the effects of the neuropeptide substance p on outcome ...the effects of the neuropeptide substance...

The Effects of the Neuropeptide Substance P on

Outcome Following Experimental Traumatic

Brain Injury in Rats

JAMES J. DONKIN B.HSc. (Hons.)

Discipline of Pathology, School of Medical Sciences

University of Adelaide

August 2006

A thesis submitted in partial fulfilment of the requirements for the degree

of Doctor of Philosophy

The brain is a wonderful organ. It starts

working the moment you get up in the

morning and does not stop until you get into

the office.

Robert Frost (1874-1963)

iii

Declaration

This work contains no material which has been accepted for the award of any other

degree or diploma in any university or other tertiary institution and that, to the best of

my knowledge and belief, the thesis contains no material previously published or

written by another person, except where due reference has been made in the text of the

thesis.

I give consent to this copy of my thesis, when deposited in the University Library, being

available for photocopying and loan.

Date James Donkin

iv

Dedication

This thesis is dedicated in memory of Ray Donkin and Ardiss Moran, who instilled in

me the determination to succeed in everything I do.

v

Publications and Presentations

The following articles have been published or accepted for publication or presentation

during the period of my PhD candidature, and sections of these articles have been

included in the present thesis.

Journal Papers:

Van Den Heuvel C, Donkin JJ, Finnie JW, Blumbergs PC, Kuchel T, Koszyca B,

Manavis J, Jones NR, Reilly PL and Vink R. (2004) Downregulation of amyloid

precursor protein (APP) mRNA and antigen expression following post-traumatic

cyclosporin-A administration. J Neurotrauma 21(11): 1562-72.

Vink R, Donkin JJ, Cruz MI, Nimmo A and Cernak I. (2004) A substance P antagonist

increases brain intracellular free magnesium concentration after diffuse traumatic brain

injury in rats. J Am Coll Nutr 23(5): 538S-540S.

Conference Papers:

Donkin JJ, Cernak I, Rodgers K and Vink R. (2004) Mild concussive head injury

results in increased brain substance P immunoreactivity. In: Proceedings of the 7th

International Neurotrauma Symposium, Adelaide, South Australia: 110

vi

Abstracts and Posters:

Vink R, Cruz MI, Donkin JJ, Nimmo A and Cernak I. (2003) A substance P antagonist

increases brain intracellular free magnesium concentration after diffuse traumatic brain

injury in rats. Magnesium Res 16(4): 315.

Donkin JJ, Cernak I, Rodgers K and Vink R. (2004) Mild Concussive head injury

results in increased brain substance P immunoreactivity. Rest Neurol Neurosci 23: 169

Howard C, Donkin JJ, Ghabriel M, Blumbergs PC and Vink R. (2004) A substance P

antagonist increases aquaporin-4 expression and reduces oedema after traumatic brain

injury. Rest Neurol Neurosci 23: 194

Donkin JJ, Nimmo AJ and Vink R. (2006) Delayed administration of a substance P

antagonist at 12 h after diffuse traumatic brain injury improves outcome.

J Neurotrauma 23: 778

Book Chapters

Donkin JJ, Turner R, Hassan I and Vink R (2006) A review of the role of

neuropeptides in traumatic brain injury. In: Progress in Brain Research. Neurotrauma:

new insights into pathology and treatment (Weber JT and Maas, AIR), Rotterdam:

Elsevier, in press.

vii

Acknowledgments

The decision to undertake this PhD has been very rewarding for me and has allowed me

to explore many exciting areas and meet many interesting people. There is a touch of

sadness to be finishing up my studying days at university where I have been for a third

of my life. It has provided me with a sense of curiosity and an attitude of determination

in all that I undertake. However to get to this point I have required the help and support

of many people. I would like to acknowledge the Faculty of Health Sciences for

awarding me the divisional scholarship to enable me to embark on the challenge.

First and foremost, to my mentor and friend, Prof Robert Vink. Your guidance as my

supervisor throughout the entire candidature has been exceptional and one which I am

forever thankful for. I thank you for sharing with me your immense research

experiences and allowing me the opportunity to undertake the PhD in your lab. I find it

amazing that with your busy schedule you were able to make time for me to bounce

ideas off, seek advice or just have a friendly chat. Your door is always open and I am

confident it always will be. I wish you all the luck for the future, especially in Eustralis.

Next, to my co-supervisor, Dr Corinna Van Den Heuvel. A friend and colleague who

has taught me the whole way through, from the undergraduate level until now. You laid

the foundation to my research life and always impart constant advice and expertise. I

thankyou, not just for your help but also your friendship, which we have developed over

the years. You’re a true legend and I wish you and the family all the best luck for the

future.

viii

To Renee, Emma, Tuyet, Islam, Felicity and more recently Naomi who have shared

PhD life with me along the way. In particular, Renee and Emma, thankyou for all your

help with surgery, we were always there to support each other, especially when things

didn’t go well. I especially enjoyed the chats and laughs during our wine meetings. I

wish you all well for the remainder of your studies and all the best for your future

careers.

To all the people who have helped me throughout my studies, in particular Christine

O’Connor, thankyou for introducing me to brain injury and teaching me around the

surgery. Your persona was fantastic around the lab, constantly having us in fits of

laughter with your firm attitudes. You were the mother of the lab and were always there

for a comforting talk when experiments weren’t working. Other people who have

helped along the way include Prof Peter Blumbergs, who offered informative advice

and help with analysing slides. Jim Manavis and Kathryn Rodgers who taught and

helped with immunohistochemistry. Ghafar Sarvestani for the confocal images and Dale

Caville for making all my figures magnificent in this thesis.

To Mum and Dad, thankyou for your tireless support and love throughout my life,

especially during my studies. You had faith in me and believed that I would succeed.

Also to the boys, thanks for the welcomed distractions along the way and the mems.

Last but not least, to Beck, who has put up with me throughout the entire PhD.

Thankyou for your love and patience, especially with my indecisiveness. I congratulate

you too on finishing your PhD and look forward to our next adventure.

ix

Abbreviations

A1 Adenosine

ACE Angiotensin-Converting Enzyme

Ach Acetylcholine

ADC Apparent Diffusion Coefficient

ANOVA Analysis of Variance

APP Amyloid Precursor Protein

APT 3-Aminopropyl Triethoxysilane

ATP Adenosine Triphosphate

BBB Blood-Brain Barrier

B0 Maximum Binding

BP Blood Pressure

Ca2+ Calcium

cAMP Cyclic Adenosine Monophosphate

CBF Cerebral Blood Flow

CBV Cerebral Blood Volume

CDC Centre for Disease control and prevention

CCI Controlled Cortical Impact

CGRP Calcitonin-gene Related Peptide

Cl- Chloride

CNS Central Nervous System

COOH-terminal Carboxyl Terminal

CPP Cerebral Perfusion Pressure

x

CREB cAMP Response Element-Binding

CSF Cerebrospinal Fluid

DA Dopamine

DAB Diaminobenzidine Tetrahydrochloride

DAI Diffuse Axonal Injury

DNA Deoxyribonucleic Acid

DPX Depex Mountant

DRG Dorsal Root Ganglion

DVI Diffuse Vascular Injury

DWI Diffusion Weighted Image

EAA Excitatory Amino Acid

EB Evans Blue

EC Endothelial Cell

EDTA Ethylenediaminetetra-acetic Acid

ELISA Enzyme-Linked Immunosorbent Assay

eNOS Endothelial Nitric Oxide Synthase

FP Fluid Percussion

FPI Fluid Percussion Injury

GABA Gamma-aminobutyric acid

GCS Glasgow Coma Scale

GI Gastrointestinal

GOS Glasgow Outcome Score

5-HT 5-hydroxytryptamine

H2O2 Hydrogen Peroxide

H3 Histamine

xi

H&E Haematoxylin and Eosin

ICAM-1 Intercellular Adhesion Molecule 1

ICP Intracranial Pressure

iNOS Inducible Nitric Oxide Synthase

I.P. Intra Peritoneal

I.V. Intra Venous

IP3 Inositol 1,4,5-triphosphate

K+ Potassium

LIS Locked In Syndrome

LOC Loss of Consciousness

MABP Mean Arterial Blood Pressure

MCS Minimally Conscious State

Mg2+ Magnesium

MHC Major Histocompatibility Complex

MRI Magnetic Resonance Imaging

mRNA Messenger Ribonucleic Acid

MTBI Mild Traumatic Brain Injury

MWM Morris Water Maze

Na2+ Sodium

NAD n-acetyl D-tryptophan

NAT n-acetyl L-tryptophan

NBM Nucleus Basalis Magnocellularis

NE Norepinephrine

NEP Neutral Endopeptidase

NHS Normal Horse Serum

xii

NK1 Neurokinin-1 Receptor



NKA Neurokinin A

NKB Neurokinin B

NMDA N-methyl-D-aspartate

nNOS Neuronal Nitric Oxide Synthase

NO Nitric Oxide

NOS Nitric Oxide Synthase

NPK Neuropeptide K

NP! Neuropeptide !

NSB Non-specific Binding

PBS Phosphate Buffered Solution

PCD Programmed Cell Death

PCr Phosphocreatine

PCS Post-Concussion Syndrome

!Pi Inorganic Phosphate Peak

PM Prospective Memory

PNS Peripheral Nervous System

PPTA Preprotachykinin A

PPTB Preprotachykinin B

PTA Post-traumatic Amnesia

PVS Persistent Vegetative State

ROS Reactive Oxygen Species

Rpm Revolutions per Minute

xiii

SD Standard Deviation

SEM Standard Error of Measurement

SP Substance P

SP-IR Substance P-Immunoreactivity

SPC Streptavin Peroxidase Conjugate

TA Total Activity

TAI Traumatic Axonal Injury

TBI Traumatic Brain Injury

TRH Thyrotropin-Releasing Hormone

TRS Target Retrieval Solution

TRIM 1-(2-trifluoromethylphenyl) imidazole

TRIMPOH 2-trifluoromethylphenol

UK United Kingdom

VR-1 Vanilloid Receptor-1

WDR Wide Dynamic Range

WHO World Health Organisation

xiv

Table of Contents

PUBLICATIONS AND PRESENTATIONS ...............................................................................................V

ACKNOWLEDGMENTS............................................................................................................................VII

ABBREVIATIONS ........................................................................................................................................ IX

TABLE OF FIGURES................................................................................................................................ XIX

ABSTRACT ................................................................................................................................................XXV

CHAPTER 1.......................................................................................................................................................1

INTRODUCTION.............................................................................................................................................1

1.1 EPIDEMIOLOGY .........................................................................................................................................4 1.1.1 Incidence and cause.........................................................................................................................4

1.2 NEUROPSYCHOLOGICAL CONSEQUENCES OF BRAIN INJURY..................................................................6 1.2.1 Mild Traumatic brain injury ...........................................................................................................7 1.2.2 Moderate Traumatic brain injury ...................................................................................................8 1.2.3 Severe Traumatic brain injury ......................................................................................................10

1.3 NEUROPATHOLOGY AND PATHOPHYSIOLOGY OF BRAIN INJURY.........................................................12 1.3.1 Primary traumatic brain injury mechanisms ...............................................................................13 1.3.2 Secondary traumatic brain injury mechanisms ...........................................................................19 1.3.3 Cell Death.......................................................................................................................................22

1.4 TRAUMATIC BRAIN OEDEMA .................................................................................................................24 1.4.1 Classification of oedema ...............................................................................................................25 1.4.2 temporal profile of oedema ...........................................................................................................27 1.4.3 Blood Brain Barrier Permeability ................................................................................................28 1.4.4 cerebral homeostasis and autoregulation ....................................................................................29 1.4.5 Treatment........................................................................................................................................31

1.5 SUBSTANCE P..........................................................................................................................................33 1.5.1 Synthesis .........................................................................................................................................33 1.5.2 Location ..........................................................................................................................................35 1.5.3 Localisation in the CNS.................................................................................................................36 1.5.4 Localisation in the PNS .................................................................................................................38 1.5.5 Enzymes ..........................................................................................................................................39 1.5.6 Receptors ........................................................................................................................................40 1.5.7 NK1 receptors .................................................................................................................................40 1.5.8 Co-localisation of tachykinins.......................................................................................................43 1.5.9 NK1 Receptor Internalisation ........................................................................................................45 1.5.10 NK1 receptor agonists..................................................................................................................46 1.5.11 NK1 receptor antagonists ............................................................................................................47 1.5.12 Effects of SP .................................................................................................................................49 1.5.14 Neurogenic Inflammation............................................................................................................53

1.6 EXPERIMENTAL TBI MODELS.................................................................................................................57 1.6.1 Fluid percussion injury..................................................................................................................59 1.6.2 Controlled Cortical Impact Injury ................................................................................................59 1.6.3 Impact-acceleration injury model (closed skull-weight drop) ....................................................60 1.6.4 Impact-acceleration injury model (closed skull-hydraulic piston).............................................61

1.7 SYNOPSIS.................................................................................................................................................62

xv

CHAPTER 2 GENERAL METHODS ........................................................................................................64

2.1 ANIMAL CARE.........................................................................................................................................65 2.1.1 Ethics ..............................................................................................................................................65 2.1.2 General ...........................................................................................................................................65

2.2 EXPERIMENTAL PROCEDURES................................................................................................................65 2.2.1 Anaesthesia.....................................................................................................................................65 2.2.2 Impact-Acceleration Injury (Weight-drop) ..................................................................................67 2.2.3 Impact-acceleration Injury (Hydraulic Piston) ...........................................................................69 2.2.4 Lateral Fluid percussion injury ....................................................................................................72 2.2.5 Perfusion.........................................................................................................................................73

2.3 DRUG TREATMENTS ...............................................................................................................................73 2.3.1 N-acetyl L-Tryptophan ..................................................................................................................73 2.3.2 1-(2-Trifluoromethyl-Phenly) Imidazole ......................................................................................74

2.4 FUNCTIONAL TESTS ................................................................................................................................74 2.4.1 Rotarod ...........................................................................................................................................74 2.4.2 Cognitive Outcome ........................................................................................................................77

2.5 OEDEMA MEASUREMENTS .....................................................................................................................82 2.5.1 Brain Water Content......................................................................................................................82 2.5.2 Nuclear magnetic resonance studies ............................................................................................82

2.6 BLOOD-BRAIN BARRIER PERMEABILITY ...............................................................................................85 2.6.1 Quantitative Evaluation.................................................................................................................85

2.7 MEASUREMENT OF SP BY ELISA ASSAY .............................................................................................85 2.8 TISSUE PROCESSING ...............................................................................................................................86

2.8.1 Paraffin embedding and sectioning ..............................................................................................86 2.8.2 Vibratome sectioning .....................................................................................................................87

2.9 HISTOLOGY AND IMMUNOHISTOCHEMISTRY.........................................................................................87 2.9.1 Haemotoxylin and Eosin staining .................................................................................................87 2.9.2 Amyloid precursor protein immunohistochemistry......................................................................88 2.9.3 Substance P Immunohistochemistry .............................................................................................88 2.9.4 Neurokinin-1 receptor Immunohistochemistry ............................................................................89 2.9.5 Qualitative evaluation of blood brain barrier permeability .......................................................89 2.9.6 Confocal Immunohistochemistry...................................................................................................90 2.9.7 Electron microscopy ......................................................................................................................90

2.10 STATISTICAL ANALYSIS .......................................................................................................................91

CHAPTER 3 CHARACTERISATION OF SUBSTANCE P RELEASE FOLLOWING

TRAUMATIC BRAIN INJURY...................................................................................................................92

3.1 INTRODUCTION .......................................................................................................................................93 3.2 METHODS ................................................................................................................................................96

3.2.1 Induction of traumatic brain injury ..............................................................................................96 3.2.3 Determination of blood brain barrier permeability ....................................................................97 3.2.4 ELISA assay....................................................................................................................................98 3.2.4 Statistical Analysis .........................................................................................................................98

3.3 RESULTS ..................................................................................................................................................99 3.3.1 Impact-Acceleration Injury (Weight-drop) ..................................................................................99 3.3.2 Lateral Fluid percussion injury ..................................................................................................106 3.3.3 Impact-acceleration Injury (Hydraulic Piston) .........................................................................106

3.4 DISCUSSION...........................................................................................................................................110

xvi

CHAPTER 4 CHARACTERISATION OF N-ACETYL L-TRYPTOPHAN IN TRAUMATIC

BRAIN INJURY ............................................................................................................................................112

4.1 INTRODUCTION .....................................................................................................................................113 4.2 METHODS ..............................................................................................................................................114

4.2.1 Induction of traumatic brain injury ........................................................................................114 4.2.3 Oedema.........................................................................................................................................116 4.2.4 Nuclear magnetic resonance studies ..........................................................................................117 4.2.4 Histology and Immunohistochemistry ........................................................................................119 4.2.5 Statistical analysis .......................................................................................................................121

4.3 RESULTS ................................................................................................................................................121 4.3.1 confocal microscopy ....................................................................................................................121 4.3.2 n-acetyl L-tryptophan optimal dosage........................................................................................121 4.3.3 Nuclear magnetic resonance studies ..........................................................................................124 4.3.4 Effects of NAT on Brain water content .......................................................................................126 4.3.5 Effects of NAT on blood brain barrier permeability .................................................................126 4.3.6 Effects of NAT on Brain Free Mg

2+............................................................................................129

4.3.7 Immunohistochemistry.................................................................................................................129 4.4 DISCUSSION...........................................................................................................................................137

CHAPTER 5 EFFECTS OF N-ACETYL L-TRYPTOPHAN ON FUNCTIONAL AND

HISTOLOGICAL OUTCOME FOLLOWING DIFFUSE TRAUMATIC BRAIN INJURY .........142

5.1 INTRODUCTION .....................................................................................................................................143 5.2 MATERIALS AND METHODS ..................................................................................................................146

5.2.1 Induction of diffuse impact-acceleration Injury.........................................................................146 5.2.2 Induction of the lateral fluid percussion Injury .........................................................................147 5.2.3 Groups ..........................................................................................................................................148 5.2.4 Treatment......................................................................................................................................148 5.2.5 Assessment of motor outcome .....................................................................................................149 5.2.6 Assessment of cognitive score .....................................................................................................149 5.2.7 Immunohistochemsitry.................................................................................................................151 5.2.8 Statistical analysis .......................................................................................................................151

5.3 RESULTS ................................................................................................................................................152 5.3.1 Motor outcome following diffuse impact-acceleration Injury ..................................................152 5.3.2 Motor outcome in lateral fluid percussion Injury......................................................................152 5.3.3 Cognitive outcome following diffuse impact-acceleration Injury.............................................155 5.3.7 Histology and immunocytochemistry..........................................................................................159

5.4 DISCUSSION...........................................................................................................................................165

xvii

CHAPTER 6 EFFECTS OF ALTERNATIVE NK1 RECEPTOR ANTAGONISTS ON

FUNCTIONAL OUTCOME FOLLOWING DIFFUSE TRAUMATIC BRAIN INJURY ..............170


6.2.1 Induction of diffuse impact-acceleration Injury.........................................................................171 6.2.2 Treatment......................................................................................................................................172 6.2.3 Roche NK1 receptor antagonist dose – response .......................................................................172 6.2.4 Assessment of motor outcome .....................................................................................................173 6.2.5 Statistical analysis .......................................................................................................................173

6.3 RESULTS ................................................................................................................................................174 6.3.1 Roche drug optimal dosage.........................................................................................................174 6.3.2 Motor outcome following diffuse impact-acceleration Injury ..................................................174

6.4 DISCUSSION...........................................................................................................................................177

CHAPTER 7 INHIBITING SUBSTANCE P RELEASE AS A NEUROPROTECTIVE

STRATEGY FOLLOWING DIFFUSE TRAUMATIC BRAIN INJURY ..........................................178


7.2.1 Induction of diffuse impact-acceleration Injury.........................................................................181 7.2.2 1-(2-trifluoromethylphenyl) imidazole dose – response............................................................182 7.2.3 Groups ..........................................................................................................................................183 7.2.4 Treatment......................................................................................................................................183 7.2.5 Assessment of motor outcome .....................................................................................................183 7.2.5 Immunohistochemsitry.................................................................................................................184 7.2.6 Statistical analysis .......................................................................................................................184

7.3 RESULTS ................................................................................................................................................184 7.3.1 1-(2-trifluoromethylphenyl) imidazole dose – response............................................................184 7.3.2 Motor outcome following diffuse impact-acceleration injury...................................................185 7.3.3 Histology and immunocytochemistry..........................................................................................188

7.4 DISCUSSION...........................................................................................................................................193

xviii

CHAPTER 8 ESTABLISHING THE THERAPEUTIC WINDOW FOR THE NK1 ANTAGONIST,

L-732,138, FOLLOWING DIFFUSE TRAUMATIC BRAIN INJURY ..............................................198


8.2.1 Induction of diffuse impact-acceleration Injury.........................................................................201 8.2.2 Treatment......................................................................................................................................202 8.2.3 Assessment of motor outcome .....................................................................................................202 8.2.4 Statistical analysis .......................................................................................................................203

8.3 RESULTS ................................................................................................................................................203 8.3.1 Motor outcome following 12 h treatment ...................................................................................203 8.3.2 Motor outcome following 24 h treatment ...................................................................................204

8.4 DISCUSSION...........................................................................................................................................207

CHAPTER 9 GENERAL DISCUSSION...................................................................................................210

9.1 CONCLUSION.........................................................................................................................................224

BIBLIOGRAPHY .........................................................................................................................................198

TABLE OF TABLES

Table 2.1 Relationship between rotational speed and seconds on the rotarod

device…………………………………….………………………………………..…75

xix

Table of Figures

Figure 1.1 Schematic representation of the biosynthesis of SP and related

peptides. (Harrison and Geppetti, 2001)....................................................34

Figure 1.2 Schematic diagram of human NK1 receptor (Regoli et al., 1994

.................................................................................................................41

Figure 1.3 Diagrammatic representation of the contribution of SP within

the peripheral nervous system (Harrison and Geppetti, 2001)...................55

Figure 2.1 Schematic representation of the impact-acceleration device used

to induce diffuse TBI. ...............................................................................68

Figure 2.2 Schematic representation of the location of the 10 mm

diameter stainless steel disc. .....................................................................69

Figure 2.3 Impact-acceleration injury model (closed skull-air piston)......71

Figure 2.4 The rotarod device consisted of a motorised rotating assembly

of 18 rods (1 mm in diameter) upon which the animals are placed............76

Figure 2.5 Barnes Maze photo .................................................................78

Figure 2.6 Object Recognition task..........................................................81

Figure 3.1 SP stained section from male Sprague-Dawley rats after sham

surgery…................................................................................................100

xx

Figure 3.2 SP stained section from male Sprague-Dawley rats following

the weight-drop model of diffuse TBI.....................................................101

Figure 3.3 Confocal imaging from the rat cortex (A) SP staining and (B)

EB staining from male, sham treated Sprague-Dawley rats. (C) SP staining

5h after moderate/severe TBI and (D) EB staining from male Sprague-

Dawley rats 5h after moderate/severe diffuse TBI. .................................103

Figure 3.4 Confocal imaging from a male Sprague-Dawley rat 5h after

injury. Injury results in co-localisation of both SP and EB at a cortical

vessel......................................................................................................104

Figure 3.5 Plasma concentration levels of SP in male Sprague-Dawley rats

following diffuse TBI. ............................................................................105

Figure 3.6 SP stained section from male Sprague-Dawley rats after sham

surgery....................................................................................................107

Figure 3.7 SP stained section from male Sprague-Dawley rats after

moderate/severe lateral fluid percussion TBI. .........................................108

Figure 3.8 SP stained section from male Sprague-Dawley rats after mild

diffuse TBI. ............................................................................................109

Figure 4.1 Confocal imaging of cortex from male, Sprague-Dawley rats

after diffuse TBI .....................................................................................122

xxi

Figure 4.2 EB brain tissue concentration in male Sprague-Dawley rats

following diffuse TBI. A concentration of 2.5mg/kg was determined to be

the optimum dosage for n-acetyl L-tryptophan. ......................................123

Figure 4.3 (A) Apparent diffusion coefficients (ADC) maps derived from

the diffusion weighted magnetic resonance images obtained from naïve

male Sprague-Dawley rats. Naïve treatment resulted in negative water

movement while (B) following diffuse TBI areas of subcortical

hyperintensity indicates the presence of vasogenic oedema. (C) When

treated with NAT this hyperintensity is decreased and oedema is resolved.

(D) ADC obtained from the brain following naïve, sham, moderate TBI and

moderate TBI + treatment in rats. ...........................................................125

Figure 4.4 Percentage cortical water content in male rats 5h following

moderate/severe TBI and treated with NAT............................................127

Figure 4.5 The level of BBB permeability in male Sprague-Dawley rats as

determined via EB penetration into the brain tissue.. ..............................128

Figure 4.6A Phosphorus MR spectrum displaying the effects of NAT on

brain free magnesium concentration.. .....................................................130

Figure 4.6B Brain intracellular free magnesium concentration following

diffuse TBI in male Sprague-Dawley rats following treatment with NAT

...............................................................................................................131

Figure 4.7 SP stained section from male Sprague-Dawley rats 5hr after

moderate/severe diffuse TBI and treated with NAT................................132

xxii

Figure 4.8 SP stained sections from male Sprague-Dawley rats 24hr after

moderate/severe diffuse TBI and treated.................................................134

Figure 4.9 SP stained sections from male Sprague-Dawley rats 3d after

moderate/severe diffuse TBI and treated with NAT................................135

Figure 4.10 AQP-4 staining within the perivascular astrocyte processes at

5hr following diffuse TBI. ......................................................................136

Figure 5.1 Motor (Rotarod) function in male shams and male animals

treated with saline, n-acetyl L-tryptophan (NAT) and n-acetyl D-tryptophan

(NAD) following TBI. ............................................................................153

Figure 5.2 Motor (Rotarod) function in male animals treated with saline

and n-acetyl L-tryptophan (NAT) following lateral FP TBI. ...................154

Figure 5.3 Cognitive (Object Recognition) function in male shams and

male animals treated with saline and n-acetyl L-tryptophan (NAT)

following TBI.. .......................................................................................156

Figure 5.4 Cognitive (Barnes Maze) function in male shams and male

animals treated with saline and n-acetyl L-tryptophan (NAT) following the

weight-drop TBI. (A) Acutely assessed up to 7days after injury (B)

Delayed assessment out to 3 weeks after injury.. ....................................158

Figure 5.5 Cognitive (Water Maze) function in male shams and male

animals treated with saline and n-acetyl L-tryptophan (NAT) following

TBI.. .......................................................................................................160

xxiii

Figure 5.6 H&E stained sections from male Sprague-Dawley rats

following injury and treatment with NAT...............................................161

Figure 5.7 H&E stained hippocampal sections from male Sprague-Dawley

rats following injury and treatment with NAT. .......................................163

Figure 5.8 APP stained sections from male Sprague-Dawley rats following

injury and treatment with NAT. ..............................................................164


following diffuse TBI. Optimal dosage of AU001 and AU002 was

determined to be 3mg/kg and 1mg/kg respectively. ................................175

Figure 6.2 Motor (Rotarod) function in male animals treated with saline,

AU001 and AU002 following TBI..........................................................176


following diffuse TBI. A concentration of 21 mg/kg was determined to be

the optimum dosage for 1-(2-trifluoromethylphenyl) imidazole..............186

Figure 7.2 Motor (Rotarod) function in male shams and male animals

treated with saline, 1-(2-trifluoromethylphenyl) imidazole (TRIM) and 2-

trifluoromethylphenol (TRIMPOH) following TBI.................................187

Figure 7.3 H&E stained sections from male Sprague-Dawley rats

following injury and treatment with TRIM and TRIMPOH. ...................189

Figure 7.4 APP stained sections from male Sprague-Dawley rats following

injury and treatment with TRIM and TRIMPOH. ...................................191

xxiv

Figure 7.5 SP stained sections from male Sprague-Dawley rats following

injury and treatment with TRIM and TRIMPOH. ...................................192

Figure 8.1 Motor (Rotarod) function in male animals treated with saline,

low dose L-732,138 (NAT-P), high dose of NAT-P and n-acetyl L-

tryptophan (NAT) 12 hr following TBI. .................................................205

Figure 8.2 Motor (Rotarod) function in male animals treated with low dose

L-732,138 (NAT-P), high dose of NAT-P and n-acetyl L-tryptophan (NAT)

24hr following TBI. ................................................................................206

Figure 9.1 Schematic illustrating the interrelationships between SP release

and inflammation....................................................................................223

xxv

ABSTRACT

Traumatic brain injury (TBI) today remains a major health and social problem for both

developed and developing countries. It is the leading cause of death and disability under

the age of 40, and despite the significance of this public health problem, no effective

therapy exists. While a number of factors have been shown to be associated with the

development of irreversible tissue injury after TBI, the formation of oedema and

opening of the blood brain barrier (BBB) have been shown to be of major significance

to outcome. Oedema formation in the brain, when left uncontrolled, results in increased

intracranial pressure that may lead to a decrease in brain tissue perfusion, localised

hypoxia and ischemia, and ultimately tissue herniation and death. The mechanisms

associated with the development of oedema are largely unclear, and accordingly, current

therapies are generally ineffective, often resulting in dehydration, hypotension and renal

problems.

This thesis describes the characterisation of neurogenic inflammation in the

development of post-traumatic brain oedema and functional deficits, and particularly the

role of substance P (SP) in mediating their development, using rodent models of both

focal and diffuse TBI. Results from this thesis demonstrate that increased SP

immunoreactivity, particularly at the level of the vasculature, is a ubiquitous feature of

TBI implying a potential role in the breakdown of the blood brain barrier (BBB)

following TBI. This was confirmed through the use of the NK1 receptor antagonists,

which attenuated BBB and oedema formation as well as deleterious morphological

events such as dark cell change and axonal injury. The NK1 receptor antagonists also

xxvi

resulted in an associated improvement in both motor and cognitive deficits, as assessed

using a battery of functional outcome tests. Possible mechanisms of action of the NK1

receptor antagonist included effects on the BBB, SP release, intracellular free

magnesium concentration and aquaporin-4 channels. Neuroprotection could be

facilitated with administration of a non-lipid permeable NK1 receptor antagonist in the

immediate postinjury period, or up to 12 h after TBI with a lipid permeable NK1

receptor antagonist, suggesting that this class of drugs have a clinically viable

therapeutic window. We conclude that SP has a significant role in the secondary injury

process following TBI and may offer a novel target for the development of

interventional pharmacological strategies.

the effects of the neuropeptide substance p on outcome ...the effects of the neuropeptide substance...

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