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C:\Documents and Settings\lproctor\Loca! Settings\Temporary Internet Files\OLK8\Copyright - thesis (2).doc T h e in f l u e n c e o f Fe t a l G r o w t h R e s t r ic t io n a n d In f e c t io n on Th e Ce r e b r a l M e t a b o l ic r e s p o n s e t o A c u t e H y p o x ia.
James Charles Dixon, M.B. Ch.B.
Thesis submitted for the Degree of Doctor of Medicine (M.D.) of the University of London.
In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion.
Donald Peebles MD (Senior Lecturer) for their encouragement and supervision, and for giving me the opportunity to undertake research as a Clinical Research Fellow within the Department of Obstetrics and Gynaecology at University College London.
I am especially grateful for the advice, assistance and technical support of Dr Em Cady Ph.D., Dr John Thornton Ph.D., Dr Andrew Priest Ph.D., Dr Alan Bainbridge Ph.D. and Professor Roger Ordidge Ph.D. in the Department of Medical Physics and Bioengineering, University College London. In addition, I wish to express my appreciation of the work of Dr Suzie Miller PhD, who performed early experiments to assess the feasibility of cerebral MRS in chick embryos.
I wish to thank Dr Gena Raivich MD, DSc and Dr Matthias Galiano MD for their assistance in the development of the histology techniques used in this research.
Intrauterine growth restriction and infection are significant risk factors for
hypothesised that these two factors increase the susceptibility of the fetal brain to hypoxic ischaemic damage by their influence on cerebral metabolism and development.
Cerebral energy and structural metabolites in a late gestation in-ovo chick embryo model of acute hypoxia and fetal growth restriction were measured noninvasively using proton and phosphorus Magnetic Resonance Spectroscopy. The influence of fetal growth restriction and acute hypoxia on cerebral metabolites was assessed. In addition, the individual and combined effects o f growth restriction, acute hypoxia and systemic infection on morphological markers of neuronal injury were assessed.
Acute hypoxia increased mean cerebral lactate/creatine (Cr) (0.58 to 1.56) and alanine/Cr (0.14 to 0.29) that gradually returned to normal after restoration of normoxia. Growth restriction was associated with an attenuated lactate/Cr response to acute hypoxia (0.32 to 0.92), and significantly increased cerebral P-hydroxybutyrate/Cr (0.57 vs. 0.25) and reduced cerebral intracellular energy reserves, inositol/Cr (1.15 vs. 1.61) and N-acetylaspartate/Cr (0.74 vs.
0.89) under normoxic conditions.
The cerebral energy and structural metabolite concentration changes associated with fetal growth restriction were consistent with impaired oxidative phosphorylation, ketosis, and altered brain development. However, despite the significantly different lactate response to hypoxia, there was no histological evidence that growth restriction affected markers of neuronal injury or exacerbated the effects of acute hypoxia.
The scientific and medical literature was reviewed to January 2004.
L ist of Figures
L ist of Tables
1.1 Fetal growth restriction
1.1.2 Evidence o f an association with brain injury
1.1.3 Influence o f fetal growth restriction on cerebral metabolism 20 1.1.4 Influence o f fetal growth restriction on cerebral development....22
1.2 Intrauterine infection
1.2.1 Evidence o f an association with brain injury
1.2.2 Interaction with hypoxia
1.3 Fetal Cerebral Metabolism
1.3.1 Energy substrates
220.127.116.11 Ketone bodies
1.3.2 Principal energy substrate
1.3.3 Response to hypoxia
1.4 Fetal cerebral perfusion
1.4.2 Acute hypoxia
1.4.3 Chronic hypoxia
1.5 Mechanisms of brain injury
1.5.2 Fetal growth restriction
1.6 Current methods of antenatal fetal assessment
2 MAGNETIC RESONANCE AND PERINATAL CEREBRALMETABOLISM________________________________________________ 50
2.1 Historical Background
2.2 Nuclear Magnetic Resonance
2.2.1 2.2.2 Factors affecting MRS spectral quality
18.104.22.168 Water suppression
2.22.2 Fat suppression
2.2.3 Computer based methods o f analysing MR spectra
2.2.4 Diffusion Weighted Imaging (DWI)
2.3 Magnetic Resonance and the Perinatal Brain
2.3.1 Magnetic Resonance Spectroscopy (MRS)
2.3.2 Perinatal Diffusion Weighted Imaging (DWI)
2.3.3 Human fetal magnetic resonance spectroscopy (MRS).................61 3 THE CHICK EMBRYO_____________________________________ 63
3.1 Physiology and development
3.3 Cardiovascular response to hypoxia
3.4 Absence of maternal influences
3.5 Model of fetal hypoxia
3.5.1 Preparation fo r acute hypoxia
3.5.2 Control o f acute hypoxia
3.5.3 Susceptib ility to hypoxia
3.6 Chick embryo model of fetal growth r estriction
4 METHODS AND MATERIALS_____________._________________ 71
4.1 Chick embryo preparation
4.1.1 Normal day 19 chick embryos
4.1.2 Growth restricted day 19 chick embryos
4.1.3 Inoculation o f day 18 chick embryos with lipopolysaccharide.... 73
4.2 Magnetic Resonance Spectroscopy (MRS) Methods
4.2.1 Sedation o f chick embryo
4.2.2 Chick embryo brain location
4.2.3 Proton (}H) spectroscopy (relative signal amplitudes)
4.2.4 Diffusion Weighted Magnetic Resonance Imaging
4.2.5 Phosphorus ( P ) spectroscopy (relative amplitudes)
4.2.6 Proton MRS cerebral metabolite quantitation method
22.214.171.124 Basic Physics:
126.96.36.199 Quantifying spectra
188.8.131.52 Proton (’H) spectroscopy (absolute concentrations)..................82
4.3 Chick embryo brain histology
4.3.1 Fixation protocol
4.3.2 Cryostat cutting protocol
184.108.40.206 Cerebral hemispheres (Anterior brain)
220.127.116.11 Optic tecti, cerebellum and brain stem (Posterior brain) 91 4.3.3 Fixation and dehydration o f tissue sections
4.3.4 Staining protocol
4.4 Nissl stained chick embryo brain cell counting protocol 92 4.4.1 Cerebral hemispheres
4.4.2 Optic tectum and cerebellum
4.4.3 Sample size
5 A COMPARISON OF THE ABSOLUTE AND RELATIVE
CONCENTRATIONS OF CEREBRAL METABOLITES OF DAY 19
NORMALLY GROWN AND GROWTH RESTRICTED CHICK
EMBRYOS IN-OVO INVESTIGATED BY PROTON AND PHOSPHORUSMAGNETIC RESONANCE SPECTROSCOPY.
5.2 Materials and Methods
6 THE CEREBRAL METABOLIC RESPONSE TO ACUTE HYPOXIA
OF THE DAY 19 CHICK EMBRYO IN-OVO MEASURED BY PROTONMAGNETIC RESONANCE SPECTROSCOPY_____________________111
6.2 Materials and Methods
18.104.22.168 Hyperstriatum ventralis, hippocampus and optic tectum........115 22.214.171.124 Cerebellum
7 A COMPARISON OF THE CEREBRAL METABOLIC RESPONSE
TO ACUTE HYPOXIA OF DAY 19 NORMALLY GROWN AND
GROWTH RESTRICTED CHICK EMBRYOS IN-OVO MEASURED BYPROTON MAGNETIC RESONANCE SPECTROSCOPY
7.2 Materials and Methods
126.96.36.199 Hyperstriatum ventralis, hippocampus and optic tectum 131 188.8.131.52 Cerebellum
8 THE EFFECT OF GROWTH RESTRICTION ON CEREBRAL
INTRACELLULAR ENERGY RESERVES IN THE CHICK EMBRYO INOVO DURING ACUTE HYPOXIA MEASURED BY PHOSPHORUSMAGNETIC RESONANCE SPECTROSCOPY.____________________ 146
8.2 Materials and Methods
9 A DIFFUSION WEIGHTED MAGNETIC RESONANCE IMAGING
STUDY OF THE EFFECT OF ACUTE HYPOXIA AND GROWTH
RESTRICTION ON CEREBRAL METABOLISM IN THE CHICKEMBRYO IN-OVO ____________________________________________ 158
9.2 Materials and Methods
10 AN EXPERIMENT TO DETERMINE THE REQUIRED DOSE OF
SALMONELLA TYPHILEPOPOLYSACCHARIDE ADMINISTERED ONDAY 18 OF INCUBATION TO CAUSE 50% MORTALITY (LD50) IN CHICK EMBRYOS BY DAY 22_____________________ 167
10.1 Introduction......................................... 16710.2 Materials and M ethods
11 AN INVESTIGATION OF THE EFFECT OF SYSTEMIC
SALMONELLA TYPHI LEPOPOLYSACCHARIDE AND ACUTE
HYPOXIA ON DAY 21 CHICK EMBRYO CEREBRAL HISTOLOGY. 17411.1 Introduction
Materials and Methods
11.3.1 Hyperstriatum ventralis, hippocampus and optic tectum 177 11.3.2 Cerebellum
12 FURTHER WORK.______________________________________ 181 13 OVERVIEW____________________________________________ 183 A p p e n d ix 1
G lo ssa ry, Apparent Diffusion Coefficient ADC ADP Adenosine diphosphate Adenosine triphosphate ATP pHB P-hydroxybutyrate CAM Chorioallantoic Membrane Confidence Interval Cl Creatine Cr Cardiotocograph CTG Diffusion Weighted Imaging DWI FID Free Induction Decay Hi Hippocampus Hypoxic Ischaemic Encephalopathy HIE Hyperstriatum ventralis HV Growth Restriction GR IL Interleukin Intelligence Quotient IQ Intrauterine growth restriction IUGR Lipopolysaccharide LPS Magnetic Resonance Spectroscopy MRS NAA N-acetylaspartate NMR Nuclear Magnetic Resonance Number of Summed echoes NS Odds Ratio OR OT Optic Tectum Partial pressure of carbon dioxide pC 02 PCr Phosphocreatine Pi Inorganic phosphate Partial pressure of oxygen p02 SD Standard Deviation SGA Small for Gestational Age T1 Spin-lattice or longitudinal relaxation time T2 Spin-spin or transverse relaxation time TE Echo Time TR Repetition Time TNF-a Tissue Necrosis Factor a VOI Volume of interest L is t o f F ig u r e s Figure 2.1 Free induction decay signal showing a decaying (e_t/T sinusoid with a 2) frequency 0)o, where T2 is the spin-spin relaxation time
Figure 3.1 A chick embryo and internal egg structures
Figure 4.1 Sagittal MRI of day 19 chick embryo in-ovo showing spine and brain.
Figure 4.2 Components required for chick embryo cerebral MRS: chick embryo in-ovo, a 2.
5 cm diameter circular surface coil, neonatal sphygmomanometer cuff (attached to opaque white tubing), fluoroptic thermistor (black wire) and 150ml plastic bag (attached to transparent green tubing)
Figure 4.3 Assembled components required for chick embryo cerebral MRS, with the chick embryo brain was fixed over the centre of the circular surface coil