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Retinopathy of Prematurity and Multiple
Postnatal Infections in Preterm Neonates: Delays
in White Matter Development with Poorer
Neurodevelopmental Outcomes.
by
Torin James Alexander Glass
A thesis submitted in conformity with the requirements
for the Degree of Master of Science
Institute of Medical Science
University of Toronto
© Copyright by Torin James Alexander Glass 2018
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Retinopathy of Prematurity and Multiple Postnatal Infections in
Preterm Neonates: Delays in White Matter Development with Poorer
Neurodevelopmental Outcomes.
Torin James Alexander Glass
Master of Science
Institute of Medical Science
University of Toronto
2018
Abstract
Preterm birth is a common cause of neurodevelopmental disorders in childhood.
Little is known about the outcome of infants with severe retinopathy of prematurity (ROP)
and multiple infections in the postnatal period. This thesis describes their associations
with abnormal brain development using multi-modal MR imaging and standardized
outcome assessments. Those infants with severe ROP had delayed brain maturation of
the posterior white matter and optic radiations, with poorer 18 month cognitive and motor
outcomes. Compared to fewer episodes of infection, three or more postnatal infections
was associated with delayed maturation of the posterior limb of the internal capsule
(PLIC), corpus callosum and the optic radiations, with poorer 36 month motor outcomes.
Our findings support that severe ROP and multiple postnatal infections in very preterm
newborns are associated with decreased brain maturation and poorer
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neurodevelopmental outcomes, and that advancements in these disorders have the
potential to improve outcomes.
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Acknowledgements
Firstly, I thank Dr. Steven Miller for his mentoring and superb insight into the needs
of my learning as a trainee and developing researcher. I have felt continually supported,
challenged and encouraged throughout completing this work. During my time at SickKids
Hospital and the University of Toronto I have learnt so much and I am grateful for the time
and energy that was invested in the development of my research and clinical skills.
Secondly, I thank Dr. Vann Chau for his unending support and friendship, and for
his commitment to my success as a clinician and researcher. It was in part due to his
encouragement, kindness and laughter in the work environment that I came to the
University of Toronto in the first place. Thank you for the words of wisdom and the
thousands of coffees that have fueled me through the last couple of years.
I also sincerely thank Dr. John Sled and Dr. Margot Taylor for their guidance and
assistance as members of my advisory committee. Their combined analytical assessment
improved greatly the quality of the work within this thesis and have made me a better
researcher.
To the members of the Miller and Tam labs and the Neonatal Neurology fellows,
nurses and NPs, thank you for making my time in Toronto some of the best of my
professional life. Your unending encouragement of my work provided me with the
inspiration to complete it and has educated me in the importance of surrounding oneself
with truly good and inspiring people. I would especially like to thank Emily Tam, Diane
Wilson, Claire Watt, Julianne Schneider, Lara Leiser, Mariam Ayed, Amr Al-Shahed,
Elana Pinchefsky, Dalit Cayam-Rand, Isabel Benavente-Fernandez and Asma Al-
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Mazroei and for their support through the years and for the laughter we have shared in
the process of my fellowship. In addition, this project would not have been possible
without the contributions of Jessie Guo, Justin Foong, Emma Duerden, Janet Rigney,
Ruth Grunau, Anne Synnes and Ken Poskitt.
Special thanks go to my family, whose continued support and encouragement has
always pushed me to be the best that I can be and in large part are the reason why I am
where I am today. My biggest thanks to my wife, Michelle, and son, Ronan, who have
joined me on this adventure to Toronto and whose strength and humility provided the
backbone upon which I was able to complete this work.
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Contributions
The author was responsible for the writing and preparation of this original thesis. All of
the work presented, including the planning, analysis and writing of the original research,
was performed by the author, with the guidance and expertise of the individuals listed
below. The following contributions to the work in this thesis are formally and inclusively
acknowledged:
Dr. Vann Chau: Provided clinical assessments and data described in Chapters 2 – 3 as
well as producing the DTI and MRSI database used in Chapters 2 and 3.
Dr. Emma Duerden and Mr. Justin Foong: Assisted in the development of the TBSS
analysis method and provided the figures in Chapters 2 and 3.
Dr. Jessie Guo: Provided the brain segmentation volumes used in Chapter 3.
Dr. Anne Synnes and Dr. Ruth Grunau: Contributed to the overall planning and
assessments for the follow-up and outcomes in Chapters 2 and 3.
Dr. Jane Gardiner: Provided the retinopathy of prematurity scoring in Chapters 2 and 3.
Dr. Ken Poskitt: Provided the MRI analysis and injury scoring in Chapters 2 and 3.
Dr. Jillian Vinall: Contributed DTI analysis to the study in Chapter 2.
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Table of Contents
Contents Page
Acknowledgments…………………………………………………………………………. iv
Contributions………………………………………………………………………………. vi
Table of Contents…………………………………………………………………………. vii
List of Abbreviations………………………………………………………………………. xi
List of Figures …………………………………………………………………………….. xiv
List of Tables ……………………………………………………………………………… xv
1 Literature Review……………………………………………………………………….. 1
1.1 Introduction………………………………………………………………………… 2
1.2 Preterm Birth………………………………………………………………………. 3
1.2.1 Overview…………………………………………………………………….. 3
1.3 Brain Injury in Preterm Infants…………………………………………………… 6
1.3.1 White Matter Injury…………………………………………………………. 6
1.3.2 Intraventricular Hemorrhage………………………………………………. 10
1.3.3 Cerebellar Hemorrhage……………………………………………………. 11
1.4 Retinopathy of Prematurity………………………………………………………. 12
1.4.1 Background…………………………………………………………………. 12
1.4.2 Pathophysiology……………………………………………………………. 15
1.4.3 Outcomes…………………………………………………………………… 17
1.5 Infection in Preterm Infants………………………………………………………. 18
1.5.1 Background…………………………………………………………………. 18
1.5.2 Classification………………………………………………………………... 18
1.5.3 Pre-Natal Infections………………………………………………………… 19
1.5.4 Postnatal Infections…………………………………………………………. 20
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1.5.5 Neonatal Inflammation……………………………………………………… 22
1.5.6 Neonatal Hypoxia-ischemic Injury…………………………………………. 24
1.6 Magnetic Resonance Imaging……………………………………………………. 26
1.6.1 Imaging of Preterm Newborn………………………………………………. 26
1.6.2 Overview of MRI basics…………………………………………………….. 27
1.6.3 MRI Sequences……………………………………………………………… 28
1.6.4 Diffusion Weighted Imaging and Diffusion Tensor Imaging ……………. 29
1.6.5 Magnetic Resonance Spectroscopic Imaging……………………………. 35
1.7 Neurodevelopmental Outcomes………………………………………………….. 39
1.7.1 Background………………………………………………………………….. 39
1.7.2 Bayley Scales of Infant and Toddler Development……………………… 39
1.7.3 Peabody Developmental Motor Scales…………………………………… 40
1.7.4 Cerebral Palsy……………………………………………………………….. 41
1.8 Hypothesis, Major Goal and Specific Aims……………………………………… 43
1.8.1 Hypothesis……………………………………………………………………. 43
1.8.2 Major Goal……………………………………………………………………. 43
1.8.3 Specific Aims…………………………………………………………………. 44
2 Severe Retinopathy of Prematurity predicts Delayed White Matter Maturation
and Poorer Neurodevelopment at 18 months CA………………………………….. 45
2.1 Introduction…………………………………………………………………………. 46
2.2 Material and Methods……………………………………………………………… 47
2.2.1 Participants…………………………………………………………………… 47
2.2.2 Clinical Characteristics……………………………………………………… 47
2.2.3 MRI Studies………………………………………………………………….. 48
2.2.4 Diffusion Tensor Imaging…………………………………………………… 48
2.2.5 Developmental Follow-up…………………………………………………… 50
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2.2.6 Data Analysis…………………………………………………………………. 50
2.3 Results………………………………………………………………………………. 51
2.3.1 Clinical Characteristics……………………………………………………… 51
2.3.2 Retinopathy of Prematurity…………………………………………………. 52
2.3.3 Brain Injury…………………………………………………………………… 54
2.3.4 White Matter Maturation…………………………………………………….. 54
2.3.5 Developmental Outcomes………………………………………………….. 57
2.4 Discussion………………………………………………………………………….. 58
2.4.1 Limitations……………………………………………………………………. 60
2.5 Conclusions………………………………………………………………………… 61
3 Multiple Postnatal Infections in Preterm Newborns are associated with delayed Maturation of
Motor Pathways at Term-equivalent age and Poorer Motor Outcomes at
3 years…………………………................................................................................ 62
3.1 Introduction…………………………………………………………………………. 63
3.2 Material and Methods……………………………………………………………… 64
3.2.1 Study Population…………………………………………………………….. 64
3.2.2 Clinical Characteristics……………………………………………………… 65
3.2.3 MR Brain Imaging……………………………………………………………. 65
3.2.4 Magnetic Resonance Spectroscopic Imaging……………………………. 66
3.2.5 Diffusion Tensor Imaging…………………………………………………… 66
3.2.6 Developmental Follow-up…………………………………………………… 68
3.2.7 Data Analysis………………………………………………………………… 68
3.3 Results……………………………………………………………………………… 69
3.3.1 Clinical Characteristics……………………………………………………… 69
3.3.2 Infection Characteristics…………………………………………………….. 70
3.3.3 MR Imaging………………………………………………………………….. 73
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3.3.4 MRSI and DTI Imaging……………………………………………………… 74
3.3.5 Developmental Outcomes………………………………………………….. 76
3.4 Discussion………………………………………………………………………….. 79
3.4.1 Limitations……………………………………………………………………. 84
3.5 Conclusions………………………………………………………………………… 84
4 Summary of Main Findings and Future Directions…………………………………… 86
4.1 Conclusions………………………………………………………………………… 87
4.2 Future Directions…………………………………………………………………… 88
4.2.1 Retinopathy of Prematurity and Neurodevelopment…………………….. 88
4.2.2 Multiple Postnatal Infections and Neurodevelopment…………………… 93
References ………………………………………………………………………………… 104
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List of Abbreviations
ω3-PUFA – ω3 Polyunsaturated fatty acids
3D – Three-dimensional
ADHD – Attention-deficit hyperactivity disorder
BPD – Bronchopulmonary dysplasia
BSID-III – Bayley Scales of Infant and Toddler Development-III
CA – Corrected age
CHO – Glycerophosphocholine + phosphocholine
CI – Confidence interval
CNS – Central nervous system
CONS – Coagulase-negative staphylococcus aureus
CR – Creatine + phosphocreatine
CSF – Cerebrospinal fluid
DCD – Developmental coordination disorder
DHA - Docosahexaenoic acid
DNA – Deoxyribonucleic acid
DTI – Diffusion tensor imaging
DWI – Diffusion weighted imaging
EPO – Erythropoietin
FA – Fractional anisotropy
GA – Gestational age
GABA – Gamma-aminobutyric acid
GBS – Group B streptococcus
IGF-1 – Insulin-like growth factor 1
IL – Interleukin
INS – Myo-inositol
IQ – Intelligence quotient
IQR – Interquartile range
IVH – Intraventricular hemorrhage
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LAC – Lactate
LMP – Last menstrual period
MD – Mean diffusivity
MEG - Magnetoencephalogram
MHz – Megahertz
MR – Magnetic resonance
MRI – Magnetic resonance imaging
MRSI – Magnetic resonance spectroscopic imaging
NAA – N-acetylaspartate + n-acetylaspartylglutamate
NEC – Necrotising enterocolitis
NICU – Neonatal intensive care unit
OCT – Optical coherence topography
OL – Oligodendrocyte
OR – Odds ratio
PDA – Patent ductus arteriosus
PDMS-2 – Peabody developmental motor scales 2
PLIC – Posterior limb of the internal capsule
PMA – Post-menstrual age
PPM – Parts per million
PRR – Protein recognition receptor
PVL – Periventricular leukomalacia
RF – Radiofrequency
RNFL – Retinal nerve fibre layer
ROI – Region of interest
ROP – Retinopathy of prematurity
ROS – Reactive oxygen species
RR – Risk ratio
SNAPPE-II – Score for neonatal acute physiology with perinatal extension-II
TBSS – Tract-based spatial statistics
TE – Echo time
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TNF-α – Tumor necrosis factor alpha
TR – Repetition time
VEGF – Vascular endothelial growth factor
WMI – White matter injury
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List of Figures Page
1.1 Number of Neonatal Morbidities and Probability of Poor Outcomes at 5 years… 6
1.2 WMI Severity in the Preterm Infant………………………………………………..... 8
1.3 Pathophysiology of Diffuse and Axonal WMI…………………………..………….. 8
1.4 Retinopathy of Prematurity scoring scale…………………………………………… 13
1.5 Retinopathy of Prematurity stages of severity……………………………………… 14
1.6 Pathogenic Mechanisms in Retinopathy of Prematurity…………………………... 16
1.7 Representation of the 3D diffusion of an ellipsoid…………………………………. 30
1.8 Diffusion Tensor Imaging colour map of a Preterm Infant Brain…………………. 32
1.9 TBSS model of Age-specific Templates……..……………………………………... 34
1.10 MRSI example from a Preterm Infant……………………………………………… 38
2.1 Flow chart of study enrollment………………………………………………………. 52
2.2 Mean FA map and results of Severe ROP………..……………….………………. 54
2.3 TBSS model of Severe ROP results…………..………………….………………… 56
3.1 Flow chart of study enrollment………………………………………………………. 70
3.2 MRSI graph results of NAA/CHO ratio in white matter and basal ganglia……… 74
3.3 TBSS results of Multiple Infections compared to no Infection group…………….. 76
3.4 BSID-III Outcomes at 36 months across groups…………………………………… 78
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List of Tables Page
2.1 Demographics and Imaging results in Infants with and without Severe ROP….. 53
2.2 Follow-up Assessments at 18 months CA…………………………………………. 57
2.3 Multivariate Linear Regression Analysis of Outcomes at 18 months CA……….. 58
3.1 Demographics and Imaging in Infants with Postnatal Infections……………….... 72
3.2 Infection Location and Organism…………..………………………………………… 73
3.3 BSID-III Outcomes at 36 months CA by Infection groups……..…………………. 77
3.4 Outcome Odds for Poorer Outcomes at 36 months CA……..……………………. 79
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Chapter 1
Literature Review
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1.1 Introduction
Retinopathy of prematurity (ROP) and postnatal infection are important factors in
children born preterm, which are often associated with poor neurodevelopmental
outcomes. The pathophysiologies of ROP and infection share several characteristics with
inflammation and oxygenation alterations likely leading to impairments of the developing
brain. Infections, in particular postnatal bacteremia and fungal infections, are an
independent risk factor in the development of severe ROP in extremely preterm infants
(Manzoni et al, 2006; Tolsma et al, 2011). This association is possibly explained by the
combined effects of systemic inflammation and hypoxia-ischemia in the susceptible
preterm infant (Chen et al, 2011; Dammann, 2010), though the complete effects of each
remains uncertain. Furthermore, infection has been shown to have effects on insulin-like
growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF), growth factors
known to have critical roles in the growth of normal and abnormal vascularization of the
developing retina (Biswas et al, 2006; Heemskerk et al, 1999).
Structural MRI brain imaging of preterm infants has the potential to predict long-
term outcomes, with abnormalities in motor pathway tracts shown to be associated with
a higher likelihood of developing neurodevelopmental impairments (de Vries et al, 2011;
Guo et al, 2017). Advanced MRI techniques, such as diffusion tensor imaging (DTI) to
measure fractional anisotropy, have described delayed development of motor pathways
in children with cerebral palsy (Thomas et al, 2005; Yoshida et al, 2010), further
strengthening the role of MRI in predicting outcomes.
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While much is known about postnatal infection and severe ROP, and their
associated developmental outcomes, descriptions of the alterations in the brain of infants
with these conditions are few. The research presented in this thesis examines the
association of severe ROP and of multiple postnatal infections with brain development of
preterm infants using multi-modal MRI methods, and describes the neurodevelopmental
outcomes of children with these disorders.
1.2 Preterm Birth
1.2.1 Overview
Every year approximately 15 million infants world-wide are born preterm (before
37 completed weeks of gestation), with complications stemming from prematurity the
number one cause of death in infants under 5 years of age (Blencowe et al, 2012).
Surviving preterm infants have higher rates of cognitive dysfunction, motor impairments,
attention-deficit hyperactivity disorder (ADHD) and autism than infants born at term
(Bhutta et al, 2002; Johnson et al, 2010; Wood et al, 2000). This leads to an increased
requirement for medical services resulting in a greater cost to the medical system than
term-born infants (Petrou, 2005).
Infants born prematurely are roughly grouped into three categories based upon the
completed weeks of gestation since the last menstrual period (LMP), approximated with
antenatal ultrasound measurements, and referred to as their “gestational age” (GA).
Infants born at <28 weeks GA are termed “extremely preterm”, 28 to <32 weeks GA as
“very preterm”, and 32 to <37 weeks as “moderate to late preterm”. The GA at birth carries
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a wealth of information about the potential risks of poor outcomes resulting from greater
immaturity of the lungs, brain and other developmental systems. More medical
interventions and an increased risk of their potential complications are consequences of
a lower GA at birth (Leviton et al, 2005) with GA being more informative than birthweight
for these risks.
Preterm birth often occurs as a result of several maternal (e.g. pre-eclampsia,
diabetes), obstetric (e.g. short cervix, placenta previa, chorioamnionitis) and/or fetal
(multiple gestations, genetic disorders) causes. Poorer outcomes in an infant are often
linked to specific causes of preterm labour, with several perinatal factors associated with
delivery, such as asphyxia, inflammation, fetal growth restriction and major birth defects
all occurring more commonly in those infants with poorer outcomes (Delorme et al, 2016;
Nelson and Blair, 2015).
There is significant epidemiological data describing the marked worldwide
variations in outcomes of infants born preterm (Hossain et al, 2015; Shah et al, 2016).
Current data supports the resuscitation of infants ≥23 weeks GA, considered by many
experts in the field to be the limit of viability for the human infant. A “gray zone” still exists
<23 weeks and <500 grams where infant survival to neonatal intensive care unit (NICU)
discharge ranges between 0% and 50%, with wide variation between centres (Rysavy et
al, 2015; Seri and Evans, 2008; Stoll et al, 2010). Neurodevelopmental outcomes of
preterm-born infants have been shown to be significantly impaired compared to their
peers born at term-age, with the greatest percentage of impairments seen within the
extremely preterm group (Marlow et al, 2005; Stoll et al, 2010). However, a significant
proportion of children born near the limits of viability at 23-24 weeks will perform within
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the expected school norms at early school age, providing meaningful reassurance that
resuscitation of these infants should be continued (Garfield et al, 2017; James et al,
2017).
In helping to understand the clinical factors that have the greatest impact upon the
long-term development of the infant born preterm, large epidemiologic studies have been
reported. One particular study of 5 year outcomes reported increased mortality and
morbidity independently with each of brain injury, bronchopulmonary dysplasia (BPD) and
retinopathy of prematurity (ROP) (Schmidt et al, 2015). They were also able to show that
in those infants with a combination of morbidities the risk for poor outcomes increased
significantly from 11% in those with none of the above morbidities to 62% in those with
all three (Figure 1.1) (Schmidt et al, 2015). What causes such a large percentage of
infants with these conditions to have poor outcomes remains an important area of
research in the care of the preterm infant.
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Figure 1.1 Number of Neonatal Morbidities and Probability of Poor Outcomes at 5
years. Probability of poor 5-year outcome in infants; 95% CI shown by error bars. Linear
line indicates predictions based on the fitted morbidity count model. Horizontal dotted
line indicates the overall probability of poor 5 year outcome. Republished from Schmidt
et al (2015) Journal of Pediatrics with permission from Elsevier.
1.3 Brain injury in Preterm Infants
1.3.1 White Matter Injury
Severe white matter injury (WMI), known today as cystic periventricular
leukomalacia (PVL), was first described by Banker and Laroche in 1962 based upon
pathological specimens of infants who were found to have lesions consisting of necrosis
and macrophage activity within the periventricular white matter (Banker and Larroche,
1962). The identified cause of PVL in the majority of these cases was severe anoxia, with
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WMI now seen as the major form of brain injury recognized in survivors of preterm birth,
with the greatest risk to infants <32 weeks PMA (Volpe, 2009).
PVL is often caused by cerebral ischemia because of hemodynamic instability with
hypoxia-ischemia or inflammation as a result of intrauterine or postnatal infections.
During the development of the cerebral vascular bed in the preterm infant, vascular
watershed regions develop. These areas are vulnerable to reductions in perfusion as they
are furthest from the arterial supply and their blood and oxygen delivery supply is at the
border of two vascular territories. In the preterm infant brain, the white matter surrounding
the periventricular region is in a major watershed zone. Thus, PVL has historically been
considered a form of watershed ischemic brain injury of the preterm infant.
During the initial phase of WMI the major cells thought to degenerate are the pre-
oligodendrocyte (OL) lineage cells. Axonal injury can also occur and is a prominent
feature of cystic WMI where necrosis is present, such as seen in classical cystic PVL.
Though in a recent series, cystic PVL comprised only about 5% of the total burden of WMI
(Haynes et al, 2008; Kinney and Back, 1998). Disturbances in the normal myelination
pathway are initiated by the selective vulnerability of the late pre-OL in the most common
form of WMI at 23 – 32 weeks GA (Back and Miller, 2014; Back et al, 2001).
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Figure 1.2 Severity of WMI. MRI examples of (A) mild (B) moderate and (C) severe white
matter injury on T1 1mm sagittal images of preterm infants. Scoring scale adapted from
Miller et al (2005) Journal of Pediatrics. Arrows highlight the areas of T1 hyperintensity
which are marked as areas of WMI.
Figure 1.3 WMI pathophysiology. Proposed pathogenic mechanisms with distinct
differences in periventricular leukomalacia [PVL] (upper pathway) and diffuse WMI (lower
pathway). More severe events of hypoxia-ischemia results in cystic PVL, with milder
events resulting in selective pre-oligodendrocyte (OL) death and myelination failure.
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Reprinted from Back and Miller (2014), Annals of Neurology with permission from
publisher John Wiley and Sons.
The predominant form of WMI in the preterm infant is diffuse WMI, which is more
commonly seen in those infants with necrotizing enterocolitis (NEC), infection, and
hypoxia-ischemia, and which results in poorer outcomes than expected for a neonates’
GA (Glass et al, 2008; Shah et al, 2008). Through hypoxia-ischemia and inflammation,
glutamate-mediated injury occurs to the pre-OL cell leading to selective cell death and
loss of maturation-dependent cellular processes (Khwaja and Volpe, 2008). It remains
unknown the extent to which the individual and combined effects of hypoxia-ischemia or
inflammation result in WMI, with white matter cellular injury described in animal models
of both (Hagberg et al, 2002). There remains significant debate in the literature
considering the effects of both inflammation and hypoxia-ischemia in WMI, with some
experts suggesting it is predominantly a result of inflammation (Gilles et al, 2017) and
others suggesting it’s predominantly from hypoxia-ischemia (Hagen et al, 2014). What
seems most likely is that the developing cells are susceptible to the combined effects of
both inflammation and hypoxia-ischemia, resulting in cellular injury (Back, 2006; Back and
Rivkees, 2004; Khwaja and Volpe, 2008; Penn et al, 2016). In addition, inflammation can
be induced by hypoxia-ischemia, resulting in a greater cellular impairment, while hypoxia-
ischemia can occur from inflammation, such as from hypotension during an infection. The
best description of these effects refers to the “upstream” mechanisms of hypoxia-
ischemia and inflammation in activating brain microglia, followed by the “downstream”
mechanisms of excitotoxicity with generation of free radicals resulting in the subsequent
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death of the vulnerable pre-OL (Khwaja and Volpe, 2008). Regardless of cause, as we
gain a greater understanding of the pathophysiology of WMI we will be better prepared to
evaluate new strategies in its prevention and treatment.
The outcomes of children with classical PVL range from CP to milder motor
impairments (Fazzi et al, 1994; Hamrick et al, 2004; Miller et al, 2000; Spittle et al, 2011).
The outcomes of diffuse WMI are less robust in the literature with motor and cognitive
impairments described (Woodward et al, 2006; Woodward et al, 2012). The outcomes of
infants following diffuse WMI are best predicted by the location of the injury, with presence
of frontal lobe WMI the strongest predictor of poor motor, language and cognitive
outcomes (Guo et al, 2017). With the development of advanced MR techniques in the
preterm infant we are gaining further information about white matter maturation (Hoon et
al, 2009; Miller et al, 2002), connectivity (Smyser et al, 2013) and regional abnormalities
(Pierson et al, 2007) seen in white matter injury of the preterm infant.
1.3.2 Intraventricular Hemorrhage
Intraventricular hemorrhage (IVH) is a condition in which there is hemorrhage in
the ventricular system of the brain, or the sub ependymal zone immediately adjacent to
it. IVH is especially common in preterm infants with a highest period of injury at <30 weeks
due to the fragility of the germinal matrix at this time. The germinal matrix is a region of
blood vessels immediately adjacent to the lateral ventricles which arises during fetal
development and usually disappears before 35 weeks GA. When born prematurely,
infants are at risk of rupturing the germinal matrix, particularly those infants requiring
resuscitation with fluctuations in blood pressure (Ballabh, 2010). IVH is categorized into
grades of severity with grade I considered mild, grade II moderate and grades III and IV
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severe. IVH grades I and II have a low possibility of long-term damage as the blood does
not cause excessive pressure or occlude the normal ventricular system. However, in
grades III and IV there is frequently blood occluding the ventricular system resulting in a
backflow of fluid, potentially irreversibly injuring the brain. Post-hemorrhagic ventricular
dilatation often develops slowly in causing injury to the brain parenchyma, and while the
optimal management of this condition remains uncertain, treatment with a CSF reservoir
and/or ventricular-peritoneal shunt remains standard therapy. Improvements in antenatal
care of the pregnancy at-risk for preterm birth has resulted in reduced rates of IVH for
those infants exposed to antenatal steroids across all gestational ages (Wei et al, 2016),
suggesting preventative therapies can effectively reduce brain injury in this population.
The extent of the injury and the resultant neurodevelopmental impairments remains a
function of the severity of the hemorrhage and the location of any parenchymal injury
(McCrea and Ment, 2008). When accompanied by parenchymal injury there is a greater
risk of cerebral palsy, low mental and motor scores and visual and hearing impairments
(Vohr et al, 2003); though without associated parenchymal injury the risk of adverse
outcomes is low (Han et al, 2002; Linsell et al, 2016; O'Shea et al, 1998).
1.3.3 Cerebellar Hemorrhage
The cerebellum is a compact and particularly important anatomical region involved
in neurodevelopment of the preterm infant, encompassing more than 3.5 neurons for
every neuron in the cerebrum (Herculano-Houzel, 2010). Its role in motor, language and
cognitive development is evident. Injury to the cerebellum, such as a hemorrhage, is
shown to result in significant adverse outcomes with greater rates of autism, motor
impairments and an abnormal neurological examination (Tam et al, 2011; Tam et al,
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2009; Ure et al, 2016; Wang et al, 2014). The size of cerebellar hemorrhage is an
important predictor of outcomes, with small hemorrhages (<4mm on MRI) not seen to be
associated with poorer outcomes (Steggerda et al, 2013). In addition, the location of the
hemorrhage is also of importance, with injury to the vermis thought to confer a greater
risk of impairments than injury to a hemisphere (Hashimoto et al, 1995). Delays in the
cerebellar development with poorer neurodevelopmental outcomes are also seen
following cerebellar hemorrhage (Lee et al, 2016; Messerschmidt et al, 2008). With the
exploration and expansion of research into the cerebellum the medical community has
learned that the cerebellum in the preterm infant is “rapidly developing, vulnerable and
clinically important” (Volpe, 2009).
1.4 Retinopathy of Prematurity
1.4.1 Background
Retinopathy of prematurity (ROP) is a vascular proliferation disorder of the
developing retina, and is the leading cause of visual impairment and blindness in infants
born preterm (Rivera et al, 2011). First described in the late 1940s, ROP appeared
suddenly in those infants who were the first to receive supplemental oxygen in closed
incubators. Initially called “retrolental fibroplasia,” meaning “proliferation of fibrous tissue
behind the lens,” ROP had severe consequences with retinal detachment leading to
blindness in the most severe cases (Hellstrom et al, 2013). As the shift away from
unrestrained oxygen therapy has been made in NICUs throughout the developed world,
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there remains a delicate balance of oxygenation between lung maturity and survival with
poorer visual outcomes (Hellstrom et al, 2013).
ROP severity is scored clinically by a pediatric ophthalmologist via a dilated eye
examination using an indirect ophthalmoscope with examinations starting in those infants
born <31 weeks GA. The first examination is normally planned for around 4 weeks
following delivery. Two components are utilized in scoring, - the zone and the grade. The
location of the abnormal vessel growth, referred to as the “zone”, is determined by
proximity to the central vision and optic nerve. Zone 1 includes abnormal vessel growth
in the central retina, whereas zone 3 is when abnormal vessel growth occurs in the
peripheral retina (Figure 1.4).
Figure 1.4 Retinopathy of Prematurity (ROP) scoring scale. Showing zone borders
and clock hours used to describe the location and extent of ROP. Figure originally
published in Pediatrics (2006), reproduced with permission from the American Academy
of Pediatrics.
The second component, the “stage,” ranges from 1 to 5, with stages 1 and 2
considered mild, and stages 3 to 5 considered severe, on which treatment is usually
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provided according to the Early Treatment for ROP trials (Early treatment of Retinopathy
of prematurity cooperative group, 2003) (Figure 1.5).
Figure 1.5 Stages of Retinopathy of Prematurity. Stage 1 is characterized by a thin
demarcation line between non-vascularized and vascularized retina, stage 2 by a ridge,
stage 3 by extraretinal fibrovascular proliferation, stage 4 by part retinal detachment, and
stage 5 by total retinal detachment. Figure originally published by Hellstrom et al (2013)
in The Lancet; reproduced with permission from Elsevier.
The current mainstay of treatment is with laser photocoagulation therapy to stop
the growth of the abnormal vessels and to prevent retinal detachment. Other approved
therapies include the intravitreal injection of anti-VEGF agent Bevacizumab (Trade name
Avastin) (Gunther and Altaweel, 2009) and cryotherapy in which reginal retinal destruction
was performed through freezing of the area (Pearce et al, 1998). Other therapies of
interest include scleral buckling, which is aimed to treat retinal detachments, propranolol,
to reduce the progression of ROP, and recombinant humanized IGF-1 to prevent
- 15 -
excessive vessel formation through reducing VEGF (Hellstrom et al, 2003; Hellstrom et
al, 2013). The neurodevelopmental outcomes following treatment for severe ROP are still
not understood.
1.4.2 Pathophysiology
During embryogenesis, the retina, ciliary body, iris and optic nerves arise from the
diencephalon, which goes on to form the caudal forebrain; thus the retina develops as
part of the central nervous system (CNS). The retina includes layers of cells with three
neural cells including the photoreceptor cells, bipolar cells and ganglion cells, and a fourth
layer of pigmented epithelial cells. These layers are complete in their development by
around 16 weeks PMA, though are not vascularized. Normal retinal vascularization
continues until term age through the balance of growth factors of insulin-like growth factor
(IGF-1), vascular endothelial growth factor (VEGF), erythropoietin (EPO) and ω3 poly-
unsaturated fatty acids (ω3 PUFA), among others. Following preterm birth, normal
vascularization is inhibited and normal vascularization may continue if the optimal
environment for retinal development is achieved. However, should there be a relative
imbalance of growth factors with increasing metabolism, hyperoxia or hypoxia, the
potential for abnormal retinal neovascularization occurs (Hellstrom et al, 2013).
Of particular importance to reducing the development of ROP is the prevention of
hyperoxia with reduced oxygen supplementation in the care of the preterm infant.
Because of improved NICU care and practices, ROP is relatively uncommon in those
infants born >30 weeks PMA, though a significant proportion of infants <28 weeks will
have at least some retinal vasoproliferation. Furthermore, infants who are small for
gestational age, and those with hyperglycemia, hyperinsulinemia, and/or postnatal
- 16 -
infections, are at greater risk for ROP and are more likely to require treatment to preserve
vision (Hellstrom et al, 2013).
Figure 1.6: Retinopathy of Prematurity Pathophysiology. (A) In-utero: normal vascular
growth with low oxygen tension. (B) Phase 1: retinal vascularization is inhibited by
hyperoxia and loss of growth-factors from the placenta. Blood-vessel growth stops and
with retinal maturation hypoxia results. (C) Phase 2: hypoxic retina stimulates expression
of oxygen-regulated factors such as erythropoietin (EPO) and vascular endothelial growth
factor (VEGF) which in turn stimulate retinal neovascularization with Insulin-like growth
factor 1 (IGF-1). (D) Resolution: retinopathy may be prevented or treated with prevention
of phase 1 or inhibition of phase 2 with laser therapy or an antibody. ω3 PUFA= ω3
polyunsaturated fatty acids. Figure originally published by Hellstrom et al (2013) in The
Lancet; reproduced with permission from Elsevier.
Low IGF-1 has a strong association with later ROP due to poor retinal vascular
growth. This is likely due to its impacts upon VEGF, which is an important factor in normal
vascularization (Hellstrom et al, 2013) (Figure 1.6). IGF-1 is also an important growth
- 17 -
factor for the fetus in-utero and remains an important regulator of glucose metabolism in
postnatal development (Cheng et al, 2000). Low IGF-1 concentrations in the early
postnatal period in babies born preterm correlates strongly with later ROP and other
comorbidities, including a slower growth rate of brain volume, a marker of cerebral
development (Hansen-Pupp et al, 2011; Hellstrom et al, 2003). Low IGF-1 concentrations
are nutrition-dependent markers and can be reduced following starvation, infection and
stress (Demendi et al, 2012). In addition, hyperglycemia within neonates, absence of ω3
PUFAs in diet and low levels of EPO are identified as other potential risks for increased
ROP (Hellstrom et al, 2013).
1.4.3 Outcomes of Infants with ROP
In assessing the neurodevelopmental outcomes of children with ROP, Schmidt et al
reported an odds ratio of 4 for death or disability at 5 years, greater than that of brain
injury or BPD (Schmidt et al, 2015) with a 3 to 4 times greater risk for non-visual
disabilities than those without ROP (Msall et al, 2000; Schmidt et al, 2014). While vision
abnormalities are common in preterm infants (O'Connor et al, 2002), visual outcomes
have improved in the CRYO-ROP (Cryotherapy for Retinopathy of Prematurity Group,
1996), early treatment for ROP (Early Treatment For Retinopathy Of Prematurity
Cooperative, 2003) and Avastin trials (Martinez-Castellanos et al, 2013; Mintz-Hittner et
al, 2011). Common visual disturbances seen in long-term follow-up of children with ROP
include strabismus, amblyopia and refraction abnormalities (O'Connor et al, 2002). The
impacts of these visual disturbances are thought to be minor, though when severe, early
visual-motor coordination and vestibular system dysregulation can effect developmental
mechanisms (Goyen et al, 2006; Prechtl et al, 2001). The association between severe
- 18 -
ROP and brain maturation and long-term neurodevelopmental outcomes remains an area
in which little is known.
1.5 Infection in Preterm infants
1.5.1 Background
In many large epidemiological studies of preterm infants worldwide, infection has
been determined to be a strong predictor of poorer outcomes compared to non-infected
infants (Kiechl-Kohlendorfer et al, 2009; Mitha et al, 2013; Rand et al, 2016; Schlapbach
et al, 2011; Stoll et al, 2004; Van der Ree et al, 2011). Rates of infection in preterm infants
range between 20 – 65% within industrialized countries, with even greater rates reported
in developing countries, where mortality rates are also significantly higher (Adams-
Chapman and Stoll, 2006; Orsi et al, 2009; Stoll et al, 2004; Zaidi et al, 2005). It is for
these reasons that infection remains an important determinant of childhood outcomes and
prevention strategies remain a key initiative of the World Health Organization.
1.5.2 Classification
Traditionally, neonatal infections are divided into two categories based upon the
timing of the infection in relation to birth; [1] early-onset infection, classified as presenting
within the first 72 hours of life, and [2] late-onset infection, presenting past 72 hours of
life. This division has been utilized to assist in the differentiation of the early-onset
infections, which are thought to be a result of maternal risk factors or transmission and
acquisition of the infection via the birth canal, in comparison to late-onset infections which
- 19 -
are more often related to acquisition in the hospital environment. It is because of these
factors that they are also referred to as “pre-natal” and “postnatal” infections.
1.5.3 Pre-Natal Infection
Infections occurring in the first 72 hours of life appear to have a different
pathophysiology than that of postnatal infections, evident by transmission via the maternal
bloodstream or via direct exposure to the organism ascending to within the uterus or from
delivery through the vaginal canal. Chorioamnionitis, the most common pre-natal
infection, involves infection of the fetal membranes, placenta or amniotic fluid, and is
commonly diagnosed in the third trimester with associated maternal fever, leukocytosis,
uterine tenderness and foul-smelling amniotic fluid with fetal tachycardia. Current
treatment of chorioamnionitis consists of appropriate intravenous antibiotic treatment
early in the maternal illness and may include postnatal antibiotics following delivery. Pre-
natal infection is a known cause for stillbirth as well as a common cause of preterm
delivery (Goldenberg et al, 2008; McClure et al, 2010). Infants delivered following a pre-
natal infection have a higher mortality rate and are commonly infected with organisms
such as Escherichia coli and group B Streptococcus (GBS), which are less commonly
seen in postnatal infections (Jiang et al, 2004; Stoll et al, 2005).
The literature with regards to whether pre-natal infection confers a greater risk of
brain injury and poorer outcomes remains unclear with some studies reporting increased
rates of cerebral palsy and cystic PVL (Dammann et al, 2002; Dammann and Leviton,
1998; Grether and Nelson, 1997; Mitha et al, 2013), while other studies report no
significant difference in WMI, brain maturation or poorer outcomes (Chau et al, 2009;
Grether et al, 2003). While the neurodevelopmental outcomes resulting from pre-natal
- 20 -
infection alone remain unclear, there is research supporting that the combined influence
of pre-natal and postnatal infections together may confer a stronger risk for cerebral injury
and cerebral palsy (Mitha et al, 2013; Yanni et al, 2017).
1.5.4 Postnatal Infection
Infections occurring after 72 hours of life are more common than pre-natal
infections and have a far greater association with poorer outcomes, with a higher
percentage of infected infants developing motor and/or cognitive impairments in follow-
up compared to uninfected infants (Jiang et al, 2004; Stoll et al, 2004). Studies reporting
long-term outcomes at school age continue to support the association of infection with
greater frequency of cognitive and motor dysfunction, as well as ADHD and other mental
health impairments (Mitha et al, 2013; Rand et al, 2016).
Published research of clinical cohorts exploring postnatal infection have
investigated the different types of infections separately, with the hypothesis that sepsis
and meningitis would be more likely to result in poorer outcomes. However, among all
infants with postnatal infections there is little difference between those with “clinical
infection alone” and those with sepsis and/or meningitis (Stoll et al, 2004). The same has
been shown for necrotizing enterocolitis (NEC), thought to convey an increased
inflammatory response, resulting in a greater risk of WMI and motor impairments (Shah
et al, 2008; Stoll et al, 2004). In multiple cohorts of preterm infants, postnatal infection
was associated with an increased risk of WMI, as well as worsened WMI seen on
subsequent MRIs, highlighting it as an important risk factor for brain injury in the preterm
newborn (Chau et al, 2009; Glass et al, 2008). In addition to increases in WMI, infants
with postnatal infection have delayed brain development using measures of white matter
- 21 -
maturation with increased average diffusivity, decreased white matter fractional
anisotropy and lower MRSI metrics of brain maturation (Chau et al, 2012). The link
between multiple postnatal infections and development of the preterm brain has not been
thoroughly investigated and remains an area of interest in the field.
Common organisms involved in postnatal infection include gram-negative and
gram-positive bacteria, as well as fungal species, with coagulase-negative
staphylococcus (CONS) species the most common (Orsi et al, 2009). Other organisms
such as Klebsiella pneumoniae, Escherichia coli, Enterococcus species, Enterobacter
species and Candida species are less commonly seen (Orsi et al, 2009). Among the
organisms implicated in postnatal infections, it has been shown that infants are at risk of
poorer outcomes as a result of any infectious organism (Stoll et al, 2004), suggesting that
the impacts on the brain are not specific to one organism, but rather related to the
inflammatory response that occurs during the infection and the impacts on the
hemodynamic circuitry and immune system of the body.
Implementing tighter infection control measures within NICUs, such as increased
hand hygiene by health care workers, reduces infection rates, particularly of hospital-
acquired infections (Schelonka et al, 2006; Won et al, 2004). The implementation of
improved infection control measures can result in reductions in the length of hospital stay
and of other co-morbidities, and is even associated with improved outcomes at 2 years in
one study (Davis et al, 2016). However, it is not fully understood whether these
procedures, and other more costly measures, need to be put in place for an entire NICU
unit, or whether the most vulnerable infants would benefit better through an individualized
approach. Additionally, there is no testing of these personalized or “precision” infection
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control measures specifically targeted at those infants at greatest risk of adverse
outcomes.
1.5.5 Neonatal Inflammation
When an infectious pathogen makes contact with a mature host’s immune system,
the innate immune system identifies and eliminates the invading pathogen through the
activation of pattern recognition receptors (PRRs). Within the preterm infant, however,
the innate immune system is not well developed, with a deficiency of PRRs, which results
in an unbalanced and potentially harmful inflammatory response (Kan et al, 2016; Van
der Poll et al, 2017). This heightened vulnerability of the preterm infant brain to infection
was first illustrated and described by Gilles in 1976 when a bacterial lipopolysaccharide,
injected systemically into newborn kittens, caused a leukoencephalopathy, though it
resulted in no effect in the mature cat (Gilles et al, 1976). The activation of toll-like
receptors within the human epithelium of the bloodstream, known to be activated by
bacterial lipopolysaccharides, is believed to be one mechanism involved in the activation
of the local host response on the blood brain barrier (Strunk et al, 2014). This results in
the production of various inflammatory mediators, with cytokines, prostaglandins and
reactive oxygen species (ROS) released into the CNS causing direct cytotoxicity on the
brain; and may occur even without direct bacterial invasion into the CNS, such as in
meningitis (Strunk et al, 2014).
Increased levels of cytokines tumor necrosis factor (TNF-α) and interleukins (IL-
1β, IL-6, IL-8, IL-10, IL-12, IL-17 and IL-18) have been investigated in the fetal and
neonatal responses to infection, with some shown to have a pro-inflammatory role within
the developing immune system (Dammann and Leviton, 1997; Leviton et al, 2005; Van
- 23 -
der Poll et al, 2017). In those infants with higher levels of these pro-inflammatory markers,
cerebral injury is more commonly seen, with greater extent of white matter injury and PVL
(Duggan et al, 2001; Viscardi et al, 2004). In animal models of fulminant infection, blocking
some of these cytokines has been shown to result in increased survival and has been
suggested as a potential therapeutic target against the hyper-immunity in sepsis (Flierl et
al, 2008).
The impact of multiple inflammatory events has been investigated in what is referred
to as the “two-hit” model, in which an initial inflammation sensitizes the vulnerable immune
system to a subsequent inflammatory event. The second inflammation event is thought
to have a threshold stimulus lower than is required for the initial event and results in the
release of excitotoxic inflammatory markers, leading to cerebral injury and potential
epigenetic alterations (Fleiss and Gressens, 2012; Fleiss et al, 2015; Yanni et al, 2017).
Both pre-natal and postnatal stimuli have been shown to sensitize the immune system
with increased levels of pro-inflammatory cytokines (Yanni et al, 2017). Pre-natal
infections, and in particular chorioamnionitis, are often associated with a fetal
inflammatory response in which pro-inflammatory markers are increased and thought to
act as an initial trigger (Wang et al, 2007), with some studies reporting a greater incidence
of WMI and cerebral palsy as a result (Leviton et al, 2010; Yanni et al, 2017). Postnatal
events, such as prolonged mechanical ventilation, NEC and postnatal infection, often
trigger similar inflammatory responses to those seen in pre-natal infections, resulting in a
significant release of pro-inflammatory markers (Aden et al, 2010; Hagberg et al, 2015;
Volpe, 2008). Furthermore, it has been shown that exposure of the developing brain to
multiple inflammatory events leads to excitotoxicity, with greater mitochondrial
- 24 -
impairment and weakened vascular integrity potentially causing direct brain injury or
interfering with normal CNS development (Boisse et al, 2004; Fleiss et al, 2015; Wang et
al, 2009; Yanni et al, 2017), and potentially altering the epigenome (Fleiss and Gressens,
2012). Multiple inflammation events have the potential to result in an altered immune
response leading to a chronic inflammatory condition.
1.5.6 Neonatal Hypoxic-Ischemia Injury
The preterm infant brain is particularly susceptible to the effects of hypoxia-
ischemia as reflected in the development of white matter injury and PVL. The
susceptibility of the OL progenitor cell is a maturation-dependent phenomenon, which is
represented by a greater resistance of the mature OL cells to the effects of oxidative
stress than the immature pre-OL cell (Back et al, 1998). What results is a critical period
of vulnerability for the preterm infant during which irreversible injury can occur to the OL
cell, resulting in myelination dysfunction. The over-expression of the immune system
during this period may contribute to the cerebral injury as a consequence of the pressure-
passive and poor auto-regulatory system of the preterm infant (du Plessis, 2009; Khwaja
and Volpe, 2008). We know that cardiovascular collapse commonly occurs during an
acute infection (Healy et al, 2004), which when during a vulnerable period, may potentiate
the hypoxic-ischemic injury (Khwaja and Volpe, 2008). Animal models have shown the
combined effects of hypoxia-ischemia insults and infection can worsen neuronal injury,
with particular periods in which there is greater susceptibility to injury (Eklind et al, 2005;
Larouche et al, 2005).
There are various mechanisms which make the preterm infant brain vulnerable to
the effects of hypoxia (Gopagondanahalli et al, 2016). Firstly, the preterm brain receives
- 25 -
a relatively reduced global and regional supply of blood compared to the term infant brain,
which may result in a limited margin for reduced supply (Altman et al, 1988; Pichler et al,
2014). Additionally, arterial extension into the developing brain is incomplete resulting in
poorly vascularized end-zone watershed regions, particularly of the periventricular white
matter regions (Altman et al, 1988; Inage et al, 2000). Include these factors with the poor
cardiac output commonly seen in the preterm infants (Kluckow and Evans, 2000), and the
poor correlation between blood pressure monitors and cerebral blood flow (Weindling and
Kissack, 2001), and it becomes clear that poor cerebral blood flow is likely
underestimated in the clinical care of the preterm infant.
The pressure passive blood pressure system of the preterm infant brain is
vulnerable to alterations in blood pressure, in which the greatest fluctuations are seen in
the smallest infants (Soul et al, 2007). The regulation of blood pressure in the preterm
infant has a poor reserve for fluctuations and is dysfunctional in response to hypotension
(Munro et al, 2004). Treatment with inotropes and fluids, and other appropriate therapies
for low blood pressure, can improve cerebral blood flow, reducing cerebral ischemia
(Munro et al, 2004). Poor cerebral autoregulation is associated with a higher frequency
of IVH and WMI, thus preserving a stable mean arterial blood pressure is paramount in
reducing injury to the developing brain (O'Leary et al, 2009; Vesoulis and Mathur, 2017).
Further potentiating the blood pressure fluctuations in the brain are the effects of
hyperoxygenation and hypocarbia, resulting in vasoconstriction of the blood vessel wall,
with greater amounts of brain injury (Fujimoto et al, 1994; Khwaja and Volpe, 2008).
Hypocarbia is also a strong factor in the development of severe ROP, with changes in the
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oxygen targets and more awareness of hyperoxygenation and hypocarbia helping to
reduce the severity and incidence of ROP (Sears et al, 2009).
1.6 Magnetic Resonance Imaging
1.6.1 Imaging the Preterm newborn
Brain imaging in the very preterm newborn has developed within the last two
decades to be the gold standard assessment for brain injury, and assists in identification
of those infants at high-risk for adverse outcomes in infancy and childhood (Anderson et
al, 2017; Woodward et al, 2006). Magnetic resonance imaging (MRI) of the preterm
newborn, in many instances without the need for sedation, is a safe and reliable technique
with a greater sensitivity for white matter abnormalities than cranial ultrasound (Inder et
al, 2003; Miller et al, 2003; Plaisier et al, 2012). Following the use of routine term-
equivalent MRI in some centres, 25-33% of infants were found to have brain
abnormalities, many of which were not detected with conventional cranial ultrasound
(Kidokoro et al, 2014; Neubauer et al, 2017). The timing of the MRI is an important
consideration in detecting injury in the preterm infant as WMI is more easily seen in the
preterm period, and may be undetectable on subsequent imaging at term-equivalent age
(Kersbergen et al, 2014; Martinez-Biarge et al, 2016). As a result of this, it is
recommended that serial imaging of the preterm brain be done as this improves the
sensitivity for WMI, providing a more definite prediction of childhood outcomes (Martinez-
Biarge et al, 2016; Sarkar et al, 2015). However, MRI does have its limitations with the
current clinical scanners unable to visualize microscopic WMI, the least severe form of
- 27 -
WMI, which remains a neuropathological diagnosis, and suggests that MRI is liable to
underestimating the full extent of WMI (Back et al, 2012; Volpe, 2017). With the addition
of advanced MR techniques, these milder injuries and delays in development may be
more readily assessed allowing a further understanding of the long-term effects of
prenatal and postnatal injuries (de Vries et al, 2011; Guo et al, 2017; Kwon et al, 2014;
Rutherford et al, 2005; Thomas et al, 2005).
1.6.2 Overview of MRI Basics
MRI utilizes the nuclear magnetic resonance properties particularly of hydrogen
protons, which when combined with radiofrequency (RF) waves, results in energy release
which can be detected, interpreted and transformed into images. The images are formed
as a result of the physical properties of the molecules and their response to the magnetic
field. When first placed within a strong magnetic field, such as that provided by the main
B0 field, the hydrogen protons within the body polarize. The RF pulse is then used to kick
spins off their axes, which results in excitation when applied at the correct resonance
frequency, flipping the natural spins of the hydrogen atoms into the anti-parallel state.
This is known as the “Larmor Frequency” and it depends on the field strength of the
magnet (42.6 MHz/Tesla). As the spins return, or relax, from the high energy anti-parallel
state back to their equilibrium they release energy in the form of an oscillation magnetic
field at the Larmor frequency.
For visualization, the vector representation is used such that when excited by the
RF pulse, the net storage of energy is represented by the tipping of the vector onto its
side, away from the z-axis and into the x-y planes, the amount referred to as the ‘Flip
angle’. Following this, the vector begins to precess around the z-axis slowly returning to
- 28 -
equilibrium through the relaxation phase. The relaxation of the protons can be divided
into two processes: T1 and T2 relaxation, described below.
1.6.3 MRI sequences
T1 relaxation, also referred to as spin-lattice relaxation, refers to the exponential
recovery of the protons to the z-axis. T2 relaxation, also known as spin-spin relaxation,
refers to the loss of phase coherence among excited protons that leads to exponential
decay of the component of the magnetization in the transverse plane. Variations in the
relaxation rates of T1 and T2 signal occurs as a result of the diverse concentrations of
molecules in the different tissues within the brain. Fat, which has tighter bonds to
surrounding structures than water, releases energy quickly; while water results in a slower
relaxation, resulting in water having a higher T1 and higher T2 signal than fat.
The signal received by the receiver coils during the relaxation phase, is acquired
in a temporary image space, also referred to as ‘k-space’, in which the digitized MR
signals are stored during data acquisition. The images are then produced using the
mathematical conversion known as the “Fourier transform”. The k-space is expressed as
a summation of overlapping sinusoids that are used to produce an anatomical image.
The repetition time (TR) and echo time (TE) play key roles in the contrast of a MR
image due to the variations in the recovery and relaxation times of different tissues. The
TR refers to the time, in milliseconds, between successive RF excitation pulses, with the
TE reflecting the time, also in milliseconds, from the application of the RF excitation pulse
and the peak signal induced by the coil. T1-weighted sequences maximize T1 contrast
- 29 -
by utilizing a short TR and short TE, increasing tissue contract. T2-weighted sequences
use a longer TE and long TR, minimizing the T1 contrast.
Two-dimensional multi-slice imaging is produced by sequentially exciting and
collecting data from one slice region at a time. The thickness of the tissue excited in a
single image slice of the MR image is determined by changes in the RF pulse and/or the
gradient strength. These images are then stacked together to produce a combined 3D
image of the brain. Three-dimensional (3D) volumetric imaging is produced by exciting a
thick slab of tissue, which is then spatially encoded by traversing the 3D k-space,
reconstructed in 3D and resliced for image review.
1.6.4 Diffusion Weighted Imaging and Diffusion Tensor Imaging
Diffusion weighted imaging (DWI) is a MR technique that measures free motion of
water in tissue, based upon measuring the random Brownian motion of water molecules
within a voxel of tissue. Unlike the free diffusion, or movement, of water inside a container,
diffusion of water inside a voxel of the brain is hindered by cellular membrane boundaries
in both the intracellular and extracellular compartments. Using these properties, DWI can
be utilized in assessing micro-structural architecture and is sensitive to cell changes as a
result of ischemia and those seen in highly cellular tumours. Images are produced by
adding extra gradients to standard MR imaging sequences, such as a T2-weighted echo
planar sequence, which sensitizes the sequences to molecular diffusion. The diffusion
gradients are equal in magnitude and centered on a 180-degree refocusing
radiofrequency pulse. DWI images are produced as static hydrogen protons are
unaffected by the diffusion gradient and will retain their signal, whereas water molecules
- 30 -
moving in the axis of the gradient will accumulate phase by the first gradient, but not the
second and hence will lose their signal.
Diffusion tensor imaging (DTI) is another quantitative MR method that uses the
same properties of diffusion of water molecules in conventional DWI imaging, though
does so using a Gaussian model of diffusion. The Gaussian distribution is defined by a
3x3 symmetric, positive definite matrix, with 3 orthogonal (mutually perpendicular)
eigenvectors and 3 positive eigenvalues in each voxel. The direction of fastest diffusion,
the principle eigenvector (λ1), is referred to as axial diffusivity, with the other eigenvectors
(λ2, and λ3) termed radial diffusivity. These eigenvectors, in combination with the 3
eigenvalues define an ellipsoid reflecting the diffusion probability within each voxel
(Figure 1.7).
Figure 1.7 Representation of the 3D diffusion ellipsoid. Presented as an ellipsoid
with three unit eigenvectors (ε1, ε2, and ε3), with corresponding lengths (λ1, λ2, and λ3),
the eigenvalues.
DTI characterizes the 3D spatial distribution of water diffusion in each voxel of the
MR image, providing an indirect measure of microstructure (Beaulieu, 2002; Miller et al,
λ1 ε1
λ2 ε2
λ3 ε3 y
z
x
- 31 -
2002; Mukherjee et al, 2002). Within each voxel it is possible to infer the axial diffusivity
(λ1), the radial diffusivity (λ2 and λ3), and mean diffusivity (MD), a measure of the average
size of diffusion in each voxel, where Dxx, Dyy and Dzz are the diagonal terms of the
diffusion tensor:
MD = λ1 + λ2 + λ3 = Dxx + Dyy + Dzz = Trace
3 3 3
Molecules diffuse differently within tissues depending on the type, integrity,
architecture and the presence of barriers generating a quantitative anisotropy. Anisotropy
refers to the diffusion of water which may be unrestricted in all directions, such as within
CSF, termed “isotropic”, or restricted to a single plane, such as in the white matter
pathways of the mature brain, characterized as “anisotropic” (Soares et al, 2013).
Fractional anisotropy (FA), the normalized measure of the fraction of the tensor’s
magnitude due to anisotropic diffusion, corresponds to the degree of directionality and
ranges from 0 (isotropic diffusion) to 1 (anisotropic diffusion), averaged between bilateral
brain regions in mean FA analyses (Soares et al, 2013). FA increases with white matter
maturation, and is thought to reflect the maturation of the oligodendrocyte lineage and
early events of myelination within the preterm brain (Drobyshevsky et al, 2005; Miller et
al, 2002). Using this technology, DTI can be displayed by condensing the tensor
information into one number, referred to as a “scalar”. The tensor can also be represented
using glyphs, a small 3D representation of the major eigenvector or whole tensor, or
presented as 4 numbers in producing colour-coded FA map. The colour FA map is
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produced with each colour representing the primary diffusion direction within each voxel
of the image (Figure 1.8) (Soares et al, 2013).
Figure 1.8 DTI colour map. DTI map of a preterm infant brain at 32 weeks PMA in the
axial plane at the level of the superior white matter (left) and basal ganglia (right). Colour
coding is reflected by red colour representing the left-to-right orientation within the image,
green the posterior-to-anterior and blue the inferior-to-superior axis of diffusion.
Another way of viewing the DTI image includes estimations of the course of the
white matter tracts through the brain, termed tractography. The most common approach,
streamline tractography, involves following the path of successive principle eigenvectors
from a given voxel origin, and is a form of “deterministic” tractography. “Probabilistic”
tractography, another widely utilised method, relies upon connection probabilities
between voxels and is more reliable at reconstructing crossing fibers than deterministic
- 33 -
tractography (O'Donnell and Westin, 2011). Despite significant promise with tractography,
both methods have their limitations and challenges with interpretation, in part as a result
of the complexity of the crossing fibres within the brain, but also as a result of the large
numbers of potential false positive or negative tracts that can result from the analysis.
Tract-based spatial statistics (TBSS; http://fsl.fmrib.ox.uk/fsl/fslwiki/TBSS), a semi-
automated and 3D assessment of FA, provides a less user-dependent measure of FA
than complementary ROI-based analyses. TBSS utilizes “voxel-based morphometry,” in
which each subject’s FA image is registered to a standard space, following which
voxelwise statistics are applied to find areas that correlate to the covariate of interest.
Through TBSS, FA data can be projected onto a group mean FA tract skeleton which can
be used to display the voxelwise cross-subject statistics as an assessment of white matter
integrity differences (Smith et al, 2006). The normalization of each image to a standard
space is a crucial step to the TBSS model, but can lead to misalignment and issues with
the normalization of the data. This is especially true for the preterm infant brain which is
undergoing rapid changes in size and shape within the normal fetal third trimester. The
FA images are aligned using voxelwise nonlinear registration, which is driven by the FA
images. Secondly, the mean of the aligned FA image is utilised to produce a skeletonised
mean FA image. Following that, each subjects’ aligned FA image is projected on the
skeleton filling the skeleton with FA values, on which cross-subject voxelwise statistics
can be performed. In producing voxelwise statistical models with a correction for multiple
comparisons, a Gaussian random field theory thresholding approach is often applied for
analysis (Smith et al, 2006). If the spatial width of this Gaussian filter is not chosen
appropriately then the signal-to-noise ratio will be reduced resulting in poorer power of
- 34 -
the model and more imperfections. Cluster-size thresholding, a permutation-based
approach to thresholding, can help to reduce this without the use of a Gaussian filter,
while still allowing the appropriate statistical test. By using a cluster size determined by
500 permutations of the cluster size using Randomise v.2.9 within the functional MRI of
the Brain Software Library (http://sel.fmrib.ox.ac.uk/fsl/fslwiki/Randomise) the familywise
error rate is lessened while maintaining the ability to search the entire FA skeleton for
regions of significant difference (Smith et al, 2006). In showing cross-subject voxelwise
significance, a threshold of P <0.05, equivalent to the 95th percentile of the distribution, is
used for the clusters, and corrected for multiple comparisons across space. Using these
techniques, a preterm infant model for assessing TBSS with the white matter skeleton
separated into four categories based upon GA at scan was developed by Duerden et al
(Duerden et al, 2015) (Figure 1.9).
Figure 1.9 Tract-based spatial statistic (TBSS) model. Age-specific models for preterm
infants are shown here. Green regions reflect the white and grey matter skeleton template
for the respective age group overlaid upon an axial T1-image.
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1.6.5 Magnetic Resonance Spectroscopic Imaging
Proton magnetic resonance spectroscopic imaging (MRSI) uses the signals
produced by the different metabolite molecules within the voxel to produce spectra of
individual peaks. These peaks are based upon the concentrations of metabolites,
represented by parts per million (ppm) on the spectroscopic map. The principle behind
MRSI is that the distribution of electrons around hydrogen protons in the different
metabolites results in slight alterations to the magnetic field. Because these metabolites
have molecular concentrations 10,000 times lower than water, the signal produced is
much lower, thus larger voxels are required and the water signal has to be suppressed
(Posse et al, 2012). The most commonly used MRSI method involves using conventional
phase-encoded imaging in which the phase encoding gradient amplitude is incremented
once per TR. Phase encoding modulates the signal phase and amplitude of the MR signal
before detection of the signal frequency, which ensures that subsequent phase and
amplitude modification due to the chemical shift is independent of spatial encoding (Posse
et al, 2012). Suppression of the water signal is then achieved by using either a chemical
shift selective or inversion recovery technique, which when the Fourier transform is
applied, separates the signal into individual frequencies. The greater the magnetic field
strength, the greater the signal-to-noise ratio, which allows smaller voxels to be used in
producing the metabolite spectra. Another technique increasingly used is echo-planar
MRSI, which has strong localization performance and large volume coverage with
improved spatial and temporal resolution (Posse et al, 2012). This process utilizes a
zigzag trajectory in k-t-space creating a series of gradient echoes that are modulated by
- 36 -
the chemical shift evolution and relaxation. Through reformatting, the data format is
equivalent to conventional phase-encoded MRSI.
As with conventional MR, the TE is an important factor in the information obtained.
With a short TE of 30 msec, metabolites with both short and long T2 relaxation times are
observed (Brandao and Domiques, 2003). A longer TE of 270 msec results in only
metabolites with a long T2 to be seen. An intermediate length TE of 144 msec is often
used as it allows the imaging of lactate as an inverted doublet at 1.3 ppm. MRSI
metabolites of importance in the brain include N-acetyl-aspartate + N-
acetylaspartylglutamate (NAA) at 2.0 ppm, lactate (LAC) at 1.3 ppm,
glycerophosphocholine + phosphocholine (CHO) at 3.2 ppm, creatine + phosphocreatine
(CR) at 3.0 ppm and myo-Inositol (INS) at 3.5 ppm (Card et al, 2013) (Figure 1.10). A
long TE spectra at 270 msec produces a spectra of primarily NAA, CR and CHO, while
the other metabolites are best seen with a short TE with a high signal to noise ratio. Other
metabolites of interest include glutamate/glutamine and gamma-aminobutyric acid
(GABA) [all at 2.2 – 2.4 ppm], which have been shown to correlate with white matter injury
(Wisnowski et al, 2013). NAA is a neuronal marker, present in high concentrations in the
brain, and synthesized in neurons (Moffett et al, 2007). CHO is a measure of increased
cellular turnover or membrane breakdown, and is often elevated in tumours and
inflammatory disorders, while CR provides a measure of brain metabolism and energy
stores. INS participates in phospholipid metabolism and plays a role in cellular message
transduction and a proposed marker for gliosis. Unfortunately, many notable metabolites
are not represented in the MRSI spectra, with neurotransmitters acetylcholine, dopamine
- 37 -
and serotonin all absent, either as a result of low concentrations or molecules that don’t
respond to conventional MRSI techniques.
The metabolites of interest are commonly presented as ratios, which helps to
control for changes in brain water content, the T1 and T2 relaxation times of water and
possible CSF fluid contamination of the voxel in use (Card et al, 2013). Using absolute
metabolite concentrations is becoming more common, though its use in vivo is difficult
and requires intensive post-processing requirements. CHO is commonly used as a
denominator in metabolite ratios due to the fact that its concentration reduces over time
as the rapid brain growth of the neonate slows in infancy. During the preterm period
dramatic increases in CR/CHO and NAA/CHO ratios are seen with decreases in INS/CR
and INS/CHO ratios (Card et al, 2013). LAC/CR and LAC/CHO ratios have been shown
to correlate with the severity of asphyxia at birth and likely reflects lactate as a marker of
anaerobic metabolism seen in tissue infarction. White matter injury has previously been
shown to result in lower NAA/CHO and NAA/CR ratios (Card et al, 2013; Chau et al,
2009).
- 38 -
Figure 1.10 Magnetic Resonance Spectroscopic Imaging (MRSI) example. Example of a
preterm infant scanned at term with a 6mm voxel located in the left basal ganglia with a
long echo time (TE) of 144 ms on a 3T magnet. The peaks of Cr2 (phosphocreatine), Cho
(choline), Cr (creatine), and N-acetylaspartate (NAA) are shown at their respective parts
per million position on the spectroscopy (ppm) on the x-axis with the MR signal peak
amplitude on the y-axis.
- 39 -
1.7 Neurodevelopmental Outcomes
1.7.1 Background
Standardized developmental assessments are important in the early detection of
developmental delays both for the eligibility requirements of early intervention programs,
but also in the evaluation of therapies in the attempt to improve childhood developmental
outcomes (Johnson and Marlow, 2006). While no assessment battery is perfect and
several standardized assessments for infants are available, the Bayley Scales of Infant
and Toddler Development and the Peabody Developmental Motor scales, along with their
revisions, are validated and widely reported assessment methods (Anderson et al, 2010).
1.7.2 The Bayley Scales of Infant and Toddler Development
The Bayley Scales of Infant and Toddler Development, 3rd edition (BSID-III) are
meant to examine all facets of a young child’s development with norm-referenced,
standardised testing that can be done for infants 1 to 42 months of age (Bayley, 2005).
Initially developed in 1969 as the first edition, changes were made in the second edition
utilized from 1993 – 2006, with the 3rd edition used since 2006. Originally standardized to
1,700 infants, toddlers and pre-schoolers in the United States of America, the scoring
scale reflects an assessment of healthy individuals and does not include at-risk or
disabled populations (Bayley, 1993; Bayley, 2005). Qualified therapists, through the
administration of a battery of developmentally appropriate tests of child interaction for
infants, toddlers and pre-school age children, calculate an individual infant’s cognitive,
language and motor composite scores. Social-emotional and adaptive behaviour
assessments are also included and conducted via parent questionnaires. The scoring
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scales for each composite have a standardized and normalized mean of 100 with
standard deviation of 15, with scores ≤85 considered below average and <70 considered
severely abnormal (Bayley, 2005). When assessing the scores across timelines and
previous literature it is important to note that there is considered to be a large difference
between the scales on the BSID-III and the previously used BSID, 2nd edition, particularly
at the lower score values (Moore et al, 2016).
Cognitive scoring is done by assessing the way the child thinks, reacts and learns
about the world around them through their interaction with familiar and unfamiliar objects.
The language scale includes two components, with the receptive communication (RC)
part assessing the ability for the child to recognize sounds and understand words and
directions. Expressive communication (EC) assesses how well the child communicates
with sounds, gestures or words. The motor scale also has two parts with fine motor (FM)
assessing how well the child uses his or her fingers to manipulate objects, while the gross
motor (GM) part looks at how well the child moves his or her body within their
environment.
1.7.3 Peabody Developmental Motor Scales
The Peabody Developmental Motor Scales, 2nd edition (PDMS-2), was first
published in 2000, and is a developmental assessment focused on the motor skills of
children from birth through to age 5 years (Folio and Fewell, 2000). The PDMS-2 was
validated on 2,003 children residing in 46 U.S. States and one Canadian province, and
matched to a normative sample of children <5 years with regard to geographic region,
sex, race, rural or urban residence, ethnicity, family income, parent education and
disability (Folio and Fewell, 2000). The testing contains six subtests which are divided
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into the following categories: reflexes, stationary, locomotion, object manipulation,
grasping and visual-motor integration. All of the sub-tests allow the formation of a total
motor quotient, considered the best estimate of overall motor abilities. The composite
scores for each of gross motor and fine motor quotients are a combination of a number
of the subsets. The PDMS-2 composite scores have high correlation with the BSID-III
composite motor score, greatest in those infants >18 months of age (Connolly et al, 2012).
1.7.4 Cerebral Palsy
Cerebral palsy (CP) is the most common movement disorder in children, with an
estimated incidence of 2.1 per 1,000 live infants (Oskoui et al, 2013). Some of the first
descriptions of CP were made by Hippocrates in the 5th century BCE, with its first
description in modern medicine in the 19th century by William John Little, and first termed
“cerebral palsy” by William Osler (Panteliadis et al, 2013). CP is defined as “a group of
permanent disorders of the development of movement and posture, causing activity
limitation, that are attributed to non-progressive disturbances that occurred in the
developing fetal or infant brain” (Rosenbaum et al, 2006). Often accompanied with the
disorders of motor function are disturbances of sensation, perception, cognition,
communication and behaviour, epilepsy and secondary musculoskeletal problems. The
diagnosis of CP is made following an assessment by a pediatrician or neurologist familiar
with the development of children. While many factors contribute to the risk of CP, preterm
birth has a strong prevalence among children with CP. With many improvements in NICU
care over the past several decades, the incidence has decreased significantly in those
infants born >1000 grams (Sellier et al, 2016), though has remained stable in those infants
born <28 weeks or <1000 grams, with rates as high as 112 (95% CI, 70-180) per 1000
- 42 -
live births (Conde-Agudelo and Romero, 2009; Oskoui et al, 2013; Robertson et al, 2007;
van Haastert et al, 2011). As a measure of severe motor impairment, several NICU
practices have been shown to reduce the likelihood of CP in the preterm infant with
antenatal betamethasone (French et al, 2004), magnesium sulfate (Nelson and Grether,
1995; Rouse et al, 2008), postnatal caffeine therapy (Schmidt et al, 2007), increased
caesarian section rate and antenatal antibiotics (van Haastert et al, 2011) all showing a
reduced incidence. Despite these improvements in NICU care, CP remains a common
lifelong condition that results in significant impairments. Postnatal infection and severe
ROP are strong predictors of CP with double the risk in those infants with infection (Stoll
et al, 2004), and a three-fold increase of motor impairments seen in those infants with
severe ROP (Schmidt et al, 2014). Efforts to reduce the incidence of CP and its impact
upon the developing child need to be continued.
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1.8 Hypothesis, Major Goal and Specific Aims
1.8.1 Hypothesis
I hypothesize that retinopathy of prematurity (ROP) and multiple postnatal
infections will be associated with delays in the brain maturation evident on multi-modal
MR imaging with associated poorer neurodevelopmental outcomes.
1.8.2 Major Goal
My goal is to identify whether retinopathy of prematurity and/or multiple infections
in the preterm infant are associated with poorer outcomes and/or delayed white matter
maturation. Through this investigation I hope to assist in altering the care of the preterm
infant to optimize developmental outcomes and provide insight into which infants are at
greatest risk of poor outcomes and would benefit most from early intervention practices.
These findings will expand the understanding of these factors in preterm infant
development following complications of preterm birth and contribute to the understanding
of these variables in the brain development of the preterm infant, while potentially
supporting further research in the field.
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1.8.3 Specific Aims
1. To characterize the brain maturation and neurodevelopmental outcomes of
extremely preterm infants with severe ROP.
2. To describe the association of multiple postnatal infections with developmental
outcomes and brain maturation of very preterm infants.
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Chapter 2
Severe Retinopathy of Prematurity predicts
delayed white matter maturation and poorer
neurodevelopment at 18 months CA
This chapter is modified from work published in the Archives of Disease in Childhood -
Fetal and Neonatal Edition: Glass TJA et al. Severe retinopathy of prematurity predicts
delayed white matter maturation and poorer neurodevelopment Archives of Disease in
Childhood - Fetal and Neonatal Edition Published Online First: 23 May
2017. doi:10.1136/archdischild-2016-312533
The work is published here with copyright permission from the BMJ Publishing Group
Ltd.
A link to the published paper can be found at:
http://fn.bmj.com/content/early/2017/05/22/archdischild-2016-312533
- 46 -
2.1 Introduction
Retinopathy of prematurity (ROP) is a proliferative disorder of retinal
vascularisation that is most severe in extremely preterm neonates born at less than 28
weeks gestational age (GA). Primary therapeutic strategies for ROP include prevention
of abnormal vessel development with tight control of supplemental oxygen therapy and,
when severe, treatment with either retinal laser photocoagulation therapy or, more
recently, intravitreal anti-vascular endothelial growth factor (VEGF) (Gunther and
Altaweel, 2009; Lee and Dammann, 2012). Several large cohort studies of preterm
neonates have shown that severe ROP is associated with lower cognitive and motor
scores in early childhood (Bassler et al, 2009; Harrell and Brandon, 2007; Hellstrom et al,
2013; Schmidt et al, 2003). Despite favourable visual outcomes, severe ROP continues
to strongly predict non-visual disabilities independent of brain injury (Schmidt et al, 2014).
Given this link between severe ROP and non-visual disabilities, we hypothesized
that severe ROP would be associated with white matter maturational delay on advanced
MRI. White matter maturation is well recognized as an important predictor of
neurodevelopmental outcomes in infants born preterm (Chau et al, 2013).
The objectives of this prospective cohort study of extremely preterm neonates
were to determine the association of severe ROP with: (1) early brain development as
measured by MR diffusion-tensor imaging (DTI) and tract-based spatial statistics (TBSS);
and (2) motor and cognitive outcomes at 18 months corrected age (CA). We tested the
hypothesis that severe ROP would be associated with abnormalities in early brain
- 47 -
microstructural development and with worse cognitive and motor development at 18
months CA follow-up.
2.2 Material and Methods
2.2.1 Participants
This study was approved by the University of British Columbia/Children’s and
Women’s Health Centre of British Columbia Research Ethics Board. As part of a larger
prospective study of neonates born 24 to 32 weeks GA, informed consent was obtained
from parents/guardians prior to recruitment of the neonates from April 2006 to September
2013 at British Columbia Women’s Hospital (BCWH), the major provincial tertiary-level
neonatal referral center. Neonates were excluded if they had (1) clinical evidence of a
congenital malformation or syndrome, (2) congenital infection, or (3) ultrasound evidence
of a large parenchymal hemorrhagic infarction (>2 cm) (Papile et al, 1978). This cohort
has been described previously addressing separate hypotheses (Adams et al, 2010;
Brummelte et al, 2012; Chau et al, 2009; Chau et al, 2013; Duerden et al, 2015). Only
extremely preterm neonates born ≤ 28 weeks GA were included in this sub-study as they
are the subset at greatest risk of severe ROP.
2.2.2 Clinical Characteristics
Neonates were screened by a pediatric ophthalmologist at BCWH as per the
International Classification of ROP and the maximal ROP severity in sequential
assessments was included in the analysis (International Committee for the Classification
of Retinopathy of Prematurity, 2005). Severe ROP was defined as ROP requiring
- 48 -
treatment as per the Early Treatment for Retinopathy of Prematurity (ETROP) study
(Early treatment of Retinopathy of prematurity cooperative group, 2003). Intravitreal anti-
VEGF treatment was not used at our institution at the time of the study. Clinical
characteristics were collected systematically by chart review, as previously described;
(Chau et al, 2012; Chau et al, 2009): with bronchopulmonary dysplasia (BPD) defined as
oxygen therapy beyond 36 weeks PMA, hypotension defined as any treatment for low
blood pressure, culture positive infection as any positive blood, urine, cerebrospinal fluid
or respiratory culture, and necrotising enterocolitis (NEC) defined as stages 2 and 3 of
Bell’s criteria (Bell et al, 1978).
2.2.3 MRI Studies
Neonates were scanned first when clinically stable in the preterm period and again
at term-equivalent age. MRI studies were completed without pharmacological sedation
on a Siemens 1.5 Tesla Avanto scanner with 3D coronal volumetric T1-weighted and axial
fast-spin echo T2-weighted images. Neonates were scanned using an MR-compatible
isolette (Lammers Medical Technology, Luebeck, Germany) and specialized neonatal
head coil (Advanced Imaging Research, Cleveland, OH). An experienced
neuroradiologist, blinded to the participant’s medical history, reviewed the images and
recorded the WMI, IVH, ventriculomegaly and cerebellar hemorrhage severity according
to scales previously described (Chau et al, 2009; Miller et al, 2005).
2.2.4 Diffusion Tensor Imaging
DTI reflects the water movement of an ellipsoid space with axial diffusion (λ1), the
preferred movement of water along the white matter tracts, and radial diffusion (λ2 and
- 49 -
λ3) reflecting the orthogonal planes. DTI characterizes the 3-dimensional (3D) spatial
distribution of water diffusion in each voxel of the MR image, providing an indirect
measure of microstructural integrity (Beaulieu, 2002; Miller et al, 2002; Mukherjee et al,
2002). Mean FA, the average directionality of diffusion increases with white matter
maturation, reflecting the maturation of the oligodendrocyte lineage and early events of
myelination (Drobyshevsky et al, 2005; Miller et al, 2002). Diffusion tensor imaging
parameters of FA, λ1, λ2 and λ3 were measured in seven manually placed white matter
regions of interest (ROI) and acquired using a multi-repetition single-shot echo planar
sequence (Chau et al, 2009). ROIs were excluded if significant motion artifact was
present. The ROI areas were acquired in the anatomical regions of the calcarine, anterior,
central and posterior white matter regions, optic radiations, splenium of the corpus
callosum and posterior limb of the internal capsule (PLIC). These regions have been
shown previously to correlate with motor, language and cognitive outcomes (Chau et al,
2009; Chau et al, 2013).
TBSS, a semi-automated and 3D DTI assessment of mean FA, provides a less
user-dependent measure of FA than complementary ROI-based analyses. TBSS was
performed using functional MRI of the Brain software library (FSL) and, after correction
for eddy currents, each DTI volume was registered to a non-DTI volume for each subject
(Smith et al, 2006). The TBSS FA data were projected onto a mean FA tract skeleton,
which was used to apply the voxelwise cross-subject statistics (Smith et al, 2006). We
used five age-based templates and were able to compare neonate groups with a
standardised analysis and a calculated voxel significance threshold of p <0.05 adjusted
for PMA at scan (Duerden et al, 2015). White matter abnormalities detected with TBSS
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at term-equivalent age have been shown to predict neurodevelopmental outcomes in
preterm neonates, with increased FA at 2 years CA being associated with improved
outcomes (Counsell et al, 2002; Duerden et al, 2015).
2.2.5 Developmental Follow-up
At 18 months CA, neurodevelopment was assessed with the Bayley Scales of
Infant and Toddler Development-III (BSID-III) cognitive, language and motor composite
scores with a mean of 100 and standard deviation of 15 (Bayley, 2005). Assessors were
qualified therapists blinded to the imaging findings of the participants. Severe cerebral
palsy (CP) was defined as any diagnosis by an experienced clinician prior to or at the 18
month assessment of CP. Hearing impairment was diagnosed when audiograms showed
hearing threshold >70 dB. Visual acuity was assessed sequentially by the treating
ophthalmologist using varied assessment techniques depending upon age at follow-up
and collected with a retrospective chart review. Visual impairment was defined as best
visual acuity <20/200. Socioeconomic status was assessed as number of years of
maternal education.
2.2.6 Data Analysis
Statistical analysis was performed using Stata 14.1 (StataCorp, 2015). Clinical
characteristics were compared using Fisher’s exact and the Kruskall–Wallis tests for
categorical and continuous data, respectively, with a statistical significance of p <0.05.
The association of severe ROP and other clinical variables with WMI was tested with
univariate logistic regression. The mean values of FA, averaged bilaterally, were
compared between neonates with and without severe ROP, in a generalized least
- 51 -
squares regression model for repeated measures, adjusting for PMA at MRI scan and
multiple ROIs with a p<0.05. We examined the relationship of ROP with FA modified by
PMA at MRI scan, considering p<0.1 as significant due to the interaction term.
2.3 Results
2.3.1 Clinical Characteristics
Of the 234 neonates born 24-32 weeks GA, 126 were born at 24-28 weeks GA.
Ninety-eight (79%) extremely preterm neonates were assessed for ROP at BCWH and
were included in the analysis; infants not assessed were older at birth but had no
difference in other demographics. Neonates were born at a median GA of 26.0 weeks
(interquartile range [IQR], 25.0−26.9 weeks). Early MRI scans were completed in the 98
neonates assessed for ROP at median 32.4 weeks (IQR, 30.3-35.0 weeks) and term-
equivalent MRI was performed in 87 (89%) at median PMA 39.8 weeks (IQR, 38.3-41.6
weeks) (Figure 2.1).
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Figure 2.1 Study flow chart. Flow chart of study enrollment, ROP treatment, MRI
scans and follow-up. Ages presented are median, with corrected age used for Bayley-III
Assessment.
2.3.2 Retinopathy of Prematurity
Of the 98 neonates in this cohort, 67 (68%) had any stage of ROP (stage 1 = 7,
stage 2 = 47, stage 3 = 13, stage 4 = 0, stage 5 = 0), and 19 (19%) neonates required
treatment for severe ROP as per criteria defined in the ETROP study at a median PMA
of 37.9 weeks (IQR, 36.1 – 39.3 weeks). Neonatal parameters associated with severe
ROP included GA at birth, birth weight, birth length, head circumference at birth, BPD,
Birth GA 24 – 28 weeks GA 26.0 weeks
N = 126
MRI #1 GA 32.4 weeks
N=98
Excluded as ROP assessment not done
N = 28
Laser Photocoagulation Treatment
GA 37.9 weeks N=19
MRI #2 GA 39.8 weeks
N=87
Bayley-III Assessment 18.6 months N=83 (85%)
Died N = 4 Withdrew = 7
Lost to follow-up = 4
- 53 -
NEC and hypotension (Table 2.1). Culture positive infection, histologic chorioamnionitis
and multiple gestation were not significantly different in those with and without severe
ROP.
Table 2.1 Demographic, clinical characteristics and imaging findings of neonates 24-28
weeks GA with and without severe ROP treated with retinal laser therapy. Number (%) or
median (Interquartile range)
No Severe ROP
n=79
Severe ROP
n=19
P-value
Male sex 38 (48%) 14 (74%) 0.07
Multiple gestation 5 (6%) 4 (21%) 0.07
Age at birth (weeks) 26.1 (25.3-27.1) 25.4 (24.7-25.9) <0.01
Weight at birth (grams) 845 (745-991) 700 (630-795) <0.01
Length at birth (cm) 34 (32-36) 32 (31-34) 0.02
Head Circumference at birth (cm)
24 (22.5-25) 22.5 (22-23) <0.01
Conventional ventilation (days) 16 (5-33) 51 (37-59) <0.01
Histologic chorioamnionitis 39 (51%) 9 (53%) >0.99
Hypotension 37/79 (47%) 15/19 (79%) 0.02
Bronchopulmonary dysplasia (BPD)
22/79 (28%) 11/19 (58%) 0.02
Necrotizing enterocolitis (NEC) 3/79 (4%) 4/19 (21%) 0.03
Culture positive infection 46/79 (58%) 15/19 (79%) 0.12
Intraventricular hemorrhage (IVH)
43/78 (55%) 10/19 (53%) >0.99
White matter injury (WMI) 24/78 (31%) 8/19 (42%) 0.42
Cerebellar hemorrhage 16/78 (21%) 2/19 (11%) 0.51
Severe WMI and/or IVH 12/78 (15%) 3/19 (16%) >0.99
- 54 -
2.3.3 Brain Injury
Ninety-eight early and 87 term-equivalent scans were scored. Findings included:
WMI in 30 (35%) neonates, severe WMI in 9 (11%), IVH in 47 (55%) neonates, severe
IVH in 5 (6%), ventriculomegaly in 27 (32%), and cerebellar haemorrhage in 16 (19%).
Severe ROP was not associated with an increased risk of WMI, IVH, ventriculomegaly or
cerebellar haemorrhage even when comparing the most severely affected forms of WMI,
IVH, and ventriculomegaly separately (all p>0.05) (Table 2.1).
2.3.4 White Matter Maturation
Mean FA in neonates with severe ROP was significantly lower for the posterior
white matter (effect size, -0.02; 95% confidence interval (CI), -0.04 to -0.004; p=0.02)
which was more pronounced over time on interaction analysis (p=0.08) (Figure 2.2). A
trend was seen in the FA of the optic radiations (effect size, -0.02; 95% CI, -0.39 to 0.001;
p=0.07) (Figure 2.2).
- 55 -
Figure 2.2 Mean Fractional Anisotropy (FA) values. Mean FA in those with and without
severe retinopathy of prematurity (ROP) by post menstrual age at scan in (A) posterior
white matter [p=0.02], (B) optic radiations [p=0.07], (C) splenium of the corpus callosum
[p=0.50], and (D) calcarine white matter [p=0.78]. (E) Diffusion tensor imaging region of
interest model map at the level of the high centrum semiovale and (F) basal ganglia:
(AWM) anterior white matter, (CWM) central white matter, (PWM) posterior white matter,
(PLIC) posterior limb of the internal capsule, (SCC) splenium of the corpus callosum, (OR)
optic radiations, (CWM) calcarine white matter.
C D
E F
- 56 -
Using voxelwise regression analysis in TBSS, white matter regions with
significantly lower FA in severe ROP included the optic radiations, PLIC and external
capsule on scans at 34-37 weeks (n=6/30) and 42+ weeks GA (n=3/24) (Figure 2.3).
Figure 2.3 Tract based spatial statistics (TBSS) model. TBSS using semi-automated
preterm and term neonate templates. White matter regions where neonates with severe
retinopathy of prematurity (ROP) differ from those with no severe ROP [p<0.05], corrected
for age at scan, shown in blue overlaid on the FA white matter skeleton in yellow. The
number of subjects in each sample with ROP is reflected by the numerator, and total
subjects in the sample, the denominator. R = right, L = left, P = posterior, A=anterior.
FA on ROI analysis in the anterior white matter (effect size, 0.001; p=0.93), central
white matter (effect size, -0.002; p=0.84), splenium of the corpus callosum (effect size, -
Axial Sagittal
L
L L
L R
R R
R A
A
P
P
- 57 -
0.010; p=0.50), PLIC (effect size, -0.01; p=0.19) and calcarine white matter (effect size, -
0.002; p=0.78) did not differ significantly in neonates with and without ROP. In the
posterior white matter and optic radiations, there was no difference in the relationship with
FA in neonates with severe ROP in the radial axes (λ2 and λ3) and the axial diffusion axis
(λ1).
2.3.5 Developmental Outcomes
A total of 83 (85%) infants were assessed at 18 months CA follow-up (Table 2);
there were no significant differences in the 7 who withdrew from the study after at least
one MRI scan or the 8 who died or were lost to follow-up.
Table 2.2 18 month corrected age (CA) follow-up assessments of neonates 24-28 weeks
GA with and without severe ROP treated with retinal laser therapy. Number (%) or median
(Interquartile range)
No Severe ROP
n=67
Severe ROP
n=16
P-value
BSID-III Cognitive score 105 (100-115) 95 (88-105) 0.02
BSID-III Motor score 94 (85-110) 85 (75-93) 0.01
BSID-III Language score 96 (83-112) 89 (83-103) 0.32
Severe cerebral palsy 6/67 (9%) 1/16 (6%) >0.99
Visual impairment 1/67 (1%) 0/16 (0%) >0.99
Hearing impairment 2/67 (3%) 0/16 (0%) >0.99
Infants with severe ROP had lower BSID-III motor and cognitive scores, with no
difference in language scoring relative to infants without severe ROP. There was no
difference in years of maternal education between groups (p=0.91). In a multivariate
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model, the relationship between severe ROP and motor and cognitive scores remained
significant when adjusting for GA at birth and severe WMI and/or IVH (Table 2.3).
Table 2.3 Multivariate linear regression analysis. 18-month corrected age Bayley
Composite scoring adjusted for GA at birth and severe white matter injury and/or
intraventricular hemorrhage.
Effect size (95% CI) P-value
BSID-III Cognitive Composite -9.22 (-17.69 - -0.75) 0.03
BSID-III Motor Composite -10.77 (-19.87 - -1.66) 0.02
BSID-III Language Composite -3.45 (-13.80 – 6.89) 0.51
One infant in the “no severe ROP” group was unable to complete the BSID-III
assessment due to severe CP and cortical visual impairment; when assigned a value of
2 standard deviations below the mean, there was no meaningful difference in the findings.
There was no difference in the rate of cerebral palsy, hearing or visual impairment in
infants with and without severe ROP (Table 2.2). Visual acuity assessments were
available in 12 (63%) newborns with severe ROP at a median age of 3.9 years (IQR, 3.1-
5.0 years). Median visual acuity was 20/30 OD and 20/40 OS in infants with ROP; no
infants met guidelines for severe visual impairment.
2.4 Discussion
In a prospective longitudinal cohort of extremely premature neonates, we found a
significant maturational delay of the posterior white matter regions with delay on TBSS
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analysis in the optic radiations, PLIC and external capsule in those neonates with severe
ROP, which to our knowledge has not been previously reported. These novel findings
occurred independently of severe white matter injury or intraventricular hemorrhage and
likely indicate similar vital mechanisms in the development of severe ROP with poor
maturation of the white matter. It is well known that circulating levels of insulin-growth
factor 1 (IGF-1) are important modifiers in the circulatory levels of VEGF and the
development of severe ROP (Harrell and Brandon, 2007). Within the brain, IGF-1 has
been shown to have a significant role in the augmentation and utilisation of glucose across
all neural cell lineages (Cheng et al, 2000; Dercole and Ye, 2008). Furthermore, mean
IGF-1 concentrations between birth and 35 weeks PMA in preterm neonates correlate
with total brain, white matter, grey matter, and cerebellar brain volumes, with poorer
cognitive development seen in those infants with the slowest rate of increase (Hansen-
Pupp et al, 2013; Hansen-Pupp et al, 2011). These factors support severe ROP as a
marker of adverse brain development, independent of visual outcomes, and are
highlighted by our 18 month CA follow-up assessments showing lower cognitive and
motor developmental scores in those with severe ROP, consistent with larger cohorts
(Bassler et al, 2009; Schmidt et al, 2003; Schmidt et al, 2015). Our study emphasizes
how adverse motor and cognitive developmental outcomes are associated with
maturational delays on DTI and TBSS independent of the traditional markers of brain
injury in the preterm neonate.
Maturational delays in the optic radiations are consistent with previous studies
using DTI and TBSS in preterm neonates that showed a relationship between severe
ROP and delayed white matter microstructural development in the optic radiations at
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term-equivalent age, independent of white matter injury (Bassi et al, 2008; Thompson et
al, 2014). Further research at 7 years of age showed delayed optic radiation
microstructure in children with a history of severe ROP compared to those with milder
ROP (Thompson et al, 2014). We have shown that these delays are present in the late
preterm period and involve non-visual pathways, highlighting an early link of severe ROP
with poor white matter maturation in preterm neonates.
When considering the importance of a retinal disorder on the white matter
development of the preterm neonate, retinal nerve fibre layer (RNFL) thickness using
optical coherence tomography (OCT) in preterm neonates can be examined. In a study
comparing RNFL thickness with 18-24 CA developmental scores, thinner RNFL thickness
across the papillomacular bundle correlated with lower cognitive and motor scores
(Rothman et al, 2015). These findings provide an understanding of the associations
between the retina and the underlying white matter pathways, and offer a “window into
the brain” of the connections that the retinal ganglion and photoreceptor cells share with
the white matter pathways. The maturational delays in the motor, visual and visual-
association pathways of the developing brain detected in neonates with severe ROP
suggests a mechanism by which these factors act on neurodevelopment, potentially due
to an imbalance of growth factors.
2.4.1 Limitations
Although we were able to compare a large number of extremely preterm neonates
with serial MRI scans, the timing of the follow-up MRI scans may have affected the
imaging results due to the development of ROP over time as a progressively acquired
disorder. We were unable to control for the timing of the ROP retinal laser therapy and its
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possible confounding of the imaging. Visual acuity follow-up was not done in conjunction
with the 18-month CA neurodevelopmental assessments and was not standardised
across the cohort.
2.5 Conclusions
Severe ROP requiring laser photocoagulation therapy is associated with delayed
maturation in the motor, visual and visual-association pathways. Infants with severe ROP
had lower motor and cognitive functioning at 18 months CA, independent of severe brain
injury and GA at birth. More research is needed to determine which potential mechanisms
in severe ROP prevent optimal neurodevelopment, and the impacts on brain maturation
of early and effective treatment of severe ROP.
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Chapter 3
Multiple Postnatal Infections in Preterm
Newborns is associated with delayed Maturation
of Motor Pathways at Term-equivalent age and
Poorer Motor Outcomes at 3 years
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3.1 Introduction
Preterm newborns born less than 32 weeks gestational age (GA) are at a
substantial risk of postnatal infection with 20 – 65% of newborns suffering from at least a
single infection during this period of significant brain development (Adams-Chapman and
Stoll, 2006; Orsi et al, 2009; Stoll et al, 2004). In a large epidemiologic study, postnatal
infection was found to double the risk of motor impairment and cerebral palsy, while also
greatly increasing the risk of cognitive impairment (Stoll et al, 2004). Similarly, a study of
very preterm infants followed to 9 years of age showed that infants who had postnatal
infection were more likely to have poorer motor development, cognitive delays, school
delays, attention-deficit hyperactivity disorder and other mental health disorders (Rand et
al, 2016). While white matter injury (WMI) is a known complication of postnatal infection,
the majority of infants with postnatal infection do not have punctate WMI on clinical
neuroimaging (Chau et al, 2012; Chau et al, 2009; Glass et al, 2008; Stoll et al, 2004).
Experimental models of WMI with hypoxia-ischemia show inflammation to have additive
effects on the injury present (Eklind et al, 2001). Microscopic WMI, present on
neuropathological studies, has been shown to be more widespread in the brain of infants
with macroscopic WMI, suggesting a more diffuse brain injury may be present, but below
the resolution of current clinical MRI techniques (Buser et al, 2012). Chau et al. reported
that very preterm newborns exposed to infection had reduced measures of white matter
development on MRI, even when adjusting for WMI, which was most prominent in brain
regions important for motor and cognitive development (Chau et al, 2012). This
impairment in the development of the white matter may reflect injury to the pre-OL lineage
cells following hypoxic-ischemic and/or inflammatory events in which the premature
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infants’ immune system, and brain, may be primed for further events, increasing their
vulnerability to injury (Hagberg and Mallard, 2005; Wang et al, 2012; Yanni et al, 2017).
Our aim was to investigate the association of multiple postnatal infections with the
white matter development and outcomes of very preterm newborns with the hypothesis
that a greater number of infections would be associated with delayed white matter
development and poorer motor outcomes at 36 months corrected age (CA) compared to
non-infected neonates.
3.2 Material and Methods
3.2.1 Study Population
The study was approved by the University of British Columbia/Children’s and
Women’s Health Centre of British Columbia Research Ethics Board and informed consent
was obtained from parents/guardians prior to recruitment. Preterm neonates 24-32 weeks
gestational age (GA) were recruited into a prospective longitudinal cohort study at British
Columbia Women’s Hospital from April 2006 to September 2013. Infants were excluded
if they had clinical evidence of a congenital malformation or syndrome, congenital
infection, or ultrasound evidence of a large parenchymal hemorrhagic infarction (>2cm),
as these conditions are strongly predictive of neurodevelopmental impairments or early
mortality. This cohort has been described previously to address different hypotheses
(Adams et al, 2010; Brummelte et al, 2012; Chau et al, 2012; Chau et al, 2013; Duerden
et al, 2015; Glass et al, 2017).
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3.2.2 Clinical Characteristics
Infection characteristics were collected by systematic chart review. Cultures that
had multiple growths with organisms consistent with contamination were excluded.
Culture positive infection was defined as any positive blood, urine, tracheal aspirate
and/or cerebrospinal fluid culture treated with ≥5 days of antibiotics with “clinical-only
infection” defined as any instance in which there was a clinical concern for infection with
negative cultures in which the antibiotic treatment duration was ≥5 days. A positive culture
with the same organism in each of two separate locations or cultures during a continued
antibiotic course was considered a single infection. Tracheal aspirates required a positive
culture and ≥4 white blood cells per field to be considered a positive culture. An “early
infection” was defined as any infection occurring at <72 hours of postnatal age, and
“postnatal infection” as any infection ≥72 hours after birth, with infections included up to
40 weeks post menstrual age (PMA). Other clinical characteristics were collected via
chart review: histologic chorioamnionitis as confirmed by clinical pathology assessment,
hypotension as any treatment for low blood pressure, patent ductus arteriosus (PDA) as
any requiring pharmacological or surgical treatment, BPD as oxygen therapy beyond 36
weeks PMA, and NEC as stages 2 and 3 of Bell’s criteria (Bell et al, 1978).
3.2.3 MR Brain Imaging
MRI scans were performed early in life when neonates were clinically stable, and
again at term-equivalent age, all without pharmacological sedation. At both time points
MRIs were carried out on a Siemens 1.5 Tesla Avanto scanner using an MR-compatible
isolette (Lammers Medical Technology, Luebeck, Germany) and specialized neonatal
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head coil (Advanced Imaging Research, Cleveland, OH). 3D coronal volumetric T1-
weighted and axial fast-spin echo T2-weighted images were performed. An experienced
neuroradiologist (K.J.P.), blinded to the participant’s medical history, reviewed the images
and recorded the severity of WMI, IVH, ventriculomegaly and cerebellar hemorrhage
according to previously described scales (Chau et al, 2009; Miller et al, 2005). The most
severe injury score seen on the preterm or term scan was used in the structural MRI
analysis as the greatest severity of injury and most likely to adversely impact
developmental outcomes and to be associated with greater amounts of microscopic WMI,
not seen on MRI. WMI volumes were calculated on the T1-weighted images with voxels
of abnormal T1 shortening identified as WMI, reviewed by two neonatal neurologists (V.C.
and S.P.M.), then manually segmented with simultaneous coronal, sagittal and axial
views of the brain using Display software (http://www.bic.mni.mcgill.ca/software/Display)
as previously reported (Guo et al, 2017).
3.2.4 Magnetic Resonance Spectroscopic Imaging
MR spectroscopic imaging (MRSI) was used as a measure of neuronal maturation
using quantitative metabolite ratios with bilaterally placed 4mm ROI voxels in six
anatomical regions: the anterior, central and posterior white matter, the caudate, lentiform
nucleus (globus pallidus and putamen), and thalamus. To reflect the overall metabolism
of the regions, we analyzed an average of the three white matter regions, and the three
basal ganglia regions, the caudate, lentiform nucleus, and thalamus regions.
3.2.5 Diffusion Tensor Imaging
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DTI is a measure of water diffusion in an ellipsoid space within each 3D voxel of
the MR image. Mean FA, the average directionality of diffusion, increases with white
matter maturation reflecting the maturation of the oligodendrocyte lineage and early
myelination (Drobyshevsky et al, 2005; Miller et al, 2002). DTI parameters of FA, λ1, λ2
and λ3 were acquired using a multi-repetition single-shot echo planar sequence, and
excluded if significant motion artifact was present (Chau et al, 2009). ROI analyses were
manually placed in seven white matter anatomical regions (anterior, central and posterior
white matter regions, optic radiations, splenium of the corpus callosum, genu of the
corpus callosum and PLIC) and three deep grey matter regions (caudate, lentiform
nucleus and thalamus).
TBSS was performed using functional MRI of the brain software library (FSL;
https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/FSL) as previously described (Duerden et al, 2015;
Glass et al, 2017). A diffusion tensor model was fit to the data at each voxel to calculate
the voxelwise FA with the spatial location of alterations in diffusion measures done using
the TBSS pipeline (Smith et al, 2006). The TBSS data were projected onto a mean FA
tract skeleton using age appropriate templates (preterm scans: 27-29 weeks, 30-33
weeks; 34-36 weeks; term scans: 37-41 weeks, ≥42 weeks), which were then used to
apply voxelwise regression cross-subject analysis. Cluster-size thresholding was applied
to the data in which the size of the cluster was determined by 500 permutations by using
Randomise v.2.9 within FSL. A threshold of P<0.05 (95% percentile of the distribution)
was set for the clusters and corrected for multiple comparisons across space in
determining the association of FA and ≥3 infections across subjects within each of the
age-based templates and projected upon the white matter skeleton.
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3.2.6 Developmental Follow-up
At 36 months CA, neurodevelopment was assessed with the Bayley Scales of
Infant and Toddler Development-III (BSID-III) cognitive, language and motor composite
scores and Peabody Developmental Motor scales 2nd edition (PDMS-2) all with a mean
of 100 and standard deviation of 15 (Bayley, 2005; Folio and Fewell, 2000). In addition to
the BSID-III motor composite, the PDMS-2 total, gross and fine motor quotient values
were used in the analyses as a more robust assessment of motor impairment at 36
months CA (Folio and Fewell, 2000). Assessments were carried out by qualified
therapists blinded to the imaging findings of the participants. Socioeconomic status was
classified by the self-reported number of years of maternal education. Cerebral palsy was
defined as a confirmed diagnosis made by an experienced pediatrician at the 36 month
assessment.
3.2.7 Data Analysis
Statistical analysis was performed using Stata V.14.2 (StataCorp, 2015). Clinical
and imaging characteristics were compared using Fisher’s exact test for categorical and
the Kruskall–Wallis test for continuous data with a statistical significance threshold of
P<0.05. The association between postnatal infections and other clinical variables with
neurodevelopmental outcomes was tested with univariate logistic regression.
The mean values, averaged bilaterally, of FA and NAA/Choline were compared
between neonates with and without postnatal infection, in a generalized least squares
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regression model for repeated measures, adjusting for PMA at MRI scan and multiple
ROIs with a P<0.05. A log-transformed outcome variable for the NAA/choline was used
to determine the percentage differences of the MRS measures (Chau et al, 2012). An
interaction term was examined describing the imaging relationships of postnatal infection
modified by PMA at MRI scan, and considered P<0.1 as significant. The 36 month
outcomes were compared using univariate analysis for multiple infections. An unadjusted
risk ratio and adjusted odds ratio were calculated in assessing the 36 month outcomes
association with the groups of infections.
3.3 Results
3.3.1 Clinical Characteristics
Of the 234 neonates born 24-32 weeks gestation recruited in the cohort, 219 (94%)
completed at least one MRI with a median birth GA of 27.9 weeks (interquartile range
[IQR], 26.0 – 29.7 weeks). Early MRI scans were completed at a median 32.1 weeks (IQR
30.4 – 34 weeks) with term-equivalent scans in 184 (84%) at median 40.2 weeks (IQR
38.7 – 42.0 weeks) (Figure 3.1).
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Figure 3.1 Flow chart. Flow chart of study enrollment, MRI scans and follow-up.
Ages presented are median, with corrected age used for Bayley-III and PDMS-2
Assessments.
3.3.2 Infection Characteristics
Of the 219 infants, 110 (50%) had one or more instances of postnatal infection and
109 (50%) had no infections in the postnatal period. Due to the small number of the
“clinical-only infection” group we grouped them with “culture-positive infection” for the
analysis and thus classified each infection event as a “postnatal infection”. There were 54
(25%) infants with one postnatal infection, 29 (13%) had two infections, 19 (9%) had three
infections, 7 (3%) had four infections and 1 (0.5%) had five infections. Infants were
Birth GA 24 – 32 weeks GA 27.9 weeks
N = 234
MRI #1 GA 32.1 weeks
N=219
Excluded as MRI not done
N = 15
MRI #2 GA 40.2 weeks
N=184
Bayley-III and PDMS-2 Assessments
35 months N=175 (82%)
Died N = 4 Withdrew/second scan
not done N= 31
Died N=1 Lost to follow-up N= 8
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grouped according to the number of infections, with three or more infections included
together in the analysis due to the small numbers in the four and five infections groups.
One and two infections were presented together in the results section as there were few
differences between the groups on clinical characteristics, neuroimaging and motor
outcomes (Table 3.1).
There were 46 (21%) infants with early infection of whom 21 had no other infection,
and 2 who had positive cultures (1 sepsis, 1 lower respiratory culture), which were
included in the septicemia and lower respiratory analyses respectively. In total there were
202 distinct episodes of postnatal infection among the total of 110 infants. There was a
greater rate of septicemia and lower respiratory infections in the three or more infections
group as well as a greater rate of infection with coagulase-negative Staphylococcus
(CONS) and Enterobacter species organisms compared to the one or two infection group
(Table 3.2). Neonatal characteristics more common among those with higher numbers of
infections included lower GA at birth, lower birth weight, lower birth length, smaller head
circumference at birth, hypotension, PDA, BPD and NEC stage ≥2 (Table 3.1).
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Table 3.1 Demographics table. Demographics, clinical characteristics and MRI findings
of newborns 24-32 weeks GA classified by the number of postnatal infections. Number
[%] or median (IQR). Early infection = any infection <72 hours of life; NEC = necrotizing
enterocolitis; IVH = intra-ventricular hemorrhage; WMI = white matter injury
No Infection N=109
One Infection N=54
Two Infections N=29
Three or more Infections
N=27
P-value
Male sex 54 [50] 30 [56] 10 [35] 19 [70] 0.12
Gestational age at birth (weeks)
29.4 (27.7-31.1)
27.3 (25.9-28.6)
25.6 (25.0-27.1)
25.7 (24.9-26.6)
<0.01
Birth weight (grams) 1190 (1020-1376)
909 (789-1190) 835 (663-950) 755 (630-896) <0.01
Birth length (cm) 38 (35-40) 35 (34-39) 33 (31-35) 33 (31-35) <0.01
Birth head circumference (cm)
27 (26-28) 25 (23-27) 24 (22-25) 24 (22-25) <0.01
Twin birth 41 [38] 19 [35] 7 [24] 9 [33] 0.69
Antenatal MgSO4 24 [22] 13 [24] 5 [17] 5 [19] 0.97
Antenatal steroids 11 [10] 7 [13] 2 [7] 4 [15] 0.75
Histologic chorioamnionitis
33 [31] 22 [42] 14 [48] 8 [31] 0.18
Early infection 21 [19] 11 [20] 7 [24] 7 [26] 0.69
Hypotension 21 [19] 23 [43] 14 [48] 23 [85] <0.01
Patent Ductus Arteriosus
35 [32] 27 [50] 21 [72] 25 [93] <0.01
Bronchopulmonary dysplasia
5 [5] 8 [15] 9 [31] 17 [63] <0.01
NEC stage ≥2 1 [1] 1 [2] 2 [7] 4 [15] <0.01
IVH grades 2-4 34 [32] 12 [34] 13 [54] 9 [35] 0.67
WMI 38 [35] 14 [26] 8 [28] 8 [31] 0.49
WMI volume (mm) 35.8 (16.3 – 272.4)
41 (11 – 275) 53 (5 – 892) 17.9 (7.6 – 83.6)
0.60
Cerebellar hemorrhage 5 [5] 6 [17] 7 [29] 7 [27] <0.01
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Table 3.2 Infection characteristics. Infection location and organism characteristics divided
by the number of infection groups. Number [%]
3.3.3 MR Imaging
WMI was present on the first MRI in 31% of infants (33 mild, 23 moderate, 12
severe) with 6 new instances of WMI on the second scans (4 mild, 1 moderate, 1 severe).
At least one infection occurred before the first MRI in 99 infants (90%). There was no
increase in WMI seen, either mild or moderate/severe, with increased numbers of
infection, with no increase in WMI when a separate analysis was done for the different
types of infection and organisms (all P>0.05). Similarly, IVH severity and
ventriculomegaly were not associated with infection (all P>0.05) (Table 3.1). Cerebellar
hemorrhage was more common among those infants with postnatal infection (Table 3.1).
One Infection
N=54
Two Infections
N=29
Three or more
Infections N=27
P-value
Clinical-only infection 7 [13] 5 [17] 8 [30] 0.21
Septicemia 16 [30] 17 [59] 17 [63] <0.01
Urinary tract infection 24 [44] 17 [59] 15 [56] 0.41
Lower respiratory infection 6 [11] 10 [34] 16 [59] <0.01
Meningitis 1 [2] 1 [3] 0 [0] >0.99
Coagulase-negative staphylococcus (CONS)
23 [43] 17 [59] 21 [78] 0.01
Enterococcus species 8 [15] 7 [24] 7 [26] 0.41
Escherichia coli (E.coli) 5 [9] 2 [7] 6 [22] 0.17
Klebsiella species 4 [7] 5 [17] 6 [22] 0.13
Enterobacter species 1 [2] 5 [17] 9 [33] <0.01
Candida species 1 [2] 2 [7] 4 [15] 0.08
Other pathogens 7 [13] 11 [38] 9 [33] 0.02
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3.3.4 MRSI and DTI Imaging
The NAA/Cho ratio was lower over time in those infants with ≥3 infections in the
white matter (coefficient, -1.4%/week; 95% CI, -2.3% to -0.5%; P<0.01) (Figure 3.2) and
basal ganglia (coefficient, -0.7%/week; 95% CI, -2.5% to -0.1%; P=0.03) (Figure 3.2),
after adjustment for WMI.
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Figure 3.2 Graphs of N-acetylaspartate/Choline (NAA/Choline) ratio. Graphs depict
associations of multiple infections with the neurulation of the basal ganglia (top) and white
matter (bottom) included in both preterm and term scans over time. Dots reflect the scatter
plot of the 3 regions included in each analysis and each subject, with the lines reflecting
the linear fit line to the data.
Those with ≥3 infections had lower mean FA over time in the PLIC (coefficient, -
0.005; 95% CI, -0.002 to -0.008, P<0.01). There were no other regions with significant
differences in the FA over time on the ROI analyses (all P≥0.10). The TBSS analysis of
the preterm MRIs 30-34 weeks PMA revealed only delayed FA located within the posterior
corpus callosum. In contrast, on the term scans at 37-42 weeks PMA lower FA was more
widespread and involved the complete corpus callosum, the optic radiations and PLIC
(Figure 3.3).
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Figure 3.3 TBSS model for multiple infections. Shown in the pre-term (30-34 weeks GA)
and term (37-42 weeks GA) model for infants with ≥3 infections compared to no infection
group on a white matter skeleton map. The number of infants with ≥3 infections are
indicated by the ‘N=’ with the denominator reflecting the total number of infants. R = right,
L = left.
3.3.5 Developmental Outcomes
Thirty-six month corrected age outcome scores were available in 175 (82% of
survivors) infants (median 35 months, IQR 34 – 37 months). There were no differences
in the rates of WMI, IVH grade ≥3 or neonatal clinical factors (all P>0.05) in the follow-up
and missed follow-up groups. Three infants were unable to complete the testing due to
severe impairment thus were assigned scores of 49 (3.3 standard deviations from the
R R L L
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mean). On univariate analysis a greater number of infections in the neonatal period was
significantly associated with poorer BSID-III motor composite score and PDMS-2 total,
gross and fine motor scores, but not language and cognitive scores (Figure 3.4)(Table
3.3).
Table 3.3 BSID-III and PDMS-2 outcomes at 36 months CA. Divided by number of
infection groups. Median (IQR) or Number [%]
No Infection N=93
One Infection N=42
Two Infections N=24
Three or more Infections
N=22
P-value
BSID-III Motor composite score
103 (97 – 115) 103 (94 – 110) 100 (91 – 107) 93 (76 – 107) 0.02
BSID-III Language composite score
109 (103 – 118)
112 (103 -115) 106 (83 – 115) 100 (94 – 112) 0.18
BSID-III Cognitive composite score
100 (95 – 110) 105 (95 – 105) 100 (95 – 105) 100 (90 – 110) 0.57
PDMS-2 Total motor 96 (90 – 102) 94 (88 – 97) 93 (88 – 97) 87 (74 – 94) <0.01
PDMS-2 Gross motor
96 (89 – 102) 94 (87 – 98) 91 (85 – 96) 84 (72 – 94) <0.01
PDMS-2 Fine motor 100 (94 – 103) 94 (91 – 97) 97 (88 – 103) 93 (85 – 97) <0.01
Cerebral Palsy 5 [5] 3 [6] 3 [12] 1 [4] 0.70
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Figure 3.4 BSID-III outcomes at 36 months CA. Divided by infection groups with motor,
language and cognitive composite scores reflected by the shades indicated in the
legend. The middle line and box reflect the median and IQR respectively, with the
1.5 IQR reflected by the whiskers and open circles as any outliers. ** p<0.05 (all
others p >0.05)
Unadjusted risk ratio (RR) and adjusted odds ratio (OR) assessments of BSID-III
scores ≤85 and PDMS-2 ≤80, considered clinically significant impairments, are shown
(Table 3.4). These illustrate that a greater number of infections was significantly
associated with lower Bayley-III motor composite scores and poorer performance on the
PDMS-2 total and gross motor testing when adjusted for potential confounding factors,
with less impairment in fine motor scores (Table 3.4). There was no significant increase
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in the incidence of cerebral palsy at 36 months CA with multiple infections (P>0.05) (Table
3.3)
Table 3.4 RR and ORs. Unadjusted RR and adjusted OR values for poor BSID-III and
PDMS-2 scores in infants with three or more postnatal infections. Odds (95% CI)
3.4 Discussion
Using multimodal MR imaging methods and follow-up assessments in a cohort of
very preterm newborns our results showed that three or more postnatal infections are
associated with delays in brain maturation, particularly within subcortical nuclei and white
matter implicated in motor function, and with poorer motor outcomes at 36 months CA.
Findings were associated with alterations in brain maturation over time, with differences
most prevalent on the term-equivalent MRI scans reflected by involvement of the PLIC,
corpus callosum and optic radiations. This is in agreement with previous research
suggesting that poorer motor outcomes and cerebral palsy are more likely in infants with
reduced FA in the PLIC, corpus callosum and white matter in cohorts of preterm infants,
implicating these important regions in motor development and highlighting the utility of
Unadjusted RR
P-value
Adjusted OR* P-value
BSID-III Motor composite <85 2.53 (1.10 – 5.85) 0.02 1.99 (1.2 – 3.2) <0.01
BSID-III Language composite <85 5.86 (1.34 – 25.6) <0.01 1.68 (0.94 – 3.00) 0.08
BSID-III Cognitive composite <85 0.55 (0.14 – 2.11) 0.37 0.55 (0.16 – 1.83) 0.33
PDMS-2 Total Motor <80 3.06 (1.27 – 7.4) <0.01 1.93 (1.21 – 3.09) <0.01
PDMS-2 Gross Motor <80 2.07 (1.06 – 4.03) 0.03 1.84 (1.18 – 2.85) <0.01
PDMS-2 Fine Motor <80 1.69 (0.58 – 4.97) 0.33 1.59 (0.88 – 2.90) 0.13
* Adjusted for Gestational age, maternal education, cerebellar hemorrhage and WMI volume
- 80 -
MRI in predicting motor outcomes (Duerden et al, 2015; Huppi et al, 2001; Thomas et al,
2005).
Very preterm newborns are at-risk of multiple postnatal infections due to many
factors. The innate and adaptive immune systems that respond to infectious antigens are
significantly impaired in the preterm newborn, leading the infant to bemore prone to
infections (Cuenca et al, 2013; Lavoie et al, 2010). Preterm infants also have reduced
trans-placental maternal antibody transfer and immature protein recognition receptors
important for bacterial antigen presentation and antimicrobial immune recognition (Kan et
al, 2016; Wynn et al, 2008). The consequence of this is an inappropriate inflammatory
response to infections with activation of toll-like receptors in the innate immune system,
that when associated with hypoxia-ischemia, can result in neural injury (Lehnardt et al,
2003). This may also may lead to priming of the immune system for subsequent events
manifested by greater concentrations of inflammatory markers and free radicals in the
cerebrospinal fluid (CSF), as seen in infants with WMI (Ellison et al, 2005; Inder et al,
2002). Neuropathology analysis of infants with severe WMI has showed that tumour
necrosis factor α (TNF-α) and interleukin-1β (IL-1β) levels are elevated in those infants
with infection, with greater levels than what is seen with hypoxia-ischemia alone, further
supporting their combined effects (Kadhim et al, 2001).
In a large epidemiologic study of over 6000 infants born at extremely low birth
weight (less than 1kg), all types of infection, not just sepsis or meningitis, increased the
risk of severe cognitive and motor impairments (Stoll et al, 2004). We also found no
major difference in the outcomes between the different types or organisms of infection.
We did not observe an increased risk for severe impairments with increasing numbers
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of infections, with a low rate of cerebral palsy overall. Rand et al. showed that long-term
neurodevelopmental outcomes at 9 years were more likely to be delayed in infants with
postnatal infection, but with no difference in severe motor outcomes (Rand et al, 2016).
Furthermore, in a small subset of infants, Rand et al. also reported that two or more
infections increased the risk for motor impairments two-fold (RR 5.7 vs 2.6) in addition
to increasing the risk of cognitive impairment (RR 2.1 vs 1.3) (Rand et al, 2016). This is
supported by the literature on the additive effects of subsequent events, as described by
Khwaja and Volpe, in which the preterm infant brain is sensitized following an initial
event of infection or hypoxia-ischemia and has at a greater likelihood to be injured
following subsequent events of inflammation and/or hypoxia-ischemia by a stimulus with
a lower threshold than was necessary for the initial response (Eklind et al, 2001;
Hagberg et al, 2012; Khwaja and Volpe, 2008; Leviton et al, 2013; Yanni et al, 2017).
The brain of the preterm newborn also has an immature blood brain barrier which
makes it especially vulnerable to hypoxia-ischemia injury, particularly of the pre-OL, the
prominent cell injured in WMI of the preterm infant (Back et al, 2001; Back et al, 2002;
Gilles et al, 1976; Wang et al, 2012). In hypoxia-ischemia models of preterm brain injury
a significant increase in pro-inflammatory markers is seen, suggesting that even in the
absence of infection, a significant immune system response occurs (Albertsson et al,
2014; Fleiss et al, 2015). Of the infants in the three or more infection group, 78% had a
CONS infection, which is not known to cause direct cerebral inflammation, but
frequently occurs in conjunction with hypotension (Mallard and Wang, 2012). In our
group of infants with three or more infections 85% had at least a single event of
hypotension, opposed to 45% and 19% in those with one or two and no infections
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respectively. Previous work in rats using a lipopolysaccharide injection with hypoxia-
ischemia injury model showed a greater duration of hypoxia was required to induce
injury in older rats than younger rats, indicating the importance of the developmental
window on the systemic response and the vulnerability of the preterm brain to injury
(Eklind et al, 2005). How hypoxia-ischemia and systemic inflammation interact in the
genesis of neonatal brain injury and dysmaturation requires further study (Back and
Miller, 2014; Fleiss and Gressens, 2012).
Another potential contributor to brain dysmaturation includes injury resulting from
hyperoxia with hypocarbia. BPD is a known complication of chronic ventilation in the
preterm baby, with hyperoxia a well-established causative factor in the development of
BPD (Buczynski et al, 2013). Of those infants with three or more infections in our cohort,
65% required long-term oxygen therapy compared to 5% for the no infection group.
Hyperoxia and hypocarbia are potent vasoconstrictors important in the pressure-passive
autoregulatory system of the preterm brain, and are independent risk factors in the
development of WMI (Shankaran et al, 2006). The impacts with which these factors also
contribute to the poorer outcomes seen in infants with multiple infection also needs
further study.
Multiple postnatal infections have been shown to be associated with progressive
WMI on subsequent MRI scans (Chau et al, 2009; Glass et al, 2008). Within our cohort
we found no difference in the rate or volume of MRI-detected WMI, ventriculomegaly or
IVH and the numbers or types of infections. There was an increased rate of cerebellar
hemorrhage among those infants with three or more infections, which may reflect their
lower gestational age at birth, making them more vulnerable to cerebellar hemorrhage.
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This has potential implications on the outcomes analysis due to the role that cerebellar
hemorrhage has in association with poor neurological outcomes (Tam et al, 2011).
Adams et al, in a smaller subset of the current cohort, showed that postnatal infection
was associated with slower increase in corticospinal FA over time than non-infected
newborns (Adams et al, 2010). These findings were expanded upon within our study to
show the maturational delays in those infants with three or more infections included the
corpus callosum and white matter regions including the PLIC on both tradition DTI and
TBSS analyses with supportive findings on MRSI analyses (Chau et al, 2012).
Follow-up at 36 months CA revealed that those infants with three or more
infections had poorer motor development than those infants with fewer infections. In
comparison to other large cohorts of preterm newborns with infection, we did not see an
increased risk of cognitive impairment in those infants with higher numbers of infections
(Rand et al, 2016; Stoll et al, 2004). Some of the differences seen from previous cohorts
may be due to improvements in clinical practices and NICU care in recent decades. In
addition, the majority of infants in this cohort are from high-resource settings which are
known to impact cognitive and language outcomes greater than motor development
(Cusson, 2003; Gross et al, 2001; Howard et al, 2011). The differences from previous
literature, with a predisposition for motor tracts and impairments seen in our study, may
be a result of the changes seen in the distribution of WMI, from PVL in older studies, to
more diffuse WMI in contemporary studies, with diffuse WMI resulting in less
widespread axonal changes (Buser et al, 2012). As the motor tracts are some of the first
areas to myelinate, they are also the most susceptible to injury in the preterm brain (Sie
et al, 1997; Welker and Patton, 2012).
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Limitations
As described previously in Chau et al (Chau et al, 2012), many of the infants who
had multiple infections would have had later MRI scans due to taking longer to become
clinically stable, which would result in differences in postnatal age at the MRI between
the infection groups. This would, however, favor the delayed images appearing more
mature in the multiple infection groups underestimating the extent of the injury and time
interaction. It should also be considered that the “clinical-only” infections could be the
result of a virus, which would also have implications on the white matter development,
immune system development and pro-inflammatory markers. However, we did not find
neuroimaging abnormalities consistent with known descriptions of viral infections, nor
were there any positive viral cultures. Overall, our findings support the need for further
monitoring of infants with postnatal infections and continued assessment of how to
improve the care of these infants.
Conclusions
Three or more postnatal infections are associated with delays in brain
maturation, particularly in areas of motor function, with poorer motor outcomes at 36
months CA. These results highlight the vulnerability of the preterm brain to multiple
postnatal infections and supports the potential for combined detrimental effects of
inflammation and hypoxia-ischemia within the NICU. Furthermore, it suggests the need
for a personalized approach to infection control in those infants with one or two
infections. Preventing further infections in this vulnerable group of preterm newborns
may have the potential to improve outcomes. More research is needed on the function
and role of the developing immune system and the impact of hypoxia-ischemia and
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infection on pro-inflammatory markers, as well as investigating potential therapies to
correct this inflammatory imbalance within the preterm newborn.
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Chapter 4
Summary of Main Findings
and
Future Directions
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4.1 Conclusions
Through the use of multi-modal MR and outcome assessments I have shown that
severe ROP is associated with delays in brain maturation with poorer outcomes at 18
months. This work supports the need for research that investigates the potential for
recovery in the most severe ROP cases through balancing of growth factors and
preservation of vision, and points to the significance of severe ROP as a marker of
outcomes. Moreover, with similar methods of assessment, we were able to show
impairments in brain maturation in widespread brain regions for those infants with greater
numbers of postnatal infections resulting in poorer motor outcomes at 3 years, thereby
stressing the strong association of infection with poorer brain maturation. This work
supports several basic science and clinical studies on impairment of brain maturation of
the preterm infant and provides clinicians and families with the knowledge to make
informed decisions about likely outcomes and areas in which outcomes can be improved.
Interventional follow-up programs remain an important monitor for those children with
developmental impairments, though their implementation varies by country, location, and
therapist with wide differences in care and follow-up practices. While much is known
about the potential risk factors and diseases that increase the likelihood of poorer
outcomes in preterm infants, many parents and clinicians are left with more questions
than answers at a critical period in development. The work presented in this thesis
supports further research in postnatal infection and severe ROP and highlights each as
a factor which may adversely influence long term outcomes.
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4.2 Future Directions
In considering the future potential for this research, several areas of this study warrant
expanded analysis and research, and this work may enhance the development of other
research disciplines.
4.2.1 Retinopathy of Prematurity and Neurodevelopment
In the care of the infant with ROP, the optimal treatment window for this disorder
remains unknown. Current therapies involve serial observations and monitoring, with
treatments provided after the development of “threshold ROP” when it is believed
treatment is needed in order to preserve vision. It is unknown at present whether even
earlier intervention, with treatment on lower stages of ROP than what is currently
provided, improves visual outcomes, neurodevelopmental outcomes and brain
development. Furthermore, the optimal therapy for the treatment of severe ROP remains
unknown. Several methods of treatment are currently in use, or in development, with laser
photocoagulation therapy still considered one of the gold standard therapies, though is
starting to fall out of favour. Intravitreous bevacizumab (Avastin), an anti-VEGF
monoclonal antibody, has been used more recently for severe ROP. Treatment failure
(Patel et al, 2012) and neurodevelopmental outcomes (Morin et al, 2016) have been
shown in one study to be worse among those treated with intravitreous bevacizumab
compared to laser therapy, which raises concerns of the impact of bevacizumab on the
brain. Another therapy of interest includes oral propranolol which was shown in a clinical
trial to reduce the progression of ROP to higher stages that require treatment, however
significant safety issues with bradycardia and hypotension may limit its use (Filippi et al,
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2013). Several other monoclonal antibodies are also currently under investigation as
potential ROP treatments, including ranibizumab (Lucentis), aflibercept (Eylea) and
pegaptanib (Macugen). Which method of treatment will be the preferred therapy, or
whether each will be used in a more personalized approach to the stage and location of
the eye disease present, will be determined as each method is validated and compared
through clinical trials.
Visual outcomes of preterm children with ROP are improving, though preterm
children continue to have poorer visual outcomes than their peers without ROP (Al-Otaibi
et al, 2012; Cryotherapy for Retinopathy of Prematurity Group, 2002; Pearce et al, 1998).
While it has been shown, that through quality improvement measures, severe ROP can
be reduced (Lee et al, 2014), severe ROP continues to be a commonly observed disorder
among preterm infants < 28 weeks GA. Prevention of ROP altogether remains an elusive
goal given the complex mechanisms involved in its development, with infection, chronic
oxygen requirements and other complications of preterm birth keys to its pathophysiology.
In each of these conditions, the implications of various growth factors in modulating the
effects on preterm brain development are largely unknown. While there remains ongoing
research into the impact of IGF-1 and VEGF as potential treatments for ROP, there is
also a field of research in using these treatments to improve brain development (Hansen-
Pupp et al, 2011). The use of IGF-1 levels as a biomarker of delayed brain maturation in
the preterm infant has been reported, with investigations underway currently using
recombinant humanized IGF-1 to see if it assists in accelerating postnatal brain
development (Hansen-Pupp et al, 2013), and whether it improves outcomes. There are
also several ongoing clinical trials using IGF-1 therapy in an attempt to improve
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neurodevelopment and outcomes in Autism (Riikonen, 2016), Rett’s syndrome (Khwaja
et al, 2014), and Duchenne’s muscular dystrophy (Malik et al, 2012), with overall positive
effects presented thus far. The effects of IGF-1 treatment in preterm infants remains
unknown at present, but with ongoing research and further investigation it may become
a staple therapy for the preterm infant in treating ROP and supporting brain growth.
Animal research of IGF-1 supports its use in neuroprotection with reduced cytokine and
tumour necrosis factor production (Sukhanov et al, 2007) and a reduced infarct volume
in animal models of stroke (DeGeyter et al, 2016). Other populations that appear worth
further investigation into whether IGF-1 can provide neuroprotection or improve brain
growth and neurodevelopmental outcomes, include infants with neonatal infarcts or
strokes, intra-uterine growth restriction (IUGR), and congenital heart disease (CHD).
While my analysis supports severe ROP as resulting in poorer outcomes, we need
further longitudinal assessment to confirm its implications into childhood and beyond.
Assessments at 18 months CA have been shown to have poor correlation with childhood
outcomes and may overestimate cognitive impairments (Hack et al, 2005), hence follow-
up at 3 years and 4.5 years will be important in determining whether these delays persist
into early childhood. Furthermore, there are significant differences in the language
abilities of infants at 18 months (Lung et al, 2009), many of which are considered within
normal range, making the interpretation of language outcomes at this age difficult,
potentially over or underestimating language impairments. It continues to be unknown
which is the best assessment method for predicting poor childhood outcomes, and when
is the optimal timing for assessing infants to accurately predict normal development in
childhood and beyond. Furthermore, significant alterations in the developmental
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trajectories of an infant can occur given the wide variation in childhood experiences and
educational supports available to infants, making more accurate predictions of outcome
difficult. With more accurate socioeconomic analysis techniques and calculating the
effectiveness of individual early intervention approaches, we may better understand the
post-NICU factors that are the most important in promoting childhood development.
Further characterization of the impairments of infants with severe ROP is needed,
with developmental visual follow-up assessments. While visual outcomes are much
improved compared to previous decades, infants with severe ROP continue to have mild
visual disturbances compared to their unaffected peers (O'Connor et al, 2002). Visual-
motor integration and visual perception scores have been shown to be highly correlated
with fine motor scores in preterm infants (Goyen et al, 2008). The extent to which these
visual-motor impairments are present in severe ROP, and the impact of these
impairments on infants’ brain development, remains unknown. Similarly, visual
impairments are also common in infants with brain injury (Pike et al, 2008), and it is
unclear to what extent that visual dysfunction impacts motor integration and performance.
Developmental coordination disorder (DCD) is a clinical childhood disorder with
severe implications for childhood cognitive and motor performance, and is commonly
associated with behavioural problems. DCD is hard to detect early in infants and remains
a poorly recognized disorder by clinicians. Despite this, DCD is a common disorder in
very preterm children at school age and is seen frequently in those infants with ROP
(Zwicker et al, 2013). Visual disturbances with poor ocular alignment, binocular vision and
refractive errors have all been shown to occur in a high prevalence in children with DCD
(Creavin et al, 2014). This further suggests that mild impairments to the visual system
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have the potential to adversely affect the motor and cognitive control of the developing
brain. To better understand the pathophysiology for these impairments, more research is
needed into the visual outcome monitoring of very preterm children in order to properly
assess the impacts of these visual outcomes on development, and to assess their true
prevalence.
Follow-up MRIs in childhood or adolescence of infants born very preterm are also
needed to monitor whether slower maturation on imaging methods DTI and TBSS
continues over subsequent analysis. This is an important step in providing information on
long-term outcomes from severe ROP, and will allow correlation over time of preterm and
term-equivalent studies. These studies will also be of importance in determining which
factors associated with childhood result in the greatest improvements in brain
development into childhood, as we continue to search for additional interventions to
improve outcomes and maximize each child’s potential.
In addition to the techniques utilized in this thesis, investigating severe ROP with
other measures of brain development such as MRSI and probabilistic tractography may
provide more information about the nature of the maturational deficits present.
Tractography and TBSS methods have been shown to correlate with visual outcome
scores in preterm infants, with delays in the optic radiations (Bassi et al, 2008), though
the link with severe ROP was not explored in this group. Emerging techniques, such as
diffusion kertosis imaging, magnetization transfer ratio, myelin water fraction and
quantitative susceptibility mapping, have all been shown to reflect features of white matter
microstructure (Groeschel et al, 2016), and are techniques of interest in determining the
method best predictive of outcomes in preterm infants. Resting state functional
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connectivity MRI at term-equivalent age has previously shown that infants with WMI have
aberrant connectivity, with the degree of variation determined by the severity of injury
(Smyser et al, 2013). The use of functional MRI in those infants with severe ROP could
be explored in this population to determine whether the maturational delays correlate with
not only alterations in the resting state networks, but also in the visual-motor integration
pathways, if utilized in older individuals. As MR methods continue to advance, it is hopeful
that we will be able to visualize better the cortex and its development. Current techniques
with volumetric analysis have shown global cerebral cortex volumes to be reduced in
preterm infants at term-equivalent age (Inder et al, 2005) and in WMI (Inder et al, 1999),
with cortical volumes reduced in cortical lobes in late childhood (Kesler et al, 2004),
though these techniques have been unable to assess regional cortical volumes in
sensitive areas in early infancy. Furthermore, alterations to the cortical volumes from
severe ROP, particularly of visual regions in the primary visual cortex and surrounding
regions, is another way in which neuroimaging can help us to understand the
pathophysiology of these maturational delays, and assist in identifying ways in which we
can improve outcomes.
4.2.2 Multiple postnatal infections and neurodevelopment
In the care of the infant with postnatal infection, infection control measures are
largely provided universally and across units in attempts to reduce the incidence of
infections. Despite this, a large number of preterm infants develop nosocomial infections
during their NICU course. Improvements in NICU infection control have been shown to
reduce infection rates and improve outcomes (Davis et al, 2016), though the specific
features of a sepsis quality improvement project that result in lowered infections is
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unknown. Increased hand hygiene results in significant decreases in nosocomial
infections, shown in several studies (Lam et al, 2004; Won et al, 2004). A large-scale
structured quality improvement project that included hand hygiene with stringent catheter-
insertion policies and early advancement of enteral feeds was shown to reduce infections
from 17% to 15% (Wirtschafter et al, 2011). While this is a significant improvement, the
high rate of infants with infection is still concerning. In identifying which factors are the
most important to reducing infection rates, and which can improve outcomes, the
relatively high cost of implementing these procedures can be reduced. Of further interest
is whether these procedures can be implemented in lower income countries where
nosocomial infections contribute to a high rate of morbidity and mortality (Zaidi et al,
2005).
Providing personalized infection control measures to those infants most
susceptible to the adverse effects of infection, is an intriguing concept of care that the
results of this thesis supports as a potential intervention to improve outcomes. Further
investigation is needed into whether, for those infants with one or two infections,
outcomes are improved by more individualized infection control measures, such as
reduced contact, more stringent guidelines for hand washing and line insertion, or other
measures. A personalized care package, such as that implemented to reduce the
incidence of intraventricular hemorrhage, has the potential to improve outcomes (Schmid
et al, 2013). How these measures can be applied, while also allowing for the other
features also important for the growth of the infant, are considerations that would need to
be taken into account.
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What also remains an important consideration in reducing the impact of infection
is determining whether there exists a specific period of vulnerability in which postnatal
infections are more likely to impact brain development. With the immature immune system
of the preterm newborn, particularly before 28 weeks, it would seem that this is a period
of particular importance. In addition, considering what we currently understand about the
pre-OL cell and its maximal period of susceptibility, between 23 to 32 weeks GA (Back et
al, 2001), it would seem reasonable to consider the period of maximal impact from
infection during this period as well. Despite this, it is not known whether the timing of the
infections is an important factor, and whether each infection has an equally cumulative
impact on the white matter development. This highlights the question whether all
infections are of equal importance in brain development and long term outcomes, and
stresses what factors need more attention when improving the care provided.
As has been discussed previously in this thesis, inflammation and hypoxia-
ischemia appear to have independent, yet contributory roles on the influence of infection
on the brain. Some of this is seen with the impact of the early physiological alterations
calculated via the Score for Neonatal Acute Physiology-II (SNAP-II), a measure of illness
severity in the first 12 hours of NICU admission which calculates a morbidity and mortality
risk score, and includes early measures of mean blood pressure, temperature,
oxygenation, serum pH among other clinical factors. A higher SNAP-II has been shown
to correlate with greater mortality (Harsha and Archana, 2015), delays in the corticospinal
tracts (Zwicker et al, 2013), and poorer cognitive, behavioural, social, educational, and
neurological outcomes at 10 years (Logan et al, 2017). Recent research in the fields of
inflammation and hypoxia-ischemia injury has improved our understanding of the
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contribution of those factors on the brain, and despite some divergence of these fields in
clinical research, there is considerable research to support their joint impact upon the
developing brain (Albertsson et al, 2014; Girard et al, 2008; Khwaja and Volpe, 2008). In
developing and assessing neuroprotective treatments and mechanisms in the preterm
brain, a greater understanding is needed of the numerous factors involved in the
pathophysiologies with which infection, inflammation and hypoxia-ischemia impact the
brain. Investigating the effects of pro-inflammatory biomarkers on preterm brain
development and white matter maturation is another area of interest, in addition to
exploring potential therapies to mitigate these effects. Furthermore, consideration should
be given to whether inflammation and hypoxia-ischemia contribute to other clinical
disorders in the preterm newborn, such as ROP, NEC and BPD.
Another growing area of research includes exploring the effects of many of the
drugs that are used in the treatment of the sick neonate. The impact of antibiotics,
commonly prescribed to infants with infections, is of increasing concern with reports of
poorer outcomes among those exposed, particularly early in life (Greenwood et al, 2014;
Kuppala et al, 2011). Postnatal exposures to hydrocortisone or dexamethasone have
been shown to be related to impaired cerebellar growth, though antenatal betamethasone
did not (Tam et al, 2011). Midazolam, a commonly prescribed drug used for sedation and
analgesia in the NICU, has been shown to result in decreased hippocampus growth, as
well as poorer outcomes (Duerden et al, 2016). Morphine, another analgesic agent, has
been shown to be associated with poorer cerebellar growth (Zwicker et al, 2016),
cognitive and motor outcomes (Grunau et al, 2009) as well as a lower IQ (de Graaf et al,
2011). As a result of these findings, many NICUs are adapting to encourage non-
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pharmacological interventions for analgesia with non-nutritive sucking, swaddling, music,
kangaroo care, swaddling and facilitated tucking all used in attempting to reduce the effect
of analgesia on the infant. While unequivocal evidence to support their use is not yet
available, there are encouraging reports of their analgesic abilities (Cignacco et al, 2007).
Sucrose is an agent that has been used extensively with non-nutritive sucking during
painful procedures and has shown favourable results in reducing the effects of pain
(Stevens et al, 2008). As we learn more about the effects of various medications
commonly used in the NICU, we will gain more insight into the areas with which we can
further improve the care provided.
One such area of research that has resulted in significant awareness and changes
in practice has been in the field of neonatal pain. With growth of awareness in the ability
for the preterm neonate to experience pain, we have learned much about how the preterm
infant responds to pain, and how these painful experiences can adversely impact brain
development (Brummelte et al, 2012; Ranger et al, 2013) as well as long term cognitive
IQ scores (Vinall et al, 2014). Increased pain scores in the preterm period results in a
slower increase in the FA of the corticospinal tract, even after adjusting for postnatal risk
factors and gestational age (Zwicker et al, 2013). Neonatal pain has also been shown to
impact the visual-perceptual abilities, using magnetoencephalography (MEG), in school
age children who had greater skin breaking procedures in the NICU period (Doesburg et
al, 2013). Similarly, neonatal pain results in alterations of the clinical responses to pain
(Grunau et al, 2001) and to the hypothalamic-pituitary-adrenal axis with lower cortisol
responses to stress (Grunau et al, 2005). The link between pain and abnormal brain
growth is seen in long term follow-up with decreased cortical thickness of the frontal and
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parietal lobes at 8 years of age in those children who had greater number of NICU painful
procedures as very preterm infants (Ranger et al, 2013). While it is clear that pain is an
important factor in the development of the preterm infant, little is known about whether
there are other factors associated with infection that potentiate these impacts.
Considering the significant number of procedures that are part of the management and
monitoring of those infants with infection, it is also unclear how much of the impacts of
multiple infections can be attributed to the increased number of painful procedures.
The microbiome of the preterm infant, which represents all the bacteria living in or
on the host, is a field with which there is growing interest in how to maximize outcomes
through investigating, what has been termed, the “microbiota-gut-brain axis”
(DiBartolomeo and Claud, 2016). Research into the microbiome of meconium in preterm
infants has shown that abnormal gut flora, likely introduced through the swallowing of
amniotic fluid, correlates with a higher likelihood of preterm birth and may contribute to
the early inflammatory response (Ardissone et al, 2014). Investigation into the healthy
microbiota, in a mouse model of behavioural abnormalities and autism, showed
improvements in the behaviours exhibited in those mice treated with probiotics (Hsiao et
al, 2013). This has coincided with an expansion into the investigation of the role of
probiotics in the preterm population, with studies reporting improvements with reduced
death and incidence of NEC (Lin et al, 2008), though without an impact upon sepsis
(Mihatsch et al, 2012) or early neurodevelopmental outcomes (Chou et al, 2010). As we
learn more about the healthy microbiota of the preterm newborn, we will be able to further
explore the impact of infection on this important system, and how treatments, such as
with probiotics, may help improve outcomes in this vulnerable population.
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Nutritional requirements and treatments is another field in which significant
research exists, yet the metabolic requirements and optimal nutritional support of the
infant with infection are not known. Increased brain growth in the NICU of the very preterm
infant, catch-up growth in particular, and increased brain volumes are associated with
improved outcomes at discharge and follow-up (Cheong et al, 2016; Franz et al, 2009).
Conversely, brain atrophy, seen on repeat imaging, is associated with poorer cognitive,
motor and behavioural scores at 3 year follow-up (Horsch et al, 2007). Early brain growth
has a direct relationship with early nutrition with improved growth seen with higher protein
intake (Cormack and Bloomfield, 2013), higher amino acid intake (Poindexter et al, 2006),
higher energy and lipid intake (Beauport et al, 2017), as well as better growth with earlier
enteral feeding (Dinerstein et al, 2006). In addition, the composition of the lipids is an
important factor in development with increased levels of docosahexaenoic acid (DHA)
and reduced levels of linoleic acid levels associated with decreased odds of IVH,
increased brain microstructural development and improved developmental scores on
follow-up (Tam et al, 2016). Suboptimal nutrition in the preterm period has also been
shown to be predictive of childhood intelligent quotient (IQ) scores and a higher frequency
of cerebral palsy in late childhood (Lucas et al, 1998). Similarly, increased intake of breast
milk in the first 4 weeks of life in very preterm infants showed significant improvements in
deep grey matter volumes at term (Belfort et al, 2016). These changes did not persist on
follow-up imaging at 7 years, but subjects with greater breast milk intake showed better
performance on IQ scores, mathematics, working memory, and motor functioning (Belfort
et al, 2016). Using advanced MRI techniques, higher energy and fat intake has been
shown to correlate with improved brain growth, particularly of the basal ganglia and
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cerebellum, as well as a positive correlation with FA in the PLIC and higher cognitive and
motor scores at 2 years CA (Coviello et al, 2017). Moreover, early establishment of full
enteral breast feeding in very preterm infants has been shown to be a protective factor in
the development of postnatal sepsis, suggesting a potentially protective effect from the
early breast milk (Ronnestad et al, 2005). Reduced rates of infection have also been
observed to be greater in breast milk fed infants than formula fed infants, further
supporting the potential immune modifying benefits of breast milk (Hylander et al, 1998).
While it is clear that early and adequate nutrition, preferably with breast milk, are important
factors in the growth of the preterm brain, we do not have good evidence on the effects
of infection upon the nutritional intake of the preterm infant. Furthermore, in the clinical
treatment of the sick infant, feeds are often held or stopped in the acute management of
infection, and switched to parenteral nutrition. While this treatment may be necessary in
the evaluation and workup for NEC, the reduced enteral feeds provided during this time
may adversely affect the brain growth during a time of particular vulnerability, and is a
potential contributing source of maturational delays and poor development in those
infants with multiple events of infection. More research is needed to further understand
this relationship.
As noted in chapter 3, a significant number of infants in the multiple infection group
had cerebellar hemorrhage. We know that cerebellar hemorrhage is an important factor
in the development of infants, and is associated with several long term outcome
developmental disorders, including motor and cognitive disabilities (Limperopoulos et al,
2007), autism (Wang et al, 2014) and cerebral palsy (Johnsen et al, 2005). What is also
well known is that there are close connections between the cerebrum and cerebellum
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with reduced volumes of the cerebellum seen in those infants with unilateral cerebral
injury (Limperopoulos et al, 2005), with impacts seen in the pons of the brainstem in those
infants with large cerebellar hemorrhages (Parodi et al, 2016). Yet, it is not well known
what is the impact of cerebellar hemorrhage on the growth and development of the brain
in the infant with infection, who may have a greater vulnerability to injury. Conversely, it
remains undetermined how cerebellar hemorrhage impacts the outcomes of the child with
infection and whether it factors into the poorer outcomes described.
The study of the epigenetics is a field of significant interest in predicting long term
outcomes in preterm infants. Epigenetics is a field that involves the study of the impacts
of environmental factors upon genetic expression. Through the processes of DNA
methylation and acetylation, genetic signaling can be reduced or enhanced, potentially
resulting in alterations in disease expression. Epigenetic factors have been shown to be
altered in other populations of children with neurodevelopmental disabilities, such as
Rett’s and Fragile X syndromes (Collins et al, 2004; Gu et al, 1996). Various other at-risk
groups of preterm infants have been shown to have methylation alterations with poorer
attention, self-regulation and quality of movements (Lester et al, 2015), traits which are
commonly observed in children born preterm. Bacterial organisms themselves have been
shown to produce “epimutations” in the epigenome with alterations that can potentially
result in disruptions to host cell immune function and pathogen identification (Bierne et al,
2012). Furthermore, in a mouse model of prenatal infection, stable alterations in the DNA
methylation within the cells of the prefrontal cortex and nucleus accumbens have been
described (Richetto et al, 2017). Those alterations were seen in several regions important
for neural function, WNT signaling, and GABA differentiation (Richetto et al, 2017). ROS
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have been shown to impact DNA methylation through the formation of oxidized DNA
lesions which are structurally similar to the methylated DNA cytosines (Lewandowska and
Bartoszek, 2011). In addition, ROS can impact the histone-modifying enzymes involved
in acetylation and methylation with direct effects upon the epigenetic fluctuations in the
cell (Simpson et al, 2012). The impact of multiple infections on the epigenome of the
preterm infant, and what epigenome alterations correspond with outcomes, is a field that
is largely unexplored.
In considering alterations to epigenetic factors, the “epigenetic clock” is a feature
of DNA methylation that has been developed as a biomarker of aging (Horvath, 2013). A
preterm infant epigenetic clock using DNA methylation has been shown to be a strong
predictor of GA in two separate birth cohorts, comparable to antenatal ultrasound
methods estimates (Bohlin et al, 2016; Knight et al, 2016). In utilizing this epigenetic clock,
higher maternal socioeconomic status and higher birthweight percentiles were associated
with an accelerated biological age in preterm infants (Knight et al, 2016). Age acceleration
using the epigenetic clock in adults has been shown to be associated with obesity
(Horvath et al, 2014), HIV infection (Horvath and Levine, 2015), Down’s syndrome
(Horvath et al, 2015), Alzheimer’s disease (Levine et al, 2015) and Parkinson’s disease
(Horvath and Ritz, 2015), and is said to predict cancer, cardiovascular and all-cause
mortality (Christiansen et al, 2016; Marioni et al, 2015; Perna et al, 2016). The preterm
epigenetic clock has been used to show that age acceleration is associated with
advanced maternal age, pre-eclampsia, previous fetal demise, lower 1-minute APGAR
score, antenatal betamethasone and female sex; and that age deceleration is associated
with insulin-treated gestational diabetes mellitus in a previous pregnancy (Girchenko et
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al, 2017). The impact of many post-natal events upon the epigenetic age of the preterm
infant remains unknown, yet this area has the potential to provide important information
on alterations to genetic expression, and may aid in the understanding of the
pathophysiology of the many neurodevelopmental impairments of the preterm infant.
There is much to be explored and investigated in the preterm infant in improving
outcomes and contributing to the literature of pathophysiology of diseases that impact
infants and children, while also assisting in the expansion of our understanding of brain
development and diseases.
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