Hadzijahic N., Freeman W.E., Ma C.K., Zhang X , Fogel R.

Myenteric Plexus Destruction alters the Morphology of Rat Intestine.


Hagger R., Finlayson C , Jeffrey!., Kumar D.

R. Hagger, C. Finlayson, I. Jefffey, D. Kumar.

Role Of Interstitial Cells Of Cajal In The Control Of Gut Motility

BIS 1997; 84: 445-450

Hambourg MA., Mignon M.,

Serum gastrin levels in hypertrophic pyloric stenosis in infancy .

Archives Disease Childhood. 1979; 54: 208,

Hayashi A.H., Giacomantonio J.M., Lau H.Y.C, Gillis DA.

Balloon catheter dilatation for hypertrophic pyloric stenosis.


Hebeib K., Kilbinger H.

Differential effects of nitric oxide donors on basal and electrically evoked release of acetylcholine from guinea-pig myenteric neurones.

Br. J. Pharm 1996; 118: 2073-2078

Heinisch HM.

Die sog: Pylorischhypertrophic

Tierversuch. Klin Wochenscher 1967; 45: 1251,

Heiss L.N., Flak T.A., Lancaster,Jr. J R. McDaniel M L., Goldman W.E.

Nitric Oxide Mediates Bordetella pertussis Tracheal Cytotoxin Damage to the Respiratory Epithelium.

Infectious Agents and Disease 1994; 2: 173-177

Hellstrom PM., Ljung T.

Nitrergic inhibition of migrating myoelectric complex in the rat is mediated by vasoactive intestinal polypeptide

Neurogastroenterol. Mot. 1996; 8: 299-306

Henderson JL., Mason Brown JJ.

Clinical observations on pyloric stenosis in premature infants.

Archives Disease Childhood., 27, 175-178

Henning S.J

Functional Development of the Gastrointestinal Tract.

Physiology of the Gastrointestinal Tract, Second Edition, Ed. L.R. Johnson, Raven Press 1987

Hershovitz E., Steiner Z , Shinwell E.S., Meizner I.

Prenatal Ultrasonic Diagnosis of Nonhypertrophic Pyloric Stenosis Associated with Intestinal Malrotation

J. Clin Ultrasound 1994; 22: 52-54

Hibbs JB., Taintor RR., Vavrin Z.

Macrophage cytotoxicity : role for L-arginine deiminaseand imino nitrogen oxidation to nitrite.

Science 1987; 235: 473-476

Hibbs JB., Vavrin Z., Taintor RR.

L-Arginine is required for expression of the activated macrophage effector mechanism causing selective metabolic inhibition of target cells.

J. Immunol. 1987; 138: 550-565

Hirschsprung H

Falle van angeborenen Pylonisstenose,

beobachtet bei Saenlingen. Jb. Kinderheilk 1888; 28: 61

His W.

Anatomie menschlicher Embryonen.

Zur Geschichte der Organe.- Leip-zig 1885

Hitchcock Bishop A.E., L. Spitz, Polak J.M.

A Quantitative study of abnormal peptide innervation in oesophageal achalasia in children.

Unpublished manuscript.

Hitchcock Pemble M.J., Bishop A.E., Spitz L., Polak J.M.

The Ontogeny and Distribution of Neuropeptides in the Human Fetal and Infant Oesophagus.

Gastroenterology 102 (3): 840-848 1992

Holzer P, Holzer-Petsche U, Leander S.

A tachykinin antagonist inhibits gastric emptying and gastroduodenal transit in the rat.

Br. J. Pharmacol 1986; 89: 453-459

Horton R.T.

Pyloric Musculature

Am J. Anatomy, 41, 2:197-225

Hoyle C H., Kamm M.A., Bumstock G., Leonard-Jones J.E.

Enkephalins modulate inhibitory neuromuscular transmission in circular muscle of human colon via opioid receptors.

J Physiology (London) 1990; 431: 465-478

Hsu SM, Raine L, Franger H

Use of avidin-biotin-peroxidase complex (ABC)in immunoperoxidase techniques

J Histochem Cytochem 1981; 29: 577-580 J Histochem Cytochem 1981; 29: 577-580

Huang P.L., Dawson T.D., Bredt D.S. Snyder SH., Fischman MC.

Targeted Disruption of the Neuronal Nitric Oxide Gene.

Cell 1993 ;75: 1273 - 1286

Huddy S.P.J.

Investigation and diagnosis of hypertrophic pyloric stenosis.

J. of the Royal College of Surgeons of Edinburgh 1991 ; 36 : 91 - 93

Hurko 0., Walsh F.S.

Human muscle-specific antigen is’restricted to regenerating myofibres in regenerating myofibres in diseased adult muscle.

Neurology 1983; 33; 737-743

Hutterer J., Banekovitch M., Fuchs D., Watcher H.

Biochemical and Clinical Aspects of Pteridines.

From Michael

Ichiyanagi S., Sugiyama T., Ochiai T., Ogawa A., Fujita N., Endo K.

Expression of NCAM IN healing gastric ulcers.

Gastroenterologica Japonica 1992; 27 : 559

Itoh N, Obatah K, Yanaihara N, Okamata H.

Human preprovasoactive intestinal polypeptide contains a novel PHI-27-like peptide, PHM-27.

Nature 1983; 304: 547-549

lyenger R., Stuehr DJ.,Marietta MA.

Macrophage synthesis of nitrite, nitrate, and nitrosamines: precursors and role of the respiratory burst.

Proc Natl Acad Sci USA 1987; 84: 6369-6373

Jackson P, Thompson RJ.

The demonstration of new brain-specific proteins by high resolution two dimensional polyacrylamide gel electrophoresis.

J. Neurol Sci 1981; 49: 429-438

Jin L., Hemperly J.J., Lloyd R.V.

Expression of NCAM in Normal and Neoplastic Human Neuroendocrine Tissues.

Am. J. Pathology 1991 ;138 : 961 - 968

Johnson C F., Peterson R.M., Koch R., Gross Friedman E.

Congenital and Neurological Abnormalities in Infants with Phenylketonuria.

American J. Mental Deficiency 1978 ; 4 : 375 - 379

Jona J.Z.

Electron microscopic observations in Infantile Hypertrophic Pyloric stenosis.

J. of Paediatric Surgery 1978 ; 13 ; 17-20

Kadota T., Mimura K., Kanabe S., Oshaki Y., Tamkauma S.

Proximal gastric vagotomy with carbon dioxide laser: Experimental studies in animals.

Surgery 1990; 107: 655-660

Keef K., Du C , Ward S., Mcregor B., et al.

Enteric Inhibitory Neural Regulation of Human Colonic Circular Muscle : Role of Nitric O xide.


Kelly E.J., Lagopoulos M., Primrose J.N.

Immunocytochemical localisation of Parietal cells and G cells in the developing human stomach.

Gut 1993 ; 34 : 1057 - 1059

Kelsey D. Stayman JW. ,McClaughlin , ED., Mebane, W.

Massive bleeding in a newborn infant from a gastric ulcer associated with hypertrophic pyloric stenosis.

Surgery 1976; 64: 979-989

Keynes, W.M.

Pyloric Stenotic Hypertrophy in Adults

Gut, 1965; 6: 240

Kimura S, Okada M, Sugita Y, Kanazawa T, Munekata E.

Novel neuropeptides, neurokinin alpha and beta, isolated from porcine spinal cord.

Proc Jap Acad 1983 59B: 101-104

Kirschgessner AL, Gerson MD.

Identification of vagal efferent fibres and putative target neurons in the enteric nervous system of the rat.

J. Comp. Neurol. 1989; 285: 38-53

Knowles R.G., Moncada S.

Nitric oxide synthases in mammals

J. Biochemistry 1994; 298: 249-258

Knowles R.

Nitric Oxide Synthases


Knox E.G., Armstrong E., Haynes R.

Changing incidence of hypertrophic pyloric stenosis.

Archives of Disease in Childhood . 1983 ; 58 : 582 - 585

Knudsen K., Mcelwee S.A., Myers L.

A Role for NCAM in myoblast interaction during myogenesis.

Dev. Biology 1990 ; 138 : 159 - 168

Kobayashi H., O'Brien D.S., Puri P.

Selective reduction in intramuscular nerve supporting cells in infantile hypertropic pyloric stnosis.

B.A.P.S. Aug 1993

Kobayashi H., O'Briain D.S.; Puri P.

Immunochemical characterisation of Neural Cell Adhesion Moecule (NCAM),Nitric Oxide Synthase and Neurofilament Protein expression in Pyloric stenosis.

Unpublished manuscript. 1994

Kobayashi H., O'Briain D.S., Puri P.

Selective Reduction in Intramuscular Nerve Supporting Cells in Infantile Hypertrophic Pyloric Stenosis

J. of Paediatric Surgery 1994; 29: 5, 651-654

Kobayashi H., O'Briain D.S., Puri P.

Defective Cholinergic Innervation in pyloric muscle of patients with hypertrophic pyloric stenosis.

Pediatric Surgery International 1994; 9 : 338-341

Koop H., Frank M., Kuly S., Nold R., Eiselle R., Rager G , Ruschoff J., Rothmund M., Arnold R.

Gastric argyrophil (enterograffin-like), gastrin, and somatostatin cells after proximal selective vagotomy in man.

Dig Dis Sci 1993; 38: 295-302

Koshland, Jr. D.E.

The Molecule of the Year.

Science 1992; 258: 1861-1863

Krause YT., Kempinnen P., Carter MS., Xu ZS , Hershey AD.

Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A.

Proc Natl. Acad. Sci USA 1987; 84: 881-885

Kressel M., Berthoud H R., Neuhber W.L..

Vagal innervation of the rat pylorus: an antegrade tracing study using carbocyanine dyes and laser scanning confocal microscopy.


Kuwayama H., Suzuki M., Inoue A., Kobayashi H., Tanaka T., Mikawa T., Sugiura M., Ebashi S.

Amino acid sequence of Myosin Light chain kinase from bovine stomach with speial references to its Actin binding Domain.

Biomedical Research 1993; 14: Supplement 2,113-116

Lamki N., Athey P.A., Round M E., Watson A.B., Pfleger M.J.

Hypertrophic Pyloric Stenosis in the neonate - diagnostic criteria revisited.

J. of the Canadian Assocn. of Radiologists 1993 ; 44 : 21 - 24.

Lammel EJ., Edmonds LD.,

Trends in pyloric stenosis incidence, Atlanta, 1968 tol982.

J.Med Genet. 1987;24: 482-487

Lancaster, Jr J.R.

Nitric Oxide in Cells.

American Scientist 1992 ; 80 ; 248 -259

Lander A.

Comparison of postpyloromyotomy feeding regimens in infantile hypertrophic pyloric stenosis .

J. Royal College of Surgeons Edingburugh 1992 ; 37 ; 137

Langer JC., Berezin I , Daniel EE.

Hypertrophic Pyloric Stenosis: Ultrastnictural Abnormalities of Enteric Nerves and the Interstitial Cells of Cajal

J. Paediatric Surgery 30; 11: 1535-1543

Larsson LT, Polak JM, Buffa R, Sundler F, Solicia E.

On the immunohistochemical localization of vasoactive intestinal polypeptide.

J. Histochem Cytochem. 1979; 27: 936-938

Lawson, D

The incidence of pyloric stenosis in Dundee.

Arch Dis Child. 1951; 26: 616-617,

Leach J.L., Johnson III. J.F.

Antral web : assoociation with hypertrophic pyloric stenosis.

Paediatric Radiology 1993 ; 23 ; 206

Lee CM., Campbell NJ., Williams BJ., Iversen LL.

Multiple tachykinin binding sites in peripheral tissues and in brain.

Eur J. Pharmacol 1986; 130: 209-217

Leib MS., Saunders GK., Moon ML., Mann MA., Martin RA., Matz ME, Nix B., Smith MM., Waldron DR.

Endoscopic Diagnosis of Chronic Hypertrophic Pyloric Gastropathy in Dogs.

J. Veterinary International Medicine 1993;7(6); 335-341

Leib MS., Saunders GK., Moon ML., Mann MA., Martin RA., Matz ME ,Nix B.

Endoscopic Diagnosis of Chronic Hypertrophic Pyloric Gastropathy in Dogs.

J. Veterinary International Medicine 1993; 7(6): 335-341

Letoumou P.C., Condic M L., Snow D M.

Interactions of Developing Neurons with the Extracellular Matrix.

The J. of Neuroscience 1994; 14: 915 - 928

Li CG.jRand MJ.

Evidence that part of the NANC relaxant response of guinea pig trachea to electrical field stimulation is mediated by nitric oxide.

BrJ. Pharmacol., 1991; 102: 91-94

Lidberg J.M., Edin R., Lundberg J.M., Dahlstrom A., Rosell S., Folkers K., Ahlman H.

The involvement of substance P in the vagal control of the feline pylorus.

Acta Physiol Scand 1982; 114 : 307 -309

Lindquist S.

Thge heat shock response

Annu Rev Biochem 55: 1151-1191

Lippert M.M.

Jaundice with hypertrophic pyloric stenosis.

J. of Paediatrics .1990 ; 117 : 168 - 169

Ljungdahl A, Hokfelt T, Nilsson G.

Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals.

Neuroscience 3: 861-943

Llewellyn-Smith IJ., Furness JB., Gibbins IL., Costa M.

Quantitative ultrastnictural analysis of enkephalin, substance P, and VTP immunoreactive nerve fibres of the guinea pig small intestine .

J. Comp. Neurol. 1988; 272: 130-148

Llewely-Smith IJ., Song Z-M., Costa M., Bredt DS., Snyder SH.

Ultrastnictural localisation of nitric oxide synthase immunoreactivity in guinea pig enteric neurons.

Brain Res 1992; 577: 337-342

Lloyd K.C.K.

Gur Hormones in Gastric Function.

Balliere's Clinical Endocrinology and Metabolism 1994; 8, 1: 111-135

Loewi O.

Uber humerorale Ubertragarkeit der Herznervenwirkung -II. Mitteilung.

Pflugers Arch Physiol 293: 201-213

Loewi O.

Uber humorale Ubertragbarkeit der Herznervenwirkkung - II Mitteilung.

Pfulgers Arch Physiol 1921; 293: 201-213

Ludtke F-E, Bertus M., Michalski S., Dapper F.D., Lepsien G.

Gastric Emptying 16-26 Years After Treatment of Infantile Hypertrophic Pyloric Stenosis

J. Paediatric Surgery 1993; Apr; 29: 523-526

Ludtke F-E, Bertus M., Michalski S., Dapper F.D., Lepsien G.

Long-term Analysis of Ultrasonic Features of the Antropyloric Region 17-27 Years After Treatment of Infantile Hypertrophic Pyloric Stenosis

J. Clin Ultrasound 1994; 22: 299-305

Lundberg JM,

Evidence for the coexistence of VIP and acetylcholine in neurons of cat exocrine glands.

Acta Physiol Scand 1981; 496; 1-57

Lynn HB.

The mechanism of pyloric stenosis and its relationship to preoperative preparation .

Arch Surg 1960; 81: 453,

Mabuchi I.

Regulation of Cytokinesis in Animal Cells: Possible involvement of Protein Phosphorylation


Macdessi J., Oates . R.K.

Clinical Diagnosis of pyloric stenosis : a declining a r t .

Br. Medical Journal 1993 ; 306 : 553 -555

MacDonald A., Rylance GW, Asplin DA, Hall K, Harris G, Booth IW

Feeding problems in yoimg PKU children

ActaPaediatr Suppl 1994; 407: 73-4.

Madarikan B.A., Rees B.I..

Hallitosis and gastric outlet obstruction in infants.

Br. J. Clinical Practice 1990 ; 44 :419

Malmfors G., Sundler F.

Peptidergic iimervation in Infantile Hypertrophic Stenosis.


Mandell, G.A.

Association of antral diaphragms and Infantile Hypertrophic Pyloric Stenosis .

J. Paediat. 1978; 47: 95-99,

Marietta M A.

Nitric Oxide Synthase Structure and Mechanism,

j. Biological Chemistry 1993, 268, 17: 12231-12234

Mason P.P.

Increasing infantile hypertrophic pyloric stenosis ? Experience in an overseas military hospital.

J. of the Royal College of Surgeons Edingbugh 1991 ; 36 : 293 - 294

Matthiesen DT., Walter MC.

Surgical treatment of chronic hypertrophic pyloric gastropathy in 45 dogs.

J. Am. Anim. Hosp Assoc 1986; 22:241-247,

Mayer BJ., Heinzel BM., Werner ER., Wachter H., Schultz G., Bohme E.

Brain nitric oxide synthase is biopterin- and flavin-containing multifunctional oxidoreductase.

Fed. Eur. Biol. Soc. 1991; 288: 187-191

Mayer E.A., Schufifler M.D., Rotter J.I., Hanna P., Mogard M.

Familial visceral neuropathy with autosomal dominant transmission.


McLain CR.

Amniography studies of the gastrointestinal motility of the human fetus.

Am. J. Obstet Gynaecol 1963; 86: 1079-1087

McConalogue K., Furness J.B.

Gastrointestinal neurotransmitters.

Balliere's Clinical Endocrinology and Metabolism 1994; 8: 1: 51-76

McDonald J.D., Bode. V.C.

Hyperphenlalanaemia in the hph-1 Mouse Mutant

Paediatric Research 1988; 23: 63 - 67

McDonald J.D., Cotton R.G.H., Jennings I., Ledley F.D., Woo S.L.C., Bode V.C.

Biochemical Defecct in the hph-1 Mouse Mutant is a Deficiency in GTP-Cyclohydrolase Activity.

J. ofNeurochemistry 1988; 50(2): 655-657

McDonald J.D., Cotton R.G.H., Jennings I , Ledley F.D., Woo S.L.C., Bode V.C.

Hyperphenylalanaemia in the hph-1 Mouse Mutant

Pediatric Research 1988; 23: 1, 63-67

McDonald J.D., Bode V.C., Dove W.F.

Pah hph-5: A mouse mutant deficient in phenylalanine hydroxylase.

Procedings National Academy Science 1990; 87: 1965 - 1967

McGreegor GP, Gibson SJ, Sabate IM, Blank MA, Christofides ND, Wall PD, Polak JM, Bloom SR

Effect of peripheral nerve section and nerve crush on the spinal cord neuropeptides in the rat; increased VIP and PHI in the dorsal horn.

Neuroscience 1984;13:207-216,

Mearin F., Mourelle M., Guarner F., Salas A., Riveros-Moreno V., Moncada S.

Patients with achalasia lack nitric oxide synthase in the gastro - oesophageal junction.

European J. of Clinical Investigation 1993; 23: 724 - 728

Mercado-Deane M.G., Burton E.M., Brawley A.V., Hatley R.

Prostaglandin - induced foveolar hyperplasia simulating pyloric stenosis in an infant with cyanotic heart disease

Paediatric Radiology 1994; 24: 45-46

Merlie J.P., J. Mudd, Chang T.C.

Myogenin and Acetylcholine Receptor alpha Gene Promotors mediate Transcriptional Regulation in Response to Motor innervation.

The J. of Biological Chemistry 1994; 269; 2461 - 2467

Michel T.

Nitric oxide synthase activity in Infantile Hypertrophic Pyloric Stenosis.

New England Journal of Medicine 1992; 327: 1690

Miettinen M., Cupo W.

Neural Cell Adhesion Molecule in Soft Tissue Tum ors.

Human Pathology 1993 ; 24 : 62 - 66

Mili F., Khoury M.J., Flanders W.D., Greenberg R.S.

Risk of childhood cancer for infants with Birth Defects.

Am . J. Epidemiology 1993 ; 137 : 629 -643

MillaP.J.

Gastric outlet obstruction in children.

New England J. Medicine 1992 ; 20 : 558 - 560

Mills T.N.

History and Introduction to Lasers

Manuscript

Missenger J.P., Bomstein J.C., Furness J.B.

Elecrophysilogical and Morphological Classification of Myenteric Neurons in the Proximal Colon of the Guinea-Pig

Neuroscience 1994; 60: 1, 227-244

Mitchell L.E., Risch N.

The Genetics of Infantile Hypertrophic Stenosis

American J. Diseases in Childhood 1993; 147: 1203-1211

Miura S., Fukumura D., Kurose I., Higuchi H., Kimura H., Tsuzukii Y., Takeharu S., Han J-Y., Tsuchiya M., Ischii H.

Roles of ET-1 in endotoxin-induced microcirculatory disturbance in rat small intestine.

Am J. Physiol 1996: G461-G469

Molenaar J.C, Tibboel D., van der Kamp A.W.M., Meijers J.H.C.

Diagnosis of Innervation Related Motility Disorders of the Enteric Nervous System Development.

Progress in Paediatric Surgery 1989; 24 : 13 -185

Moncada S., Palmer Higgs E.A.

Nitric Oxide ; Physiology, Pathology, and Pharmacology.

Pharmacological Review 1991; 43 ; 109-142

Morgagni GB.

Adult Hypertrophic Pyloric Stenosis

Mayo Clinic Proc 1971; 46: 692-694

Moss S., Legon S., Bishop AEB., Polak JM., CalamJ.

Effect of Helicobacter pylori on gastric somatostatin in duodenal ulcer disease

Lancet 1992; 340: 930-932

Muayed R., Zaber K., Young DG., Raine PAM.

Pyloric Stenosis in a sick premature infants.

Lancet, ii, 1984; 344-345,

Murray J. A., Clark E.D.

Characterisation of Nitric Oxide Synthase in the Opossum Esophagus


Najmanldin A., Tan H.L.

Early Experience with Laparoscopic Pyloromyotomy for Infantile Hypertrophic Pyloric Stenosis

J. Paediatric Surg 1995; 30 (1):37-38

Nandha K.A., Bloom S.R.

Neuromedin U - An Overview

Biomedical Research 1993; 14: 3, 71-76

Nichols K., StainesW., Krantis A.

Nitric Oxide Synthase Distribution in the Rat Intestine: A Histochemical Analysis


Nonomura Y.

Multifunctional role of Myosin Molecules in various kinds of cells


Nour S., Mangall Y., Dickson J.A.S., Pearse R., Johnson A.G.

Measurement of gastric emptying in infants with pyloric stenosis using applied potential timography.

Archives of Disease in Childhood 1993 ; 68 : 484 -486

Nowicki PT.

The Effects of Ischaemia-Reperfusion on Endothelial Cell Function in Postnatal Intestine.

Pediatric Research 1996; 39 (2): 267-273

Nussier A.K., Billiar T. R

Inflammation, immunoregulation, and inducible nitric oxide synthase.

J. of Leukocyte Biology 1993; 54: 171 - 178

ODoimell C., Liew E.

Immunological Aspects of Nitric Oxide


Okazaki T., Yamataka A., Fujiwara T., Nishiye H., Fujimoto T., Miyano T.

Abnormal Distribution of Nerve Terminals in Infantile Hypertrophic Pyloric Stenosis

J. of Paediatric Surgery 1994 29: 5, 655-658

Okorie N.M., Dickson J.A.S., Carver R.A., Steiner G.M.

What happens to the pylorus after pyloromyotomy?

Archives of Disease in Childhood, 1988; 63: 1339-1340

Olson E.N.

Signal transduction pathways that regulate skeletal muscle gene expression.

Molecular Endocrinology 1993; 93: 1369 - 1378

Orrego F

Criteria for the identification of central neurotransmitters and their application to studies with some nerve tissue preparations in vitro.

Neuroscience 4: 1979; 1037-1057

Osborne NN.

Evidence for co-transmission by specific neurones.

In: Osborne NN (ed), Dale's principle and communication between neurones. Pergamon press, Oxford, New York. pp. 49-68

Palmer RMJ., Ferrige AG., Moncada S.

Nitric oxide release accounts for the activity of endothelium derived relaxing factor

Nature 1987; 327: 524-526

Parag HA, Raboy B, Kulka RG

Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system.

EMBO 1987; J 6: 55-61

Parodi JE, Cho N, Zenilman ME, Barteau JE.

Substance P stimulates the Opposum sphincter of oddi in vitro.

J. Surg Research 1990; 49: 197-204

Patten BM.

Human Emryology

New York Philadelphia 1957

Pearson H.

Pyloric Stenosis in the dog

The Veterinary Record 1979; 105; 393-394

Peeters ME.

Pylonisstenose bij de bond: ontwikkelingen in de chinirgische behandeling en retropectief onderzoek bij 47 patienten. Pyloric stenosis in the dog: developments in surgical treatment and a retrospective study in 47 patients.

Tijdschr. Diergeneeskd., 1991; deel 116, afl.3

Peled N., Dagan O , Babyn P., Meredith M., Barker MD., Heilman J., Scolnik D., Korean G.

Gastric outlet obstruction induced by prostaglandin therapy in neonates .

New England J. of Medicine 1992 ; 327 : 505 - 510

Peter N., AronofFB.

Decrease in Growth Cone-Neurite Fasciculation by Sensory or Motor Cells in vitro Accompanies Downregulation of Aplysia Cell Adhesion Molecules by Neurotransmitters.

The J. ofNeuroscience 1994; 14: 1413 - 1421

Polak J.M., Van Noorden . S.

Immunocytoshemistry.

Immunocytochemistry . Springer Verlag 1989

Prichard J.

Fetal swallowing and amniotic fluid volume.

Obstet Gynecol 1966; 28: 606-610

Prieto A.L., Crossin K.L., Cunningham B.A., Edelman G.M.

Localization of mRNA for neural cell adhesion molecule (N-CAM) polypeptides in neural and nonneural tissues by in situ hybridisation.


Prince R.G.

Rising interest in nitric oxide synthase.

TIBS Feb 1993; 35-36

Pobstmeier R., Fahrig T., Spiess E., Schachner M.

Interactions of the Neural Cell Adhesion Molecule and the Myelin -associated Glycoprotein with collagen Type I .Involvement in Fibrillogenesis

J. CeliBiologyl992;116; 1063-1070

Probstmeier R., Blitz A., Schneider-Schaulies J.

Expression of the Neural Cell Adhesion Molecule and Polysialic Acid During Early Mouse Embryogenesis.

J. of Neuroscience Research 1994; 37: 324 - 335

Publicover N.G., Hammond E.M., Sanders K.M.

Amplification of Nitric Oxide signaling by interstitial cells isolated from canine colon.

Proc. Natl. Acad. Sci. USA 1993; 90: 2087-2090

Pullock, W.F., Norris, W.J., Gordon, H E.,

The management of Infantile Hypertrophic Pyloric Stenosis at the Los Angeles Children Hospital: a review of 1,422 cases.

Am J. Surgery 1957: 94, 335

Qureshi A., Leahy A., Darzi A., Kay E., Grace P., Geraghty J., Bouchier - Hayes D.

Beneficial effects of defocussed carbon dioxide laser irradiation in experimental peptic ulceration.

Br. J. Surg. 1992; 79 .1219

Qureshi A., Darzi A., Leahy A., Bouchier - Hayes D.

The effect of defocussed carbon dioxide laser irradiation on gastric acid secretion.


Radomski MW., Palmer RMJ., Moncada S.

An L-Arginine/nitric oxide pathway present in human platelets regulates aggregation.

Proc Natl Acad Sci USA 1987; 87: 5193-5197

Raine PAM., Goel KM., Young DGET AL

Pyloric Stenosis and transpyloric feeding.

Lancet. 1982; 2; 821-822

Rao N., Youngson G.G.

Wound sepsis following Ramstedt pyloromyotomy.

Br. J. Surgery 1989 ; 76 : 1144 - 1146

Rasmussen L., Green A., Hansen LP.

The epidemiology of Infantile Hypertrophic Pyloric Stenosis in a Danish Population, 1950-84.

Int J. Epidemiol. 1989; 18: 413-417

Rasmussen L., Hansen LP., Qvist N., Pedersen SA.

Infantile Hypertrophic Pyloric Stenosis and Subsequent Ulcer Dyspepsia

Acta Chir Scand 1988; 154: 657-658

Rattan S., Chakder S.

Role of nitric oxide as a mediator of internal anal sphincter relaxation.

Am J. Physiol 1992; 262: G107-G112

Ravitch M.M.

The Story Of Pyloric Stenosis

Surgery 1960; 48: 1117-1143

Reinecke M, Schluter P, Yanaihara N, Forssman N

VIP immunoreactivity in the enteric nerves and endocrine cells of the vertebrate gut.

Peptides 1981; 2(Suppl. 2): 149-156

Rieger F., Grumet M., Edelman G.M.

N-CAM at the Vertebrate Neuromuscular Junction

J. Cell Biology 1985; 101: 285-293

Rodrigo J., Polak JM., Fernandez L., Ghatei MA,. Muldberry P., Bloom SR.

Calcitonin Gene related peptide immunoreactive sensory and motor nerves of the rat, cat, and monkey oesophagus.


Rogers IM., Drainer IK.,

Plasma gastrin levels in congenital hypertrophic pyloric stenosis : a hypothesis disproved?

Archives Disease Childhood. 1975; 50:467,

Rollins M.D., Shields M.D., Quinn R.J.M., Wooldridge M.A.W.

Pyloric stenosis : congenital or acquired .

Archives of Disease in Childhood 1989 ;64 : 138 - 147

Rollins M.D., Shields M.D., Quinn R.J.M., Woolridge MAW.

Value of ultrasound in differentiating causes of persistent vomiting in infants.

Gut 1991 ;45 : 612-614

Rollins M.D., Shields M.D.

Clinical diagnosis of pyloric stenosis.

British Medical Journal. 1993 ;306 : 1065 - 1066

Romanska H.M., Bishop A.E., Brereton R.J., Spitz L., Polak J.M.

Increased expression of muscular Neural Cell Adhesion Molecule in Congenital Aganglionosis.


Romanska H.M., Bishop A.E., Brereton R.J., Spitz L., Polak J.M.

Immunocytochemistry for neuronal markers show deficiencies in Conventional Histology in the treatment of Hirschsprung's Disease.


Romanska - Knight H. M., Bishop A.E., Brereton R.J., Spitz L., Polak J.M.

Increased expression of NCAM in congenitally aganglionic bowel.


Romanska H., Bishop A.E., Moscoso G., Spitz L., Polak J.M.

NCAM expression in nerves and muscle of the developing human large bowel.

Manuscript accepted for publication by Gastroenterology.

Romeo G., Ronchetto P., Leo Y.

Point mutations affecting the tyrosine kinase domain of the RET proto-oncogene in Hirschsprung's disease.

Nature 1994; 367-377

Roth B., Statz A., Heinisch H.

Jaundice with hypertrophic pyloric stenosis : a possible manifestation of Gilbert's syndrome .

J. of Paediatrics . 1990 ; 116 : 1003

Rothstein RD, Johnson E, Ouyang A.

Substance P: mechanisms of action and receptor distribution at hte feline ileocaecal sphincter region.

Am J. Physiol 1989; 49; 197-204

Rothstein RD., Johnson E., Ouyang A.

Distribution and Density of Substance P Receptors in theFeline gastrointestinal tract using autoradiography

Gastroenterology 1991; 100 : 1576-1581

Rougon G., Dubois C , Buckley N., Magnani J.L., Zollinger W.

Monoclonal antibody against Meningococcus Group B Polysaccharids Distinguishes Embryonic from Adult NCAM .

J. Cell Biology 1986 ; 103 : 2429 -2437

Rutishauser U., Acheson A., Hall A.K., Mann D M., Sunshine J.

The Neural Cell Adhesion Molecule (NCAM) as a regulator of Cell-Cell interactions.

Science 1987; 240: 53-57

Said SI.

Vasoactive Intestinal Polypeptide (VIP)

Raven Press New York. 1982

Salenius P.

Ontogenesis of the Human gastric epithelial cells.

Acta Anat 1962; 50; 7-70

Sams V.R., Bobrow L.G., Happerfield L., Keeling J.

Evaluation of PGP 9.5 in the Diagnosis of Hirschsprung's disease.

J. of Pathology 1992; 168: 55 - 58

Sandler A.D., Scmidt C , Richardson K., Murray J., Maher J.W., Donahue P.C., Bass B.L., Mulvihill S.J.

The regulation of distal oesophageal blood flow - the Roles of Nitric Oxide and Substance P .

Surgery 1993 ; 114 : 285 -294

Saria A., Troger J., Kirchmair R., Fischer-Colbrie R., Hogue-Angeletti R., Winkler H.

Secretoneurin Releases Dopamine from Rat striatal slices : a Biological effect of a peptide derived from Secretogranin II ( Chromogranin C )

Neuroscience 1993 ; 54 : 1 - 4

Scharli, A.F., Leditsche, J.F.,

Gastric motility after pyloromyotomy in infants: a reappraisal of postoperative feeding.

Surgery 1968; 64: 1133-1137

SchleifFer R., Raul F.

Prophylactic administration of L-arginine improves the intestinal barrier function after mesenteric ischaemia

Gut 1996; 39: 194-198

van der Schouw Y.T., van der Velden M.T.W., C. Hitge-Boetes, A.L.M. Verbeek, S.H.J. Ruijs

Diagnosis of Hypertrophic Pyloric Stenosis : Value of Sonography when used in Conjunction with Clinical Findings and Laboratory Data

American J. Roentology 1994; 163: 905-909

Schurmann G., Bishop AEB., Facer P.,Eder U., Fischer-Colber R., Winkler H., Polak JM.

Secretoneurin: a new peptide in the human enteric nervous system

Histochem Cell Biol 1995; 104: 11-19

Schwab M E..

Experimental aspects of spinal cord regeneration.

Neurology and Neurosurgery 1993; 6: 549 - 553

Scorpio R.J., Beasley S.W.

Pyloromyotomy: why make an easy operation difficult?

J. Royal College of Surgeons Edingburgh 1993; Oct; 38(5): 299-301

Scriver C.R, Kaufman S., Woo S.L.C.

The Hyperphenylalaninemias

Chapter 15 ? RPMS Library textbook

Schwyzer

A Case of Congenital Hypertrophy and Stenosis of the Pylorus

New York Med. J., 1896; 64: 674,

Sessa W.C.

The Nitric Oxide Synthase family of Proteins.

J. Vascular Surgery 1994; 31: 131-143

Shaw A.

Physical examination for ' initial evaluation ' of hypertrophic pyloric stenosis

Am. J. Diseases in Childhood 1990 ; 144 : 139

Shaw R.B.

A Simple Technique for Assuring the Complteness of a Pyloromyotomy

J. American College Surgery 1994; Mar; 178(3); 303-304

Shen Z , Klover - Stahl B., Larson L.T., Malmfors G., Ekbland E., Sandier F.

Peptide containing neurons remain unaffected after intestinal autotransplantation : an experimental study in the p ig let.

European J. Pediatric Surgery 1993 ; 3 : 271 - 277

Shim, WKT., Campbell, A., Wright, SW

Pyloric Stenosis in the racial groups of Hawii

J. Paediat 1970; 76: 89-93.,

Shu Y., Ju G., Fan LZ

The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system

Neurosci Lett 1988; 85: 169-171

Sikes RI, Birchard S, Patnaik A.

Chronic hypertrophic pyloric gastropathy: a review of 16 cases.

J. Am. Anim. Hosp Assoc. 1986; 22: 99-104

Sikkema DA., Layton C.

What is your Diagnosis?

JAVMA201;4: 631-632

Da Silva AL, Petroianu A.

Generalized Smooth Muscle Hypertrophy

Rev Paul Med Jul/Aug 1992; 110 (4): 180-182

Souquet JC, Grider JR, Bitar KN.

Receptors for mammalian tachykinins on isolated smooth muscle cells.

Am J. Physiol 1985; 249: G533-G538

Skofitsch G., Jacobwitz DM.

Metabolism of Substance P and bradykinin by human neutrophils.

Biochem Pharmacol 1985; 41: 1335-1344

Matthiesen D.T

Chronic Gastric Outflow Obstruction.

Textbook of Small Animal Surgery

Sobue K., Hayashi K., Yano H., Haruna M.

Molecular approach for Smooth Muscle Differentiation: Association of caldesmon isoformal interconversion with phenotypic modulation of Smooth Muscle Cells


Soediono P., Belai A., Bumstock G.

Prevention of Neuropathy in the Pyloric Sphincter of Streptozocin - Diabetic induced rats by Gangliosides.

Gastroenterology 1993; 104 : 1072 - 1082

Solowiejczyk M., Holtzifian M., Michowitz M.

Congenital Hypertrophic Pyloric Stenosis: a long-term follow-up of 41 cases.

Am Surgeon 1980; 46:567-571

Spicer R.D.

Early postoperative feeding - a continuing controversy in pyloric stenosis .

J. of the Royal Society of Medicine 1990 ; 83 : 58

Spitz L., Kaufinann J.C.E.

The neuropathological changes in congenital hypertrophic pyoric stenosis.

South Afncan J. Surg. 1975 ; 13 : 239 -242

Spitz L., Zail S.S.

Serum gastrin levels in congenital hypertrophic pyloric stenosis.

J. of Paediatric Surgery 1976 ; 11 : 33

Spitz L.

Vomiting after pyloromyotomy for infantile hypertrophic pyloric stenosis.

Archives of Disease in Childhood . 1979 ; 54 ; 886

Spitz L.

Vomiting after pyloromyotomy for infantile hypertrophic pyloric stenosis.

Archives of Disease in Childhood 1979 ; 54 : 886 - 889

Spitz L., Batcup G..

Haematemesis in infantile hypertrophic pyloric stenosis.

Hr. J. Surg. 1979 ;66 :827-828

Spitz L., MacKinnon A.E.

Posture in the postoperative management of infantile hypertrophic pyloric stenosis .

B r. J. of Surgery 1984 ; 643

Spitz L.

Infantile Hypertrophic Pyloric Stenosis and Pyloric Mucosal Diaphragm.

Shwartz S I , Ellis H eds. ' Maingot's Abdominal Operations . ' Appleton ■ Century - Croft , Connecticut : 63 - 41

Spitz L., Abel R.M.

Infantile Pyloric Stenosis and Pyloric Mucosal Diaphragm.

Accepted by Maingot's textbook of Abdominal Surgery

SpringallD., Riveros-Moreno V., Buttery L., Suburo A., Bishop AEB., Merrett M., Moncada S., Polak JM.

Immunological detection of nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms.

Histochemostiy 1992; 98: 259-266

Stack WA., Filipowicz B., Hawkey CJ.

Nitric oxide donating compounds stimulate human colonic ion transport in vitro

Gut 1996; 39: 93-99

Stark M E., Szurszewski J.H.

Role of Nitric Oxide in Gastrointestinal and Hepatic Function and Disease.

Gastroenterology 1992 ;103 : 1929 - 1949

Stringer M.D., Brereton R.J.

Current management of infantile hypertrophic pyloric stenosis.

Br. J. Hospital Medicine 1990 ; 43 : 266 - 272

Stringer M.D., Kiely E.D., Drake D.P.

Gastric retention of Swallowed coins after pyloromyotomy.

Br. J. Clinical Practice 1991 ; 45 : 66 - 67

Stringer M.D., Spitz L., Abel R., Kiely E., Drake D.P.D., Agrawal M., Stark Y., Brereton R.J.

Management of alimentary duplications in children.

British Journal of Surgery 1995; 82: 74-78

Strombeck D R., Guilford N.G.

Chronic Gastritis, Gastric Retention, Gastric Neoplasms, and Gastric Surgery

Small Animal Gastroenterology 2nd Edition 1990 Stonegate Publishing Co.

Stuehr D.J.

Mammalian Nitric Oxide Synthases

Advances in Enzymology 1992 ; 65 : 287 -346

Sun WM., Doran S., Lingenfelser TH., Hebbard GS., Morley JE., Dent JE., Horowitz M.

Effects of glyceryl trinitrate on the motor response to intraduodenal triglyceride infusion in humans.

Eur. J. Clin. Inv. 1996; 26: 657-664

Surprenant A.

Control of the Gastrointestinal Tract by Enteric Neurons.

Annual Review Physiology 1994, 56: 117-140

Swan, XT.

Congenital Pyloric Stenosis in the African Infant.

Br Med. J. 196.1; 1: 545-7,

Swift P.G.F., Prossor J.E.

Modem Management of pyloric stenosis.

Archives of Disease in Childhood 1991 ; 66 : 667

Szele F.G., Dowling J.J., Gonzales C , Thevanieu M., Rougon G , Chesselet M.F.

Pattern of Expression of Highly Polysialylated Neural Cell Adhesion Molecule in the Developing and Adult Rat Striatum

Neuroscience 1994; 60: 1, 133-144

Tack E D., Perlman J.M., Bower R.J., McAlister W.

Pyloric stenosis in a sick premature infan t.

American J. of Disease in Childhood 1988 ; 142 : 68 - 70

Taguchi T., Guo O , Rahman MS., Nakao M., Suita S.

Distribution of Nitric Oxide Neurons in Rat Small Intestinal Transplantation

Transplatation Proceedings 1996; 28 (5): 2560

Takayanagi S., Hanai H., Kaneko E.

Ca2+/Calmodulin,and Protein Kinase C dependent signal transduction affect serotonin uptake in rat jejunal enterocytes

Biomedical Research 1993; 14; Supplement 2, 103-105

Takeuchi S., Tamate S., Nakahira M., Kadowaki. H.

Esophagitis in children with Hypertrophic Pyloric Stenosis : a Sorce of Haematemesis.


Takeuchi K., Ohuchi T., Okabe S..

Endogenous Nitric Oxide in Gastric Alkaline Response in the Rat Stomach After Damage.

Gastroenterology 1994; 106; 367 - 374

Tatemoto K, Lundberg JM, Jomwall H, Mutt V

Neuropeptide K: isolation, structure and biological activities of a novel brain tachykinin

Biochem Biophys Res Commun 1985; 128: 947-953

Tallerman KH.

Discussion on the treatment of congenital hypertrophic pyloric stenosis .

Proc Roy Soc Med., 1951; 44:1055-1057,

Tam P.K.H., Saing H., Koo J., Wong J. Ong GB.

Pyloric function five to eleven years after Ramstedt's Pyloromyotomy.


Tam P.K.H.

An immunochemical study with Neuron - Specific - Enolase and Substance P of HumanEnteric Innervation - the normal developmental pattern and abnormal deviations inHirschprung's disease and Pyloric Stenosis.


Tam P.K.H., Carty H.

Non surgical treatment for Pyloric stenosis.

Lancet 1989 ; 2 : 393

Tam P.K.H, Chan J.

Increasing incidence of hypertrophic pyloric stenosis.


Tan H.L., Najmaldin A.

Laparoscopic pyloromyotomy for infantile hypertrophic pyloric stenosis .

Paediatric Surgery International 1993; 8 : 376 - 378

Tanner MS

Arch Dis Child 1976; 51: 837

Tare M., Parkington H.C., Coleman H.A., Neild T O., Dusting G.J.

Hyperpolarisation and muscle relaxation of arterial smooth muscle caused by nitric oxide derived from the endothelium.

Nature 1990 ; 346 : 69 - 70

Tatemoto M , Lundberg JM., Jomwall H., Mutt V.

Neuropeptide K: isolation, structure and biological activities of a novel brain tachykinin.

Biochem Biophys Res Commun 1985; 128: 947-953

Taylor T.V., Bhandarkar D.S.

Laparoscopic Vagotmy: an operation for the 1990s?

Annals of the Royal College of Surgeons 1993; 75:, 385 - 386

Thiery J-P., Duband J-L., Rutishauser U., Edelman G.M.

Cell adhesion molecules in early chicken embryogenesis.


Thirlby R.C., Pearson P.J.

Nitric oxide is or is not everywhere.


Thompson RJ, Doran JF, Jackson P. Shillon AP and Rode J

PGP 9.5 - new marker for vertebrate neurons and neuroendocrine cells.

Brain Res 1983; 278: 224-228

Thomson J

On Congenital Pyloric Spasm .

Scot Med Surg 1897; 1: 511,

Thor G , Probstmeier R., Schachner M.

Characterisation of the cell adhesion molecules L1,N-CAM, and J1 in the mouse intestine.

The - ? Journal 1987; 6; 2581-2586

Timmermans J., Barbiers M., Scheuermann D.W., Bogers JJ., Adriaensen D., Fekete E., Mayer B., Van Marck., De Groodt-Lasseel MHA.

Nitric Oxide immunoreactivity in the enteric nervous system of the developing human digestive tract.


Torgensen J.,

Acta radiol. 1942; supple 45,

Tottrup A , Ny L., Aim P., Larsson B., Forman A., Andersson K-E.

The role oe the L - Arginine / Nitric oxide pathway for relaxation of the human lower oesophageal sphincter.

Acta Phsiologica Scand 1993; 149 451-459

Uddman R, Luts A, Absood A, Arimura A, Ekelund P, Desai H, Hakanson R, Hambreaus G, Sundler F.

PACAP a VIP like peptide in neurons of the oesophagus.

Regul peptides 1991; 36:415-422

Uvnas-Moberg K., Bruzelius G , Alster P.

Vagally mediated release of gastrin and cholecystokinin following sensory stimulation.

Acta Physiol Scand 1992; 146: 349-356

Vallance P.

Nitric Oxide in the Clinical Arena


Vanderwinden J.M., Mailleux P., Schiffman S.N., Vaderhaegen J.J., De Laet M.

Nitric oxide synthase activity in Infantile Hypertrophic Pyloric Stenosis.

New England Journal of Medicine 1992; 327; 511 - 515

Vanderwinden JM., Liu H., Conreur JL., Laet MH., Vanderhaegen JJ.

The Pathology of Infantile Hypertrophic Pyloric Stenosis after Healing

J. Paediatric Surgery 1996; 31(11): 1534-1534

VanderwindenJ. M., De LaetM.H., SchififtnanS.N.

Nitric oxide synthase distribution in the Enteric Nervous System of Hirschpsrung's.


Villa P., Sensenbrenner M., Pettmann B..

Synthesis of Specific Proteins in Trophic Factor-Deprived Neurons Undergoing Apoptosis.

J. Neurochemistry 1994; 62: 1468-1475

Vilmann P., Hjortrup A., Altmann P., Andersen F.H., Sorensen C.

A Long-term Gastroiintestinal Follow-up in Patients Operated on for Congenital Hypertrophic Pyloric Stenosis

Acta Paediatr Scand 1986; 75: 156-158

VincentSR., Hope BT.

Localisation of nitric oxide synthase in the rat intestine.

Trends in Neurooscience 1992; 15; 108-113

Voelker CA., Miller MJS., Zhang X., et al.

Perinatal Nitric Oxide Synthase Inhibition Retards Neonatal Growth by Inducing Hypertrophic Pyloric Stenosis

Ped Res 1995; 38, 5 :768 -774

Vogalis F., Sanders K.M.

Excitatory and inhibitory neural regulation of canine pyloric smooth m uscle.

Am. J. Physiology 1990 ;259 ; G125 -G133

Vogalis F., Ward S.M., Sanders K.M.

Correlation between electrical and morphological properties of canine pyloric circular m uscle.

Am. J. Physiology 1991 ; 260 : G390 - G398

von Euler VS, Gaddum JH

An unidentified depressor substance in certain tissue extracts.

J. Physiol 1931; 72: 577-583

Wallgren A

Preclinical Stage of Infantile Hypertrophic Pyloric Stenosis

.Am. J. Disease. Childhood 1948 ; 72: 371-376

Walpole IR.

Some epidemiological aspects of pyloric stenosis in British Columbia .

Am J. Med. Genet. 1981;10: 237-244

Walter MC, Goldschmidt MH, Stone EA.

Chronic Hyperrtrophic pyloric gastropathy as a cause of pyloric obstruction in the dog.

J. Am. Vet. Med Assoc 1985; 186:157-161

Walter M.C., Matthiesen DT.

Acquired antral pyloric hypertrophy in the dog.

Veterinary Cliniics of North America: Small Animal Practice 1993; 23;3: 547-555

Wanscher B., Jensen H-E.

Late follow up Studies after Op[eration for Congenital Pyloric Stenosis

Scand J. Gastroenterol 1971; 6:597-599

Ward SM., Sanders KM.

Pacemaker activity in septal structures of canine colonic circular muscle.

Am J. Physiol 1990; 259; 0264-273

Ward SM., Xue C , Shuttleworth C W , Bredt DS., Snyder SH., Sanders KM.

NADPH diaphorase and Nitric Oxide Synthase colocalization in enteric neurons of the canine proximal colon.

Am J. Physiol., 1992; 263; G277-G284

. Ward M., Xue C., Sanders K.M.

Localisation of nitric oxide synthase in canine ileocolonic and pyloric sphincers.


Walsh F.S., Dickson G., Moore S.E.,.Howard C Barton D.

Unmasking NCA M .

Nature 1989 ;3 3 9 ; 516

Wastell C.

The Stomach and Duodenum

A Short Practice of Surgery Eds. A. J.H. Rains , H.D. Chapman Hall, 1984

Wattchow D.A., Cass D.T., Furness J.B., Costa M., O'Brien P.E., Little K.E., Pitkin J.

Abnormalities of peptide containing Nerve Fibres in Infantile Hypertrophic Pyloric Stenosis.


Wattchow D.A., Furness J.B., Costa M..

Distribution and coexistance of Peptide in Nerve Fibres of the External Muscle of the Gastrointestinal trac t.


Weaver C , Bishop AEB., Polak JM.

Pancreatic Changes Elicited by Chronic Administration of Excess L-Arginine

Experimental and Molecular Pathology 1994; 60,71-87

Webb A.R., Lari J., Dodge J.A.

Infantile hypertrophic pyloric stenosis in South Glamorgan .


Werlin SL., Grand RJ., Drum DE.

Congenital pyloric stenosis : the role of gastrin reevaluated.

Paediatrics 1978; 61: 833,

Wharton J., Gordon L., Walsh F.S., Flanigan T.P., Moore S.E., Polak J.M.

Neural cell adhesion molecule exxpression during cardiac development in the rat.

Brain Research 1989; 483: 170-176

Wilkinson KD, Lee KM,Deshpande S, Duerksen-Hughes P, Boss JM and Pohl J

The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase.

Science 1989; 246: 670-673

Wollstein M.

Healing of Hypertrofic stenosis after the Fredet-Ramstedt operation

Am. J. Dis. Child 1922; 23: 511-517

Woolley, MM., Felscher BF., Asch MJ., Carpio N., Isaacs H.

Jaundice, hypertrophic pyloric stenosis and hepatic glucuronyl transferase.

J. Paediatric Surgery 1974; 9: 539-543.,

Wheeler R.A., Najmaldin A.S., Stoodley N., Griffiths D M., Burge D M., Atwell J.D.

Feeding regimens after pyloromyotomy.

Br. J. Surgery 1990 ; 77 : 1018 - 1019

Yamaguchi K., Furuya K.

Regulation of Neurite -Outgrowth by intracellular calcium in cultured mammalian neuron


Yunker A.M., Galligan J.J.

Extrinsic Denervation increases NADPH diaphorase staining in myenteric nerves of guinea pig ileum.

Neuroscience Letters 1994; 167: 51 - 54

The Ontogeny of the Peptide Innervation of the Human Pylorus, With Special Reference to Understanding the Aetiology and Pathogenesis

of Infantile Hypertrophic Pyloric StenosisBy Robin Michael Abel

London, England

# Pyoric stenosis (PS) is a common condition in infancy, which is associated with sm ooth muscle hypertrophy tha t results in pyloric outlet obstruction. The author examines the ontogeny of the peptide innervation of the pylorus in fetal tissues and an experimental model in mice and evaluates the histochemical and morphological changes in the pylorus. The data suggest tha t PS is an intrauterine lesion tha t occurs by 12 w eeks' gestation. This is associated with diminished nitric oxide in human tissues and reduced enzyme activity (resulting from a deficiency in an enzyme cofactor) in mice. Increased vasoactive intestinal polypeptide expression in pyloric myenteric ganglia may be an intrinsic mechanism for resolving this condition.Copyright © 1996by W.B. Saunders Company

INDEX WORDS: Hypertrophic pyloric stenosis, nitric oxide synthase, fetal pylorus, vasoactive intestinal polypeptide.

IN FA N TILE H Y PE R T R O PH IC pyloric stenosis (PS) was first described in 1646 by Fabricius

Hildanus. It is the most common cause of emergency abdominal surgery in infancy.^ A related condition occurs in brachycephalic dogs. The aetiology is unknown. Before the development of R am stedt’s pyloromyotomy in 1911 and the development of safe paediatric anaesthesia, some cases were treated successfully by conservative m anagement.

Many investigators have described the histological changes underlying PS,^'^ but the ontogeny and structure of the normal infant pylorus has not been described previously. To date, none of the biochem ical or histochemical studies of PS have accounted for the apparent dilutional reduction in neural tissue that occurs after muscle hypertrophy.

Although animal models of PS have been reported,^-^^ none has been verified or quantified, and

M r Robin A be l was the Research Fellow at the Institute o f Child Health, London, England.

Presented a t the 42nd A nnual International Congress o f the British A ssociation o f Paediatric Surgeons, Sheffield, England, July 25-28, 1995.

This essay was the prize-winning award fo r the BA PS Trainees Prize. M r A b e l’s supervisors were Professor L. Spitz (Institute o f Child Health, L ondon) and Professor J.M. P olak (The Royal Postgraduate M edical School, London).

A ddress reprint requests to M r Robin M ichael Abel, D epartm ent o f Surgery, Birmingham C hildren’s Hospital, N H S Trust, L adyw ood M iddleway, Birmingham B 16 SET, England.

Copyright © 1996 by W.B. Saunders Com pany0022-3468l96 l3104-0006$03 .00 l0

none of the reports has described the chronological changes underlying pyloric hypertrophy.

The objectives of this project were threefold: (A) to examine the ontogeny of the peptide innervation of the pylorus; (B) to quantify the histochemical and morphological changes in PS; and (C) to identify an experimental model of PS.

A. THE DEVELOPMENTAL STUDY

A i m s

1. To study the ontogeny of the intrinsic and extrinsic innervation of the pylorus.

2. To identify the developmental stage at which PS develops.

Tissue was collected from 35 hum an fetuses (age range, 8 to 23 weeks’ gestation) and 20 children (age range, 1 week to 8 months). To trace the vagal innervation of the pylorus, the carbocyanine compound D il was u s e d . T h i s is a lipophilic crystal that is absorbed by nerves. D il passively diffuses and autofluoresces in aldehyde-fixed tissue, which allows selective neuronal tracing postmortem.

To minimise the transport time of D il along the vagus to the pylorus, and thus the tissue decomposition time, a selective vagotomy was perform ed on four foetal stomachs (age range, 10 to 23 weeks). A crystal of D il was placed on the severed surface of the nerve. Control specimens had D il placed directly on the serosa of the pylorus. The specimens were incubated for 6 months.

To identify the craniocaudal and tem poral patterns of development, serial sections were cut at 200-jxm intervals through the pylorus. The sections were stained with haematoxylin and eosin and immunostained using antisera to the general neural m arker protein gene product 9.5 (PGP 9.5), vasoactive intestinal polypeptide (VIP), the neural isoform of nitric oxide synthase (NOS), calcitonin gene-related peptide (CG RP), substance P (SP), the foetal isoform of neural cell adhesion molecule (fNCAM ), the neural isoform of neural cell adhesion molecule (nNCAM), the neurom uscular isoform of neural cell adhesion molecule (nmNCAM ), and actin.

R e su lts

The tem poral pattern of expression of the antigens 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. Tem poral Pattern of A ntigen Expression (no. o f w eek s)

Mucosa SubmucosaMuscularis

Propria Adventitia

PGP 9.5 11 8 8 8VIP 12 12 11 11NOS 15 12 11 13SP 23 11 8 8CGRP 23 23 12 —

fNCAM 12 8 8 12nNCAM 12 12 8 12nmNCAM 12 12 8 —

Actin 14 12 8 —

had a craniocaudal pattern of expression within the muscularis propria. The highest concentration of immunoreactivity was toward the duodenum. Only PGP 9.5, VIP, and nNCAM had a craniocaudal pattern of expression in the submucosa. The craniocaudal changes in VIP expression in the pylorus are consistent with those described in the small intestine.

The fetal pylorus had developed the morphology and histochemical structure of the infant pylorus by 23 weeks of gestation.

E x tr in s ic In n e rv a tio n

The vagal innervation of the pylorus was first dem onstrated by D il autofluorescence and was confirmed by VIP immunoreactivity at 11 weeks. (VIP is expressed by vagal flbres.^^)

B. THE QUANTITATIVE STUDY

A im s

1. To quantify the morphological changes underlying PS.

2. To characterise the histochemical changes underlying PS in humans, by quantifying the p r o p o r

tio n of the total neural tissue expressing antigen, thus accommodating the dilutional reduction in total neural tissue owing to muscle hypertrophy.

Twenty pyloric muscle biopsy specimens from children who underwent pyloromyotomy for PS were studied, along with 20 age- and sex-matched control specimens. The tissues were immunostained for PGP 9.5, VIP, NOS, SP, and CGRP. Five nonserial sections were examined for each antigen, A computer- assisted image analysis system was used to quantify 25 images for each param eter m easured and in each of tissue studied. The mean of these values was used for statistical analysis. Overall, 49,350 images were analysed.

R e su lts

The relative changes in the proportion of nerves expressing antigens is summarised in Table 2. The

proportionate numbers of ganglia expressing SP, CGRP, and NOS were reduced significantly. The size of these ganglia was unchanged. However, ganglia expressing VIP increased in num ber and size.

The longitudinal muscle layer was hypertrophied, as was the circular muscle layer (Fig 6).

C. EXPERIMENTAL ANIMAL MODEL

A im s

1. To identify an animal model of PS.2. To quantify the tem poral pattern of morphologi

cal changes underlying PS.A colony of hph-1 mice deficient in tetrahydrobiopterin has been developed as a model of phenylketonuria.15,16 U ntreated phenylketonuria is associated with PS.'^ Tetrahydrobiopterin is a cofactor to NOS and phenylalanine hydroxylase.

Six specimens were gathered from control and diseased animals (age range, 2-week-old foetuses to 180-day-old animals). The pylorus was examined immunohistochemically. The width of the pylorus, and its muscle layers, was measured using a graticule.

R e su lts

The circular, longitudinal, and muscularis mucosae layers had significant hypertrophy up to 40 days of age. Qualitatively, there was no change in im munoreactivity to PGP 9.5, VIP, NOS, SP, CGRP, or nNCAM. The relationship between body weight and the width of each muscle layer is illustrated in Figs 7-10.

DISCUSSION

NOS has been implicated in the pathogenesis of PS since the description that only the circular muscle is hypertrophied and the diminished expression of NOS is localised to this tissue.^^ The present study shows that longitudinal muscle hypertrophy is a characteristic of PS, and that NOS expression is diminished in the circular and longitudinal muscle layers as well as in the myenteric plexus. That the proportionate reduction in expression of NOS and VIP is the same in the circular muscle and myenteric plexus is biologically significant, because the peptides are colocalised to the same nerves in these s tru c tu re s .O n ly NOS expression was diminished in all the tissues studied.

Table 2. R elative C hanges in Proportion of N erves E xpressing A ntigen

A ntigen C ircular M uscle M yenteric P lexus Longitudinal M uscle

VIP i 1 1 1 tNOS i i f i i i iSP ; iCGRP i i

NOTE. The relative changes are illustrated in Figs 1-5.

Fig 1. Estim ates of differences in m edians of proportion of nerves expressing antigen in th e circular m uscle.

Fig 2. Estim ate of differences in median and 95% confidence intervals of proportion of nerves ex pressing antigen in the m yenteric plexus Anticen

Fig 3. Estim ated differences in m edian proportions of nerves expressing antigen in the longitudinal m uscle.

Fig 4. Estimate of differences in median antigen staining per ganglion.

Fig 5. Differences in m edian numbers of ganglia expressing antigen.

S 0.08

Fig 6. Width of the longitudinal m uscle in control (■ ) and d iseased (□ ) patients.

494 ROBIN MICHAEL ABEL

30

r15

10

10 Fig 7. W eight and m uscularis m ucosae trend.

It is notew orthy that SP expression is not dim inished in the circular muscle but is so in the m yenteric plexus and longitudinal muscle layers. SP has a potent contractile effect on enteric sm ooth muscle. C G R P expression is reduced only in the m yenteric plexus. The significance of the reduced expression of CORP*-"* dem onstrated by this study is unclear. It is rem arkable that the same proportion of nerves in the m yenteric plexus express all the antigens in PS (Fig 11).

Several investigators have a ttribu ted various m orphological changes to the nerves in PS. It has been proposed that compression of the nerves may account for the histochemical changes^^; however, the present study shows that the only significant morphological change is a shortening of the nerves in the longitudi

nal muscle, and thus this cannot account for the histochemical changes.

Recently, a NOS-gene deleted “knockout m ouse” m odel was described,*^’ * in which the only abnorm ality is gasric outlet obstruction due to pyloric hypertrophy. Because nitric oxide is required by erectile tissues, it is strange that the knockout mice were described as being just as fertile as normal mice. Furtherm ore, these mice did not lose weight or behave abnorm ally despite having gastric outlet obstruction and gastric distension. A fter a 3-year study of the genetic lesion underlying PS, no evidence was found to suggest that an aberration of NOS gene expression underlies PS in humans.^’ T herefore, the significance of the knockout m ouse as an animal model of PS is unclear.

Fig 8. W eight and circular m uscle trend.

10

- O — HPHl CM/DP


Fig 9. W eight and longitudinal muscle trend.

20

The hph-1 model identified in this study is significant for several reasons. (1) This is the first animal model of PS that was originally created and verified as a model of another human condition known to be associated with PS. (2) hph-1 mice behave abnormally, are less fertile, and lose weight after birth, then regain it by 40 days as the pyloric smooth muscle hypertrophy resolves. (3) That the hypertrophy is transient is consistent with reports of successful conservative managem ent of PS. (4) That the hypertrophy occurs in fetal hph-1 mice is consistent with reports of the condition being found in prem ature babies.

Because pyloric hypertrophy develops in prem ature babies and hph-1 fetal mice, the lesion underlying 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, and that both are expressed at approximately 12 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 histochemical changes seen in children with PS, suggests that these may be secondary changes in the 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 unclear. It is rem arkable that the ontogeny of NCAM in the pylorus is different from that in the large bowel,^^ where fNCAM is expressed by the fetal muscularis mucosae, but not in the fetal pylorus. NCAM has been implicated in cell-cell adhesion and neurom us

cular differentiation.^^ In H irschsprung’s disease, fNCAM is reexpressed by the muscularis mucosae^^; however, in PS, nNCAM is expressed by muscle cells.25 Presently this finding is being further characterised by immuno-electron microscopy.

The increase in size and num ber of VIP expressing ganglia is functionally consistent with that of a reduc-

C

8

1Series!

-20

Age (Days)

Fig 10, Estimated differences in m eans of proportionate circular m uscle width to pylorus diameter.

496 ROBIN MICHAEL ABEL

Fig 11. Antigen expression by nerves of th e m yenteric plexus.

tion in m etE nkephalin expression in PS^^: endogenous endorphins tonicly inhibit V IP release.^^’® The increase in V IP expression by the ganglia may rep re sent a com pensatory m echanism by which the condition naturally regresses: V IP release results in the release of nitric oxide from sm ooth m uscle cells.^^

These data suggest tha t pyloric sm ooth muscle hypertrophy is an in trau terine lesion tha t becom es clinically apparen t as “ infantile hypertrophic stenosis” after feeding is established. T he in trau terine lesion occurs by 12 weeks of gestation. T he condition is characterised by hypertrophy of the longitudinal and circular muscle layers. D im inished nitric oxide,

caused by a reduction in the am ount of nitric oxide synthase presen t in all tissue layers (hum ans) or by a reduction in the functional activity of the enzyme secondary to a cofactor deficiency (tetrahydrobiopterin in hph-1 m ice), underlies the condition. Increased V IP expression by the ganglia may represen t a m echanism by which the condition naturally resolves.

The present study identified two potential pa th ways for possible nonoperative trea tm en t of PS: enhancing nitric oxide production by the use of nitric oxide donors or NOS cofactors, or augm enting VIP activity (eg, by opiate antagonists).

REFERENCES

1. Knox EG, Armstrong E, Haynes R: Changing incidence of hypertrophic pyloric stenosis. Arch D is Child 58:582-585,1983

2. D ieler R, Schroder JM, Skopnik H, et al: Infantile hypertrophic pyloric stenosis: Myopathic type. A cta N europathol 80:295- 306,1990

3. D ieler R, Schroder JM: M yenteric plexus neuropathy in infantile hypertrophic pyloric stenosis. Acta Neuropathol 78:649-661,1989

4. Malmfors G, Sundler F: Peptidergic innervation in infantile hypertrophic stenosis. J Pediatr Surg 21:303-306,1986

5. M ichel T: Nitric oxide synthase activity in infantile hypertrophic pyloric stenosis. N Engl J M ed 327:1690,1992

6. Spitz L, Kaufmann JCE: The neuropathological changes in congenital hypertrophic pyloric stenosis. South Afr J Surg 13:239- 242,1975

7. Tam PKH: An immunochemical study with neuron-specific enolase and substance P of human enteric innervation— The normal developm ental pattern and abnormal deviations in Hirschsprung’s disease and pyloric stenosis. J Pediatr Surg 21:227-232, 1986

8. Vanderwinden JM, D e Laet MH, Schiffman SN, et al: Nitric oxide synthase distribution in the enteric nervous system of Hirschsprung’s. Gastroenterol 105:969-979,1993

9. Bredt D , Huang P, Dawson T, et al: Nitric oxide synthase

gene knockout mice: M olecular and morphological characterisation. Endothelium 19:S6 ,1993 (suppl)

10. D odge JA, Karim AA: Induction of pyloric hypertrophy by pentagastrin. Gut 17:280-284,1976

11. Huang PL, Dawson TD , Bredt DS, et al: Targeted disruption o f the neuronal nitric oxide gene. Cell 75:1273-1286,1993

12. Gotz M, Novak N, Bastymer M, et al: Membrane-bound m olecules in rat cerebral cortex regulate thalamic innervation. Developm ent 116:507-519,1992

13. Kressel M, Berthoud HR, Neuhber WL: Vagal innervation of the rat pylorus: An antegrade tracing study using carbocyanine dyes and laser scanning confocal microscopy. Cell Tissue Res 275:109-123,1994

14. Facer P, Bishop AE, M oscoso G, et al: Vasoactive intestinal polypeptide gene expression in the developing human gastrointestinal tract. Gastroenterology 102:77-55,1992

15. M cDonald JD, Cotton RGH, Jennings I, et al: Biochemical defect in the hph-1 m ouse mutant is a deficiency in GTP- cyclohydrolase activity. J Neurochem 50:655-657,1988

16. M cDonald JD, Cotton RG H , Jennings I, et al: Hyperphenyl- alanaemia in the hph-1 mouse mutant. Pediatr Res 23:63-67,1988

17. Johnson CF, Peterson RM, Koch R, et al: Congenital and neurological abnormalities in infants with phenylketonuria. Am J M ental D ef 4:375-379,1978


18. V anderwinden JM, M ailleux P, Schiffm an SN , et al: Nitric oxide synthase activity in infantile hypertrophic pyloric stenosis. N E n glJ M ed 327:511-515,1992

19. Furness JB, Bornstein JC, M urphy R , et al: R o les o f peptides in transm ission in the enteric nervous system. Trend N eurosci 15:66-71,1992

20. Stringer M D , Brereton RJ: Current m anagem ent o f infantile hypertrophic pyloric stenosis. Br J H osp M ed 43:266-272,1990

21. G ardiner M: Personal com m unication. University C ollege, London, England

22. R om anska H, B ishop A E , M oscoso G, et al: N C A M expression in nerves and m uscle o f the developing human large bowel. G astroenterology (in press)

23. Cunningham BA: C ell adhesion m olecules and the regulation o f developm ent. A m J O bstet G ynecol 164:939-948,1991

24. Rom anska H M , B ishop A E , Brereton RJ, et al: Increased expression o f m uscular neural cell adhesion m olecule in congenital aganglionosis. G astroenterology 105:1104-1109,1993

25. A bel RM , Bishop A E , Spitz L, e t al: T he expression o f neural cell adhesion m olecule in infantile hypertrophic stenosis. Presented at the 41st A nnual International C ongress o f the British A ssociation o f Paediatric Surgeons, R otterdam , T he N etherlands, June 29-July 1 ,1 9 9 4

26. W attchow D A , Cass D T , Furness JB, e t al: A bnorm alities o f peptide containing nerve fibres in infantile hypertrophic pyloric stenosis. G astroenterology 92:443-448,1987

27. H oyle CH, Kamm M A, Burnstock G, et al: E nkephalins m odulate inhibitory neurom uscular transm ission in circular m uscle o f human colon via op ioid receptors. J Physiol (L ondon) 431:465-4 7 8 ,1990

28. G rider JR, M akhlouf GM: Suppression o f inhibitory neural input to colonic m uscle by opioid peptides. J Pharmacol Exp T her 243:205-210,1987

29. G rider JR, M urthy K N , Jin JG, e t al: Stim ulation o f nitric oxide from m uscle cells by VIP; Prejunctional enhancem ent o f VIP release. A m J Physiol 262:G 774-G 778,1992

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