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Michael Shevell and Steven Miller

Acquired Brain Injury in
ACQUIRED BRAIN INJURY IN the Fetus and Newborn
THE FETUS AND NEWBORN

The leukodystrophies are serious, progressive demyelination disorders, manifesting themselves in infancy or
early childhood. Some progress rapidly, leading to loss of sight, hearing, speech, and ambulation, and early
death.

This book is the only up-to-date, comprehensive guide to the genetics and pathogenesis of these disorders,
as well as their clinical features, diagnosis and therapy. Its purpose is to summarize for the reader all aspects
of the inherited disorders of myelin in children and adults. After a thorough overview of the role of oligo-
dendrocytes, astrocytes and microglia in white matter disease, chapters are then devoted to individual
disorders, covering their biochemical and molecular basis, genetics, pathophysiology, clinical features,
diagnosis, treatment and screening. The final chapters discuss the development of treatments for these
disorders and present a clinical approach to diagnosis in children and adults.

The book was conceived by Hugo Moser, whose research led to major developments in the treatment of
adrenoleukodystrophy, and is dedicated to him by his colleagues.

Mac Keith Press

EDITED BY MICHAEL SHEVELL AND STEVEN MILLER

I
C International Review
of Child Neurology Series
A
Mac Keith Press
 International Review of Child Neurology Series

Acquired Brain Injury in

the Fetus and Newborn

Edited by Michael Shevell and Steven P. Miller

© 2012 Mac Keith Press
6 Market Road, London N7 9PW

Editor: Hilary Hart

Managing Director: Ann-Marie Halligan
Production Manager: Udoka Ohuonu
Project Management: Prepress Projects Ltd

The views and opinions expressed herein are those of the authors and do not necessarily represent those of
the publisher

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or
transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise,
without the prior permission of the publisher

First published in this edition 2012

British Library Cataloguing-in-Publication data

A catalogue record for this book is available from the British Library

ISBN: 978-1-907655-02-9

Typeset by Prepress Projects Ltd, Algo Business Centre, Glenearn Road, Perth, UK

Printed by Latimer Trend & Company, Plymouth, Devon, UK

Cover image is of a fractional anisotropy map of the developing brain at term-equivalent age, showing the
major white matter pathways.
 International Review of Child Neurology Series

Acquired Brain Injury in

the Fetus and Newborn

Edited by

michael shevell
Chairman, Department of Pediatrics; Professor (with Tenure), Departments
of Pediatrics and Neurology/Neurosurgery, McGill University; Pediatrician-
in-Chief, Montreal Children’s Hospital, McGill University Health Centre,
Montreal, QC, Canada; Harvey Guyda Chair in Pediatrics

and

steven p. miller
Head, Division of Neurology, The Hospital for Sick Children; Bloorview
Children’s Hospital Foundation Chair in Paediatric Neuroscience; Professor,
Department of Pediatrics, University of Toronto, Toronto, ON; Affiliate
Professor, University of British Columbia, Vancouver, BC, Canada; Adjunct
Associate Professor, University of California, San Francisco, CA, USA

2012
Mac Keith Press
 International Review of Child Neurology Series

SENIOR EDITOR Lieven Lagae

Department of Paediatric Neurology
Charles RJC Newton University Hospitals KULeuven
University of Oxford Department of Psychiatry Leuven, Belgium
Oxford, UK
Makiko Osawa
EMERITUS SENIOR EDITOR Department of Pediatrics
Tokyo Women’s Medical University
Peter Procopis Tokyo, Japan
The Children’s Hospital at Westmead
Sydney, NSW, Australia Ingrid Tein
Division of Neurology
FOUNDING EDITOR Hospital for Sick Children
University of Toronto
John Stobo Prichard Toronto, ON, Canada

EDITORIAL BOARD Jo Wilmshurst

Department of Paediatric Neurology
Peter Baxter Red Cross Children’s Hospital
Department of Paediatric Neurology School of Child and Adolescent Health
Sheffield Children’s NHS Trust, University of Cape Town
Sheffield, UK Cape Town, South Africa

Paolo Curatolo
Department of Paediatric Neurology and
Psychiatry
Tor Vergata University
Rome, Italy

iv
Contents

Authors’ appointments vii

FOREWORD x

PREFACE xi

ACKNOWLEDGMENTS xii

Section I The Fetus

1. BRAIN INJURY IN THE FETUS 1

Adre du Plessis

2. Imaging the Fetal Brain 18

Catherine Limperopoulos

Section II The Preterm Infant

3. Mechanisms of Acute and Chronic Brain Injury in the Preterm Infant 29

Stephen A. Back

4. Clinical assessment of the Preterm Infant including Near-infrared

spectroscopy, amplitude-integrated Electroencephalography, and
Electroencephalography53
Lena Hellström-Westas and Frank van Bel

5. Imaging the Brain of the Preterm Infant 66

Gareth Ball, Mary A. Rutherford and Serena J. Counsell

6. Protecting the Brain of the preterm infant 81

Christopher D. Smyser and Terrie E. Inder

7. Seizures in the Preterm Infant 95

Hannah C. Glass

v
 Contents

8. Outcomes After Brain Injury in the Preterm Infant 99

Marilee C. Allen

Section III The Term Infant

9. Mechanisms of brain Neurodegeneration in the Term infant 121

Frances J. Northington, Raul Chavez-Valdez and Lee J. Martin

10. Clinical Approach to Term Encephalopathy 154

Anastasia Dimitropoulos, Steven P. Miller and Jerome Y. Yager

11. Imaging term infants with suspected hypoxic–ischemic encephalopathy  165

Kenneth J. Poskitt, Vann Chau and A. James Barkovich

12. Protecting the Brain in term infants 183

Fernando F. Gonzalez and Donna M. Ferriero

13. Seizures in the term newborn infant 198

Mona C. Toet and Linda S. de Vries

14. Outcomes after Brain Injury in the Term infant 209

Beatrice Latal

Section IV Specialized Topics

15. Neonatal Neurology in the Developing World 221

Nicola J. Robertson

16. PERINATAL STROKE 237

Kendall B. Nash and Yvonne W. Wu

17. Brain Injury in Newborn infants with Congenital Heart Disease 251
Patrick S. McQuillen, Steven P. Miller and Annette Majnemer

18. Metabolic Brain Injury in the fetus and the neonate 268
Linda De Meirleir

19. The Nutritionally Deprived Fetus and Newborn infant 277

Raghavendra Rao and Michael K. Georgieff

20. Ethical Considerations in Fetal and Neonatal Neurology 288

Lucie Wade, Michael Shevell and Eric Racine

Index306

vi
Authors’ Appointments

Marilee C. Allen The Johns Hopkins Hospital, Baltimore, MD, USA

Stephen A. Back Associate Professor of Pediatrics and Neurology, Oregon Health and
Science University, Clyde and Elda Munson Professor of Pediatric
Research, Director, Neuroscience Section, Papé Family Pediatric
Research Institute, Portland, OR, USA
Gareth Ball Centre for the Developing Brain, King’s College London, London, UK
A. James Barkovich Departments of Radiology and Neurology, University of California, San
Francisco, CA, USA
Frank van Bel Department of Neonatology, Wilhelmina Children’s Hospital, University
Medical Center, Utrecht, the Netherlands
Vann Chau British Columbia Children’s Hospital, Department of Pediatrics –
University of British Columbia, Vancouver, BC, Canada
Raul Chavez-Valdez Department of Pediatrics, Johns Hopkins University School of Medicine,
Baltimore, MD, and Department of Pediatrics, Division of Neonatology,
Texas Tech University – Health Sciences Center, Odessa, TX, USA
Serena J. Counsell Centre for the Developing Brain, King’s College London, London, UK
Linda De Meirleir Department of Pediatric Neurology and Metabolics, Universitair
Ziekenhuis Brussel, Brussels, Belgium
Anastasia Dimitropoulos Division of Pediatric Neurology, BC Children’s Hospital and University of
British Columbia, Vancouver, BC, Canada
Donna M. Ferriero Department of Pediatrics and Neurology, University of California, San
Francisco, CA, USA
Michael K. Georgieff Department of Pediatrics, Division of Neonatology, Institute of Child
Development, Center for Neurobehavioral Development, University of
Minnesota, Minneapolis, MN, USA
Hannah C. Glass Department of Pediatrics and Neurology, University of California, San
Francisco, CA, USA
Fernando F. Gonzalez Department of Pediatrics, University of California, San Francisco, CA,
USA

vii
 Authors’ Appointments

Lena Hellström-Westas Department of Women’s and Children’s Health, Uppsala University,

Uppsala, Sweden
Terrie E. Inder Departments of Neurology and Pediatrics and Mallinckrodt Institute of
Radiology, Washington University, Saint Louis, MO, USA
Beatrice Latal Child Development Center, University Children’s Hospital, Zurich,
Switzerland
Catherine Limperopoulos Associate Professor of Pediatrics; Director, MRI Research of the
Developing Brain; Director, Advanced Pediatric Brain Imaging
Research Laboratory, Children’s National Medical Center, Washington,
DC, USA
Patrick S. McQuillen Departments of Pediatrics and Neurology, Benioff Children’s Hospital
and University of California, San Francisco School of Medicine, San
Francisco, CA, USA
Annette Majnemer Professor, Director, and Associate Dean, School of Physical and
Occupational Therapy, McGill University and Montreal Children’s
Hospital, Montreal, QB, Canada
Lee J. Martin Professor of Pathology and Neuroscience, Departments of Pathology
and Neuroscience, Division of Neuropathology, Johns Hopkins University
School of Medicine, Baltimore, MD, USA
Steven P. Miller University of Toronto and The Hospital for Sick Children, Toronto, ON,
and University of British Columbia and British Columbia Children’s
Hospital, Vancouver, BC, Canada
Kendall B. Nash Department of Neurology, Division of Child Neurology, University of
California, San Francisco, CA, USA
Frances J. Northington Professor of Pediatrics; Director, Neurosciences Intensive Care Nursery,
Department of Pediatrics, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
Adre du Plessis Children’s National Medical Center, Center for Neuroscience
Research; George Washington University School of Medicine and
Health Sciences, Washington, DC, USA
Kenneth J. Poskitt British Columbia Children’s Hospital, Departments of Radiology and
Pediatrics – University of British Columbia, Vancouver, BC, Canada
Eric Racine Neuroethics Research Unit, Institut de recherches cliniques de
Montréal; Biomedical Ethics Unit, Division of Experimental Medicine,
Department of Neurology/Neurosurgery, McGill University; Departments
of Medicine and Social and Preventive Medicine, Bioethics Programs,
University of Montreal, Montreal, QC, Canada
Raghavendra Rao Department of Pediatrics, Division of Neonatology, and Center for
Neurobehavioral Development, University of Minnesota, Minneapolis,
MN, USA
Nicola J. Robertson Professor in Perinatal Neuroscience and Honorary Consultant
Neonatologist, Institute for Women’s Health, University College London,
London, UK

viii
 Authors’ Appointments

Mary A. Rutherford Centre for the Developing Brain, King’s College London, London, UK
Michael Shevell Departments of Neurology/Neurosurgery and Pediatrics, McGill
University; Division of Pediatric Neurology, Montreal Children’s Hospital,
McGill University Health Centre, Montreal, QC, Canada
Christopher D. Smyser Departments of Neurology and Pediatrics, Washington University, Saint
Louis, MO, USA
Mona C. Toet Department of Neonatology, Wilhelmina Children’s Hospital, University
Medical Center, Utrecht, the Netherlands
Linda S. de Vries Department of Neonatology, Wilhelmina Children’s Hospital, University
Medical Center, Utrecht, the Netherlands
Lucie Wade Neuroethics Research Unit, Institut de recherches cliniques de
Montréal; Biomedical Ethics Unit and Division of Experimental
Medicine, McGill University, Montreal, QC, Canada
Yvonne W. Wu Department of Neurology, Division of Child Neurology, University of
California, San Francisco, CA, USA
Jerome Y. Yager Director of Research, Department of Pediatrics, Pediatric
Neurosciences, Stollery Children’s Hospital and University of Alberta,
Edmonton, AB, Canada

ix
Foreword
Over the last decade there have been considerable
advances in understanding the neurologic conditions of
the newborn infant, particularly those conditions that start
during fetal life and eventually manifest after delivery.
Although there are now considerable insights into the
genetic basis of brain development and the maldevelop-
ment leading to migrational disorders, the understanding
of disorders that result from injury to the brain during the
fetal and neonatal period are relatively unexplored, and
are often not covered by textbooks and other books on
neonatal and child neurology.
Thus, Acquired Brain Injury in the Fetus and
Newborn is a timely book, in which internationally rec-
ognized clinical scientists have written state-of-the-art
reviews in their areas of expertise. Edited by two world
authorities on neonatal neurology, it is a book that pro-
vides the scientific basis of these conditions and yet it is
sufficiently pragmatic to be useful for the clinician. This
approach, from the basic science to the bedside, provides
the knowledge and insight to introduce readers to this
rapidly expanding field.
x
Fetal and neonatal neurology is becoming increas-
ingly complex, with the interaction between the genetic
predisposition to specific forms of brain damage and the
wide variety of insults to which the developing brain is
exposed. Studying injury of the fetal brain is difficult
because the fetus is relatively inaccessible to investiga-
tion; as a result, it has been difficult to diagnosis many
fetal conditions or predict the outcome. The advent of
more sophisticated investigations, particularly imaging of
the brain, has provided the tools to advance fetal neurol-
ogy. This book provides chapters with excellent reviews
of the imaging and physiologic tests that are available for
examining the fetal and neonatal brain. The final section
of the book addresses special populations and situations,
which are often not included in standard books on child
neurology.
This comprehensive book on acquired brain injury
in the fetus and neonate will not only be very useful for
pediatric neurologists and neonatologists, but it should
also appeal to the many disciplines interested in the fetal
and neonatal brain.
Charles Newton
Scott Family Professor of Psychiatry
University of Oxford Department of Psychiatry
Oxford, UK
Preface
Over the last decade, the field of neonatal neurology has
undergone tremendous advances. Advances in magnetic
resonance imaging technology now provide an unprec-
edented view of the brain in health and in critical ill-
ness, from the fetal period through childhood. With the
widespread uptake of hypothermia for the treatment of
hypoxic–ischemic encephalopathy, neuroprotection is
now a reality. Most recently, the integrative, collabora-
tive, high-tech and protocol-driven approach to neonatal
neurology has seen the emergence of neonatal neurologi-
cal intensive care with collaboration among neurologists,
neonatologists, neurophysiologists, neuroradiologists,
psychologists, and the rehabilitation specialties.
In the face of these advances, our approach to Acquired
Brain Injury in the Fetus and Newborn Infant is pragmatic
and focuses on specific populations encountered regularly
by the clinician. Given the increasing demand for fetal
neurology and the interpretation of fetal imaging studies,
this book begins by addressing fetal neurology. We then
follow a ‘bench to bedside’ approach to acquired brain
injury in the preterm and term newborn infant. Preterm
births have been increasing, and at earlier gestations,
placing these infants at higher risk of brain injury. With
improvements in intensive care, the risk of cerebral palsy
is beginning to decline, yet this, and a high prevalence of
other cognitive deficits, remains a considerable burden.
xi
Our understanding of the relationship between critical
illness and brain development is catalyzing new opportu-
nities to further reduce the burden of neurodevelopmental
impairments in the preterm newborn infant. Important
advances have been made in our understanding of the
underlying etiology and prognosis in the term newborn
infant with encephalopathy. The application of hypother-
mia in this population has ushered in a new era of brain
protection. Advances in the recognition and treatment
of neonatal seizures offer new opportunities to further
improve outcomes. Congenital heart disease, now increas-
ingly diagnosed in utero, is increasingly recognized as a
significant risk for brain injury and neurodevelopmen-
tal impairments. The final chapters of the book address
advances related to special populations and concerns.
We are very grateful to the authors of these chapters
– internationally recognized clinician scientists – who
synthesized the state-of-the-art understanding and clini-
cal approach in their chapters. The advances made in our
understanding of acquired brain injury in the fetus and the
newborn infant has allowed us to move from a focus on
vulnerability to resilience and recovery, and from diag-
nosis to therapy and, ultimately, prevention. We sincerely
hope that the next decade brings us further in our care of
affected newborn infants so that books such as this are no
longer needed.
Steven Miller and Michael Shevell
Toronto and Montreal
September 2012
Acknowledgments
The editors are grateful to their teachers, mentors, col-
leagues, fellows, residents, and students, as well as their
patients and their families, for fostering and enabling
their clinical and research interests in neonatal neurol-
ogy throughout their careers. The editors are especially
grateful for their spouses and children who have ensured
that the ‘life’ portion of the ‘work–life’ balance has been
so amply filled.
xii
The author of Chapter 3 is grateful to Dr Roger
Hohimer for his helpful comments and suggestions and
to Dr Art Riddle for advice and assistance with Figure 3.4.
The authors of Chapter 10 thank Meisan Brown-Lum
for her helpful comments and her support with editing
the chapter.
The authors of Chapter 19 thank Kristin Koppen for
her help with preparing the manuscript.
1
Brain Injury in the Fetus
Adre du Plessis
Introduction
The consequences of injury to the fetal brain are influ-
enced by factors unique to this initial phase of the lifes-
pan. Fetal brain development unfolds across gestation
through a sequence of overlapping phases, each with a
specific period of peak activity. These events occur in
different cell types and different regions of the brain in a
complex, highly programmed manner. The regions with
the most active development under normal conditions
are also those that are at greatest risk for injury under
adverse conditions. Regional injury during critical phases
of development may derail subsequent developmental
events, in and around the region of injury, as well as
remotely in future projection fields of the injured area
(Limperopoulos et al 2005, Limperopoulos and du Plessis
2008). In summary, the topography, and consequently the
long-term manifestations, of brain injury depend not only
on the nature of the insult but also on its timing; ‘when’ is
as important as ‘what’. Although the developing brain is
more susceptible to injury, its immature state also under-
lies its sometimes remarkable ability to compensate for
injury through the incompletely understood phenomenon
of ‘plasticity’. Thus, the mechanisms of injury to the fetal
brain, as well as the long-term structural and functional
sequelae, are inextricably linked to normal developmental
events in the brain. Although these developmental events
will be reviewed briefly to provide context, the reader
is referred to excellent published reviews for additional
detail (e.g. Johnston et al 2009).
Normal fetal brain development may be disrupted
by primary disturbances in its genetic blueprint, or by
internal and external environmental factors. Antenatal
influences may also predispose the fetal brain to injury
during the intrapartum period. A review of primary brain
dysgenesis is beyond the scope of this chapter (Volpe
2008a), and intrapartum brain injury has been reviewed
exhaustively elsewhere (du Plessis 2005, Volpe 2008b).
1
The focus of this chapter is therefore confined to fetal
brain injury occurring prior to intrapartum events.
Overview of normal fetal brain development
Nervous system development in the fetus progresses
through a series of events, starting several days after con-
ception. During the embryonic period the principal phases
of development are definition of the neural axis and for-
mation of the neural tube (dorsal induction). The neural
tube then comes under the influence of regional gene
product gradients, which promote certain developmental
processes and suppress others (Jessell 2000). After the
neural tube closes at around 4 weeks after conception,
three vesicles begin to form at its rostral end. These are
the prosencephalon, mesencephalon, and rhombencepha-
lon that will form the future forebrain, midbrain, and
hindbrain respectively. This rostral region of the neural
tube then goes through a series of folds in the sagittal
plane forming the cervical, pontine, and cephalic flexures.
The central canal, which, at its rostral end, will form
the ventricular system of the brain, is surrounded by a
layer of cells that form the primary neuroepithelium, the
origin of all neuronal and glial cell lineages (Rakic et al
2007, Bystron et al 2008). The neuroepithelium becomes
divided into dorsal and ventral segments. The dorsal neu-
roepithelium is the source of excitatory pyramidal neurons
(that will form the future projection pathways), as well as
the radial glial cells. The ventral neuroepithelium devel-
ops two thickened regions, the ganglionic eminences,
which give rise to the future interneuronal population,
which become critical for localized cortex-to-cortex cir-
cuits. The main neurotransmitter of these interneurons is
gamma-aminobutyric acid (GABA), which has an initial
excitatory influence, but ultimately becomes the principal
inhibitory neurotransmitter in the brain. Neurons reach the
developing cerebral cortex via two major paths of neuro-
nal migration. Neurons from the dorsal ventricular zone
 Section I: The Fetus
undergo radial migration along radial glial cells acting as
guide-wires to the surface of the brain. During this migra-
tion the six-layered neocortex is formed in an inside-out
manner, with later waves of migration passing through
previous layers to settle on their outer-side. A critical part
of this process is development and subsequent regression
of the subplate zone (Kostovic and Jovanov-Milosevic
2008). The transient subplate zone acts as a ‘waiting sta-
tion’ below the future cortex, where it is thought to ‘fine
tune’ the itinerary of axons of thalamic neurons seek-
ing to make appropriate connections in the developing
cortex. Successful completion of these thalamocortical
pathways establishes the fundamental neural scaffolding
connecting the internal and external environment to the
sensory cortex, and in so doing enables the development
of conscious experience.
During initial formation of the cerebral hemispheres
an overabundance of neural structures are formed, includ-
ing neurons, dendrites, dendritic spines, and synapses.
This occurs under the influence of ‘spontaneous’ bursts
of electrical activity that are endogenously generated,
i.e. are not activated by incoming stimuli. Once devel-
opment of the neural apparatus connects the peripheral
sensory nerves to the cerebral cortex through connection
in the thalamus, a reorganization of the cortex occurs
under experience-driven influences. This phase coincides
with normal regressive events occurring in the develop-
ing brain. Specifically, ‘unstable’ synapses are pruned
back, unless they are stabilized by an appropriate level
of activation. Such neural activation also releases local
growth factors and activates gene programs that support
the survival of neurons. On a ‘use-it-or-lose-it’ basis,
redundant neurons are culled by active energy-dependent
cell death, or apoptosis. These regressive events and the
early abundance of neural structures might underlie the
compensatory plasticity of the immature nervous system
after injury. Myelination is a relatively late phase of devel-
opment and by term gestation has proceeded only into the
brainstem, cerebellum, and the posterior limb of the inter-
nal capsule. The subsequent pattern of myelination does
not commence uniformly across the cerebral hemispheric
white matter, but rather it proceeds in a predictable spatial
and temporal sequence (Kinney et al 1988, Drobyshevsky
et al 2005).
At a cellular level, the consequences of an insult
are heavily influenced by the maturational level of the
neuronal and glial lineages at the time of the insult. As
a broad statement, neurons in regions of the most active
development at any one time have a physiology that pro-
motes depolarization, making it most sensitive to incom-
ing stimuli that then activate receptors responsible for
controlled influx of enzyme-activating calcium. In order
2
to finely titrate this process and to keep it localized to
establish the most regionally discrete connectivity, the
neurotransmitter is then rapidly cleared from the syn-
aptic cleft by reuptake channels. During insults such
as hypoxia and hypoglycemia, this arrangement turns
hostile as neurotransmitter release becomes uncontrolled
and reuptake mechanisms fail, resulting in sustained
neurotransmitter activity at the synapse with neurotoxic
levels of calcium influx. From this brief outline it can be
seen that during critical periods of development the fetal
brain might be particularly vulnerable to injury, which, in
turn, might disrupt the complex sequence of subsequent
developmental events.
The oligodendrocyte lineage goes through a series of
maturational steps, from a progenitor phase to the mature
myelin-generating form. Across this period, the oligoden-
drocyte lineage passes through a developmental phase (the
late oligodendrocyte progenitor, or preOL) phase, during
which it is highly vulnerable to hypoxia–ischemia and
other insults (Back et al 2007, Segovia et al 2008); before
and after this preOL phase the oligodendrocyte lineage is
relatively resistant to insults. Data from animal models
correspond with the peak period of white matter injury in
preterm infants between 26 and 32 weeks’ gestation (Baud
et al 2004, Back et al 2007). In fact, both the temporal
and topographic distribution of the preOL overlaps with
the timing and topography of preterm birth-related white
matter injury (Buser et al 2010). During oligodendrocyte
development there is a shift in glutamate receptor and
transporter density, limitation of antioxidant defenses, and
cytotoxic cytokine receptors (Back 2006, Volpe 2009),
making the preOL particularly vulnerable to insults such
as hypoxia–ischemia and infection–inflammation. A syn-
opsis of published data suggests that hypoxia has differ-
ent effects on the oligodendrocyte lineage at different
stages of development. In the very early oligodendrocyte
precursors, hypoxia may result in accelerated matura-
tion (Akundi and Rivkees 2009). In the somewhat more
mature preOL, chronic hypoxia causes either delayed
preOL degeneration or developmental arrest in the vul-
nerable preOL phase, and thus it is primed for injury
from subsequent insults (Segovia et al 2008). Finally, in
its mature myelinating phase the oligodendrocyte has a
relatively elevated threshold to injury.
Similar phases of increased vulnerability occur dur-
ing neuronal development. Immature neurons in rapidly
developing brain regions have membranous and intracel-
lular features that facilitate depolarization and promote
influx of calcium for activation of growth-promoting
enzymes. During earlier phases of development, neu-
rons are maintained in a relatively hypopolarized state
by the neurotransmitter GABA. At this early stage of
 Brain Injury in the Fetus
development, GABA has an excitatory influence (as
opposed to its later inhibitory action) related to the
ambient chloride gradients across the immature neu-
ronal membrane (Staley et al 1995, Dzhala et al 2005).
Furthermore, a paucity of reuptake transporters allows
GABA to accumulate in the extracellular space, where
it then acts in a more diffuse paracrine manner by main-
taining a field of hypopolarization, which in turn releases
the magnesium blockade at the N-methyl-d-aspartic acid
(NMDA) receptor channel, permitting greater calcium
influx for maturational processes. However, during
insults such as hypoxia–ischemia both the hypopolar-
ized membranous state and high density of glutamate
receptors in regions of accelerated brain development
predispose to excitotoxic injury with necrotic and/or
apoptotic cell death.
The maturational development of astroglial cells is
also relevant to the current discussion. Specifically, the
typical response to brain injury seen in later stages of
development and in the mature brain, i.e. reactive astro-
gliosis, does not occur before about 20 to 24 weeks ges-
tational age (Kinney and Armstrong 1997). Consequently,
tissue destruction occurring prior to this point in gestation
triggers very little cicatricial response. As a result, the
resulting lesions may have minimal gliosis and resemble
malformations rather than encephaloclastic lesions.
The nature and severity of environmental insults may
disrupt brain development in a number of different ways.
Milder insults may trigger subtle pathologic processes
through epigenetic programming pathways. Progressively
more severe insults may result in arrested or disrupted
development, selective cellular injury and loss, and frank
pancellular destruction (infarction) of the structural scaf-
folding required for normal brain development. These
different pathways likely act in concert during and after
insults. Furthermore, below the threshold of injury, insults
may also increase (sensitize) or decrease (pre-condition-
ing) sensitivity to subsequent insults.
The timing and nature of insult and injury might
also influence the subsequent efficiency of compensa-
tory processes, so-called plasticity. In the same way that
early injury may disrupt subsequent brain development,
so too may it affect the normal regressive processes of
‘pruning’ back and reorganization during later phases
of brain development. As discussed above, earlier pro-
cesses in fetal brain development produce an excess of
neural structures, which are subsequently ‘pruned back’
by energy-dependent apoptosis, a process that occurs in
part through competition for trophic factors. One theory of
plasticity is that regional brain injury reduces competition
for trophic factors and substrate, allowing surviving tissue
to compete successfully.
3
Normal function of the maternal placenta–fetal
interface
During the antenatal period the principal contact between
the fetus and the environment is through the placenta. The
placenta serves as the only nutrient and clearance system
for the fetus. In addition, the placenta serves as a barrier
to potentially noxious agents. The placenta also has a
critical endocrine role in fetal growth and development.
Placental development begins with ‘placentation’ several
days after implantation of the embryo. The normal physi-
ology of implantation includes trophoblast invasion of the
endovascular layers of the spiral arteries, with disruption
of the muscular media (Nanaev et al 1995), and con-
version of the normally small caliber spiral arteries into
distended flaccid vessels with limited vasoconstrictive
capability. In so doing the uteroplacental system is con-
verted to a low-resistance, low-pressure, and high-volume
circulation. Exchange across the placenta occurs across
an interface composed of the syncytiotrophoblast, a basal
membrane, and the fetal endothelium. Such exchange
occurs in several different ways: bulk flow down hydro-
static and osmotic gradients; diffusion down concen-
tration gradients; transporter protein-mediated transfer
(e.g. glucose, amino acids); and endo- or exocytosis (e.g.
immunoglobulin G). Therefore, in addition to perfusion
of the uteroplacental and fetoplacental circulations, the
supply of nutrients across the placenta is also dependent
upon the functional surface area, as well as the membrane-
bound transporter activity on either side of the maternal–
fetal interface. Transporter-mediated transfer is adaptively
regulated via cellular homeostatic mechanisms, which
change transporter function in response to substrate lev-
els, and thereby maintain placental supply coupled with
fetal demand. Transporters may be upregulated and may
increase their efficiency during decreased circulatory sup-
ply, but, like other fetal compensatory mechanisms, this is
a temporizing response and cannot be sustained over long
periods (Constancia et al 2002, 2005). These responses
may explain why the fetal:placental weight ratio (placen-
tal transfer efficiency) may be greater in growth restricted
infants than in appropriate for gestational age infants.
Mechanisms of fetal brain injury
Fetal Substrate Deprivation
Fetal oxygen-substrate deprivation eventually leads to
growth restriction, which in turn increases the risk of peri-
natal and long-term complications (Brodsky and Christou
2004). Fetal brain development is dependent upon the
appropriate delivery of nutritional elements for structural
accretion, and of energy substrate to support enzyme func-
tion. The appropriate availability of energy substrate is
 Section I: The Fetus
particularly important during the third trimester, when
energy-dependent neuronal activation is critical for estab-
lishing and consolidating neuronal circuitry.
Restriction of specific nutrients essential for nervous
system development
The classic association between specific nutrient defi-
ciency and disturbed neurodevelopment is that between
folate deficiency and disturbances in neural tube closure.
Maternal folate deficiency may result from inadequate
dietary intake as well as malabsorption conditions (after
gastric bypass surgery) (Haddow et al 1986). The pre-
cise cellular mechanisms by which folate supplementa-
tion prevents neural tube defects remains unknown; the
current understanding is reviewed in detail elsewhere
(Haddow et al 1986). Disturbances in cholesterol avail-
ability have been implicated in disruption of prosence-
phalic development. Specifically, the Sonic hedgehog
(SHH) protein plays a central role in the development of
the face, brain, and genitalia. In the brain the SHH gene
product is critical for ventral induction and patterning,
with formation of the cerebral hemispheres and the mid-
line structures, most notably the corpus callosum. For the
SHH protein to be activated it must bind to cholesterol.
When cholesterol availability is limited, lesions such as
holoprosencephaly (Fig. 1.1) and agenesis of the corpus
callosum may develop. The classic example of cholesterol
deficiency is 7-dehydrocholesterol reductase deficiency,
the autosomal recessive Smith–Lemli–Opitz syndrome
(ACOG 2000).
Restricted energy substrate for normal brain
development
Under normal conditions the developing brain enjoys a
privileged supply of oxygen and energy substrate. In fact,
normal oxygen and glucose delivery to the fetal brain
(a)
Fig. 1.1 Holoprosencephaly semilobar with large dorsal cyst in a 36-week gestational age fetus: (a) MRI midline sagittal and (b)
coronal T2 images.
4
significantly exceeds demands. If this supply decreases,
a number of systemic and cerebral compensatory mech-
anisms are activated to optimize cerebral supply and
demand and to maintain a normal cerebral metabolic rate
until hypoxemia is severe (Richardson 1993). In fetal
sheep with a 50% decrease in oxygen delivery to the pla-
centa, cerebral oxygen consumption was maintained for
at least 24 hours (Bocking et al 1992). In another experi-
mental model in which maternal oxyhemoglobin satura-
tion was maintained below 30% (by decreasing inspired
oxygen concentration), cerebral oxidative metabolism
was maintained for more than 4 days (Richardson 1993).
In animal models of fetal hypoxemia, induced by decreas-
ing uterine artery blood flow, the first fetal response was
an increase in umbilical blood flow and an increase in
oxygen extraction, followed by increased shunting of
umbilical venous return from the placenta through the
ductus venosus and foramen ovale. Sympathetic activa-
tion causes peripheral vasoconstriction, while intrinsic
autoregulatory vasodilation in the brain reduces resistance
and increases cerebral blood flow. These adaptations in
the fetal circulation divert the most highly oxygenated
perfusion to vital organs including the brain (‘central-
ization’ or the so-called ‘brain-sparing effect’). In addi-
tion, there are physiologic responses aimed at decreasing
energy demand. Myocardial energy utilization is reduced
through a chemoreceptor-mediated fetal bradycardia.
Cerebral metabolism is decreased by active suppression
of neuronal activation; this is achieved by adenosine, an
adenosine triphosphate (ATP) breakdown product that
inhibits synaptic activity by blocking the presynaptic A1
receptor (Blood et al 2003). Chronic compensated fetal
hypoxemia may cause epigenetic changes in fetal pro-
gramming and, by sublethal neuronal suppression, may
disrupt activity-driven processes in brain development.
Pure hypoxemia (with intact perfusion) may be tolerated
(b)
 Brain Injury in the Fetus
for sustained periods of time, by adjustments in demand,
redirected blood flow, and alternative energy pathways
such as anaerobic metabolism and utilization of alterna-
tive energy sources (e.g. lactate and ketones). The effi-
cacy of these compensatory mechanisms at preventing
destructive brain injury is dependent upon multiple factors
such as the ‘dose’, nature, and delivery of the insult, the
maturational state and sex of the fetus, and pre-exist-
ing conditions in the fetal milieu (e.g. preceding energy
restriction, infection). These and other processes confine
the destructive brain injury caused by substrate restriction
to a ‘very narrow window between intact survival and
death’ (Bennet and Gunn 2009). When hypoxemia and
hypoperfusion occur in combination (i.e. hypoxia–isch-
emia) these compensatory mechanisms rapidly collapse,
in part because the interruption of glucose supply limits
anaerobic metabolism and lactate formation, and destruc-
tive pathways are unleashed.
Mechanisms of fetal energy substrate deprivation
Limitation of substrate supply to the fetal brain may origi-
nate at the maternal, uteroplacental, fetoplacental, or fetal
level. Maternal starvation level deprivation is uncom-
mon in the developed world but remains a problem in
underdeveloped regions and those ravaged by natural and
man-made disasters. Although substrate concentrations
in the maternal circulation may restrict fetal supply, the
more common scenario is limitation of uteroplacental
perfusion, by lesions such as abnormalities of placenta-
tion, infarction, and hemorrhage. Impaired uteroplacental
perfusion may stem from abnormalities at the level of the
uterine arteries, the spiral arteries, or the uteroplacental
vascular bed. Failure of spiral artery transformation and
vasodilation results in impaired perfusion of the placental
intervillous spaces, setting the stage for compromised
fetal oxygenation. Failure of normal placentation may
result in spontaneous miscarriage, isolated fetal growth
restriction (FGR), and pre-eclampsia with or without
growth restriction. In pre-eclampsia the muscular media
not only persists but may even hypertrophy.
Placental mechanisms of fetal substrate restriction
constitute a major pathway for morbidity during the
fetal period, with effects that extend through the neo-
natal period and beyond. However, the system has con-
siderable reserve, and fetal growth is not impaired until
approximately 30% of placental function is lost. Placental
dysfunction, leading to FGR, may result from a spectrum
of different etiologies, leading to a common end result.
Broadly speaking, these placental pathologies may be
considered in three categories: abnormal vascular devel-
opment, inflammatory processes, and acquired degen-
eration, usually with thrombotic changes. Inflammatory
5
changes in the intervillous space of the placenta may be
caused by infections, such as toxoplasmosis, cytomegalo-
virus, and other presumed viral infections, as well as auto-
immune conditions, such as the antiphospholipid antibody
syndrome. However, fibrin deposition and intervillous
thrombi may also be seen in up to half of placentas from
normal-outcome pregnancies. In addition, some studies
have failed to identify an association between maternal or
neonatal thrombophilic polymorphisms and an increased
risk of FGR (Infante-Rivard et al 2002, Infante-Rivard
et al 2005).
The fetoplacental circulation normally receives
almost half the fetal cardiac output. Signaling between
fetal and maternal placental vessels couples fetal to utero-
placental blood flow (Talbert and Sebire 2004). However,
this coupling may be disrupted by vasoconstrictive or
occlusive placental lesions. The major fetal vessels in the
chorionic plate perfuse large segments of the placenta,
called cotyledons. The vascular territories of these large
arteries do not overlap; the cotyledons have no collat-
eral supply, and these vessels are neither innervated nor
do they autoregulate. Fetoplacental blood flow is locally
controlled entirely through fetal endocrine and placen-
tal paracrine systems (Poston 1997, Benoit et al 2008).
Fetoplacental vascular pathology may cause significant
elevation in fetal peripheral vascular resistance. In fact,
sustained hypoperfusion of the fetoplacental circula-
tion may actually lead to constriction of these vessels
(Rockelein et al 1990). Fetoplacental thromboinflamma-
tory lesions associated with adverse neurodevelopmen-
tal outcomes include fetal thrombotic vasculopathy and
villitis of unknown etiology, which is associated with
chronic inflammation and, ultimately, avascular distal
villi (Redline 2004). Longstanding meconium exposure
may cause vascular necrosis through apoptotic death of
vascular smooth muscle cells and vasospasm.
Intrinsic fetal conditions may adversely affect fetal
brain development. As discussed above, the normal fetal
circulation is arranged such that there is an optimal oxy-
gen-substrate delivery to the developing brain. In certain
forms of fetal cardiac malformation this arrangement may
be disrupted with potential restriction of cerebral oxygen-
substrate delivery. For example, the oxygen content of aor-
tic (and hence brain) blood flow is decreased in conditions
such as transposition of the great arteries. Conversely,
volumetric cerebral blood flow may be compromised by
conditions such as aortic and left ventricular hypoplasia,
which, in severe cases, leaves the brain dependent on
retrograde perfusion from the ductus arteriosus across
the aortic isthmus. A study comparing fetal volumetric
brain growth in fetuses with heart lesions with controls
showed that despite similar brain volumes at the end of the
 Section I: The Fetus
second trimester, there occurred a significant and progres-
sive fall-off in brain growth among the congenital heart
disease fetuses (Limperopoulos et al 2010a). This brain
growth failure was most pronounced among fetuses with
the greatest expected oxygen delivery to the brain, as well
as with the presence of cerebral lactate on fetal magnetic
resonance spectroscopy, suggesting the development of
anaerobic metabolism in these most affected participants
(Catherine Limperopoulos, Children’s National Medical
Center, personal communication, 2010).
The oxygen-carrying capacity may also become
impaired in conditions that cause severe anemia, such
as Rhesus incompatibility and fetal infections, especially
with parvovirus (see below).
Assessing perfusion of the uteroplacental and feto-
placental circulations by Doppler ultrasound measures of
blood flow velocity and vascular resistance is now stan-
dard in the management of suspected FGR. When FGR is
associated with placental infarction and/or hemorrhage on
the maternal side of the circulation, uterine artery Doppler
indices will show an increase in resistance. Similarly, in
the fetoplacental circulation, a characteristic sequence
of perfusion and resistance changes develop during pro-
gressive placental failure. Particularly concerning is the
development of decreased or reversed diastolic flow in
the umbilical arteries, which becomes apparent only when
about 50% or more of placental function is lost.
The so-called brain-sparing effect is misleading
because, in many cases, the attempted compensatory
response does not spare the brain. Fetuses with evidence
of brain sparing on Doppler studies are almost always
growth restricted (Arduini et al 1987).
Abnormal development of brain structure in fetal growth
restriction
In animal studies, placental insufficiency has a broad
range of effects on the developing brain, often with
decreased gray matter volumes and impaired myelina-
tion (Mallard et al 1998). Experimental models of FGR
have shown regional decreases in growth factor levels in
the fetal brain (Duncan et al 2004), which in turn leads
to apoptosis and activation of pro-apoptotic pathways.
It has been proposed that the patterns of structural
brain abnormality in human fetuses with FGR are depen-
dent on the gestational age, although there is lack of con-
sensus on this issue (Bassan et al 2011).
In humans with FGR the range of structural brain
changes is broad; findings between studies are not consis-
tent. It is likely that many different factors influence this
relationship, including gestational age at onset of FGR,
gestational age at delivery, and postnatal age at the time
of study. Microcephaly is a known complication of severe
6
FGR. Some studies of preterm growth-restricted fetuses
have suggested a preferential catch-up of head growth
over the first few years (Jordan et al 2005, Westerberg
et al 2010). At follow-up, many individuals have head
sizes similar to appropriately grown ex-preterm infants.
Padilla et al (2010) found no difference between growth-
restricted and appropriately grown preterm infants in head
circumference, total brain volume, or the volumes of gray
or white matter (by three-dimensional magnetic resonance
imaging [MRI]) at 12 months corrected age. Other stud-
ies of preterm FGR have shown decreased intracranial
volumes and decreased cortical gray matter volumes after
preterm birth, persisting at term, but no significant dif-
ferences with appropriately grown ex-preterm infants at
later ages (Tolsa et al 2004, Dubois et al 2008). Although
overall brain size may not differ significantly, neuroimag-
ing studies have detected lobar or regional decreases in
the frontal lobe (Geva et al 2006a,b, Figueras et al 2008a;
Hernandez-Andrade et al 2008), hippocampus (Geva et al
2006a, Lodygensky et al 2008), and insular lobes (Padilla
et al 2010) in preterm growth-restricted infants.
Other more subtle differences in cerebral cortical
development gyrification (Dubois et al 2008, Esteban
et al 2010); with decreased gray matter density (Tolsa
et al 2004, Lodygensky et al 2008), have been described.
Magnetic resonance spectroscopy studies have detected
elevated cerebral lactate (Leth et al 1996, Kok et al 2002,
Wolfberg et al 2007) (suggestive of anaerobic metabo-
lism), and elevated inositol–choline ratios (Sanz-Cortes
et al 2010), suggestive of reactive astrogliosis. Diffusion-
weighted imaging has shown significantly higher apparent
diffusion coefficient values in the pyramidal tracts (Sanz-
Cortes et al 2010).
Abnormal development of brain function in fetal growth
restriction
A number of studies have demonstrated the increased
risk for adverse neurodevelopmental and behavioral out-
come in survivors of FGR (Low et al 1992, Kok et al
1998, Monset-Couchard et al 2002, Tideman et al 2007).
However, there are major inconsistencies in the reported
prevalence and manifestations of these sequelae. Both
preterm and term (Oros et al 2010) growth-restricted
fetuses appear to be at risk; some studies have suggested
that the clinical profile of these two groups may differ
(Figueras et al 2008b), but this has not been consistent
(Bassan et al 2011). Padilla et al (2010) suggested that
preterm FGR was associated with worse neurodevelop-
mental outcome, especially in the fine motor domain.
FGR in late preterm and term infants may be associated
with a distinct clinical picture with impaired cognition
and executive function (Fattal-Valevski et al 1999, Geva
 Brain Injury in the Fetus
et al 2006a,b, 2008). FGR in preterm infants increases the
risk for neurodevelopmental sequelae in some, but not all,
studies (Lodygensky et al 2008, Padilla et al 2010).
For term growth-restricted infants the risk for cerebral
palsy increases four- to sixfold compared with those born
between the 25th and 75th centiles (Larciprete et al 2005).
Others have shown a significantly greater risk of subse-
quent cerebral palsy for growth-restricted infants born
between 34 and 37 weeks’ gestation compared with those
born before 33 weeks (Blair and Stanley 1990, 1992).
Monset-Couchard et al (2002) showed an almost twofold
increase in behavioral abnormalities when preterm infants
born growth restricted compared with those appropriately
grown. The need for later special education services was
significantly greater for growth restricted than for appro-
priately grown preterm infants in some (Kok et al 1998),
but not other (Schaap et al 1999) studies.
Fetal Cerebrovascular Injury
Currently, the majority of cerebrovascular injury diag-
nosed in newborn infants is considered to be of peri-
natal origin. Although both ischemic and hemorrhagic
lesions are more easily diagnosed by modern fetal imag-
ing, distinguishing between antepartum, intrapartum, and
neonatal-onset stroke may be difficult when imaging is
delayed. Arterio-occlusive stroke of the immature brain
has a relatively limited acute presentation, with seizures
being the most obvious clinical change. If seizures are the
heralding sign of stroke, these usually occur over a period
of days, then recede whether or not they have been treated.
Hereafter, there is commonly a latent period with a pau-
city of physical signs until around 4 to 6 months, when
normally emergence of purposeful movements reveals
motor asymmetry. Early stroke, including intrauterine
stroke, without detected seizures, may have delayed motor
asymmetry as the first indication of a focal brain lesion.
By this chronic phase of the injury even advanced MRI
may be unable to distinguish between antenatal, perinatal,
or neonatal-onset stroke. Early intrauterine stroke occur-
ring before mid-gestation may show little, if any, residual
evidence of tissue destruction as reactive gliosis is usually
minimal (see above). In fact, the resulting lesion may be
misdiagnosed as a primary dysgenetic lesion. Current
clinical fetal MRI techniques remain relatively limited in
their ability to detect fetal hemorrhagic lesions.
For all of the above reasons our understanding of the
true incidence and pathogenesis of fetal arterial stroke
remains poorly characterized (Curry et al 2007). Reported
associations have included twin pregnancies (especially
monochorionic with co-twin demise), fetal congenital
heart disease, thrombophilias, and intrauterine infections.
Strokes occurring in the perinatal period have a strong
7
predilection for the middle cerebral artery distribution,
especially on the left. Pregnancy is a naturally occurring
hypercoagulable state, resulting from elevated circulating
prothrombotic factors, decreased natural anticoagulants,
and reduced fibrinolytic activity. The placenta has been
implicated as a source for embolic phenomena in neona-
tal stroke, and thus presumably also fetal stroke (Burke
et al 1997, Kraus and Acheen 1999). The most commonly
implicated placental lesions include fetal thrombotic vas-
culopathy and fetal vasculitis (in intrauterine infection).
As a major proportion of the normal fetal venous
return from the placenta passes through the foramen ovale
and from there into the major cerebral arteries, a throm-
boembolic source in the placenta has direct access to
the fetal brain arteries. Such thromboembolic sources
in the placenta may result from several different pro-
cesses, including inflammatory processes such as infec-
tion, thrombosis, and abnormal arteriovenous connec-
tions between monochorionic twins, especially in cases
of co-twin demise and twin–twin transfusion syndrome
(Fig. 1.2). Monochorionic twin pregnancies have placen-
tal connections; the twin circulations remain balanced in
all but 10% to 15%. Stroke risk is significantly increased
in twin–twin transfusion syndrome; when there is co-twin
demise (which is probably underestimated) thromboplas-
tin material is transferred from the dead twin.
An association between maternal thrombophilias
and adverse pregnancy outcome was first suggested by
Kupferminc and colleagues in 1999 (Kupferminc et al
1999). Proposed thrombophilia-related complications
have included recurrent miscarriages, fetal demise,
intrauterine growth retardation, pre-eclampsia, and fetal-
neonatal stroke. Thromophilias have been implicated in
up to 70% of neonatal arterial strokes (Volpe 2008c),
including factor V Leiden, prothrombin 20210A muta-
tion, MTHFR mutation, protein C deficiency, protein
S deficiency, antithrombin deficiency, antiphospholipid
antibody syndrome, and elevated lipoprotein A (Golomb
et al 2001, Mercuri et al 2001, Curry et al 2007, Suppiej
et al 2008, Simchen et al 2009). Conversely, a recent study
for a broad range of genetic thrombophilia polymor-
phisms failed to show an association with arterial stroke
in newborn infants (Miller et al 2006). In many cases
with an association between thrombophilia and neona-
tal stroke, it has been in the setting of other potentially
prothrombotic conditions such as sepsis, chorioamnion-
itis, and pre-eclampsia. Infection is known to predispose
to a hypercoagulable state, in part through endothelial
injury and cytokine generation, with downregulation
of thrombomodulin. In summary, the association (espe-
cially a causative association) between thrombophilia and
arterial stroke in the fetus remains inconsistent at best
 Section I: The Fetus
(a)
Fig. 1.2 Ischemic brain injury in recipient in twin-to-twin transfusion syndrome (coronal and axial T2-weighted MRI scan).
(Infante-Rivard et al 2002, Rey et al 2003, Rodger et al
2008, Lynch 2009).
Patterns of intracranial hemorrhage in the fetus
resemble those described in the preterm newborn infant.
These hemorrhages are likely due to an underlying ana-
tomic and physiologic immaturity, with an intrinsic fragil-
ity of the vasculature (reviewed in more detail elsewhere)
(Volpe 2008d). Hemorrhages in the fetus include the typi-
cal germinal matrix-intraventricular hemorrhage lesion,
and its complications including periventricular hemor-
rhagic infarction (Fig. 1.3) and posthemorrhagic hydro-
cephalus (Fig. 1.4). Similarly, cases of fetal cerebellar
hemorrhage have been reported that probably result from
rupture of the fragile vessels in the germinal matrices of
the immature cerebellum. Such hemorrhages may result
in disruptions of cerebellar development (Glenn et al
2007), and may mimic primary dysgenetic lesions such
as cerebellar clefts (Poretti et al 2009) and Dandy–Walker
Fig. 1.3 Periventricular hemorrhagic infarction (white star) in
30-week gestation fetus (coronal T2-weighted MRI).
8
(b)
spectrum lesions (Fig. 1.4 and 1.5) (Limperopoulos et al
2010b). Fetal intracranial hemorrhage has been asso-
ciated with fetal thrombocytopenia (e.g. in parvovirus
infection and alloimmune thrombocytopenia; Fig. 1.6),
maternal ingestion of agents such as aspirin and cocaine,
and thrombophilic conditions including factor V Leiden
and MTHFR mutations (Petaja et al 2001, Aronis et al
2002, Ramenghi et al 2005). Another proposed mecha-
nism underlying fetal intracranial hemorrhage is cerebral
venous thrombosis, which tends to recanalize rapidly,
leaving only features of hemorrhage and its complications.
Fig. 1.4 Fetal brain showing the distended fourth and lateral
ventricles with low-signal hemorrhage layering along the depen-
dent side of the dilated right lateral ventricle (arrow). Axial MRI
single-shot fast spin-echo T2-weighted image.
 Brain Injury in the Fetus

(b)

(c)
(a)

(d)

Fig. 1.5 Macroscopic autopsy findings of brain shown in Figure 1.4. (a) Ventral view of the brain showing multifocal intracortical
and focal subarachnoid hemorrhages. (b) Dorsal-inferior view of the brain with brainstem reveals membranous degeneration of the
inferior vermis and calcification in the wall of the lateral ventricle (arrow). (c) Dorsal view of the brainstem and cerebellum showing
the wispy tissue remnants of the cerebellar vermis and parts of the cerebellar hemispheres with punctate hemorrhages. (d) Coronal
section at the level of the caudothalamic groove showing marked ventricular distention, thinning of the cerebral mantle, and blood
products of varying ages in the floor of the lateral ventricles.

Maternal Toxins maternal drug abuse is complicated by confounding psy-

A multitude of substances may have toxic effects in the
developing nervous system, with both teratogenic and
destructive consequences. Only select examples are dis-
cussed here, and are confined to drugs of abuse (alcohol
and cocaine) and the effect of phenylalanine on the fetus in
asymptomatic maternal phenylketonuria. Understanding
the mechanisms of fetal brain injury associated with
Fig. 1.6 Massive cerebral hemorrhage in 32-week gestational
age fetus with alloimmune thrombocytopenia (axial T2-weighted
MRI).
chosocial factors and frequent multisubstance abuse.
Alcohol remains the most commonly implicated
teratogenic toxin. Fetal alcohol exposure may result in a
spectrum of outcomes, related in large part to the timing
and dose of alcohol exposure. The classic fetal alcohol
syndrome has well-known facial and somatic features
(Clarren and Smith 1978, Erb and Andresen 1978), and
almost universal microcephaly and mental impairment.
In addition to the full-blown fetal alcohol syndrome,
lower fetal exposure may be associated with less obvi-
ous somatic features but with some level of cognitive
impairment. The precise mechanisms by which alcohol
induces its effects on the developing brain are not well
established, and probably include a variety of mechanisms
related to the maturational stage at exposure, as well as
the confounding socioeconomic, nutritional, and multi-
toxin exposures. Putative mechanisms have included the
following: a decrease in uterine blood flow, possibly due
to vasoconstriction of the uterine arteries leading to fetal
hypoxemia; fetal hypoglycemia; fetal zinc deficiency; an
effect on NMDA receptors with pro-apoptotic effects; and
impaired vitamin A synthesis. Fetal alcohol exposure has
been implicated in disruption of all the major phases of
brain development. The most common lesions described

9
 Section I: The Fetus
have been neural tube defects, and disturbances in neural
proliferation and migration, with cell–cell adhesion dis-
turbances being implicated. Examples of neuropathologic
lesions include schizencephaly and polymicrogyria, as
well as disturbances in midline prosencephalic devel-
opment, including agenesis of the corpus callosum and
septum pellucidum.
Cocaine is another maternal intoxicant with poten-
tially devastating impacts on the fetal brain. Fetal cocaine
exposure has been associated with a spectrum of struc-
tural and functional neurologic sequelae. Cocaine readily
crosses the placenta and fetal blood–brain barrier. Once in
the fetal brain, cocaine blocks presynaptic catecholamine
reuptake, leading to sustained catecholaminergic activity
in the developing brain. Under normal conditions, the
immature nervous system is in autonomic imbalance with
delayed parasympathetic maturation favoring relatively
unopposed sympathetic tone, a scenario further amplified
by the action of cocaine.
Disturbances in cognition, affect, attention, visual–
motor and visual–spatial function, and behavioral regula-
tion have been described after fetal cocaine exposure,
even in the absence of obvious structural lesions. Several
mechanisms have been proposed for these effects, includ-
ing neurochemical, cerebrovascular, and non-specific
‘stress’ effects. During fetal life endogenous catechol-
amines form part of a regulating signal system that influ-
ences many of the major processes of brain development,
including neurogenesis, neuronal differentiation, neural
migration, and cortical organization (Gressens et al 1992,
Garg et al 1993, Lipton et al 1999, Lidow and Song 2001).
The unregulated neurotransmitter increase during cocaine
exposure may disrupt development at any or all of these
developmental pathways, as seen in the spectrum of
developmental brain lesions associated with fetal cocaine
exposure ranging from microcephaly, neuronal migration
defects, disorders of prosencephalic development (includ-
ing agenesis of corpus callosum and septo-optic dyspla-
sia), and neuronal migration abnormalities (Dominguez
et al 1991, Handler et al 1991, Gieron-Korthals et al
1994, Addis et al 2001, He and Lidow 2004, Salisbury
et al 2009). Cocaine and its metabolites may also cause
vasoconstriction in the maternal, placental, umbilical
(especially umbilical vein), and fetal (Zhang and Dyer
1991, Schreiber 1995, Patel et al 1999, Robinson et al
2000) circulation disrupting fetal oxygen-substrate sup-
ply. Although this mechanism of cocaine-mediated brain
injury has been challenged, hypoxic–ischemic/reperfusion
injury and hemorrhagic destructive brain lesions are well
described in these infants (Fig. 1.7). Another proposed
pathogenetic mechanism of developmental cocaine toxic-
ity relates to the features it shares with the general fetal
10
Fig. 1.7 Major cerebral ischemic and hemorrhagic injury in
a newborn infant after major fetal cocaine exposure. Of note,
negative diagnostic testing for congenital infections. Axial
T1-weighted MRI.
stress response (Lester and Padbury 2009). The early part
of the fetal response to cocaine, specifically the elevation
of catecholamines, is common to the fetal stress response.
Therefore, it is possible that the more downstream events
known to occur after fetal stress also play a role in sur-
vivors of fetal cocaine exposure. Specifically, the sec-
ondary effects of catecholamine on the hypothalamic–
pituitary–adrenal axis include an elevation in circulating
glucocorticoids, which in turn have potent effects on fetal
and placental genetic programming, with potential health
effects into adult life and across generations (Lester and
Padbury 2009).
Maternal phenylketonuria may have major toxic
effects on the fetal brain, with more than 75% of offspring
having an intellectual disability. During normal develop-
ment phenylalanine hydroxylase expression begins as
early as the sixth week of gestation; however, early func-
tion of the immature enzyme may be incapable of handling
a phenylalanine level that is elevated but asymptomatic in
the mother. Elevated phenylalanine levels in the fetal cir-
culation may have dose-dependent teratogenic effects on
the fetal brain (Levy and Ghavami 1996, Levy et al 1996),
including microcephaly, hypomyelinated white matter,
and callosal dysgenesis. Although a precise mechanism(s)
for these fetal effects remains unclear, possible pathways
include disruption of essential nutrient transport by the
placenta or phenylalanine’s direct oligodendrocyte toxic-
ity resulting in hypomyelination.
Fetal infections
The developmental neuropathology of fetal infection may
be categorized broadly into two, often overlapping, forms.
Specifically, fetal encephalitis may cause disruption of
 Brain Injury in the Fetus
normal pathways for brain development and/or destructive
brain lesions, often with prominent inflammatory features.
It is likely that multiple mechanisms may operate with
direct viral injury, vascular and inflammatory injury, and
possibly viral disruption of genetic mechanisms. It may
be difficult to establish congenital infection early in gesta-
tion as neither humoral inflammatory nor reactive astro-
glial responses in the fetus becomes evident until mid-
gestation. Although many clinical and imaging features
are common to the fetal encephalitides, certain features
are more suggestive of certain agents (Bale 2009). For
example, microcephaly is common to congenital encepha-
litis due to cytomegalovirus (CMV), rubella, herpes sim-
plex (HSV) type II, and varicella zoster (VZV) infections,
whereas congenital toxoplasmosis may be associated with
hydrocephalic macrocephaly. Congenital infections are
commonly associated with ocular findings, with chorio-
retinitis occurring in approximately 75% of infants with
congenital toxoplasmosis, but infrequently in congenital
CMV (20%) (Bale 2009). Sensorineural hearing loss is
common in congenital rubella and CMV (Fowler et al
1997, Grosse et al 2008), and less common in congenital
toxoplasmosis. Neuroimaging findings common to fetal
encephalitis include periventricular hyperechoic and/or
cystic lesions with atrophic ventriculomegaly and intracra-
nial calcifications. The distribution of calcifications tends
to be periventricular in congenital CMV, more scattered
in fetal toxoplasmosis, and basal ganglia–thalamic in fetal
HSV encephalitis (Hutto et al 1987). While cerebral corti-
cal malformations such as schizencephaly, pachygyria/
lissencephaly, and hydranencephaly may develop after
fetal HSV, CMV, and VZV encephalitis (Wright et al 1997,
Bonthius et al 2007, Bale 2009), polymicrogyria is highly
suggestive of CMV, particularly when associated with
cerebellar hypoplasia (Bonthius et al 2007, Volpe 2008e).
In this chapter we focus on the neurologic sequelae of
fetal CMV and parvovirus infections because they illus-
trate certain themes and are more common than other
forms of fetal viral infections with neurologic complica-
tions. Most cases of HSV encephalitis are acquired in the
perinatal and neonatal periods, with only about 10% of
HSV-II infections acquired in utero (Kimberlin 2004a,b).
Congenital rubella syndrome, a potentially catastrophic
transplacental infection associated with necrotizing
encephalopathy with marked inflammation, has become
rare in developed countries since the advent of widespread
vaccination.
Congenital CMV infection remains the most common
viral infection affecting the fetus, with approximately 1%
of neonates in the USA infected at birth; of these 10% (or
4000 individuals) per year will be symptomatic at birth,
with a significant mortality (Istas et al 1995). Humans
11
are the only reservoir and transfer is through salivary
or genital secretions and breast milk. Fortunately, fetal
transfer rates are low, occurring in only 2% of individuals.
Most fetal CMV infections occur after primary maternal
infections, but may also occur after maternal reinfection
or reactivation. The best predictor of adverse outcome in
fetal CMV is neuroimaging evidence for brain involve-
ment. The CMV is tropic to rapidly proliferating cells
and to endothelial cells. The tropism toward rapidly pro-
liferating cells likely underlies the microcephaly, often
progressive. Cerebellar hypoplasia is present in around
50% of individuals with symptomatic CMV, which is
likely to be related to the protracted development of the
cerebellum with its primary and secondary germinal
matrices for cell proliferation. Inflammatory changes are
prominent and diffuse in CMV encephalitis, and involve
the periventricular white matter, germinal matrices, and
cerebral cortex. The white matter pathology may mimic
periventricular leukomalacia (PVL) with its predilection
for hypomyelination and periventricular cysts, particu-
larly in a parietal distribution (Fig. 1.8a). However, unlike
PVL, CMV encephalopathy commonly also involves the
anterior temporal white matter (Fig. 1.8b). Another fea-
ture that distinguishes CMV from other forms of fetal
encephalitis is the often striking involvement of gray mat-
ter, ranging from neuronal migration defects, particularly
when the infection occurs earlier in gestation. In fact, in
individuals with white matter abnormalities but normal
cortical gray matter, the likely onset of CMV encephalitis
is in the third trimester (Barkovich and Girard 2003). The
cortical gray matter lesions include heterotopias, schizen-
cephalies, pachygyria–lissencephaly (suggesting infec-
tion at between 16 and 18 weeks), and polymicrogyria
(suggesting infection at between 18 and 24 weeks). The
predilection of CMV for endothelial cells underlies the
mineralizing vasculopathy (‘candelabra sign’, Fig. 1.9)
of the basal ganglia–thalamus vessels evident in one-third
of individuals with congenital CMV encephalitis. The
prominent inflammatory changes may underlie the atro-
phic ventriculomegaly, porencephaly, hypomyelination,
and cystic changes of the periventricular and subcortical
white matter in congenital CMV encephalitis.
Parvovirus B19 infection has a spectrum of poten-
tially catastrophic effects during pregnancy, including
neurodevelopmental disability in one-third of survivors
(Nagel et al 2007). ‘Fifth disease’ is the most com-
mon form of parvovirus infection in childhood, and is
transmitted by respiratory droplets and blood products.
Transmission of the virus from mother to fetus is verti-
cal. Parvovirus is tropic for erythroid lineage cells and
may cause severe fetal anemia and thrombocytopenia,
resulting in anemic hypoxia and hemorrhage, as well as
 Section I: The Fetus
(a)
Fig. 1.8 (a) T1-weighted MRI axial views in a 3-month-old infant with cytomegalovirus encephalitis showing diffuse white matter
injury resembling preterm birth-related white matter disorder. (b) Anterior temporal white matter cysts.
non-immune hydrops and fetal death, especially when
transmitted between 17 and 24 weeks’ gestation. Fetal
brain involvement ranges from perivascular calcifica-
tions in the cerebral cortex, subcortical gray matter,
and germinal matrix layers to ventriculomegaly (Katz
et al 1996), polymicrogyria, cerebellar hemorrhage, and
hypoplasia of the cerebellar hemispheres and vermis
(Glenn et al 2007, Nagel et al 2007, Pistorius et al 2008).
In animal models, fetal parvovirus infection is associ-
ated with destruction of the external granular layer and
cerebellar hypoplasia.
Fetal errors of metabolism
Inborn errors of metabolism in the fetus are usually due
to enzyme or co-factor deficiencies.
These conditions may exert injurious effects on the
developing brain through accumulation of neurotoxic
by-products or/and a deficiency in substances essential
for normal brain development (Nissenkorn et al 2001).
Fig. 1.9 CMV encephalitis with arrow showing mineralizing
vasculopathy (‘candelabra sign’). Angled parasagittal ultrasound.
12
(b)
Broadly speaking, the neuropathology in these condi-
tions may reflect disruption of cellular energetics, dis-
turbed development of the cell membrane, and abnormal
signaling between cells. The fetal brain lesions may
manifest in several ways. First, the metabolic defect
may cause ‘acquired’ disruption of brain development
or destructive lesions, often in combination. In certain
conditions (e.g. sulfite oxidase deficiency, nonketotic
hyperglycinemia, pyruvate dehydrogenase deficiency)
this combination of developmental disruption and
encephaloclastic changes may be prominent (Dobyns
1989, Schiaffino et al 2004).
A broad range of nervous system malformations
(Nissenkorn et al 2001, Prasad et al 2007, Prasad et al
2009) has been described in infants with inborn met-
abolic errors. Certain developmental lesions are more
common in certain metabolic conditions. This reflects
a stage-dependent interaction between the ‘product’ of
the metabolic defect and the concurrent events in brain
development. Malformations of the developing nervous
system from the early stages of neurulation, through pros-
encephalic and callosal development, to the late stages
of cerebral cortical development have been described in
children with inborn errors of metabolism.
All phases of brain development during the fetal
period (rapid neuronal proliferation, differentiation and
neuronal migration, and the synaptically mediated devel-
opment of normal circuitry) are, to some extent, energy
dependent. It is therefore not surprising that inborn distur-
bances in energy metabolism, such as pyruvate dehydroge-
nase deficiency (Shevell et al 1994, Nissenkorn et al 2001)
and mitochondrial respiratory chain defects (Shevell et al
1994, Rotig and Munnich 2003, von Kleist-Retzow et al
 Brain Injury in the Fetus
2003, Sarnat and Marin-Garcia 2005), are associated with
a spectrum of developmental brain anomalies. In these
cases intrauterine somatic and cerebral growth restriction
is common. White matter lesions, often cystic, ranging
from germinal matrix and other periventricular cysts to
extensive cystic encephalopathy with calcifications have
been described, together with ventriculomegaly and cal-
losal dysgenesis (Samson et al 1994). Gray matter lesions
have included cerebral cortical malformations such as
polymicrogyria and heterotopias in these disorders of
energy metabolism, as have a variety of cerebellar lesions,
including cerebellar (von Kleist-Retzow et al 2003) and
pontocerebellar hypoplasia (Lincke et al 1996), and
‘Dandy–Walker malformation’. Callosal dysgenesis may
be seen in a wide variety of inborn metabolic disorders
including pyruvate dehydrogenase deficiency, nonketotic
hyperglycinemia, as well as mitochondrial and peroxi-
somal disorders. White matter lesions may occur as areas
of cystic destruction and/or hypomyelination due to cyto-
toxic death of oligodendrocyte precursors with subsequent
failure of myelination.
Certain inborn metabolic conditions exert their effect
on brain development by disrupting normal cell-to-cell
signaling. One example is the Smith–Lemli–Opitz syn-
drome, a multisystem dysmorphic condition resulting
from 7-dehydrocholesterol reductase deficiency (Porter
2003, 2008), and hence very low cholesterol levels. The
SHH gene plays a central role in development of the
brain, cranium, face, and other organs (Opitz et al 1987,
Penchaszadeh 1987, Porter 2008). The effect of SHH is
mediated by a cholesterol-dependent gene product that is
critical for cell–cell membrane signaling (Ingham 2001).
Insufficient cholesterol results in failed SHH signaling
and results in the lesions described in SLOS, such as
(a)
Fig. 1.10 Cytomegalovirus encephalitis in a 36-week gestation infant showing striking white matter abnormality and cortical malforma-
tion, with prominent polymicrogyria particularly in the fronto-parietal and Sylvian regions: axial (a) and coronal (b) T2-weighted MRI.
13
craniofacial (e.g. cyclopia and cebocephaly) and fore-
brain development (failure of cerebral prosencephalic
‘cleavage’ or holoprosencephaly) (Porter 2003).
Congenital disorders of glycosylation (CDG) result in
multisystem malformation syndromes including the brain
and face (Krasnewich and Gahl 1997, Baric et al 1998).
The most common subtype, CDG type 1a, is associated
with cerebellar hypoplasia, hypotonia, fetal hypokinesia,
and intellectual disability (Krasnewich and Gahl 1997,
Baric et al 1998), as well as inverted nipples, aberrant
fat distribution, and disturbed coagulation and endocrine
function (Baric et al 1998). An associated cardiomyopathy
may result in non-immune hydrops fetalis (Hertz-Pannier
et al 2006, van de Kamp et al 2007, Malhotra et al 2009).
Peroxisomal disorders in the Zellweger syndrome spec-
trum are associated with the whole spectrum of neuronal
migration anomalies including lissencephaly, pachygy-
ria, polymicrogyria, and heterotopias, as well as callosal
hypoplasia and cerebellar anomalies (Fig. 1.10) (Volpe
and Adams 1972, Barkovich and Peck 1997). Nonketotic
hyperglycinemia is associated with callosal dysgenesis,
dysmyelination, and cortical anomalies (Press et al 1989,
Paupe et al 2002).
Conclusions
The fledgling clinical field of fetal neurology has
advanced dramatically in recent years. Factors play-
ing a major role in these developments have been the
advances in fetal imaging and the accelerated under-
standing of neurogenetic mechanisms underlying nor-
mal and abnormal fetal brain development. Advances
in these areas have provided invaluable insights into
the phenotype–genotype associations in fetal neurol-
ogy. Likewise, as highlighted in this chapter, a broad
(b)
 Section I: The Fetus
spectrum of environmental influences may affect brain
development. The ability of advanced in vivo fetal brain
imaging to detect and measure the effect of these ‘insults’
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2
Imaging the Fetal Brain
Catherine Limperopoulos
Introduction
Fetal magnetic resonance imaging (MRI) is becoming a
powerful tool with which to assess the developing fetal
brain. The advent of accelerated single-shot MRI tech-
niques is opening a new window into the timing, rate, and
limits of variability of normal brain development in vivo.
Better understanding of the dynamic and highly elabo-
rate developmental processes that underlie normal brain
maturation in vivo is providing important insights into the
onset and progression of acquired fetal brain injury. This
chapter will briefly review techniques of fetal ultrasound
and MRI. Advantages and disadvantages of each modality
are summarized, followed by an overview of the MRI fea-
tures associated with normal fetal brain development, and
a review of the role of MRI for acquired brain injury in the
fetus. Finally, this chapter will explore the advancing role
of fetal MRI and its application to the compromised fetus.
Imaging the fetal brain: ultrasound versus magnetic
resonance imaging
Fetal ultrasonography has been the primary imaging
modality for prenatal diagnosis of fetal brain anoma-
lies. The principal advantages of ultrasound include its
low cost, portability (can be performed at the bedside),
and widespread use. Fetal ultrasound imaging quality
has improved dramatically as a result of high frequency
three-dimensional transducers, as well as transvaginal
sonography. Despite these advances, sonographic evalua-
tion of the fetal brain continues to be limited by decreased
resolution of the side of the brain near the transducer,
acoustic shadowing resulting from ossification which
obscures visualization of posterior fossa structures, and
limited visualization of the developing cortex and subtle
parenchymal abnormalities (Levine 2001, 2002, Pugash
et al 2008). Moreover, multiplanar views may be difficult
to obtain with ultrasound because of fetal position or
advanced gestational age.
18
Fetal MRI was first introduced in the early 1980s
using T1-weighted inversion recovery and proton den-
sity sequences at low field magnet strength. These initial
imaging acquisitions were long (i.e. several minutes in
duration) and were highly susceptible to motion, necessi-
tating fetal and/or maternal sedation. The advent of ultra-
fast T2-weighted sequences (described below), together
with improved MRI hardware, enabled the acquisition
of MRI sequences in less than 20 seconds, eliminating
the need for sedation (Huisman et al 2002). Fetal MRI
offers three-dimensional resolution, multiplanar imaging
capabilities, large field of view, and robust image quality.
Additionally, fetal MRI overcomes challenges of fetal
ultrasonography resulting from reduced amniotic fluid
volume, the position of the fetus, and acoustic shadowing
(Glenn and Barkovich 2006). Compared with ultrasound,
ultrafast MRI has been repeatedly shown to have greater
sensitivity in up to 50% of cases for detecting fetal brain
lesions/injury including nodular heterotopias, periven-
tricular leukomalacia, multicystic encephalomalacia, and
germinal matrix and intraventricular hemorrhages (Levine
et al 1997, 1999, Simon et al 2000, Wagenvoort et al 2000,
de Laveaucoupet et al 2001, Whitby et al 2004, Glenn and
Barkovich 2006, Girard et al 2009, Peruzzi et al 2010).
The remainder of this chapter will focus on the role of
MRI for studying acquired injury in the fetus.
Imaging the fetal brain: the role of conventional
magnetic resonance imaging
Fetal MRI is performed on a 1.5-T scanner, generally
from about 18 to 20 weeks’ gestation onward. Prior to
20 weeks, MRI resolution is poor overall because of the
normally enlarged ventricles, thin cerebral mantle, and
thick germinal matrix (Girard et al 2009). Ultrafast MRI
techniques, known as single-shot fast spin-echo (SSFSE)
or half-Fourier acquired single-shot turbo spin-echo
(HASTE), have enhanced the study of the developing
 Imaging the Fetal Brain
brain in vivo. Using these ultrafast techniques, a single
T2-weighted image can be acquired in less than 1 second,
and thus dramatically reduces the exposure to fetal motion
(Glenn 2009). Routine clinical studies include the acquisi-
tion of multiple stacks of slices acquired in different plans
to enable complementary views of the developing brain
anatomy.
Typically, SSFSE T2-weighted images form the basis
of an MRI study and are used to assess the morphology
and signal intensity of the fetal brain. Additionally, gra-
dient-echo echo-planar T1-weighted images or fast mul-
tiplanar gradient recalled-echo T1 techniques are used to
detect hyperintense lesions such as hemorrhage, lipoma,
subependymal nodules, or calcifications. T1-weighted
imaging also offers information about the myelination
process (Girard et al 2006a, Glenn 2009). T2* sequences
are used to detect blood breakdown products and have
been recently explored in the fetus. Diffusion-weighted
imaging is performed using single-shot, echo-planar dif-
fusion imaging, which can be acquired in less than 15 to
20 seconds and assists in identifying destructive brain
lesions. Advanced MRI techniques, such as three-dimen-
sional volumetric MRI, diffusion tensor imaging (DTI),
and magnetic resonance spectroscopy, have also recently
been successfully applied to the living fetus, though their
development is still in the early stages. It is anticipated
that these advanced imaging tools will shortly provide
valuable diagnostic and prognostic information. These
techniques are described later.
Normal fetal brain development on conventional
magnetic resonance imaging
Although a comprehensive review of normal fetal brain
development is beyond the scope of this chapter, a brief
overview is warranted. Prior knowledge of normal brain
anatomy and the corresponding changes of the magnetic
resonance signal in relation to increasing gestational age
are essential. The appearance of the supratentorial (Garel
2004a, Parazzini et al 2008, Tilea et al 2009) and infraten-
torial (Garel 2004a, Schneider et al 2007, 2009, Tilea et al
2009) structures of the fetal brain using conventional MRI
have been well established with corresponding normative
reference values.
The cortical mantle has a multilayered appearance on
MRI until about 28 weeks’ gestation (Girard and Raybaud
1992, Girard et al 1995, Chong et al 1996, Brisse et al
1997, Kostovic et al 2002, Garel et al 2003, Garel 2004a,
Prayer et al 2006a). This multiple layer appearance rep-
resents the different layers of the developing fetal brain
(Kostovic et al 2002, Rados et al 2006), which include
the ventricular zone, periventricular zone, subventricu-
lar and intermediate zone, subplate zone, and cortical
19
plate (Fig. 2.1). Briefly, the ventricular zone (or germinal
matrix) corresponds to the innermost layer of the fetal
cerebral hemisphere. Early in gestation the ventricular
zone is very broad and has the appearance of a smooth
band of high T1–low T2 signal lining the lateral ven-
tricles. The ventricular zone regresses with increasing
gestational age, and by 27 to 29 weeks gestational age, a
single layer of ependymal cells will replace the ventricular
zone and line the wall of the ventricles (Kinoshita et al
2001, Bystron et al 2008). The periventricular zone is
located just superficial to the ventricular zone and is a
thin area of increased T2 signal and decreased T1 signal.
The subventricular zone contains the germinal matrix and
the intermediate zone, which encompasses the fetal white
matter located superficially to the periventricular zone.
The subventricular zone appears as a relatively homo-
geneous strip of slightly low T2 and high T1 signal. The
subplate comprises neurons, profuse hydrophilic extra-
cellular matrix, and evanescent synapses (Bystron et al
2008). Although the role of the subplate remains poorly
understood, it is presumed to be a waiting compartment
for the development of thalamocortical connections and
other cortical afferent pathways (Sur and Rubenstein
2005, Huang et al 2009, Kostovic and Judas 2010, Tau
and Peterson 2010). The subplate is thickest toward the
end of the second trimester, and progressively disappears
at around 32 to 36 weeks gestational age as axons leave
the subplate and migrate to their destined targets in the
cortex. Its appearance on MRI is characterized by a low
T1–high T2 signal. Finally, the cortical plate has a similar
intensity to the germinal matrix high T1–low T2.
Fig. 2.1 Coronal T2-weighted single-shot fast spin-echo MRI
of a 25-week gestational age fetus, illustrating the multilayered
appearance of the developing brain. The germinal matrix is the
deepest layer and is of low signal intensity (white arrowhead).
Adjacent to the germinal matrix is the periventricular zone
(white dotted arrow) with corresponding high signal intensity.
Adjacent to the periventricular zone is the subventricular zone
(double white arrows), which is of low signal intensity, and just
superficial to this layer is the high signal intensity subplate zone
(single white solid arrow) and the cortical plate (black arrow),
represented with low signal intensity.
 Section I: The Fetus

Normal brain development is characterized by expan-
sion and folding of the cerebral cortex. The emergence of
sulci follows a consistent spatial and temporal program.
Primary sulci are the first to appear followed by the forma-
tion of secondary and tertiary sulci. Overall, a sulcus first
appears as an initial smooth, shallow, and wide indenta-
tion on the brain surface that progressively deepens and
narrows, ultimately forming the secondary and tertiary
sulci (Glenn 2009). Importantly, sulcation is considered
to be one of the most accurate ways of dating a fetus by
pathologists. The appearance of sulci on two-dimensional
fetal MRI has been well described, and the sulcation land-
marks appear on magnetic resonance images in the order
predicted on neuropathology. However, the timing has
been reported to lag behind that observed on fetal autopsy
specimens by an average of 2 weeks (range 0–8 weeks)
compared with MRI visualization (Chi et al 1977, Levine
and Barnes 1999, Garel 2004b). A comparison of the
appearance of major sulci by gestational age on autopsy
versus MRI is summarized in Table 2.1. More recently,
advanced MRI techniques are beginning to quantify cere-
bral cortical development in vivo and are offering exciting
insights into normal and aberrant fetal cortical develop-
ment (described later).
The cerebellar hemispheres appear multilayered as
early as 21 weeks (evident by a central area of low T2 and
high T1 signal), and the cerebellar vermis is completely
formed by 20 weeks of gestation (Adamsbaum et al 2005,
Limperopoulos et al 2006). The primary fissure is seen on
midline sagittal images by 25 to 26 weeks gestational age,
but can be seen as early as 21 weeks. The dorsal pons and
dorsal medulla have a high T1 and low T2 signal, which
is evident as early as 23 weeks gestational age. A com-
prehensive understanding of normal brain development on
MRI is important in order to appreciate the MRI features
of acquired brain injury, which will vary accordingly.
Fetal brain injury in the fetus on conventional
magnetic resonance imaging
Of all fetal organ systems, study of the fetal brain has
undoubtedly benefited most from the superior image qual-
ity and anatomical detail offered by MRI. The high resolu-
tion afforded by fetal MRI can identify critical yet subtle
changes in central nervous system landmarks, especially
early in gestation. Acquired brain injury represents the
third most frequent indication (up to 20%) for fetal MRI
(Girard et al 2001, 2009, Girard and Huisman 2005) com-
prising a major thrust for antenatal diagnosis.
Cerebral insults can result from a number of fetal–
maternal conditions. Risk factors and underlying patho-
genetic mechanisms of fetal brain injury are described
in detail in Chapter 1. Cerebral insults that ensue early
in gestation are frequently associated with embryo/fetal
demise or developmental malformations (Gilles and
Gomez 2005). The selective vulnerability of the devel-
oping gray versus white matter also depends on the

Table 2.1
Comparison of major sulci appearance by gestational age (weeks) by fetal MRI versus autopsy

Detected by autopsy Detected by fetal MRI

Sulci (25–50% of cases)a (75% of cases)b
Sylvian 14 16–17
Callosal 14 22–23
Parieto-occipital 16 22–23
Calcarine 16 24–25
Cingulate 18 26–27
Central 20 26–27
Superior temporal 23 27
Precentral 24 26–27
Postcentral 25 28–29
Superior frontal 25 29
Inferior frontal 28 29
Inferior temporal 28 32
Insular 34 32–33

Adapted from Glenn (2009).

aChi et al (1977).

bLevine and Barnes (1999), Garel (2004b).

20
 Imaging the Fetal Brain
(a)
Fig. 2.2 Intraventricular hemorrhage with mild unilateral ventriculomegaly, which is dark on T2-weighted (a) and bright on
T1-weighted (b) fetal (gestational age of 31 weeks) MRI, and corresponding bright signal intensity on the diffusion-weighted image (c).
gestational age at which the injury occurred. White mat-
ter injury is more prevalent than gray matter injury in the
fetus. When present, gray matter injury most commonly
occurs in the upper brainstem and thalami, followed by
lesions in the convexity border zone resulting in sclerotic
microgyria (Girard et al 2009).
The MRI presentation of acquired brain injury in the
fetus varies depending on whether the injury is acute or
chronic. Acute brain injury may manifest as hemorrhage
(intra- or extra-axial), edema, thrombosis, focal ischemia,
or loss of lamination of the brain parenchyma until the end
of the second trimester (Girard et al 2006a, 2009, Prayer
et al 2006b). The chronic response to acquired fetal brain
injury is more commonly detected by prenatal MRI than
is the acute response, and may be characterized by a host
of abnormalities including ventricular enlargement and/
or ventricular distortions (e.g. irregular margin, abnormal
shape); abnormal gyration; parenchymal abnormalities
(e.g. small hemisphere); calcifications; laminar necrosis;
cerebral and cerebellar disruptions; intracranial space-
occupying lesions; or cerebral atrophy (Girard et al 2006a,
2009, Prayer et al 2006b). A combination of an acute and
chronic response is also commonly seen in response to
fetal brain injury (Girard et al 2009). Moreover, lesions
that ensued at different gestational ages may coexist and
likely reflect ongoing responses after injury (Girard et al
2006a). The MRI features of these acquired lesions are
presented below.
Hemorrhage
Hemorrhagic injury in the fetus may occur in the setting
of fetal hypoxia or infection (e.g. chorioamnionitis) sec-
ondary to anticoagulant activity and cytokine-mediated
endothelial cell damage (Dammann and Leviton 1998).
Hemorrhagic injury can be confined to the germinal matrix
or may be intraventricular (Fig. 2.2). Intraventricular
hemorrhage can be complicated by ventricular dilation
or hydrocephalus. Associated venous infarction may be
21
(b) (c)
seen as a hemorrhagic parenchymal lesion (Fig. 2.3).
In addition to intraventricular hemorrhage, intracranial
hemorrhages in the form of sudural, subarachnoid, and
supratentorial parenchymal hemorrhages can be pres-
ent. Hemorrhagic injury usually appears as an area of
dark signal on T2 and bright signal on T1. The signal
intensity can vary depending on the stage of hemorrhage.
Moreover, small hemorrhages in the subependyma may
be difficult to differentiate because of the similar signal
intensity of blood and the normal intensity of the germinal
matrix (Girard et al 2006a, 2009, Prayer et al 2006b). T2*
weighted gradient echo and echoplanar sequences can be
used to confirm the presence of blood, as hemorrhage
appears more hypointense than the germinal matrix using
these sequences (Glenn and Barkovich 2006, Girard et al
2009).
Posterior fossa hemorrhages also appear bright on
T1 and dark on T2, and MRI is helpful in localizing the
topography of injury. Fetal MRI can be used to identify
the location of the hemorrhage (i.e. intra- vs. extra-axial
hemorrhage) and can assess the integrity of the cerebellar
Fig. 2.3 Multiple small periventricular cysts adjacent to the
right frontal horn with abnormal white matter signal consis-
tent with right periventricular hemorrhagic infarction on axial
T2-weighted single-shot fast spin-echo MRI (imaged fetus of
gestational age 33 weeks).
 Section I: The Fetus
hemispheres, vermis, and brainstem (Gorincour et al
2006).
Cerebral Edema
Acute edema is difficult to visualize on fetal MRI. White
matter injury in the fetus is often characterized by acute
edema, which may be transient or lead to focal necrosis,
in which the topographic predilection is in the parieto-
occipital and frontal regions (Girard et al 2009). However,
this is not frequently visible on MRI. The absence of the
intermediate layer of the white matter can be the sole find-
ing on MRI in young fetuses. Briefly, risk factors for focal
necrosis include placental vascular anastomoses, funisi-
tis, and purulent amniotic fluid (Grafe and Kinney 2002,
Gilles and Gomez 2005). Infection and inflammation are
established mediators of white matter damage (Dammann
and Leviton 1998, Chew et al 2006, Sen and Levison
2006). The application of diffusion-weighted acquisitions
can facilitate the detection of edema, which appears as a
bright signal on diffusion-weighted MRI and a dark signal
on the corresponding apparent diffusion coefficient (ADC)
images. Loss of lamination of the brain parenchymal may
also be evident on MRI (Girard et al 2009).
Cerebral Infarction and Thrombosis
Arterial infarction may be associated with a number of
conditions including vascular occlusive disease, infection,
trauma or arteriovenous malformations, hypercoagulabil-
ity and twin-to-twin transfusion syndrome (Girard et al
2009). The MRI features of cerebral infarction often illus-
trate a loss of gray and white matter parenchyma within the
vascular territory distribution (Fig. 2.4), as well as ventric-
ular dilation. Venous thrombosis is less common and may
be associated with prothrombotic events and be present as
a parenchymal hemorrhage with a corresponding low T2
and high T1 signal. Acquiring T1 images with fat suppres-
sion may assist in ruling out a lipoma, which may show a
Fig. 2.4 Axial T2-weighted single-shot fast spin-echo MRI at
26 gestational weeks demonstrates bilateral middle cerebral
infarction.
22
similar signal intensity (Gicquel et al 2000). Noteworthy,
fetal strokes that occur early in gestation (before 20 weeks)
may present with limited to no corresponding tissue injury
on MRI and may be subsequently misdiagnosed as a pri-
mary dysgenetic lesion on follow-up imaging.
Ventriculomegaly
Ventriculomegaly can be divided into two categories:
atrophic ventriculomegaly and hydrocephalic ventricu-
lomegaly (described below). Ventricular dilation is fre-
quently the result of prior hemorrhage. Enlarged ventricle
size (usually unilateral) is a common (chronic) response
after fetal brain injury (Fig. 2.5), whereas bilateral ven-
triculomegaly is typically present in the setting of cere-
bral malformations. Brain atrophy may be documented
in cases of enlargement of the ventricles and/or the outer
cerebrospinal fluid spaces (de Laveaucoupet et al 2005),
which may result from a number of different primary
pathologies (de Laveaucoupet et al 2001, 2005, Barkovich
and Girard 2003, Brunelle 2003, Prayer et al 2006b).
Abnormal ventricular shape/margin or irregular germinal
matrix (Barkovich and Girard 2003, Girard et al 2006a)
is often present in the chronic phase of injury. A common
MRI finding is a thickened irregular ventricle present after
30 weeks gestational age (Girard et al 2009). Interestingly,
this MRI finding has been shown to correspond with epen-
dymal abrasion post mortem, resulting from brain atrophy,
subventricular gliosis, and fetal ependymal inflammation
(Sarnat 1995).
Hydrocephalic ventriculomegaly may result from an
acquired injury such as posthemorrhagic hydrocephalus,
after infection, or compression of the aqueduct by a space-
occupying lesion. Hydrocephalus results in enlarged ven-
tricles secondary to disruption/distension of cerebrospinal
fluid circulation (Fig. 2.6).
Fig. 2.5 Axial T2-weighted single-shot fast spin-echo
MRI at 26 gestational weeks shows mild isolated unilateral
ventriculomegaly.
 Imaging the Fetal Brain
Fig. 2.6 Coronal T2-weighted single-shot fast spin-echo MRI
showing severe hydrocephalus secondary to aqueductal stenosis
in a 29-week gestational age fetus.
White Matter Injury/Gliosis
Although fetal white matter injury is not commonly
identified with conventional MRI acquisitions, DTI and
proton magnetic resonance imaging (described below)
have the potential to identify gliosis in vivo. For example,
increased creatine on spectroscopic imaging may be a
marker for astrocytes (Girard et al 2006b). The applica-
tion of diffusion-weighted acquisitions may also facili-
tate the identification of edema, which appears as a high
signal on diffusion-weighted MRI and low signal on the
corresponding ADC image. Acutely, white matter injury
can be associated with edema with a corresponding low
T1-weighted signal, particularly in the frontal and parietal
regions. The absence of the intermediate layer of the white
matter can be the only MRI finding in young fetuses.
Nodules may also present as a high signal on T1 and a
low signal on T2-weighted MRI, and have been reported
in the parieto-occipital and frontal regions in individuals
with twin-to-twin transfusion syndrome (Larroche et al
1994). Edema in the white matter may be transient or lead
to necrosis. In the chronic stage, loss of brain volume is
usually evident, which manifests as enlarged lateral ven-
tricles and subarachnoid spaces, with or without abnormal
signal on MRI (Girard et al 2009).
Fetal Abscess
Fetal abscess is relatively uncommon but may be observed
in the setting of toxoplasmosis and cytomegalovirus (Kim
et al 2007). The corresponding MRI feature is a high
signal on T1 and low signal on T2 MRI. Noteworthy, a
diagnosis of abscess is commonly made retrospectively
with the presentation of calcifications on MRI, which may
be isolated or multiple.
Cystic Cavitation
Cystic cavitation in the fetal cerebral or cerebellar paren-
chymal may be the result of focal ischemic or hemor-
rhagic injury, or leukomalacia, and is often observed in the
23
chronic phase of injury. Destructive processes may lead
to porencephalic cysts, which may or may not lead into
the ventricular system and subarachnoid spaces (Girard
et al 2009). At its extreme, hydranencephaly (of ischemic
origin) may be present with limited to no cerebral tissue
remaining, and the cranial cavity is filled almost entirely
with cerebrospinal fluid.
Calcifications
Calcifications are usually seen within the cortex, germinal
matrix, periventricular areas, and basal ganglia and white
matter. They appear as a bright signal on T1 and a low
signal on T2, and are most frequently seen in individuals
with in utero infections.
Disruptions
Disruptions are often difficult to distinguish from primary
dysgenetic lesions. Extrinsic factors that can disrupt nor-
mal brain development may include infection, hemor-
rhage, and hypoxic–ischemic events (Reardon and Donnai
2007, Poretti et al 2008a, Limperopoulos et al 2010a) that
result in cortical malformations. For example, polymi-
crogyria can be identified on fetal MRI characterized by
abnormal infoldings (excessive number of small gyri) of
the cortex (Fig. 2.7) (Glenn et al 2005). Environmental
insults associated with the subsequent development of
polymicrogyria include intrauterine infection (e.g. cyto-
megalovirus), toxoplasmosis, syphilis, and intrauterine
ischemia (e.g. twin–twin transfusion). Schizencephaly can
also be seen on fetal MRI, which may be characterized by
a unilateral or bilateral cleft of the cerebral hemispheres
and communication between the ventricle and pericere-
bral subarachnoid spaces (Fig. 2.8). The walls of the cleft
may be separated (open-lip schizencephaly) or closely
adjacent (closed-lip schizencephaly) and the cortex sur-
rounding this cleft is polymicrogyric (Guerrini 2005).
The etiology of schizencephaly is likely a confluence of
genetic and acquired causes, including a local failure of
Fig. 2.7 Coronal T2-weighted single-shot fast spin-echo MRI
showing mild bilateral ventriculomegaly and diffuse polymi-
crogyria characterized by irregular shallow sulci in a 28-week
gestational age fetus.
 Section I: The Fetus
Fig. 2.8 Axial T2-weighted single-shot fast spin-echo MRI illus-
trating right open-lip schizencephaly in a 35-week gestational
age fetus.
induction of neuronal migration or focal ischemic necrosis
with destruction of the radial glial fibers during early ges-
tation (Barkovich and Kjos 1992, Guerrini 2005).
Prenatal cerebellar disruptions include unilateral or
bilateral tissue loss or volume reduction, global cerebel-
lar hypoplasia, unilateral cerebellar hypoplasia (Fig. 2.9),
cerebellar agenesis, and unilateral cerebellar clefts (Poretti
et al 2008b, 2009, Limperopoulos et al 2010b). The etiol-
ogy of these findings includes genetic as well as acquired
disruptive causes. Cerebellar hemorrhage may mimic a
primary cerebellar dysgenetic lesion (i.e. Dandy–Walker
malformation) (Limperopoulos et al 2010c). Therefore,
it is important to consider the possibility of a cerebel-
lar disruption resulting from hemorrhagic or posthemor-
rhagic injury and associated hypoxic–ischemic changes in
individuals who seemingly meet criteria for a diagnosis
of Dandy–Walker.
In summary, although larger fetal brain lesions can
be identified by conventional T1/T2-weighted fetal MRI,
more subtle disturbances, e.g. impaired volumetric growth
of brain tissue types, microarchitectural disorganization,
Fig. 2.9 Coronal T2-weighted single-shot fast spin-echo MRI
showing right unilateral cerebellar hypoplasia after in utero cer-
ebellar hemorrhage in a 32-week gestational age fetus.
24
and brain metabolite alternations, will likely require
advanced MRI techniques, which are summarized below.
Advanced magnetic resonance imaging techniques in
the ex utero infant
Innovative applications of quantitative MRI techniques
have revolutionized the in vivo study of brain develop-
ment in the ex utero infant. Specifically, volumetric three-
dimensional (3D) MRI has advanced our understanding
of normal and abnormal cerebral cortical development
and the developmental changes in specific brain tissue
subtype (Huppi et al 1998, Dubois et al 2008). Similarly,
DTI studies have shed new light on the impact of early
injury on subsequent brain microstructural organization.
These seminal studies have provided new insights into the
potential mechanisms that underlie disturbed brain injury
in the high-risk infant, and the immature nervous system’s
adaptive response after early fetal brain injury. It is impor-
tant to emphasize that although the presence of impaired
structural brain development, connectivity, and metabo-
lism is often not detectable by conventional MRI, it has
been shown that these findings are associated with adverse
neurodevelopmental outcomes (Rademaker et al 2006,
Shah et al 2006, Bassi et al 2008, Counsell et al 2008,
Kesler et al 2008, Thompson et al 2008, Soria-Pastor et al
2009). Notably, impaired brain growth and development
may also be associated with remote, regional secondary
growth disturbances not evident on conventional MRI
(Limperopoulos et al 2005, Limperopoulos et al 2010b).
These quantitative brain MRI changes in ex utero preterm
infants have led to a vigorous pursuit of the same capabili-
ties in the fetus, which are summarized below.
Acquired fetal brain injury and advanced in vivo
fetal magnetic resonance imaging techniques
Three-dimensional Volumetric Magnetic
Resonance Imaging
The recent successful application of quantitative MRI
techniques to the living fetus is offering exciting oppor-
tunities to advance our understanding of the timing and
progression of insults that disrupt normal brain develop-
ment at a level that is below current ‘lesion detection’
on conventional MRI. Motion correction algorithms and
3D reconstruction techniques developed specifically for
fetal MRI (Rousseau et al 2006, Jiang et al 2007, Kim
et al 2010) are facilitating the measurement of global and
regional brain growth and providing previously unavail-
able in vivo fetal brain growth trajectories (Habas et al
2010, Clouchoux et al 2011, Rajagopalan et al 2011).
Similarly, studies are providing the first description of
gyral development in the human fetal brain during the
 Imaging the Fetal Brain
critical period of rapid cortical development in the second
and third trimester (Clouchoux et al 2011, Habas et al
2011).
These data provide a strong impetus for the use of
quantitative techniques to study the impact of acquired
injury on the developing fetal brain. Investigators are
beginning to apply these advanced techniques to the
compromised fetus. A recent study (Limperopoulos et al
2010a) showed in vivo evidence of progressive impair-
ment of brain growth measured by 3D volumetric MRI
in fetuses with congenital heart disease compared with
healthy control fetuses (Fig. 2.10). Surprisingly, the
majority of fetuses with congenital heart disease in this
study have a structurally normal brain on conventional
MRI, demonstrating that advanced MRI techniques can
detect injury that is not apparent using conventional imag-
ing. Similarly, another study reported decreased brain
volume in growth-restricted fetuses (Duncan et al 2005),
demonstrating that the brain sparing that is evident using
sonographic measurement of fetal head circumference
masks a reduction in brain volume identified by advanced
MRI volumetry. These preliminary data suggest that 3D
volumetric MRI can reliably identify impaired fetal brain
growth even in the absence of structural injury evident on
conventional MRI, or available biometric measurements.
Proton Magnetic Resonance Spectroscopy
Proton magnetic resonance spectroscopy (1H-MRS) is
an exciting noninvasive technique for measuring meta-
bolic substrates and monitoring cerebral metabolism in
the fetal brain. The most commonly studied metabolites
in the living fetus include (1) N-acetyl aspartate (NAA),
350
300
250
TBV (mL)
200
150
100
50
26 28 30 32 34 36 38
Gestational age (weeks)
Fig. 2.10 Progressive and significant decrease in third-trimester
total brain volume (TBV) in fetuses with congenital heart dis-
ease (diamonds) compared with controls (black circles) over
the same gestational age period (adapted from Limperopoulos
et al 2010c).
25
a neuroaxonal marker reflecting the development of den-
drites and synapses and oligodendrocyte proliferation and
differentiation; (2) creatine (Cr), responsible for cellular
energy metabolism; (3) choline (Cho) involved in mem-
brane synthesis, degradation, and myelination; and (4)
lactate, a marker of anaerobic metabolism (Girard et al
2006b, Limperopoulos and Clouchoux 2009). To date,
fetal 1H-MRS studies have described the developmental
appearance and succession of these metabolites over the
second and third trimester of pregnancy, offering critical
metabolic normative data from which to examine altered
metabolic profiles in the compromised fetus.
Available fetal 1H-MRS data in high-risk pregnan-
cies are primarily derived from case reports and series.
Lactate (Robinson et al 2004, Azpurua et al 2008) and
decreased NAA/choline (Azpurua et al 2008) have been
demonstrated in growth-restricted fetuses. Increased
inositol:choline ratios have also been reported in small
for gestational age fetuses corroborating ex utero neonatal
studies of hypoxic–ischemic encephalopathy, in which
acute increases in inosotol/creatine are associated with
an abnormal developmental outcome at 1 year of age
(Robertson et al 2001).
In one of the largest fetal 1H-MRS studies to date
(Limperopoulos et al 2010a). NAA:choline ratios in
fetuses with congenital heart disease were significantly
and progressively lower with increasing gestational age
in the third trimester than in healthy controls (Fig. 2.11).
Lactate was present in 20% of fetuses with congenital
heart disease, while no control fetuses had detectable cere-
bral lactate. Interestingly, fetuses with cerebral lactate had
the lowest gestational age and total brain volume-adjusted
1.0
Controls
0.8
0.6
NAA:Cho
0.4
CHD
0.2
0.0
26 28 30 32 34 36 38
Gestational age (weeks)
Fig. 2.11 Significantly slower rate of increase in NAA:choline
ratio in fetuses with congenital heart disease (diamonds) com-
pared with normal fetuses (black circles) with increasing gesta-
tional age (adapted from Limperopoulos et al 2010c).
 Section I: The Fetus
NAA:choline ratio. Follow-up studies are needed to
assess the long-term significance of these acute metabolic
derangements in the high-risk fetus.
Diffusion-weighted Imaging
Diffusion-weighted imaging has been used primarily to
identify focal areas of reduced or increased diffusion after
acute ischemic injury (described earlier in Righini et al
2003, Schneider et al 2007, 2009). In small for gestational
age versus appropriate for gestational age fetuses, signifi-
cant higher ADC values have been reported in the pyra-
midal tract, suggesting delayed brain development and
decreased microstructural organization (Sanz-Cortes et al
2010). Similarly, higher diffusivity has been described
in fetuses with congenital heart disease in the periatrial
white matter and thalamus regions (Berman et al 2011),
suggesting an important role for this imaging modality in
the compromised fetus.
Diffusion Tensor Imaging
DTI assesses the impact of early injury on subsequent
white matter microstructural architecture (Basser et al
1994) by evaluating axonal/fiber formation and connec-
tivity in the developing brain (Huppi et al 1998, Berman
et al 2005, Partridge et al 2005, Bui et al 2006, Anjari
et al 2007, Dubois et al 2008, Huang 2010). The visual-
ization and quantification of white matter fiber direction
is accomplished by measuring fractional anisotropy. The
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3
Mechanisms of Acute and Chronic Brain
Injury in the Preterm Infant
Stephen A. Back
Magnitude of the problem and spectrum of brain
injury in preterm survivors
Although major advances in the care of preterm infants
have resulted in striking improvements in the survival of
very low birthweight (VLBW) infants (<1.5kg), improved
survival has been accompanied by a significant increase
in the number of preterm survivors with long-term neu-
rologic deficits (Wilson-Costello et al 2005). In many
parts of the world, ~10% to 15% of preterm survivors
sustain permanent motor impairment (i.e. cerebral palsy)
that ranges from mild to profound motor deficits (Hack
et al 2005, Miller SP et al 2005, Liu et al 2008, Beaino
et al 2010, Mercier et al 2010). By school age, 25% to 50%
manifest a broad spectrum of cognitive, visual, social–
behavioral, attention, and specific learning disabilities
(Jacobson and Dutton 2000, Litt et al 2005, Glass et al
2008a, Soria-Pastor et al 2008, Anderson et al 2011).
Cerebral white matter injury (WMI) is the major form
of brain injury recognized in survivors of preterm birth
(Volpe 2009). In preterm survivors, magnetic resonance
imaging (MRI)-defined WMI but not gray matter injury
manifests in the first months of life as abnormal move-
ments that are predictive of cerebral palsy (Constantinou
et al 2007, Spittle et al 2008, 2009). The impact of WMI
can be appreciated from a recent large population-based
study of children with cerebral palsy. Perinatal WMI,
including periventricular leukomalacia (PVL), was the
most common finding, seen in almost half (42.5%) of
affected children (Bax et al 2006). Moreover, preterm
birth alone is associated with a greater risk for reduction
in both cerebral white and gray matter volume, which is
associated with poorer cognitive development (Peterson
et al 2000, Loeliger et al 2006, Anderson and Doyle 2008,
Kesler et al 2008, Aarnoudse-Moens et al 2009, Delobel-
Ayoub et al 2009, Scafidi et al 2009, Soria-Pastor et al
29
2009). As VLBW infants comprise about 1.5% of the
four million live births in the USA alone each year, the
worldwide social and economic burden is considerable.
The average lifetime costs per person with cerebral palsy
is estimated to be around $1 million in the USA (CDC
2004).
Brain injury in preterm survivors has an unexplained
predilection for cerebral white matter. The period of high-
est risk for WMI is ~23 to 32 weeks postconceptional
age. Preterm infants with WMI are at markedly increased
risk for several other forms of brain injury, notably intra-
ventricular hemorrhage (IVH) and intraparenchymal
hemorrhage (Volpe 2008) that are also observed in the
nonhuman primate (Dieni et al 2004). Whereas medical
interventions have resulted in a pronounced decrease in
the incidence of IVH (Volpe 2001a, Fowlie and Davis
2003), the incidence of WMI is not decreasing (Ballabh
2010). Thus, WMI is now the major neurologic problem
that affects VLBW infants.
WMI is not exclusively associated with preterm birth
and is increasingly appreciated in term infants (Pagliano
et al 2007, Li et al 2009, Lasry et al 2010). Infants with
complex congenital heart disease (CHD) are at particular
risk for WMI and delayed brain maturation (Wernovsky
et al 2005, Miller et al 2007, Licht et al 2009). These
infants show an increased predilection for a pressure pas-
sive circulation (Bassan et al 2005). Although the risk for
WMI would be expected to be lower, as these infants are
often full term at birth, WMI is now the major neurologic
lesion associated with CHD (Galli et al 2004, Kinney et al
2005). The basis for this propensity for WMI is unknown,
but recent studies support that white matter pathology can
precede surgical repair of heart lesions (McQuillen and
Miller 2010). This suggests that CHD itself may be a risk
factor for WMI.
 Section II: The Preterm Infant

Patholologic features of brain injury in preterm
infants
Cystic Necrotic White Matter Injury
Cystic necrotic WMI has been widely appreciated since
the classical descriptions of PVL by Banker and Larroche
50 years ago (Banker and Larroche 1962, DeReuck et al
1972, Rorke 1982, Leviton and Gilles 1984, Haynes et al
2003, Iida et al 1995, Robinson et al 2006). The early
descriptions of PVL commonly defined lesions that were
symmetrically located adjacent to the anterior and poste-
rior horns of the lateral ventricles. Regions of particular
predilection for injury were the frontal and parietal-occip-
ital periventricular white matter (PVWM); the latter often
involving the optic radiations of the visual system. Figure
3.1 shows the typical appearance of severe PVL, where
extensive cystic necrotic lesions are accompanied by
enlargement of the lateral ventricles due to white matter
atrophy. These frank necrotic lesions have the appearance
of an infarct with marked degeneration of both glia and
axons and infiltration by reactive microglia and phago-
cytic lipid-laden macrophages.
Non-cystic Focal or Diffuse White Matter
Injury
As originally described, PVL refers to necrotic forms of
WMI, and thus comprises one subset of all perinatal WMI.
It is important to distinguish PVL from non-necrotic forms
of WMI that are currently the predominant form in preterm
survivors. In fact, significant PVL was more common a
decade or more ago, whereas now, with recent advances
in neonatal intensive care, such lesions are infrequently
encountered. Rather, WMI is now recognized to include a
spectrum of cerebral injury that ranges from PVL to diffuse
myelination disturbances (diffuse WMI) where necrosis is
not observed. Recent neuroimaging studies have found
that the incidence of PVL is markedly declining, whereas
non-necrotic focal or diffuse WMI is emerging as the pre-
dominant lesion (Counsell et al 2003, Inder et al 2003,
Miller et al 2003, Hamrick et al 2004). Although cranial
ultrasound is the preferred bedside imaging technique for
diagnosing PVL, it has limited sensitivity for diagnos-
ing focal non-necrotic WMI (Maalouf et al 2001, Inder
et al 2003, Miller et al 2003), which was probably under-
recognized. In several recent series, cystic PVL accounted
for less than 5% of cases (Maalouf et al 2001, Counsell
et al 2003, Inder et al 2003, Miller et al 2003, Hamrick et al
2004, Groenendaal et al 2010). Figure 3.2 shows the typi-
cal spectrum, appearance, and distribution of lesions that
often are seen by MRI. It should be emphasized that there
remains unexplained variability in the nature of lesions
detected at different centers, which may reflect differences
in clinical management, clinical acuity, or the modes of
detection by neuroimaging.
This inherent variability also may be related to the
wide spectrum of injury responses that can be defined in

(a) (b)

Fig. 3.1 Neuropathology of periventricular leukomalacia. (a) Gross autopsy specimens from an infant who died of complications of
preterm birth. Coronal views of frontal (upper specimen) and parietal (lower specimens) periventricular white matter show multiple
focal areas of severe cystic necrosis (arrowheads). (b) A coronal tissue section of the frontal periventricular white matter (stained with
hematoxylin and eosin) shows a focus of necrosis (arrowheads) adjacent to the lateral ventricle. The inset shows a high-power detail
of the edge of the lesion (arrows) where marked rarefaction of the tissue can be appreciated adjacent to a region of gliosis at lower
left. (Courtesy of Dr Marjorie Grafe, Oregon Health and Science University.)

30
 Mechanisms of Acute and Chronic Brain Injury in the Preterm Infant

Fig. 3.2 Severe white matter injuries in a preterm newborn infant born at 28 weeks gestational age and scanned with MRI at 30 weeks
postmenstrual age and again at 42 weeks postmenstrual age. The T1-weighted images demonstrate multifocal areas of T1 hyperintensity
throughout the periventricular white matter of both cerebral hemispheres that are most evident on the earlier study. The T2-weighted
images demonstrate areas of hypointensity in the subependymal region consistent with mild germinal matrix hemorrhage. The white
matter injury is most readily apparent on the thin-section T1-weighted images. A head ultrasound at the time of the first magnetic
resonance scan revealed the germinal matrix hemorrhage with normal-appearing white matter.

human chronic WMI. Reactive astrogliosis is the most
consistent pathologic feature associated with chronic WMI
(Kinney and Back 1998). However, there is no consensus
on the optimal approach to distinguish reactive astrocytes
from resting ones. We recently found by unbiased quan-
tification of glial fibrillary acidic protein (GFAP)-labeled
astrocytes that chronic human white matter lesions were
much more diffuse than appreciated by standard histo-
pathologic approaches (Buser et al 2012). The presence of
elevated but intermediate astroglial response in perilesion
areas supported the presence of graded injury responses
that extended beyond the predicted lesion boundaries.
Variability in the extent of WMI, as detected by MRI at
different centers, may reflect differences in lesion burden
as well as the timing of detection of gliotic lesions in
evolution (Riddle et al 2011a).
What is the Role of Microscopic Necrosis in
the Overall Burden of Cerebral Injury in
Preterm Survivors?
As noted above, MRI studies support that the majority of
preterm survivors display non-necrotic focal or diffuse
WMI. It has been proposed that these less destructive
forms of WMI are dominated by microscopic necrosis
that is detected by histopathology but is not readily iden-
tified by neuroimaging (Volpe 2008, 2009). However,
the extent to which microscopic necrosis (microcysts)
occurs in WMI is an important but controversial ques-
tion. Microcysts are small lesions (<1mm in diameter),
for which reason their clinical impact may be minimal
unless they occur at high density in critical locations.
Microscopically, they comprise focal collections of mac-
rophages and activated microglia (Riddle et al 2011a).
Astroglial elements are markedly reduced in the core of
the lesions but are seen at the periphery, consistent with
the notion that microcysts represent small discrete focal
regions of necrosis. The pathogenesis of these lesions is
unclear, but they occur in response to hypoxia–ischemia.
We followed the evolution of microcysts in a preterm
fetal sheep model of global ischemia (Riddle et al 2006).
Microcysts were not detected at 1 week after ischemia, but
by 2 weeks appeared to evolve to discrete lesions visual-
ized by MRI at high field strength (Riddle et al 2011a).
Progress to define these lesions in preterm survivors has
been hampered because they have not been recognized
by prior MRI studies of WMI. Definition of the extent
of focal microscopic injury may be precluded at the field
strengths currently in clinical practice.
The extent to which microscopic necrotic injury con-
tributes to the overall burden of WMI is unclear, but is
clinically important. The importance resides in the addi-
tional burden of gray matter injury that potentially occurs

31
 Section II: The Preterm Infant
in association with necrosis-associated axonal degenera-
tion. As gray matter injury may contribute to the burden
of cognitive, learning, and social disabilities in preterm
survivors, it is important to define the relationship of
microscopic necrosis to cerebral gray matter injury. Gray
matter injury was seen in association with microscopic
necrosis, but not in cases where diffuse WMI occurred
without necrosis (Pierson et al 2007). This study found a
significant incidence of microscopic lesions that occurred
in multiple regions of cerebral white matter, but the den-
sity of microcysts was not determined. Recently, we quan-
tified the density of microscopic necrosis in individuals
with chronic WMI. They were observed in ~30% of cases
but comprised <5% of the total lesion area (Buser et al
2012). Microscopic necrosis was also a minor component
of hypoxic–ischemic WMI in fetal sheep (see above), in
which they constituted only ~1% of total WMI burden
(Riddle et al 2011a). Microscopic necrosis was detected
at high field strength by ex vivo MRI and was shown to
correspond to small focal necrotic lesions enriched in
reactive microglia/macrophages but relatively deficient in
astrocytes and axons. In summary, microscopic necrosis
is associated with focal WMI and retrograde neuronal
degeneration (Haynes et al 2008, Andiman et al 2010).
However, neuronal loss may not be a prominent feature of
WMI if the burden of such necrosis is low. Future studies
are needed to determine the potential of high field strength
MRI to define the incidence of microscopic necrosis and
its relationship to gray matter volume loss in preterm
survivors.
Magnetic Resonance Imaging–Histopathology
Gap to Define Pathologic Features of the
Current Spectrum of White Matter Injury
Although MRI is the optimal imaging modality to define
WMI in preterm survivors (Ment et al 2009, Miller and
Ferriero 2009, Mathur et al 2010, Rutherford et al 2010),
the histopathologic features of MRI signal abnormalities
have mostly been defined for WMI where PVL predomi-
nates (Hope et al 1988, Schouman-Claeys et al 1993,
Felderhoff-Mueser et al 1999, Childs et al 2001, Inder
et al 2005, Lodygensky et al 2011). With the pronounced
shift to milder forms of human WMI defined by quantita-
tive and diffusion-weighted MRI, there is a need to define
the cellular features of these lesions. We have recently
analyzed diffuse lesions in a preterm fetal sheep model,
in which animals survived for 1 or 2 weeks after global
cerebral ischemia. This preparation generated a spectrum
of WMI very similar to that observed from human autopsy
studies, as well as a reduction in cerebral white matter
volume similar to that observed in preterm survivors
(Ment et al 2009, Mathur et al 2010, Rutherford et al
32
2010). We developed registration algorithms to analyze
the histopathologic features of three classes of MRI-
defined lesions (Riddle et al 2011a). Each lesion type
displayed unique astroglial and microglial responses that
corresponded to distinct forms of necrotic or non-necrotic
injury. At 1 week after injury, for example, high-field MRI
(12 Tesla) identified a novel hypointense signal abnor-
mality on T2-weighted images with high sensitivity and
specificity for lesions with astrogliosis. This unexpected
finding suggests that current clinical MRI field strength
may be a limiting factor to detect diffuse gliosis as well
as microscopic necrosis (see above). Additional clini-
cal–pathologic studies are needed to determine whether
high-field MRI can provide greater sensitivity to iden-
tify diffuse WMI than is currently feasible at lower field
strengths.
Pathophysiology of cerebral white matter injury
Infection and Cerebral White Matter Injury
Both experimental and clinical studies have found that
neuroinflammatory mediators are detected in association
with WMI (Yoon et al 1996, 1997, Hagberg et al 2002,
Folkerth et al 2004a, Ellison et al 2005, Wang et al 2009,
Lin et al 2010) and can disrupt white matter development
(Favrais et al 2011). However, a role for neuroinflam-
matory mediators in the pathogenesis of cerebral palsy
is unclear. Inflammatory-mediated mechanisms related
to maternal fetal infection in near-term and term infants
are strongly correlated with risk for later cerebral palsy
(Nelson 2002, Shatrov et al 2010). However, recent evi-
dence supports that whereas postnatal infections are asso-
ciated with increased risk for WMI, prenatal risk factors
for infection such as chorioamniotis do not correlate with
increased risk (Kaukola et al 2006, Glass et al 2008b,
Chau et al 2009). Several studies have also found no
increased risk for cerebral palsy in preterm survivors by
analysis for intrauterine exposure to infection or mea-
surement of inflammatory cytokines in neonatal blood
(Grether et al 2003, Nelson et al 2003, Massaro et al
2009). These studies failed to find an association between
cerebral palsy and elevated tumor necrosis factor alpha
(TNF-α) or interleukin 1 beta (IL-1β) in acute PVL lesions
or cerebrospinal fluid. It is also unknown whether bacterial
endotoxin crosses the fetal blood–brain barrier, although
it is associated with breakdown of the blood–brain bar-
rier (Hutton et al 2007). Recently some proinflammatory
cytokines were shown to cross the neonatal murine barrier
by saturable transport (Threlkeld et al 2010).
Multiple lines of investigation support that altera-
tions in neuroinflammatory mediators exacerbate isch-
emic WMI. Endotoxins or cytotoxic cytokines are well
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you—but it hurts me so.”
Peggy sprang up and began to dress, puffing and whistling with
desperation. As soon as she was dressed she ran to the door and
opened it. All was still, a world of green and white. “The fire is almost
out,” she reported, “and I can see Mr. Smith’s horse’s tracks.”

It was about ten o’clock when a couple of horsemen suddenly

rounded the point of the forest and rode into the clearing. One of
them, a slender, elderly man with gray, curly beard and a skin like
red leather, dismounted and came slowly to the door, and though his
eyes expressed surprise at meeting women in such a place, he was
very polite.
“Mornin’, ma’am,” he said, with suave inflection.
“Good morning,” Peggy replied.
“Fine snowy mornin’.”
“It is so.” She was a little irritated by the fixed stare of his round,
gray eyes.
He became more direct. “May I ask who you are and how you
happen to be here, ma’am?”
“You may. I’m Mrs. Adams. I came up here with my husband,
Professor Adams.”
“Where is he?”
“He has gone up the trail toward Frémont. He is a botanist.”
“Is that his horse’s tracks?”
Alice called sharply, “Peggy!”
Mrs. Adams turned abruptly and went in.
The stranger turned a slow gaze upon his companion.
“Well, this beats me. ’Pears like we’re on the wrong trail, Bob. I
reckon we’ve just naturally overhauled a bunch of tourists.”
“Better go in and see what’s inside,” suggested the other man,
slipping from his horse.
“All right. You stay where you are.”
 As he stepped to the door and rapped, Peggy opened it, but Alice
took up the inquiry.
“What do you want?” she asked, imperiously.
The man, after looking keenly about, quietly replied: “I’m
wonderin’ how you women come to be here alone, but first of all I
want to know who made them tracks outside the door?”
Alice ignored the latter part of his question and set about
satisfying his wonder. “We came up here with a geological survey,
but my horse fell on my foot and I couldn’t ride, so the men had to
leave me behind—”
“Alone?” sharply interrogated the man.
“No; one man stayed.”
“What was his name?”
“I don’t know. We called him Smith.”
“Was he the man that rode away this morning?”
“What does that matter to you?” asked the girl. “Why are you so
inquisitive?”
He maintained his calm tone of mild authority. “I’m the sheriff of
Uinta County, ma’am, and I’m looking for a man who’s been hiding
out in this basin. I was trailin’ him close when the snow came on
yesterday, and I didn’t know but what these tracks was his.”
Peggy turned toward Alice with an involuntary expression of
enlightenment, and the sheriff read it quickly. Slipping between the
two women, he said:
“Jest a minute, miss. What sort of a looking man was this Smith?”
Alice took up the story. “He was rather small and dark—wasn’t he,
Peggy?”
Peggy considered. “I didn’t notice him particularly. Yes, I think he
was.”
The man outside called: “Hurry up, Cap. It’s beginning to snow
again.”
The sheriff withdrew toward the door. “You’re both lying,” he
remarked without heat, “but it don’t matter. We’ll mighty soon
overhaul this man on the horse—whoever he is. If you’ve been
harboring Hall McCord we’ll have to take you, too.” With that threat
as a farewell he mounted his horse and rode away.
 Peggy turned to Alice. “Did you know that young fellow was an
outlaw?”
“Yes: I saw his picture and description on a placard in the railway
station. I recognized him at once.”
“Why didn’t you tell me?”
“Well, I liked his looks, and, besides, I wanted to find out if he
were really bad or only unfortunate.”
“What has he done?”
“They say he held up a train!”
“Merciful Heavens! a train-robber! What’s his real name?”
“The name on the placard was Hall McCord.”
“And to think he was in the same room with us last night, and you
were chumming with him! I can’t understand you. Are you sure he is
the robber?”
“Yes. He confessed to having tried to rob the express car.”
“He seemed such a nice fellow. How did he come to do it?”
Alice concluded not to honor the other girl by bringing her into the
discussion. “Oh, it is hard to say. Need of money, I suppose. Poor
boy, I pity him.”
“They’ll get him, sure. They can follow his tracks as easy as
anything. I don’t suppose I ought to say it, but I hope he’ll get away.
Don’t you?”
“Yes, I do!” was Alice’s fervent response. “But see! it’s snowing
again. It may cover his trail.”
Peggy went to the door and gazed long and keenly at the peaks.
When she turned her face was solemn. “Allie, this is getting pretty
serious for us. If the men don’t come to-day they may get snowed up
entirely.”
Alice stifled a wail. “Oh, if I were only able to walk I wouldn’t mind.
I could help gather fuel and keep the fire going.”
“There’s plenty of wood for another day, but I’m worried about the
men. Suppose they are up on that glacier?”
“I’m not worried about them, but I know they are worrying about
us. They’ll surely start back this morning; but they may not be able to
reach us till night.”
 The light of the morning had turned gray and feeble. The air was
still and the forest soundless, save now and then when a snow-laden
branch creaked with its burden.
There was something majestic as well as menacing in this all-
pervading solemn hush.
Peggy went about her duties as cheerfully as she could, but with a
wider knowledge of mountaineering than Alice had. She was at heart
quite terrified. “We’re going to miss our nice outlaw,” she remarked.
“He was so effective as a purveyor of wood.” Then she went to the
door and looked out. “That sheriff will never keep his trail,” she said.
“What’s that?” suddenly asked Alice.
Both listened. “I hear it!” whispered Peggy. “It’s a horse—there!
Some one spoke.”
“It’s Freeman!” Alice joyously called out. “Coo-hoo!”
No one replied, and Peggy, rushing to the door, met the young
outlaw, who appeared on the threshold with stern, set face.
“Who’s been here since I left? Your party?”
Peggy recoiled in surprise and alarm, and Alice cried out, “Why
did you come back?”
“Two men on horseback have been here since I left. Who were
they?” His voice was full of haste.
“One of them said—he was the—the sheriff,” Alice replied, faintly.
He smiled then, a kind of terrifying humor in his eyes. “Well, the
chances are he knew. They took my trail, of course, and left in a
hurry. Expected to overhaul me on the summit. They’ve got their
work cut out for ’em.”
“How did they miss you?” the girl asked, huskily.
“Well, you see, when I got up where I could view the sky I was
dead sure we were in for a whooping big snow-storm, and I just
couldn’t leave you girls up here all alone, so I struck right down the
cañon in the bed of the creek—the short cut. I don’t like to backtrail,
anyway; it’s a bad habit to get into. I like to leave as blind a trail as I
can.” His face lightened up, grew boyish again. “They’re sure up
against a cold proposition about now. They’ll lose my track among
the rocks, but they’ll figure I’ve hustled right on over into Pine Creek,
and if they don’t freeze to death in the pass they’ll come out at
Glover’s haymeadow to-morrow night. How’s the wood-pile holding
out?”
“Please go!” cried Alice. “Take your chance now and hurry away.”
“I’m not used to leaving women in such a fix. The moment I saw
that blizzard was beginning all over again I turned back.”
“You haven’t had any breakfast?” said Peggy.
“Nothing to speak of,” he replied, dryly. “I wasn’t thinking of
breakfast when I pulled out.”
“I’ll get you some.”
Alice could not throw off the burden of his danger. “What will you
do when my people return?”
“I don’t know—trust to luck.”
“You are very foolish. They are certain to come to-day.”
“They won’t know who I am if you women don’t give me away.”
“I’m sure Freeman—Professor Ward—will know you, for he also
saw the placard.”
“That’s no sign. Suppose he does—maybe he won’t think it is his
job to interfere. Anyway”—here his voice became decisive—“I won’t
leave you in such a fix as this.” His eyes spoke to her of that which
his tongue could not utter. “I wanted an excuse to come back,
anyway,” he concluded. “No matter what comes now, my job is here
to protect you.”
She did not rebuke him, and Peggy—though she wondered at his
tone—was too grateful for his presence even to question Alice’s
motive in permitting such remarks.
As for Alice, she felt herself more and more involved in the
tangled skein of his mysterious life. His sudden and reckless
abandonment of the old love which had ruined him, and the new and
equally irrational regard which he now professed for her, filled her
with a delicious marveling.
He appealed to a woman’s imagination. He had the spice of the
unknown. In her relationship with Ward there was no danger, no
mystery—his courtship narrowly escaped being commonplace. She
had accepted his attentions and expected to marry him, and yet the
thought of the union produced, at its warmest, merely a glow of
comfort, a sense of security, whereas the hint of being loved and
protected by this Rob Roy of the hills, this reckless Rough Rider of
the wilderness, was instinct with romance. Of course his devotion
was a crazy folly, and yet, lying there in her rough bunk, with an
impenetrable wall of snow shutting out the rest of the world, it was
hard not to feel that this man and his future had become an
inescapable part of her life—a part which grew in danger and in
charm from hour to hour.
Full two miles above the level of her own home, surrounded by
peaks unscalably wild and lonely, deserted by those who should care
for her, was it strange that she should return this man’s adoring gaze
with something of the primal woman’s gratitude and submission?
The noon darkened into dusk as they talked, slowly, with long
pauses, and one by one the stirring facts of the rover’s life came out.
From his boyhood he had always done the reckless thing. He had
known no restraint till, as a member of the Rough Riders, he yielded
a partial obedience to his commanders. When the excitement of the
campaigns was over he had deserted and gone back to the round-up
wagon and the campfire.
In the midst of his confidences he maintained a reserve about his
family which showed more self-mastery than anything else about
him. That he was the black sheep of an honorable flock became
increasingly evident. He had been the kind of lad who finds in the
West a fine field for dare-devil adventure. And yet there were
unstirred depths in the man. He was curious about a small book
which Alice kept upon her bed, and which she read from time to time
with serene meditation on her face.
“What is that?” he asked.
“My Bible.”
“Can I see it?”
“Certainly.”
He took it carefully and read the title on the back, then turned a
few of the leaves. “I’m not much on reading,” he said, “but I’ve got a
sister that sends me tracts, and the like.” He returned to the fly-leaf.
“Is this your name?”
“Yes.”
 “‘Alice Mansfield,’” he read; “beautiful name! ‘New York City’!
That’s pretty near the other side of the world to me.” He studied the
address with intent look. “I’d like to buy this book. How much will you
take for it?”
“I’ll trade it for your weapon,” she replied.
He looked at her narrowly. “You mean something by that. I reckon
I follow you. No, I can’t do that—not now. If I get into business over
the line I’ll disarm, but in this country a fellow needs to be protected.
I want this book!”
“For the fly-leaf?”
He smiled in return. “You’ve hit it.”
She hesitated. “I’ll give you the book if you’ll promise to read it.”
He clapped the covers together and put the volume in his pocket.
“It’s mine! I’ll read every word of it, if it takes an age, and here’s my
hand on it.”
She gave him her hand, and in this clasp something came to her
from his clutching fingers which sobered her. She drew her hand
away hastily and said: “If you read that book—and think about it—it
will change your whole world.”
He, too, lost his brightness. “Well, I’m not so anxious to keep up
this kind of life. But if anybody changes me it will be you.”
“Hush!” she warned with lifted finger.
He fell back, and after a little silence went out to wait upon the fire.
“It seems to me,” said Peggy, reprovingly, “that you’re too gracious
with this mountaineer; he’s getting presumptuous.”
“He doesn’t mean to be. It’s his unsophisticated way. Anyhow, we
can’t afford to be captious to our host.”
“That’s true,” admitted Peggy.
The night shut down with the snow still falling, but with a growing
chill in the air.
“The flakes are finer,” the outlaw announced, as he came in a little
later. “That is a good sign. It is growing colder and the wind is
changing. It will pinch hard before sun-up, and the worst of it, there’s
no way to warm this cabin. We can’t have the door open to-night. I’m
worried about you,” he said to Alice. “If only those chumps had left a
man-size ax!”
 The two women understood that this night was to bring them into
closer intimacy with the stranger than before. He could not remain
outdoors, and though they now knew something of his desperate
character, they had no fear of him. He had shown his chivalry. No
one could have been more considerate of them, for he absented
himself at Peggy’s request instantly and without suggestion of
jocularity, and when he came in and found them both in bed he said:
“I reckon I’ll not make down to-night—you’ll need all your blankets
before morning”; and thereupon without weighing their protests,
proceeded to spread the extra cover over them.
Alice looked up at him in the dim light of the candle and softly
asked: “What will you do? You will suffer with cold!”
“Don’t worry about me; I’m an old campaigner. I still have a
blanket to wrap around my shoulders. I’ll snooze in a corner. If you
hear me moving around don’t be worried; I’m hired to keep the fire
going even if it doesn’t do us much good inside.”
The chill deepened. The wind began to roar, and great masses of
snow, dislodged from the tall trees above the cabin, fell upon its roof
with sounds like those of soft, slow footfalls. Strange noises of
creaking and groaning and rasping penetrated to Alice’s ears, and
she cowered half in fear, half in joy of her shelter and her male
protector. Men were fine animals for the wild.
She fell asleep at last, seeing her knight’s dim form propped
against the wall, wrapped in a blanket Indian-wise, his head bowed
over the book she had given him, a candle smoking in his hand.
She woke when he rose to feed the fire, and the current of cold air
which swept in caused her to cover her mouth with the blanket. He
turned toward her.
“It’s all over for sure, this time,” he said. “It’s cold and goin’ to be
colder. How are you standing it? If your feet are cold I can heat a
stone. How is the hurt foot?” He drew near and looked down upon
her anxiously.
“Very much easier, thank you.”
“I’m mighty glad of that. I wish I could take the pain all on myself.”
“You have troubles of your own,” she answered, as lightly as she
could.
 “That’s true, too,” he agreed in the same tone. “So many that a
little one more or less wouldn’t count.”
“Do you call my wound little?”
“I meant the foot was little—”
She checked him.
“I didn’t mean to make light of it. It sure is no joke.” He added,
“I’ve made a start on the book.”
“How do you like it?”
“I don’t know yet,” he answered, and went back to his corner.
She snuggled under her warm quilts again, remorseful, yet not
daring to suggest a return of the blanket he had lent. When she
woke again he was on his feet, swinging his arms silently. His candle
had gone out, but a faint light was showing in the room.
“Is it morning?” she asked.
“Just about,” he replied, stretching like a cat.
The dawn came gloriously. The sun in far-splashing splendor
slanted from peak to peak, painting purple shadows on the snow and
warming the boles of the tall trees till they shone like fretted gold.
The jays cried out as if in exultation of the ending of the tempest, and
the small stream sang over its icy pebbles with resolute cheer. It was
a land to fill a poet with awe and ecstatic praise—a radiant, imperial,
and merciless landscape. Trackless, almost soundless, the mountain
world lay waiting for the alchemy of the sun.

VI

The morning was well advanced when a far, faint halloo broke
through the silence of the valley. The ranger stood like a statue,
while Peggy cried out:
“It’s one of our men!”
Alice turned to the outlaw with anxious face. “If it’s the sheriff stay
in here with me. Let me plead for you. I want him to know what
you’ve done for us.”
The look that came upon his face turned her cold with fear. “If it is
the sheriff—” He did not finish, but she understood.
 The halloo sounded nearer and the outlaw’s face lightened. “It’s
one of your party. He is coming up from below.”
Impatiently they waited for the new-comer to appear, and though
he seemed to draw nearer at every shout, his progress was very
slow. At last the man appeared on the opposite bank of the stream.
He was covered with snow and stumbling along like a man half dead
with hunger and fatigue.
“Why, it’s Gage!” exclaimed Peggy.
It was indeed the old hunter, and as he drew near his gaunt and
bloodless face was like that of a starved and hunted animal. His first
word was an anxious inquiry, “How are ye?”
“All well,” Peggy answered.
“And the crippled girl?”
“Doing nicely. Thanks to Mr. Smith here, we did not freeze. Are
you hungry?”
The guide looked upon the outlaw with glazed, protruding eyes.
“Hungry? I’m done. I’ve been wallerin’ in the snow all night and I’m
just about all in.”
“Where are the others?” called Alice from her bed.
Gage staggered to the door. “They’re up at timber-line. I left them
day before yesterday. I tried to get here, but I lost my bearin’s and
got on the wrong side o’ the creek. ’Pears like I kept on the wrong
side o’ the hog-back. Then my horse gave out, and that set me afoot.
I was plum scared to death about you folks. I sure was.”
Peggy put some food before him and ordered him into silence.
“Talk later,” she said.
The outlaw turned to Alice. “That explains it. Your Professor Ward
trusted to this man to take care of you and stayed in camp. You can’t
blame him.”
Gage seemed to have suddenly become old, almost childish. “I
never was lost before,” he muttered, sadly. “I reckon something must
have went wrong in my head. ’Pears like I’m gettin’ old and foolish.”
Alice exchanged glances with the outlaw. It was plain that he was
in no danger from this dazed and weakened old man who could think
of nothing but the loss of his sense of direction.
 As the day advanced the sun burned clear. At noon it was warm
enough to leave the door open, and Alice, catching glimpses of the
flaming world of silver and purple and gold, was filled with a desire to
quit her dark corner.
“I’m going to get up!” she exclaimed. “I won’t lie here any longer.”
“Don’t try it!” protested Peggy.
“I’m going to do it!” she insisted. “I can hobble to the door if you
help me.”
“I’ll carry you,” said the outlaw. “Wrap her up and I’ll get her a
seat.”
And so, while Mrs. Adams wrapped her patient in a blanket, the
outlaw dragged one of the rough, ax-hewn benches to the door and
covered it with blankets. He put a stone to heat and then re-entered
just as Alice, supported by Peggy, was setting foot to the floor.
Swiftly, unhesitatingly, and very tenderly he put his arms about her
and lifted her to the bench in the doorway before the fire.
It was so sweet to feel that wondrous body in his arms. His daring
to do it surprised her, but her own silent acquiescence, and the
shiver of pleasure which came with the embarrassment of it,
confused and troubled her.
“That’s better,” he said as he dropped to the ground and drew the
blankets close about her feet. “I’ll have a hot stone for you in a
minute.”
He went about these ministrations with an inward ecstasy which
shone in his eyes and trembled in his voice. But as she furtively
studied his face and observed the tremor of his hands in tender
ministration she lost all fear of him.
After three days in her dark corner of the hut the sunshine was
wondrously inspiring to the girl, although the landscape on which she
gazed was white and wild as December. It was incredible that only a
few hours lay between the flower-strewn valley of her accident and
this silent and desolate, yet beautiful, wilderness of snow. And so, as
she looked into the eyes of the outlaw, it seemed as though she had
known him from spring to winter, and her wish to help him grew with
every hour of their acquaintanceship.
 She planned his defense before Ward and Adams. “When they
know how kind and helpful he has been they can but condone his
one rash deed,” she argued in conclusion.
He was sitting at her feet, careless of time, the law, content with
her nearness, and mindful only of her comfort, when a distant rifle-
shot brought him to his feet with the swiftness of the startled stag.
“That’s your expedition,” he said, “or some one who needs help.”
Again the shots rang out, one, two, three—one, two, three.
“It’s a signal! It’s your party!”
Peggy uttered a cry of joy and rushed outside, but Alice turned an
unquiet gaze on the outlaw. “You’d better fly!”
“What is the use?” he answered, bitterly. “The snow is so deep
there is no show to cross the range, and my horse is weak and
hungry.”
Gage appeared at the door. “Lemme take your gun, stranger; I
want to answer the signal.”
“Where’s your own?”
“I left it on my horse,” the old man answered, sheepishly.
The young fellow looked at Alice with a keen glitter in his eyes. “I’ll
make answer myself,” he said; “I’m very particular about my
barkers.”
Alice, as she heard his revolver’s answering word leap into the
silent air and bound and rebound along the cliffs, was filled with a
sudden fear that the sheriff might be guided back by the sound—and
this indeed the fugitive himself remarked as he came back to his
seat beside her.
“If he’s anywhere on this side of the divide he’ll sure come back.
But I’ve done my best. The Lord God Almighty has dropped the
snow down here and shut me in with you, and I’m not complaining.”
There was no answer to be made to this fatalism of utterance, and
none to the worship of his eyes.
“Lift me up!” commanded Alice; “I want to look out and see if I can
see anybody.”
The outlaw took her in his arms, supporting her in the threshold in
order that she might see over the vast sea of white. But no human
being was to be seen.
 “Take me back—inside,” Alice said to the man who had her in his
arms. “I feel cold here.”
Once again, and with a feeling that it was, perhaps, for the last
time, he carried her back to her bench and re-enveloped her in her
blankets.
“Stay here with me now,” she whispered to him, as she looked up
into his face.
And the outlaw, filled with gladness and pride, threw himself on
the floor beside her.

VII

The signal pistol-shots came nearer and nearer, but very slowly;
and as the outlaw sat beside Alice’s couch he took her Bible from his
pocket and said:
“I made a stab at reading this last night.”
She smiled. “I saw you. How did you like it?”
“I didn’t exactly get aboard someway.”
“What was the trouble?”
“I guess it was because I kept thinking of you—and my own place
in the game. Three days ago I didn’t care what became of me, but
now I want a chance. I don’t see any chance coming my way, but if I
had I’d make use of it.” He looked at her a moment in silence, then
with sudden intensity broke forth. “Do you know what you mean to
me? When I look at your face and eyes I’m hungry for you.”
She shrank from him and called to Mrs. Adams.
He went on. “Oh, you needn’t be afraid. I just wanted to say it,
that’s all. If there was only some other way to straighten myself—but
I can’t go to jail. I can’t stand up to be clipped like a poodle-dog, then
put on striped clothing and walk lock-step—I can’t do it! They’ll put
me in for ten years. I’d be old when I got out.” He shuddered. “No, I
won’t do that! I’d rather die here in the hills.”
She grew white in sympathy. “It is a frightful price to pay for one
insane act, and yet—crime should be punished.”
“I’m getting my punishment now,” he replied, with darkly brooding
glance. “There’s a good old man and two women, my sisters, waiting
for me down the slope. If I could reach home I’d try to live straight,
but it’s a long and dangerous trail between here and there.”
Peggy now ran into the cabin. “It’s the expedition,” she
announced. “I can see Freeman.”
“I reckon this is where I get off,” said the outlaw in a tone of
mingled relief and dismay.
“No, no!” Alice entreated. “Stay till Freeman comes. He will help
you. Let me explain to him. I know he will not betray you.”
He looked at her again with that intent, longing worship in his
eyes, and answered, “I accept the chance for the sake of one more
hour with you.”
The outlaw stepped to the door, and he saw a man at the head of
his train mid-leg deep in snow, leading his horse, breaking the way
for his followers, who were on foot, crawling, stumbling, and twisting
among the down-timber, unmindful of the old trail.
At sight of that big and resolute leader, with flowing black beard
and ruddy face, the outlaw was filled with jealous sadness. To find
Ward a man of superb physical prowess, the kind that measures
peaks for the fun of it, was disturbing, and without defining his feeling
he was plunged into melancholy musing. And when later Ward
entered, and, stooping over the couch, kissed Alice, the end of his
idyl seemed to him announced.
In the bustle of the moment, in the interchange of anxious, hurried
inquiries, the outlaw stood aside in the corner, unnoticed, till Alice
caught Ward’s arm and said:
“Freeman, this is Mr. Smith, to whom we owe a great deal. He has
taken the utmost care of us. We would have frozen but for him.”
Ward shook hands with the outlaw, but wonderingly asked of
Alice, “But where was Gage?”
The outlaw answered, “Gage got lost and only turned up a couple
of hours ago.”
Ward turned to Alice in horror. “Good Lord! And you were here
alone—crippled—in this storm?”
“No—that’s what I’m telling you. Mr. Smith came and took care of
us. He brought our wood, he cooked for us, he kept our fire going.
He gave up his bed, even his blankets, for us. You should be very
generous to him.”
Ward again reached a hearty hand. “I’m tremendously obliged to
you.”
The outlaw quailed under all this praise. “There was mighty little to
do,” he answered. “I only shared my fire with them.”
Ward studied him closer. “Haven’t we met before?”
“No, I reckon not.”
“I’m quite sure I’ve see you somewhere. What are you doing up in
here?”
Alice interposed. “What are we going to do?”
Ward turned to the outlaw. “What would you advise? I’ve only had
one idea, and that was to reach this cabin. Now what would you do?”
The outlaw was ready. “I would send a part of the men with the
horses down the valley to grass and I’d wait here till Miss Mansfield
is able to ride.”
“Will this snow go off?”
“That’s my notion.”
“It’s certain we can’t camp here—the horses must have grass.”
“I’ll be able to ride in a day or two,” Alice said, bravely.
“We could frame up a portable bed and carry you,” suggested the
outlaw; “but it can’t be done to-night, so you’d better send your outfit
down to the marsh to camp. The horses are worn out and so are the
men.”
“Will you guide them to grass and help them find shelter?”
The outlaw hesitated for an instant, and Alice interposed: “No, no!
Let Gage do that. I want Mr. Smith to remain here.”
Ward perceived in her entreaty something of anxiety and fear, and
after the men and horses had started down the slope he turned to
the outlaw and said: “I’m mighty grateful to you, Mr. Smith. It must
have surprised you to find these women here.”
The outlaw dryly replied, “It did!”
Alice added: “It was in the middle of the night, too; but Mr. Smith
was very nice about it. He slept outdoors without a word of
complaint.”
 Ward had figured the situation to conclusion: “Smith is a poacher,”
and though he had a savage dislike of these illicit game-
slaughterers, he could not but be glad of the presence of this
particular outlaw, and resolved to overlook his trade in gratitude for
his cabin and service.
The outlaw helped Adams and Ward to clear away the snow for a
tent, and Alice, seeing the three men thus amicably joined in her
defense, could not find it in her heart to condemn one of them as a
criminal. Here in the white isolation of the peaks the question of
crime and its punishment became personal. To have this man’s fate
in her hand was like grasping the executioner’s sword for herself.
“If women had to punish criminals themselves, with their own
hand,” she asked, “how many of them would do it?”
Peggy came in and whispered to her: “No one else seems to have
recognized him. He may get away safely. I hope he will. Shall we tell
the men who he is?”
“Yes, we shall have to do that soon, but I’m afraid they won’t take
the sentimental view of him that we do. I tremble to think of what
they will do when they know.”
Ward explained to Adams: “Our friend Smith here is a poacher—
but as our account stands I don’t feel it my duty to report him, do
you?”
“No; Peggy tells me he has acted like a gentleman all through.”
In this spirit they made themselves comfortable for the night.
The sun set gloriously, but the air bit ever sharper, and while
Peggy went about her cooking, assisted by her husband and the
outlaw, Alice pulled Ward down to her bedside and hurriedly began:
“You remember that placard we read in the station—the one about
the train-robber?”
“Yes!”
“Well, this is the man—our Mr. Smith.”
Ward looked at her a moment with reflective eyes, then
exclaimed: “You’re right! I thought I’d seen him somewhere.”
“And the sheriff is after him. He was here yesterday morning.”
“Here?”
 “Yes. You see, Mr. Smith stayed with us till he thought the storm
was over, then rode away, intending to cross the divide, but when the
snow began again he turned back. He said he couldn’t leave us
alone. He left us just before dawn, and four or five hours afterward
the sheriff came. Of course he saw the poor fellow’s trail and
instantly set off after him.”
“But why didn’t they meet?”
“Because Mr. Smith came back a different way and then the
blizzard came on and covered up his tracks. He thinks the sheriff has
gone on over the divide. You must help him, Freeman. Help him to
get away and find some way to give him a start. Nobody could have
been more considerate, and I can’t see him taken by these cold-
blooded men who want that two thousand dollars’ reward. He really
could have escaped, only for us. He came back to protect us.”
Ward pondered. “The problem is not so easy of solution. A train
robbery is a pretty serious matter. I’m very grateful to him, but to
connive at his escape is itself a punishable act. Why did you tell me?
I could have passed it over—”
“Because I’m afraid the sheriff may come back at any moment.”
Ward’s brow was troubled. “I could ignore his deed and pretend
not to know who he is, but definitely to assist a bandit to escape is a
very serious matter.”
“I know it is; but remember he gave up his chance to cross the
divide in order to keep us from suffering.”
“I wish you hadn’t told me,” he repeated, almost in irritation. “If the
sheriff only keeps on over the range Smith can take care of himself.”
As the outlaw re-entered the cabin Alice acknowledged in him
something worth a woman to love. In the older man was power,
security, moral, mental, and physical health, the qualities her reason
demanded in a husband; but in the other was grace and charm,
something wildly admirable. He allured as the warrior, intrepid and
graceful, allured the maiden, as the forest calls the householder.
Something primordial and splendid and very sweet was in her feeling
toward him. There could be no peaceful wedlock there, no security of
home, no comfort, only the exquisite thrill of perilous union, the
madness of a few short weeks—perhaps only a few swift days of
self-surrender, and then, surely, disaster and despair. To yield to him
was impossible, and yet the thought of it was tantalizingly sweet.
When she looked toward Ward she perceived herself sitting
serenely in matronly grace behind a shining coffee-urn in a well-
ordered, highly civilized breakfast-room, facing a most considerate
husband who nevertheless was able to read the morning paper in
her presence. When she thought of life with the outlaw all was dark,
stormy, confused, and yet the way was lit by his adoring eyes. A
magical splendor lay in the impulse. His love, sudden as it seemed,
was real—she was certain of that. She felt the burning power, the
conjury of its flame, and it made her future with Ward, at the
moment, seem dull and drab.
“Why, why could not such a man and such a passion come with
the orderly and the ethical?” she asked herself.
At the best he was fitted only for the mine or the ranch, and the
thought of life in a lonely valley, even with his love to lighten it, made
her shudder. On one side she was a very practical and far-seeing
woman. The instant she brought her reason to bear on the problem
she perceived that any further acquaintance with this man was
dangerous. They must part here at this moment, and yet she could
not let him go without in some way making him feel her wish to help
him.

VIII

Ward and the outlaw were discussing plans for getting out of the
basin when Adams came in to say, “A couple of other weary
wanderers are turning up.”
“The sheriff!” instantly exclaimed Alice, her face whitening in swift
dismay.
In that moment the forester was transformed. With a weapon in
his hand he stood aside, his eyes on the door, a scowl of battle on
his face. He resembled a wolf with bared fangs ready to die
desperately.
Ward, quick to read his purpose, interposed. “Wait!” he
commanded. “Stay here; I’ll see them. Don’t be rash.”
 As he passed out into the firelight the outlaw, without relaxing his
vigilance, said in a low voice, “Well, girl, I reckon here’s where I say
good night.”
“Don’t resist,” she pleaded. “Don’t fight, please! Please! What is
the use? Oh, it’s too horrible! If you resist they will kill you!”
There was no fear in his voice as he replied: “They may not; I’m
handy with my gun.”
She was breathless, chilled by the shadow of the impending
tragedy. “But that would be worse. To kill them would only stain your
soul the deeper. You must not fight!”
“It’s self-defense.”
“But they are officers of the law.”
“No matter; I will not be taken alive.”
She moaned in her distress, helplessly wringing her hands. “O
God! Why should I be witness of this?”
“You won’t be. If this is the sheriff I am going to open that door and
make a dash. What happens will happen outside. You need not see
it. I’m sorry you have to hear it. But I give you my word—if you must
hear something I will see to it that you hear as little as possible.”
The latch clicked—he stepped back, and again stood waiting,
silent, rigid, ready to act, murderous in design.
Mrs. Adams entered quickly, and, closing the door behind her,
hurriedly whispered: “It’s the sheriff. Hide! The men will hold them as
long as they can. Hide!”
The outlaw looked about and smiled. “Where?” he asked, almost
humorously. “I’m not a squirrel.”
“Under the bunk. See, there is room.”
He shook his head. “No, I refuse to crawl. I won’t sneak. I never
have. I take ’em as they come.”
“For my sake,” pleaded Alice. “I can’t bear to see you killed. Hide
yourself. Go to the door,” she said to Peggy. “Don’t let them in. Tell
Freeman—” She rose and stood unsteadily, forgetful of her own
pain.
Mrs. Adams urged her to lie down, but she would not. The
moments passed in suspense almost too great to be endured.
“Listen!” commanded the outlaw. “They’re coming in.”
 As they harkened Ward’s voice rose clearly. “You can’t miss the
camp,” he was saying, as if speaking to some one at a distance.
“Just keep the trail in the snow and you’ll find them. I’m sorry we
can’t put you up—but you see how it is.”
“They’re going!” exclaimed Alice. “Thank God, they’re going!”
“It can’t be they’ll go without searching the shack,” the fugitive
muttered, in no measure relaxing his attitude of watchful menace.
“They’re playing a game on us.”
Again the latch clicked, and this time it was Ward who confronted
the outlaw’s revolver mouth.
“It’s all right,” Ward called, instantly understanding the situation.
“They’re gone. The old man was about played out, for they’ve been
fighting snow all day, but I told him we couldn’t take care of them
here and they have gone on down to the camp. He thinks you got
over the divide. You are all right for the present.”
“They’ll come back,” replied the other. “It only puts the deal off a
few hours. They’ll return, trailin’ the whole camp after them. What
can I do? My horse is down there in the herd.”
“That’s bad,” exclaimed Ward. “I wonder if I could get him for
you?”
“If I had him he’s weak and hungry, and the high places are feet
deep in drifts. It doesn’t signify. I’m corralled any way you look at it,
and the only thing left is to fight.”
“There’s our trail to the glacier,” Ward musingly suggested; “it’s a
pretty deep furrow—you might make it that way.”
A spark of light leaped into the man’s eyes. “How far up does it
run? Where does it end?”
“In Glacier Basin, just at timber-line.”
The outlaw pondered, speaking his thoughts aloud. “From there
across to the Indian reservation there isn’t a wolf track.... It’s a man’s
job crossing there, almost sure death, but it’s my only show.” He had
replaced his weapon in his belt and was weighing his chance, his
eyes fixed on Alice’s face. To leave this shelter, this warm circle of
light, this sweet girlish presence, and plunge into the dark, the cold,
and the snow, was hard. No one but a man of unconquerable
courage would have considered it. This man was both desperate and