Handbook of Clinical Neurology, Vol.

162 (3rd series)

Neonatal Neurology
L.S. de Vries and H.C. Glass, Editors
https://doi.org/10.1016/B978-0-444-64029-1.00010-2
Copyright © 2019 Elsevier B.V. All rights reserved

Chapter 10

Neonatal encephalopathy and hypoxic–ischemic encephalopathy

ALISTAIR J. GUNN1* AND MARIANNE THORESEN2,3
1
Departments of Physiology and Paediatrics, University of Auckland, Auckland, New Zealand
2
Department of Physiology University of Oslo, Oslo, Norway
3
Neonatal Neuroscience, Translational Health Sciences, University of Bristol, Bristol, United Kingdom

Abstract
Acute hypoxic–ischemic encephalopathy around the time of birth remains a major cause of death and life-
long disability. The key insight that led to the modern revival of studies of neuroprotection was that, after
profound asphyxia, many brain cells show initial recovery from the insult during a short “latent” phase,
typically lasting approximately 6 h, only to die hours to days later after a “secondary” deterioration char-
acterized by seizures, cytotoxic edema, and progressive failure of cerebral oxidative metabolism. Studies
designed around this framework showed that mild hypothermia initiated as early as possible before the
onset of secondary deterioration and continued for a sufficient duration to allow the secondary deteriora-
tion to resolve is associated with potent, long-lasting neuroprotection. There is now compelling evidence
from randomized controlled trials that mild to moderate induced hypothermia significantly improves sur-
vival and neurodevelopmental outcomes in infancy and mid-childhood.

INTRODUCTION Neonatal encephalopathy per se is of course not just

Birth represents a dramatic switch in life, from a fetus
that relies on the placenta for respiratory exchange and
nutrition, to an infant who must breathe for him- or her-
self. While the majority of births are completely normal,
complications that impair oxygen and glucose exchange
can occur before or at birth at any gestational age. Acute
brain injury around birth leading to disturbance of brain
function (i.e., neonatal encephalopathy) is associated
with nearly half of all cases of cerebral palsy; of these,
the Western Australian Cerebral Palsy register found that
approximately 15% of cases were born at term and had
had acute encephalopathy at birth (Reid et al., 2016).
Hypoxic–ischemic encephalopathy (HIE) occurs in the
developed world in approximately 2–3/1000 live births
(Vannucci, 2000). Worldwide, HIE at birth and during
the first 28 days of life represents the single greatest con-
tribution to overall disability worldwide and accounts for
one-10th of all disability adjusted life years (Lee
et al., 2013).
due to hypoxia–ischemia. Seizures are one of the most
obvious and common manifestations of encephalopathy
and can be used to help identify cases of encephalopathy.
In a large contemporary cohort of infants (of whom 88%
were term), the most common causes of neonatal seizures
were HIE (38%), stroke (18%), and intracranial hemor-
rhage (11%) (Glass et al., 2016). Genetic epilepsy
(6%) and metabolic problems such as hypoglycemia
(4%) were relatively uncommon. As well, hypoglycemia
is a common complication of perinatal HIE and is asso-
ciated with worse outcomes (Basu et al., 2016). It is
important to note that both neonatal encephalopathy
and seizures are very common in preterm infants but
the seizures are most often subclinical (Glass et al.,
2017). Stroke, hemorrhage, hypoglycemia, infection,
and preterm brain injury are discussed in other chapters
in this volume (see Chapters 6, 7, 8, and 11). This chapter
will therefore focus on the pathophysiology and treat-
ment of HIE at term and near-term.

*Correspondence to: Alistair J. Gunn, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019,
Auckland, New Zealand. Tel: +64-9-3737599x96763, E-mail: aj.gunn@auckland.ac.nz
218 A.J. GUNN AND M. THORESEN
In infants who have evidence of an acute, perinatal
event, the key link between exposure to asphyxia and sub-
sequent neurodevelopmental impairment is the early onset
of HIE. Newborns with mild encephalopathy are normal at
follow-up around 2 years of age, although they may be at
risk of academic problems (Robertson et al., 1989; Odd
et al., 2011). Nearly all infants with severe (Stage III)
encephalopathy die or have severe disability, whereas
only half of those with moderate (Stage II) HIE develop
disability by 18 months to 2 years of age (Robertson
et al., 1989; Gluckman et al., 2005). However, studies
from before the introduction of therapeutic hypothermia
suggest that even infants who do not develop neurologic
impairment are at risk of more subtle neuromotor and
academic problems (Robertson et al., 1989).
Causes of pathologic hypoxia–ischemia
A number of events may result in asphyxia around the
time of birth. Broadly, these may be grouped as repeated
hypoxia, acute catastrophic asphyxia, and chronic hyp-
oxia (Westgate et al., 1999). Immediate, catastrophic
asphyxia contributes approximately 25% of cases of
HIE. This can be associated with cord prolapse, placental
abruption, uterine rupture, vasa previa, fetal entrapment,
such as shoulder dystocia, and occasional cases of cord
entanglements and true knots in the cord. The impact
of asphyxia during placental abruption may be greatly
potentiated by fetal blood loss leading to hypotension.
Next, repeated but relatively short periods of deep
hypoxia are associated with approximately two-thirds
of cases of HIE at term (Westgate et al., 1999). As recently
reviewed, this pattern is a direct function of the inherent
intermittent “asphyxia” of labor (Lear et al., 2018). Intra-
partum uterine contractions impair gas exchange, leading
to transient fetal hypoxemia, hypercapnia, and metabolic
acidemia (Bax and Nelson, 1993). This is mediated by
impaired uteroplacental perfusion during contractions,
as shown by reduced uterine artery blood flow velocity
(Fleischer et al., 1987; Janbu and Nesheim, 1987; Brar
et al., 1988; Li et al., 2003) and reduced placental and
intervillous perfusion (Ramsey, 1968; Sato et al., 2016;
Sinding et al., 2016). Frequent and long-lasting uterine
contractions, for example related to oxytocin augmenta-
tion, will greatly reduce placental perfusion and so impair
fetal oxygenation (Lear et al., 2018).
Further, maternal hyperthermia directly affects fetal
temperature and risk of neural injury. In pregnant
baboons, raising the maternal temperature to between
41 and 42°C increased uterine activity and increased
the normal temperature gradient between fetus and
mother from +0.47 to +0.75°C at 42°C (Morishima
et al., 1975), and was associated with severe fetal acido-
sis and hypoxia, hypotension, and tachycardia. Although
there are no randomized controlled trials of temperature
control in labor, fever during labor has been indepen-
dently associated with neonatal morbidity, including
death, neonatal seizures, encephalopathy and neonatal
stroke in multiple studies, e.g., (Impey et al., 2008;
Greenwell et al., 2012; Spain et al., 2015; Martinez-
Biarge et al., 2016).
Finally, approximately 10% of cases of moderate to
severe neonatal encephalopathy have been reported to
be associated with abnormal fetal heart rate recordings
before the start of labor, and so potentially may have been
associated with preceding fetal compromise (Westgate
et al., 1999; Jonsson et al., 2014). Chronic, moderate
hypoxia is not typical of labor; rather it is an antenatal
pattern. It may be associated with decreased fetal hemo-
globin (e.g., feto-maternal or feto-fetal hemorrhage),
placental insufficiency, and maternal causes such as pre-
eclampsia. Naturally, there is potential for antenatal and
intrapartum insults to be combined. Studies using mag-
netic resonance suggest that the great majority of infants
with acute HIE do not have established brain injury or
atrophy, suggesting that injury still occurred relatively
soon before or during delivery (Cowan et al., 2003).
CHARACTERISTICS OF PERINATAL
ASPHYXIAL ENCEPHALOPATHY
Perinatal HIE has a number of distinct characteristics
that limit extrapolation from studies of the neonatal or
adult brain. First of all, the etiology of the insult is gen-
erally global, affecting the whole fetus. Thus the fetal
systemic and cardiovascular responses are critical to
understanding the pathogenesis of injury. Second, the
insult is generally reversible, whether spontaneously or
therapeutically (e.g., delivery and resuscitation) and so
can be associated with an evolving pattern of cerebral
dysfunction and delayed injury after the insult. Third,
although the injury may be a single acute episode, it is
commonly due to repeated insults. Fourth, many of the
insults occur in the stable and warmer thermal environ-
ment of the uterus. Finally, the maturity of the brain
has a considerable effect on how neurons and glia
respond to asphyxia.
It is now understood that the fetal response to
asphyxia is not stereotypical, but rather depends upon
both the nature of the insult and the condition of the fetus
(Gunn and Bennet, 2009). In fact, it appears that the fetus
is spectacularly good at defending itself against hypoxia,
and injury occurs only in a very narrow window between
intact survival and death.
 NEONATAL ENCEPHALOPATHY AND HYPOXIC–ISCHEMIC ENCEPHALOPATHY
THE PATHOGENESIS OF CELL DEATH
What initiates neuronal injury?
At the most fundamental level, injury requires a period
of insufficient delivery of oxygen and substrates such
as glucose (and other substrates in the fetus) such that
neurons and glia cannot maintain homeostasis. If the
neurons’ supply of high-energy metabolites such as
adenosine triphosphate (ATP) is not able to be main-
tained during hypoxia, then there is failure of the
energy-dependent mechanisms of intracellular homeo-
stasis, such as the Na+/K+ ATP dependent pump. Neu-
ronal depolarization then occurs, leading to sodium and
calcium entry into cells. This creates an osmotic and
electrochemical gradient that in turn favors further cat-
ion and water entry, leading to cell swelling (cytotoxic
edema). If sufficiently severe, this may lead to immedi-
ate lysis (Johnston, 2005). The swollen neurons may
still recover, at least temporarily, if the hypoxic
insult is reversed or the environment is manipulated.
Evidence suggests that several additional factors act
to increase cell injury during and following depolariza-
tion. These include the extracellular accumulation of
excitatory amino acid neurotransmitters due to impair-
ment of energy-dependent reuptake, which promotes
further receptor-mediated cell swelling, excessive intra-
cellular calcium entry, and the generation of oxygen
free radicals and inflammatory cytokines (Wassink
et al., 2014).
Nevertheless, it is critical to appreciate that these fac-
tors appear to be injurious almost entirely in the presence
of hypoxic cell depolarization. For example, glutamate is
far more toxic during hypoxia (or mitochondrial dys-
function) than during normoxia (Deng et al., 2006). Con-
versely, in vitro, hypoxia can still trigger cell death
despite glutamate receptor blockade, through apoptotic
mechanisms (Niquet et al., 2006).
Neural adaptation to hypoxia
If blood oxygen content is reduced, but blood flow and
substrate concentrations are not impaired (i.e., moderate
hypoxia that did not impair fetal blood pressure or brain
perfusion), neural adaptations complement the cardio-
vascular adaptations to hypoxia to delay and often
completely avoid cell depolarization. First, the brain
can reduce nonobligatory energy consumption, with a
transient switch to a high-voltage low-frequency electro-
encephalographic (EEG) state that requires less oxygen
consumption (Boddy et al., 1974; Blood et al., 2003).
Next, as hypoxia–ischemia becomes more severe, neuro-
nal activity ceases completely at a threshold above that
219
which causes neuronal depolarization (Hunter et al.,
2003). This regulated suppression of metabolic rate
before energy stores are depleted during hypoxia or
ischemia, termed adaptive hypometabolism, is actively
mediated through inhibitory neurotransmitters such as
gamma-aminobutyric acid (GABA) and adenosine
(Hunter et al., 2003), and protects the brain by delaying
the onset of cell depolarization. The duration of neuronal
depolarization in turn determines the severity of injury
(Li et al., 2000).
Brain cells can use anaerobic metabolism to support
production of high-energy metabolites for a limited
time. This is extremely inefficient since whereas aero-
bic glycolysis produces 38 ATP with complete oxida-
tion of glucose, anaerobic glycolysis produces only
two ATP plus lactate, and so glucose reserves are rap-
idly consumed, with a concomitant metabolic acidosis.
Once fetal oxygen delivery has been restored, the fetus
is able to consume this circulating lactate. Many fetal
tissues, such as the heart, get a high proportion of their
substrate from sources other than glucose, particularly
lactate (Fisher et al., 1982), and the brain can oxidize
lactate when its concentration is elevated in fetal
sheep (Turbow et al., 1995) and very likely in human
newborn infants (Harris et al., 2015). However, as
lactate requires oxygen to be metabolized, this
strategy is only possible if there is sufficient time for
re-oxygenation between intermittent episodes of
hypoxia and is not possible during sustained, severe
asphyxia.
Neural responses to severe hypoxia–ischemia
In contrast, under conditions where both oxygen and
substrate delivery are reduced, the options for the
neuron are much more limited, since less oxygen
and less glucose are available to support anaerobic
metabolism. This may occur during pure ischemia
(reduced tissue blood flow, such as occurs in stroke)
and of immediate clinical relevance, during conditions
of hypoxia–ischemia, i.e., a combination of reduced
oxygen content with reduced tissue blood flow. In
the fetus, hypoxia–ischemia commonly occurs due
to hypoxic cardiac compromise, which leads to sec-
ondary hypotension and hypoperfusion. Under these
conditions, depletion of cerebral high-energy metabo-
lites will occur much more rapidly and profoundly,
while at the same time there may actually be less
acidosis, both because there is much less glucose avail-
able to be metabolized to lactate and because the insult
evolves more quickly.
These concepts help to explain the consistent observa-
tion that most cerebral injury after acute perinatal insults
220 A.J. GUNN AND M. THORESEN
occurs in association with hypotension and consequent
tissue hypoperfusion. In contrast, although asphyxial
brain injury by definition requires exposure to an anaer-
obic environment, there is only a crude correlation
between the severity of systemic acidosis and the severity
of injury in any paradigm (Gunn and Bennet, 2009).
Clinical studies confirm a similar imprecise relationship.
For example, in a single center cohort study (Vesoulis
et al., 2017), 412 of 27,028 infants had an arterial cord
blood pH
7.10. Of these, 35 of 85 infants with arterial
cord blood pH <7.00 and 34 of 327 infants with pH
between 7.00 and 7.10 developed HIE. Thus, screening
infants with pH
7.1 for HIE was highly specific
(98.7%) but only moderately sensitive (74.2%). Criti-
cally, over 25% of infants with moderate to severe HIE
had milder acidosis than this threshold and so diagnosis
of HIE was delayed.
Asphyxia is defined as the combination of impaired
respiratory gas exchange (i.e., hypoxia and hypercapnia)
accompanied by the development of metabolic acidosis.
When we think about the impact on the brain of clinical
asphyxia, it will be critical to keep in mind that this def-
inition tells us much about things that are relatively easily
measured (fetal blood gases and systemic acidosis) and
nothing about the fetal blood pressure and perfusion of
the brain, the key factors that most directly trigger brain
injury.
SYSTEMIC AND CARDIOVASCULAR
ADAPTATIONS TO ASPHYXIA
The systemic adaptations of the fetus to whole body
asphyxia are critical to outcome. Most of the experimen-
tal studies of the pathophysiology of fetal asphyxia have
been performed in the chronically instrumented fetal
sheep, in utero. The sheep is a highly precocial species,
whose neural development broadly approximates that
of term human infants around 0.8–0.85 of gestation
and of 27–30-week preterm infants at 0.7 of gestation
(McIntosh et al., 1979). The reader should note that
the baseline heart rate of the fetal sheep is approxi-
mately 20 beats per minute higher than that of the
human fetus.
Fetal adaptations and defense mechanisms
The fetus is highly adapted to intrauterine conditions,
which include low partial pressures of oxygen and
relatively limited supply of substrates compared with
postnatal life. There are no material fetal reserves of
oxygen. Although tissue myoglobin could in theory act
as an oxygen store, in practice the fetus does not
have appreciable tissue myoglobin levels except in
the heart (Longo et al., 1973). Myocardial myoglobin
concentrations do increase in hypoxic fetuses, consistent
with previous observations in postnatal animals. This
appears to represent an intracellular compensatory mech-
anism for sustaining short-term mitochondrial oxygen
delivery in a critical organ with high oxygen consump-
tion (Guiang et al., 1993).
Thus the fetus is largely dependent on a steady supply
of oxygen, and consequently it has many adaptive fea-
tures, some unique to the fetus, which help it to maximize
oxygen availability to its tissues even during moderate
hypoxia (Table 10.1). Thanks to these adaptations, the
fetus normally exists with a surplus of oxygen relative
to its needs, which provides a significant margin of safety
when oxygen delivery is impaired. These adaptive fea-
tures include: higher basal blood flow to organs, the
higher affinity of fetal hemoglobin for oxygen, greater
sensitivity of hemoglobin to acid promoting release to
tissues, the amount of oxygen that can be extracted at typ-
ical fetal oxygen tensions, the capacity to significantly
reduce energy-consuming processes, greater anaerobic
capacity in many tissues, and the capacity to redistribute
blood flow toward essential organs away from the
periphery.
Additional structural features of the fetal circulation
further support these adaptive features including the
systems of “shunts,” such as the ductus arteriosus, and
preferential blood flow streaming in the inferior vena
cava to avoid intermixing of oxygenated blood from
Table 10.1
Cardiovascular and cerebrovascular adaptations to
asphyxia
● The healthy fetus has considerable aerobic and anaerobic
reserves to successfully adapt to transient or mild hypoxia
● The fetal defenses against hypoxia depend on the type and
severity of the insult, maturation, and fetal well-being
● During mild hypoxia the fetus switches to low-frequency,
high-amplitude quiet sleep with reduced oxygen
consumption
● During moderate hypoxia, blood flow is maintained or
increased to key organs such as the heart, brain, and adrenal
gland to enable normal brain metabolism. Blood flow is
redirected within the brain toward the structures important
for autonomic function such as the brainstem
● During severe asphyxia of rapid onset, the adaptations are
similar but more extreme. Blood flow to the brain as a whole
may not be increased but is typically maintained so long as
fetal blood pressure is normal or raised
● Progression of severe asphyxia results in failure of
adaptation and progressive hypotension and hypoperfusion.
The severity of brain injury is consistently related to the
severity and duration of hypotension
 NEONATAL ENCEPHALOPATHY AND HYPOXIC–ISCHEMIC ENCEPHALOPATHY
the placenta and deoxygenated blood in the fetal venous
system. These features ensure maximal oxygen delivery
to essential organs such as the brain and heart (Reuss
et al., 1981). During mild to moderate placental restric-
tion, fetal oxygen consumption can be maintained at nor-
mal levels until uteroplacental blood flow falls below
50% (Wilkening and Meschia, 1983), with normal
removal of waste products of metabolism, mainly carbon
dioxide, water, and heat (Gilbert et al., 1985; Giussani
et al., 1993).
Cerebral oxygen consumption is even more protected
and is little changed even if arterial oxygen content falls
as low as 1.5 mM (compared with about 4 mM in the nor-
mal fetus), thanks to compensating increases in both
cerebral blood flow (CBF) and oxygen extraction
(Peeters et al., 1979). Nitric oxide (NO) and adenosine
have been shown to play a role in mediating the local
increase in CBF during hypoxia (Green et al., 1996;
Blood et al., 2003).
Etiology of asphyxia
ACUTE PROFOUND ASPHYXIA
Studies of asphyxia by definition involve both hypoxia
and hypercapnia with metabolic acidosis (e.g., see
Table 10.2). It is important to appreciate that such stud-
ies of asphyxia typically use much greater depth of
hypoxia than is possible using maternal inhalational
hypoxia. Further, asphyxia can be induced relatively
abruptly, limiting the time available for adaptation.
Complete clamping of the uterine artery or umbilical
cord leads to a rapid reduction of fetal oxygenation
over the first minute (Bennet et al., 1998). In contrast,
gradual partial occlusion induces a slow fetal metabolic
deterioration without the initial fetal cardiovascular
responses of bradycardia and hypertension, reflecting
the greater time available for fetal adaptation and lesser
degree of hypoxia (de Haan et al., 1993). During
profound asphyxia, corresponding with a severe reduc-
tion of uterine blood flow to 25% or less and a fetal
arterial oxygen content of less than 1 mmol/L, the
fetal cardiovascular responses are substantially dif-
ferent from those during moderate hypoxia or asphyxia
(Parer, 1998).
Three broad phases of the cardiovascular responses of
the fetus to such severe events can be distinguished: the
initial, rapid chemoreflex-mediated adaptations; sus-
tained adaptation that is supported by circulating,
humoral factors; and the final period of progressive hyp-
oxic decompensation ultimately terminated by profound
systemic hypotension and cerebral hypoperfusion, as
recently reviewed in depth (Bennet, 2017).
221
Table 10.2
Acidosis and the pathogenesis of cell death.
● There is no intrinsic, physiologic relationship between the
amount of systemic anaerobic metabolism (as reflected by
metabolic acidosis) and the development of neuronal injury.
The crude clinical correlation between the two simply
reflects that hypoxic–ischemic damage occurs under
anaerobic conditions
● Systemic acidosis during asphyxia is primarily related
to peripheral vasoconstriction, which acts to redistribute
blood flow to essential organs. Even when a proportion
of whole body anaerobic metabolism is central, so long
as sufficient glucose is available to neurons to support
basal energy metabolism, injury will not occur. Thus
profound arterial metabolic acidosis may still
accompany successful protection of the brain, with a
normal outcome
● During very prolonged exposure to intermittent
asphyxia, with ongoing lactic acidosis, very severe
acidosis may compromise fetal adaptation, including
cardiac contractility and blood flow redistribution,
promoting hypotension and thus causing cerebral
hypoxia–ischemia
● During acute severe insults that lead to
hypotension-impaired tissue perfusion, less glucose is
delivered to neurons and so less lactate is produced.
Thus injury may occur despite relatively modest levels
of acidosis
● Acute or chronic insults, where preexisting reduced
fetal metabolic reserve is followed by sudden onset of
deeper hypoxia, have the potential to lead to injury after
relatively short periods of acute asphyxia and moderate
acidosis
Uterine contractions and brief repeated asphyxia
Although the fetus can be exposed to a wide range of
insults during labor, the key, distinctive characteristic
of labor is brief, intermittent, repetitive episodes of
asphyxia, which are almost entirely related to uterine
contractions (Westgate et al., 2007). During normal
labor there is intermittent reduction of placental gas
exchange that is associated with a consistent fall in pH
and oxygen tension, and a rise in carbon dioxide and base
deficit in normal, uncomplicated labor (Wiberg et al.,
2006). Typically, the second stage of normal labor will
be the time of greatest hypoxic stress for the fetus and
is accompanied by a more rapid decline in pH and trans-
cutaneous oxygen tension (Modanlou et al., 1974;
Peebles et al., 1994) and rise in transcutaneous carbon
dioxide tension (Katz et al., 1987). Thus, in a technical
sense, essentially all fetuses may be said to be exposed
to “asphyxia” during labor. Fortunately, it is usually
mild and well tolerated by the fetus. Unfortunately,
222 A.J. GUNN AND M. THORESEN
both the lay public and many clinicians associate the term
asphyxia with the development of severe metabolic
acidosis, postasphyxial encephalopathy, and other end-
organ damage or death. In our haste to avoid using the
term, the normal nature of labor and its effects on the
fetus are often not fully appreciated.
Most fetuses enter labor with a large reserve of
placental capacity that helps accommodate the repeated
brief reductions in oxygen supply during contractions.
Contraction strength, frequency, and duration are the
key factors that determine the rate at which fetal asphyxia
develops during labor. Critically, the proportion of
time the uterus spends at resting tone compared with
contracting tone will determine the extent to which fetal
gas exchange can be restored between contractions
(Huch et al., 1977). Thus, any intervention that increases
the frequency and/or duration of uterine contractions
clearly places the fetus at increased risk of compromise.
For example, studies using near-infrared spectroscopy
(NIRS) show a progressive fall in cerebral oxygen satu-
ration when contractions occur more frequently than
every 2.3 min (Peebles et al., 1994). The effects of
repeated hypoxia may be amplified in vulnerable fetuses,
for example in those with preexisting placental insuffi-
ciency (Westgate et al., 2005; Wassink et al., 2013) or
fetal inflammation leading to sensitization of the brain
to hypoxia–ischemia (Eklind et al., 2005; Osredkar
et al., 2014). Conversely, even a normal fetus with nor-
mal placental function may be unable to fully adapt to
tonic contractions or uterine hyperstimulation related
to oxytocin infusion used for induction or augmentation
or prostaglandin preparations for induction of labor
(Winkler and Rath, 1999).
PATHOPHYSIOLOGIC DETERMINANTS
OF ASPHYXIAL BRAIN INJURY
Recent studies using well-defined experimental para-
digms of asphyxia in the near-term fetal sheep have
explored the relationship between the distribution of neu-
ronal damage and the type of insult. These studies sug-
gest that, while local cerebral hypoperfusion due to
hypotension is required to cause injury, a number of fac-
tors including the pattern of repetition of insults as well as
fetal factors such as maturity, preexisting metabolic state,
and cerebral temperature markedly alter the impact of the
insult on the brain (Gunn and Bennet, 2009).
Hypotension and the watershed distribution
of neuronal loss
The development of hypotension is highly associated
with neural injury during acute asphyxia. This is readily
understood since reduced perfusion will reduce supply of
glucose for anaerobic metabolism, compounding the
reduction in oxygen delivery and concentration. The
real-life importance of hypotension is supported by both
the correlation of injury with arterial blood pressure
across multiple paradigms, and by the common patterns
of neural damage.
During severe asphyxia, mean arterial blood pressure
(MAP) initially rises due to intense peripheral vasocon-
striction (Bennet, 2017). At this time cerebral blood flow
is maintained. If asphyxia is continued, MAP eventually
falls, typically after 7 min in term fetal sheep, as a func-
tion of impaired cardiac contractility and failure of
peripheral redistribution. Once MAP falls below base-
line, carotid blood flow falls in parallel, consistent with
the known, relatively narrow, cerebral autoregulatory
range in the fetus (Parer, 1998).
In the near-term fetus neural injury has been com-
monly reported in areas such as the parasagittal cortex,
the dorsal horn of the hippocampus, and the cerebellar
neocortex after a range of insults including pure ische-
mia, prolonged single complete umbilical cord occlu-
sion, and prolonged partial asphyxia and repeated brief
cord occlusion (e.g., as illustrated in Fig. 10.1, bottom
panel) (Gunn et al., 1992, 1997; Mallard et al., 1992;
de Haan et al., 1993, 1997a). These areas are watershed
zones within the borders between major cerebral arteries,
where perfusion pressure is least. In both adults and chil-
dren lesions in these areas are typically seen after sys-
temic hypotension (Torvik, 1984).
There are some data suggesting that mild white or
gray matter injury can occur even in the absence of sig-
nificant hypotension (de Haan et al., 1993; Ikeda et al.,
1998), particularly when hypoxia is very prolonged
(Rees et al., 1998). There may have been local or relative
hypoperfusion in these studies even if overall perfusion
was maintained. Nevertheless, there is a strong correla-
tion between either the depth or duration of hypotension
and the amount of neuronal loss within individual studies
of acute asphyxia (Gunn et al., 1992; de Haan et al.,
1997a, b; Ikeda et al., 1998). This is also seen between
similar asphyxial paradigms causing severe fetal acidosis
that have been manipulated to either cause fetal hypoten-
sion (Gunn et al., 1992) or not (de Haan et al., 1993). In
fetal lambs exposed to prolonged severe partial asphyxia,
as judged by the degree of metabolic compromise, neu-
ronal loss occurred only in those in whom one or more
episodes of acute hypotension occurred (Gunn et al.,
1992). In a study where an equally severe insult was
induced gradually, but titrated to maintain normal or ele-
vated blood pressure throughout the insult, no neuronal
loss was seen except in the cerebellum (de Haan
et al., 1993).
 NEONATAL ENCEPHALOPATHY AND HYPOXIC–ISCHEMIC ENCEPHALOPATHY
Fig. 10.1. The distribution of neuronal loss assessed after
3 days recovery from two different patterns of prenatal
asphyxia in near-term fetal sheep. The left panel shows the
effects of brief (1 or 2 min) cord occlusions repeated at fre-
quencies consistent with established labor. Occlusions were
terminated after a variable time, when the fetal blood pressure
fell below 20 mmHg for two successive occlusions. This insult
led to damage in the watershed regions of the parasagittal cor-
tex and cerebellum (de Haan et al., 1997a). The right panel
shows the effect of 5-min episodes of cord occlusion, repeated
four times, at intervals of 30 min (de Haan et al., 1997b). This
paradigm is associated with selective neuronal loss in the puta-
men and caudate nucleus, which are nuclei of the striatum. The
cornu ammonis and the dentate gyrus are regions of the hippo-
campus. Mean  SD.
The pattern of injury: Repeated insults
of moderate duration
One apparent exception to a general tendency to a water-
shed distribution after global asphyxial insults in the
near-term fetus is the selective neuronal loss in striatal
nuclei (putamen and caudate nucleus, Fig. 10.1, top
panel), which develops when relatively prolonged
periods of asphyxia or ischemia are repeated (Mallard
et al., 1995; de Haan et al., 1997a). Whereas 30 min of
continuous cerebral ischemia was associated with pre-
dominantly parasagittal cortical neuronal loss, with only
moderate striatal injury, 3  10 min periods of ischemia
223
led to a greater proportion of striatal injury relative to cor-
tical neuronal loss (Mallard et al., 1995). Intriguingly,
significant striatal involvement was also seen after pro-
longed partial asphyxia in which distinct episodes of bra-
dycardia and hypotension occurred (Gunn et al., 1992).
Given that both parasagittal and basal ganglia predomi-
nant patterns of injury are seen on early MRI scans in
term infants with encephalopathy (Miller et al., 2005),
intermittent severe insults are likely to be clinically
important.
The striatum is within the territory of the middle cere-
bral artery and not a watershed zone. Thus it is likely that
the pathogenesis of striatal involvement in the near-term
fetus is related to the precise timing of the relatively pro-
longed episodes of asphyxia and not to more severe local
hypoperfusion. Speculatively, the apparent vulnerability
of striatal medium-sized neurons to this type of insult
may be related to a greater release of glutamate into
the extracellular space after repeated insults compared
with a single insult of the same cumulative duration.
Consistent with this, immunohistochemical techniques
have shown that inhibitory striatal neurons were primar-
ily affected (Mallard et al., 1995).
Cerebral injury: An evolving process
The key concept to emerge from both experimental and
clinical studies is that brain cell death does not necessar-
ily occur during hypoxia or ischemia, but rather they may
precipitate a cascade of biochemical processes leading to
delayed cell death hours or even days afterwards (the sec-
ondary phase, Fig. 10.2). It is now well established in
term infants and animals that there can be considerable
cell survival and recovery of oxidative metabolism after
severe HI in a so-called latent phase, followed by pro-
gressive secondary failure of oxidative metabolism and
evolution of cell death over hours to days (Wyatt et al.,
1989; Lorek et al., 1994).
During the immediate period of hypoxia–ischemia
(which may be termed the primary phase of the insult)
high-energy metabolites are depleted, with progres-
sive depolarization of cells, severe cytotoxic edema
(cell swelling, e.g., see Fig. 10.3) (Gunn et al.,
1997) and extracellular accumulation of excitatory
amino acids due to failure of reuptake by astroglia
and excessive depolarization mediated release (Tan
et al., 1996). Although neurons may die during a
sufficiently prolonged period of ischemia or asphyxia,
many neurons initially recover, at least partially, from
the insult in a so-called latent phase, only to die many
hours or even days later (secondary or delayed cell
death). Studies using magnetic resonance spectro-
scopy showed that many infants with evidence of
224 A.J. GUNN AND M. THORESEN
Phases of cerebral injury
Hypothermia

Insult Latent Secondary Tertiary

Hypoxic From 1 to 6 h From ~6 h to 3 d Months
depolarization Recovery of oxidative Mitochondrial failure Remodeling
EAAs metabolism vs residual
Seizures/EAAs Chronic
mitochondrial injury
OFRs/NO Cytotoxic edema inflammation
Programmed cell death
Calcium entry Final cell death Astrogliosis
2° inflammation
Cell lysis
Receptor hyperactivity

Reperfusion
Fig. 10.2. Flowchart illustrating the relationship between the mechanisms active in the pathophysiologically defined phases of
cerebral injury after a severe reversible hypoxic–ischemic insult. During the immediate reperfusion period, lasting approximately
30 min, cellular energy metabolism is restored, with resolution of the acute hypoxic depolarization and cell swelling. This is
followed by a latent phase, with near-normal oxidative cerebral energy metabolism, as shown by magnetic resonance spectros-
copy, but depressed electroencephalogram (EEG) activity, and often a delayed period of reduced CBF. The latent phase is believed
to be associated with induction of the intracellular components of the apoptotic cascade. This may be followed by secondary dete-
rioration with delayed seizures and cytotoxic edema, extracellular accumulation of potential cytotoxins (such as the excitatory
neurotransmitters), and, 4–15 h after the asphyxia, failure of oxidative metabolism and damage. The changes in the secondary
phase may take 3 days or more to resolve.

moderate to severe asphyxia show initial, transient
recovery of cerebral oxidative metabolism after birth,
followed by secondary deterioration as shown by
delayed cerebral energy failure from 6 to 15 h after
birth (Azzopardi et al., 1989). The severity of the
secondary deterioration is closely correlated with neu-
rodevelopmental outcome at 1 and 4 years of age
(Roth et al., 1997), and infants with encephalopathy
who do not show initial recovery of cerebral oxidative
metabolism have extremely poor outcomes (Azzopardi
et al., 1989).
An identical pattern of initial recovery of cerebral oxi-
dative metabolism followed by delayed (secondary)
energy failure is also seen after hypoxia–ischemia in
the piglet, rat, and fetal sheep and is closely correlated
to the severity of neuronal injury (Lorek et al., 1994;
Blumberg et al., 1997; Bennet et al., 2006). The timing
of energy failure after hypoxia–ischemia is tightly
coupled with the appearance of histologic brain damage
(Vannucci et al., 2004), implying that it is primarily a
function of evolving cell death.
It is this delay before secondary deterioration that
offered the tantalizing possibility that therapeutic inter-
vention after hypoxia–ischemia might be possible. More
recent studies have provided a detailed time course of the
evolution of injury. Because oxidative synthesis of ATP
is mediated through the mitochondrial electron transport
chain, loss of the terminal electron acceptor, cytochrome
oxidase (CytOx), is a close surrogate for mitochondrial
failure and loss of ability to produce high-energy phos-
phates (Tsuji et al., 1995). Continuous, noninvasive mea-
surements with NIRS demonstrate that after severe
asphyxia in fetal sheep there is initial recovery of CytOx
values, followed by a progressive fall, starting from
approximately 3–4 h and continuing until approximately
48–72 h after asphyxia (Bennet et al., 2006). Delayed
loss of mitochondrial activity was associated with a
marked increase in relative intracerebral oxygenation
consistent with impaired ability to use oxygen (Bennet
et al., 2006).
Characteristic pathophysiologic changes may be
distinguished during this critical latent phase compared
with the secondary phase (Figs. 10.2–10.4). After resto-
ration of circulation and oxygenation, the initial hypoxia-
induced impairments of cerebral oxidative metabolism,
cytotoxic edema and accumulation of excitatory
amino acids resolve over approximately 30–60 min
(Tan et al., 1996; Gunn et al., 1997). Despite normaliza-
tion of oxidative cerebral energy metabolism and mito-
chondrial activity (Bennet et al., 2006), mean
electroencephalogram (EEG) activity remains depressed
(Keogh et al., 2012), while CBF initially recovers, fol-
lowed by a transient secondary fall (Gunn et al., 1997;
Bennet et al., 2006). During the subsequent secondary
deterioration starting many hours later (typically approx-
imately 6–15 h after moderate to severe hypoxia–
ischemia) delayed seizures develop and then continue
for several days (Gunn et al., 1997; Bennet et al., 2006),
 NEONATAL ENCEPHALOPATHY AND HYPOXIC–ISCHEMIC ENCEPHALOPATHY
Fig. 10.3. Changes in fetal extradural temperature (top panel),
cortical impedance (middle) and electroencephalogram (EEG)
power (bottom), after 30 min of global cerebral ischemia in the
near-term fetal sheep. Impedance is a measure of cell swelling
in the parietal cortex. Fetuses received either sham-ischemia plus
sham cooling (), or ischemia with sham cooling (●) or cerebral
hypothermia ( ) started 3 h after reperfusion and continued until
72 h (shown by blue bar). The end of cerebral ischemia is shown
by vertical arrow at time 0. The hypothermia group shows
complete suppression of the secondary rise in impedance
(i.e., it prevented delayed cell swelling), and substantially
better recovery of EEG intensity after resolution of delayed
seizures. Secondary hypoperfusion is extended by hypothermia
but resolves spontaneously despite continued cooling for 72 h
(not shown). Data derived from Davidson, J.O., Draghi, V.,
Whitham, S., et al., 2018b. How long is sufficient for optimal
neuroprotection with cerebral cooling after ischemia in fetal
sheep? J Cereb Blood Flow Metab 38, 1047–1059; Davidson,
J.O., Wassink, G., Yuill, C.A., et al., 2015b. How long is too
long for cerebral cooling after ischemia in fetal sheep? J Cereb
Blood Flow Metab 35, 751–758. Mean  SEM, *P < 0.05.
accompanied by secondary cytotoxic edema (cell
swelling) (Gunn et al., 1997), accumulation of excito-
toxins (Tan et al., 1996), failure of cerebral mitochondrial
activity (Lorek et al., 1994; Bennet et al., 2006), and,
225
ultimately, cell death (Gunn et al., 1997). In contrast,
secondary edema and seizures are not seen after milder
insults that do not cause cortical injury (Williams
et al., 1991).
It is important to appreciate that the severity of
HI markedly affects the speed of evolution of cell
death. For example, in 21-day-old rat pups (broadly
equivalent to late infancy in humans), 15 min of hyp-
oxia–ischemia was associated with selective neuronal
death in an apoptotic morphology from 3 days after
hypoxia (Beilharz et al., 1995). By contrast, after
60 min of hypoxia–ischemia, some cells showed DNA
degradation by 10 h after hypoxia, and widespread cor-
tical necrosis developed after 24 h. Moreover, as well
as evolving over time, cell death spreads outward from
the most severely affected regions toward less severely
affected regions (Thornton et al., 1998). In piglets
exposed to transient HI, the cerebral apparent diffusion
coefficients (ADCs) normalized almost completely by
2 h after resuscitation, followed in the majority of ani-
mals by a fall in ADC beginning in the parasagittal cortex
and then spreading through the brain. This pattern likely
reflects both severity of injury and active mechanisms
including opening of astrocytic connexin hemichannels
on the cell surface, which can facilitate waves of spread-
ing depression that can trigger cell death in less injured
tissues (Davidson et al., 2015a).
The secondary phase resolves over 3 days after
severe hypoxia–ischemia into a tertiary phase of ongoing
injury, involving repair and reorganization that may last
weeks to months and even years (Hagberg et al., 2015;
Bennet et al., 2018). Chronic inflammation and epige-
netic changes lasting for weeks to months after injury
may impair optimal neurorepair and functional recovery
(Fleiss and Gressens, 2012; Bennet et al., 2018; Galinsky
et al., 2018). Key neuroprotection strategies in this phase
include alleviating chronic local inflammation and
stimulation of endogenous factors that can support pro-
liferation, migration, and maturation of glia and neurons
(Hagberg et al., 2015; Bennet et al., 2018). Treatments
such as stem cell therapy potentially may be beneficial
in this phase, given their multimodal effects in
reducing neural inflammation and promoting release of
trophic factors (Fleiss et al., 2014; van den Heuij et al.,
2017; Bennet et al., 2018). Further, there is a clear
role for postnatal neurorehabilitation for optimizing
development of the neural network (Pitcher et al.,
2009; Maitre, 2015). Importantly, as part of the chal-
lenges to improving overall outcomes after HIE, early
and accurate diagnosis of movement disorders such as
cerebral palsy will make a significant difference in
providing the right neurorehabilitation treatment, at the
right time (Novak et al., 2017).
226 A.J. GUNN AND M. THORESEN
These concepts, that an acute, global insult can
trigger evolving injury and that characteristic events
are seen at different times after the insult, are central
to understanding the causes and treatment of neonatal
encephalopathy.
THERAPEUTIC HYPOTHERMIA
Compelling experimental evidence from multiple pre-
clinical paradigms (Gunn and Thoresen, 2006) led to
multiple randomized controlled trials (RCTs) of mild
induced hypothermia for moderate to severe neonatal
HIE. The first large animal neonatal study, for example,
showed that mild hypothermia, reducing core tempera-
ture from 38.5 to 35°C, started quickly after hypoxia–
ischemia in newborn piglets and continued for 12 h,
prevented the delayed loss of high-energy phosphates
that developed in normothermic animals. This protection
persisted to 72 h after hypoxia–ischemia (Fig. 10.4)
(Thoresen et al., 1995).
A systematic meta-analysis of 11 RCTs of either
selective head cooling or whole body cooling initiated
within 6 h of birth, involving 1505 term and late preterm
infants with moderate/severe HIE, found highly consis-
tent beneficial effects after hypothermia (Jacobs et al.,
2013). Mild hypothermia was associated with reduced
mortality or major neurodevelopmental disability by
18 months of age (relative risk (RR) 0.75 (95% confi-
dence interval (CI) 0.68–0.83)). Cooling reduced mortal-
ity (RR 0.75 (95% CI 0.64–0.88), 11 studies, 1468
infants), with reduced neurodevelopmental disability in
survivors (typical RR 0.77 (95% CI 0.63–0.94), 8 stud-
ies, 917 infants).
Long-term follow-up of these studies is ongoing; the
available evidence suggests a similar improvement in
outcomes in middle childhood after mild induced hypo-
thermia for HIE (Guillet et al., 2012; Shankaran et al.,
2012; Azzopardi et al., 2014). For example, the Total
Body Hypothermia for Neonatal Encephalopathy Trial
has shown that significantly more children in the mild
hypothermia group survived with an IQ score of 85 or
more compared to the control group (52% vs 39%, RR
1.31, P ¼ 0.04), and that more children in the hypother-
mia group survived without neurologic abnormalities
than in the control group (45% vs 28%, RR 1.60, CI:
1.15, 2.22) (Azzopardi et al., 2014). Further, there was
a significant reduction in the risk of cerebral palsy
(21% vs 36%, P ¼ 0.03) and of moderate or severe dis-
ability (22% vs 37%, P ¼ 0.03). Moreover, recent cohort
studies of infants cooled for HIE showed a lower inci-
dence of epilepsy at 2 years of age compared with the
cooling trials (Liu et al., 2017) as well as reduced severity

Fig. 10.4. Mild hypothermia prevents secondary energy failure. One-day-old anesthetized term newborn pigs underwent bilateral
carotid occlusion with global mild hypoxia until ATP levels, as analyzed by spectroscopy, had fallen to at least 30% of normal
values (Lorek et al., 1994; Thoresen et al., 1995). Proton and phosphorous spectroscopy was undertaken at preset time-points for up
to 72 h. The pigs were ventilated and fed intravenously inside the bore of the magnet during this time. There were three groups,
anesthetized sham controls without an insult (triangles, dotted line), hypoxia–ischemia followed by normothermic recovery for
48 h (open circles, dashed line), and hypoxia–ischemia followed by hypothermia for 12 h (core temperature reduced by 3.5°C)
starting after reoxygenation and reperfusion, then normothermic recovery after hypothermia for a total of 64 h (black diamonds,
solid line); 12 h of hypothermia ameliorated the secondary energy failure seen during normothermic recovery.
 NEONATAL ENCEPHALOPATHY AND HYPOXIC–ISCHEMIC ENCEPHALOPATHY
of cerebral palsy (Jary et al., 2015). The reader should
note that it is not possible to exclude the possibility that
infants with less severe HIE were recruited once thera-
peutic hypothermia became standard care. Nevertheless,
these studies used the same entry criteria for hypothermia
as the original cooling trials, including amplitude inte-
grated EEG monitoring.
IS IT POSSIBLE TO FURTHER OPTIMIZE
THERAPEUTIC HYPOTHERMIA?
Despite significantly reducing death and disability, cur-
rent protocols for therapeutic hypothermia are only par-
tially neuroprotective, with a number needed to treat of 7
((95% CI 5–10), 8 studies, 1344 infants) (Jacobs et al.,
2013). As reviewed, the experimental efficacy of hypo-
thermia is highly dependent on the timing of initiation,
depth, and duration of cooling (Wassink et al., 2018).
Thus, potentially, it may be possible to further optimize
regimens for therapeutic hypothermia.
At the same time, it is important to appreciate that
there is some evidence that outcomes may have
improved further now that therapeutic hypothermia is
routine care for moderate to severe HIE. In a recent ran-
domized controlled trial of cooling strategies, the rate of
death or disability at 18 months of age in infants treated
with cooling to 33.5°C for 72 h was 29.3% (Shankaran
et al., 2017) compared with 44% in infants receiving
the same cooling protocol and recruited using the same
criteria in a previous trial (Shankaran et al., 2005). The
factors behind this apparent improvement are not known.
Speculatively, it might be related to recruiting infants
with less severe HIE as shown by less severe Apgar
scores (Thoresen et al., 2013), but it might also partly
be related to earlier initiation of cooling, with increasing
use of passive cooling while infants are assessed for
active cooling (Lemyre et al., 2017). A generic clinical
hypothermia protocol is shown in Supplementary
Material in the online version at https://doi.org/
10.1016/B978-0-444-64029-1.00010-2.
The timing of initiation of hypothermia
There is highly consistent evidence that hypothermia
must be started as early as possible within the latent
phase for optimal benefit. This concept is highly consis-
tent with the finding of progressive mitochondrial failure
during the latent phase demonstrated by magnetic reso-
nance spectroscopy after moderate to severe hypoxia–
ischemia in human infants (Azzopardi et al., 1989;
Roth et al., 1997) and piglets (Thoresen et al., 1995;
Fig. 10.4). Similarly, in preterm fetal sheep, levels of
oxidized mitochondrial cytochrome oxidase levels were
normal to elevated in the first 3 h after severe asphyxia
227
and then progressively fell (Bennet et al., 2006). Imme-
diate initiation of hypothermia has been shown to be
protective in many studies (Wassink et al., 2018). For
example, in anesthetized piglets exposed to either hyp-
oxia with bilateral carotid ligation or to hypoxia with
hypotension, either 12 h of mild whole body hypother-
mia (35°C) or 24 h of head cooling with mild systemic
hypothermia started immediately after hypoxia pre-
vented delayed energy failure, reduced neuronal loss,
and suppressed posthypoxic seizures (Thoresen et al.,
1995; Tooley et al., 2003).
A sufficiently prolonged duration of hypothermia
may be protective despite delayed initiation. In the
near-term fetal sheep, moderate hypothermia induced
90 min after reperfusion from a severe episode of cerebral
ischemia, in the early latent phase, and continued until
72 h after ischemia prevented secondary cytotoxic edema
and improved electroencephalographic recovery (e.g.,
see Fig. 10.3) (Gunn et al., 1997). There was a concom-
itant substantial reduction in cortical infarction and
improvement in neuronal loss scores in all regions
(Fig. 10.5). Similar neuroprotective effects were seen
when treatment was delayed until 3 h after the end of
ischemia (Davidson et al., 2018b). However, when the
start of hypothermia was delayed until just before the
onset of secondary seizures (5.5 h after reperfusion) only
partial neuroprotection was seen (Gunn et al., 1998).
With further delay until after seizures were established
(8.5 h after reperfusion), there was no electrophysiologic
or overall histologic protection with cooling (Gunn
et al., 1999).
Similar results have been reported in a wide range
of species and paradigms. For example, in unanesthe-
tized 21-day-old rat pups mild hypothermia (2–3°C
decrease in brain temperature) for 72 h after hypoxia–
ischemia prevented cortical infarction, whereas cooling
delayed until 6 h after the insult had an intermediate,
nonsignificant effect (Sirimanne et al., 1996). More
recently, in 7-day-old rat pups, hypothermia was found
to be notably protective after a “moderate” duration of
hypoxia–ischemia (90 min) (Sabir et al., 2012), such
that immediate induction of hypothermia after moderate
hypoxia–ischemia significantly reduced the area of
cortical infarction (P < 0.05), with a linear loss of
effect with greater delay in starting cooling, up to 6 h
of delay (Fig. 10.6A). This window of opportunity is
strikingly similar to the studies in fetal sheep noted
previously (Fig. 10.5). By contrast, even immediate
hypothermia did not improve outcomes after very pro-
longed hypoxia–ischemia (150 min, Fig. 10.6B). This
illustrates the critical point that the window of opportu-
nity for therapeutic hypothermia is critically dependent
on the severity of the primary period of hypoxia–
ischemia.
228 A.J. GUNN AND M. THORESEN

Fig. 10.5. Time-dependent neuronal rescue with delayed cerebral hypothermia. Effect of early (90 min) or delayed (5.5 h) or late
(8.5 h) cerebral cooling after global cerebral ischemia for 30 min on microscopically assessed neuronal loss in different brain
regions at 5 days after ischemia in near-term fetal sheep. A significant reduction (P < 0.001) in neuronal loss was seen in all regions
in fetuses treated with early cerebral cooling compared with sham cooled fetuses, demonstrating that delayed neuronal death can be
prevented. Cooling delayed until 5.5 h after ischemia, just before the start of the secondary phase, was associated with partial pro-
tection. Note: In the early hypothermia group, only fetuses in which the extradural temperature was maintained below 34°C for the
first 12 h are shown (Gunn et al., 1997, 1998, 1999). Mean  SEM.

Clinical evidence supports the importance of initiat-
ing hypothermia as early as possible. In a cohort study,
infants who were treated within 3 h after birth had signif-
icantly better motor outcomes than those treated after 3 h
(Thoresen et al., 2013). However, in a controlled trial,
hypothermia was only started in 12% of infants within
4 h of birth; at that time many infants were already show-
ing electrographic seizures (Gluckman et al., 2005). This
is earlier onset of seizures than typically seen in preclin-
ical studies (Williams et al., 1990), consistent with clin-
ical evidence that many hypoxic–ischemic insults evolve
over time before birth and so the timing of the insult is
often not clearly known (Westgate et al., 1999). More-
over, accurate staging of encephalopathy can be difficult
in the critical latent phase of injury, and so diagnosis may
be delayed until after the onset of seizure activity, by
which time the efficacy of hypothermia is greatly
reduced.
How deep is too deep?
The critical depth of hypothermia required for protection
may be affected by multiple factors, including the delay
before initiation and the severity and nature of the insult.
There is some evidence from studies of moderate to
severe ischemia that when cooling was delayed until
6 h, in both adult rodents and fetal sheep, greater func-
tional and histological neuroprotection was seen with a
5°C reduction than with 3°C (Gunn et al., 1997;
Colbourne et al., 1998). By contrast, in 7-day-old rat pups,
cooled to 33.5, 32, 30, 26, or 18°C for 5 h after hypoxia–
ischemia, there was no additional protection with temper-
atures below 33.5°C (Wood et al., 2016). Similarly, in
neonatal piglets, whole body hypothermia with a reduc-
tion in body temperature of either 3.5°C or 5°C was
associated with significant (and highly similar) overall
neuroprotection, whereas a reduction of 8°C was
associated with loss of protection after global cerebral
ischemia (Alonso-Alconada et al., 2015). Finally, a large
randomized controlled clinical trial has confirmed that
cooling infants with moderate to severe HIE to 32°C
instead of 33.5°C did not further reduce death or moderate
or severe disability at 18 months of age (Shankaran et al.,
2017). These consistent clinical and preclinical findings
suggest that there is a relatively broad range of tempera-
tures beneficial for the brain after hypoxia–ischemia, and
that, reassuringly, it should not be generally necessary to
reduce core temperatures by more than 3.5°C.
If some is good, is more better?
There is now compelling animal evidence that continu-
ing cooling for approximately 72 h provides optimal neu-
roprotection (Davidson et al., 2015b, 2018b). Broadly,
and critically for clinical practice, the greater the delay
before starting cooling, or the more severe the insult,
the greater the duration of cooling required for protec-
tion. For example, in adult gerbils when the delay before
 NEONATAL ENCEPHALOPATHY AND HYPOXIC–ISCHEMIC ENCEPHALOPATHY 229

Fig. 10.6. Effect of delay before initiating hypothermia and severity of hypoxia–ischemia on hypothermic neuroprotection. This
figure shows area loss in the ligated hemisphere after 1 week recovery in 7-day-old rats undergoing unilateral carotid ligation
followed by global inhalational hypoxia for defined times (Sabir et al., 2012). Five hours of hypothermia was initiated immediately,
or 3, 6, or 12 h after HI. Panel (A, top) shows time-dependent neuroprotection with postinsult hypothermia after a moderate period
of HI (8% O2 for 90 min with the environmental temperature maintained at 36°C), such that the sooner hypothermia was started, the
greater the reduction in area of infarction. Panel (B) shows that when the rat pups were exposed to a severe period of HI (8% O2 for
150 min with the environmental temperature maintained at 37°C), there was increased mortality and no neuroprotection from hypo-
thermia. Indeed, hypothermia started 12 h after the insult was associated with increased loss of brain area.

initiating a 24-h period of cooling was increased from 1
to 4 h, neuroprotection in the CA1 region of the hippo-
campus after 6 months recovery fell from 70% to 12%
(Colbourne and Corbett, 1995). Subsequent studies dem-
onstrated that protection could be restored by extending
the duration of moderate (32–34°C) hypothermia to 48 h
or more, even when the start of cooling was delayed until
6 h after reperfusion (Colbourne et al., 2000). Similarly,
in the near-term fetal sheep, cooling started after 5.5 h
and continued until 72 h was still partially protective
(Gunn et al., 1998) (Fig. 10.5). Finally, in the same par-
adigm, although delayed cooling, from 3 h after ischemia
until 48 h, was partially protective, it was substantially
less effective for both recovery of EEG power and neu-
ronal survival than cooling for 72 h (Davidson
et al., 2018b).
Given this compelling evidence that hypothermia must
be continued for at least 72 h in large animals and humans,
there has been interest in whether further prolonging the
duration of hypothermia may be associated with greater
benefit. However, in the term-equivalent fetal sheep when
delayed hypothermia starting 3 h after ischemia was pro-
longed from 3 to 5 days, there was no further improvement
in electrophysiologic recovery or neuronal survival or
230 A.J. GUNN AND M. THORESEN
reduction in cortical microglial induction (Davidson et al.,
2015b). Indeed, extended cooling was associated with a
small reduction in neuronal survival in the parasagittal cor-
tex and the dentate gyrus. Consistent with these experi-
mental data, a recent clinical trial of prolonged duration
and increased depth of therapeutic hypothermia was aban-
doned due to lack of effect and safety concerns (Shankaran
et al., 2014, 2017). The adjusted risk ratio for death in the
neonatal intensive care unit after cooling for 120 h com-
pared to 72h was 1.37 (95% confidence interval (CI):
0.92, 2.04) and for 32°C compared to 33.5°C was 1.24
(CI: 0.69, 2.25). Further, there was no significant overall
effect of longer or deeper cooling on death or disability
at a mean age of 18 months (Shankaran et al., 2017).
These studies suggest that current protocols of cooling
to 33.5°C for 72 h are reasonably close to optimal. Thus
the most effective way to further improve outcomes from
therapeutic hypothermia in infants with HIE is to initiate
cooling earlier, as soon as possible in the first 6 h after
birth. Alternatively, cooling plus other pharmacologic
neuroprotective agents may enable further improvements
in outcome. There is currently considerable interest in
such options, based on the endogenous induction of
potentially neuroprotective compounds in the body as
well as exogenous agents (Davidson et al., 2018a).
Is there benefit from cooling more than 6 h
after birth?
The preclinical and clinical studies reviewed previously
consistently suggest that hypothermia should be started
as early as possible in the first 6 h of life to achieve opti-
mal outcomes. However, some infants are unable to be
started within this time window, because of late diagnosis
or being outborn in areas that cannot provide support for
cooling. Even though it is not optimal, should these
infants be offered therapeutic hypothermia after 6 h of
life? A recent RCT conducted by the Neonatal Research
Network centers in the United States compared 83 term
infants who were cooled starting 6–24 h after birth at
mean  SD 16  5 h, and 85 noncooled infants (range,
36.5–37.3°C). Death or moderate to severe disability
were seen in 24.4% of cooled infants, compared to
27.9% of noncooled infants. This difference is not signif-
icant (P ¼ 0.23) (Laptook et al., 2017). The authors sug-
gest, using a Bayesian analysis that there was a 76%
probability of reduced death or disability, with a small
benefit of 1%–3%. Given the very small possible effect
size and that potential harm was not quantified, cooling
after 6 h cannot be recommended.It remains critical to
focus on initiating treatment as early as possible within
the first 6 h after birth.
Should we cool infants with “mild” HIE?
The large RCTs of therapeutic hypothermia excluded
infants who had “mild” HIE in the first 6 h of life in order
to increase the rates of unfavorable outcome, and so the
potential benefit of treating these infants with therapeutic
hypothermia is unknown. There is now evidence from
cohort studies that some infants with mild HIE as defined
using clinical criteria only in the first 6 h of life have
some risk of disability. The exact results have been rather
variable, likely because of variable criteria for “mild,”
retrospective identification, less formal neurologic
examinations than used in the prospective trials, or not
using aEEG criteria. It is critical to appreciate that some
of these infants would very likely have been classified as
having evolved to Stage II (moderate) encephalopathy by
Sarnat and Sarnat by 24 h (Sarnat and Sarnat, 1976),
which was based on longitudinal assessment of neuro-
logic progress until hospital discharge or death plus mul-
timodal assessment, typically including a formal EEG
and imaging. Thus, at present, it is not really possible
to accurately define mild HIE in the first 6 h of life.
It is also important to appreciate that the definition of
mild HIE varied between studies. The CoolCap and
TOBY studies simply specified the clinical Sarnat cri-
teria on a gestalt basis, but also required that infants have
moderate to severe changes on an aEEG recording before
randomization (Gluckman et al., 2005; Azzopardi et al.,
2009). Interestingly, the NICHD trial, which used only
clinical criteria, effectively excluded infants who
did not have at least three criteria that are considered
to be moderate/severe signs of encephalopathy
(Shankaran et al., 2005). Thus, infants with 1 or 2 mod-
erate or severe criteria were defined as having “mild”
HIE. This suggests that some of these infants actually
had “moderate” HIE even though they were excluded
from the trial, and this raises the important possibility that
it may be possible to refine the clinical criteria to more
reliably identify infants who are at risk of disability.
Alternatively, potentially adding aEEG criteria might
also improve the reliability of assessment.
One meta-analysis of studies with well-defined HIE
grading at birth and standardized neurodevelopmental
assessment at 18 months or older suggested that
86/341 (25%) of infants with “mild” HIE in the first
6 h of life had an adverse outcome (Conway et al.,
2018). Adverse outcome was defined as death, cerebral
palsy, or neurodevelopmental test scores that were more
than 1 standard deviation below the mean. Although
most of these studies recruited infants solely on clinical
criteria, a prospective cohort study of infants who were
not treated with therapeutic hypothermia suggested that
infants with mild HIE, determined by both early EEG and
 NEONATAL ENCEPHALOPATHY AND HYPOXIC–ISCHEMIC ENCEPHALOPATHY
clinical examination, had adverse cognitive and neuro-
motor outcomes at 5 years of age compared to healthy
controls (Murray et al., 2016). Although survival was
much greater after mild than moderate or severe HIE, sur-
vivors showed no significant difference in cognitive out-
comes between those who had had mild compared to
moderate HIE.
Given that this population of infants with “mild” HIE
in the first 6 h is heterogeneous, the balance of clinical
risk and benefit is unclear. Treating all cases of “mild”
HIE would lead to a considerable increase in numbers
of infants being separated from their parents for at least
3 days, receiving invasive treatments such as central
lines, invasive respiratory support, sedation, and delayed
oral feeding.
It is reasonable to reflect that there is evidence from
young rodents that therapeutic hypothermia seems to
be more protective after milder hypoxia–ischemia
(Wassink et al., 2014). Moreover, there is evidence that
neuronal degeneration develops more slowly after a
shorter, less severe period of hypoxia–ischemia com-
pared with severe insults leading to infarction
(Beilharz et al., 1995). In adult rodents, early initiation
of cooling within 1 h and continued for 12–24 h after
brief ischemia in adult rodents substantially reduced
CA1 necrosis at 10 and 30 days and improved learning
in an open field and T-maze after 6 months recovery
(Colbourne and Corbett, 1994, 1995). By contrast, in
adult gerbils, when the delay before initiating a 24-h
period of cooling was increased from 1 to 4 h after cere-
bral ischemia, neuroprotection in the CA1 field of the
hippocampus at 6 months of recovery fell from 70% to
12% (Colbourne and Corbett, 1995). Neuroprotection
could be almost completely restored by extending the
interval of moderate hypothermia to 48 h plus, even when
the start of cooling was delayed until 6 h after reperfusion
(Colbourne et al., 2000).
Taken as a whole, these data strongly support that
established protocols for therapeutic hypothermia are
likely to even more effectively reduce cell loss in infants
with milder clinical HIE in the first 6 h of life than those
who were included in the trials. Speculatively, the data
also suggest the intriguing hypothesis that a shorter
interval of 24 h of therapeutic hypothermia might be
beneficial for mild HIE, provided that it could be initi-
ated shortly after birth. Alternatively, it is possible that
early treatment with neuroprotective agents such as
erythropoietin that are already in clinical trial might
be effective as monotherapy for mild HIE (Wu et al.,
2016). Given that there are roughly as many infants with
mild HIE as there are with moderate to severe HIE, it is
critical that the benefits of treatment for this group
should be formally tested.
231
THERAPEUTIC TARGETS FOR
NEUROPROTECTION
The clinical and experimental data reviewed previously
suggest that the latent phase between the initial insult and
secondary energy failure corresponds with the poten-
tially reversible activation of cell death processes. Major
initial triggers of the delayed death cascade during expo-
sure to hypoxia–ischemia include exposure to oxygen
free radical toxicity, excessive levels of excitatory amino
acids, and intracellular calcium accumulation down the
concentration gradient due to failure of energy-
dependent pumps during hypoxia and opening of chan-
nels linked to the excitatory neurotransmitters (Johnston,
2005). However, these events rapidly resolve during
reperfusion from the insult and thus cannot readily be
related to the effects of postinsult interventions such as
cooling. It is striking that even in vitro neuronal degen-
eration can be prevented by cooling initiated well after
exposure to an insult (Bruno et al., 1994). Thus, the crit-
ical effects of hypothermia must be on secondary conse-
quences of hypoxia–ischemia, such as the intracellular
progression of programmed cell death (apoptosis), the
inflammatory reaction, and abnormal receptor activity
(Wassink et al., 2014).
Programmed cell death
There is good histologic evidence that activation of pre-
existing programmed cell death pathways is a significant
contributor to posthypoxic cell death in the developing
human brain (Thornton et al., 2017). We now know that
posthypoxic–ischemic cell death is not purely apoptotic,
but rather includes elements of both apoptotic and
necrotic processes, with one or the other being most
prominent depending on factors such as maturity and
the severity of insult (Northington et al., 2007). Consis-
tent with the hypothesis that apoptotic processes are a
key therapeutic target, postinsult hypothermia started
after severe hypoxia–ischemia was reported to reduce
apoptotic cell death, but not necrotic cell death, in the
piglet (Edwards et al., 1995). Similarly, protection with
posthypoxic–ischemic hypothermia in fetal sheep has
been closely linked with suppression of activated
caspase-3 (Bennet et al., 2007).
Encouragingly, a phase II double-blinded, placebo-
controlled randomized trial of multiple dose treatment
with recombinant erythropoietin (rEpo, 1000 U/kg intra-
venously, at 1, 2, 3, 5, and 7 days of age) or placebo in
infants with moderate to severe HIE receiving therapeu-
tic hypothermia suggested additional benefit (Wu et al.,
2016). Infants receiving rEpo had significantly reduced
brain injury scores on MRI at a mean of 5.1 days and less
232 A.J. GUNN AND M. THORESEN
frequent moderate to severe injury in multiple regions,
with improved motor scores at 1 year of age. Phase 3
studies to confirm these findings are in progress.
Inflammatory second messengers
Brain injury leads to induction of the inflammatory cas-
cade with increased release of cytokines (Hagberg et al.,
2005). These compounds are believed to exacerbate
delayed injury, whether by direct neurotoxicity and induc-
tion of apoptosis or by promoting stimulation of leukocyte
adhesion and infiltration into the ischemic brain. Experi-
mentally, cooling can potently suppress this inflammatory
reaction (Gunn and Thoresen, 2006). For example,
in vitro, hypothermia inhibits proliferation, superoxide,
and nitric oxide production by cultured microglia, and
in adult rats, hypothermia suppresses the posttraumatic
release of interleukin-1b and accumulation of polymor-
phonuclear leukocytes. Similarly, neuroprotection with
postinsult hypothermia was associated with suppression
of microglial activation in fetal sheep (Roelfsema et al.,
2004; Bennet et al., 2007; Davidson et al., 2015b).
Other receptor and nonreceptor-mediated toxic fac-
tors are likely to contribute to neural injury in the latent
phase. For example, there is some evidence from the pre-
term fetal sheep for delayed production of oxygen free
radicals after hypoxia–ischemia (Welin et al., 2005),
which may particularly be associated with death of
oligodendroglia.
Excitotoxity after hypoxia–ischemia
Pathologically elevated levels of extracellular excitatory
amino acids such as glutamate are seen in biphasic
pattern. The initial increase occurs during hypoxia–
ischemia, which resolves rapidly after reperfusion to
control values (Tan et al., 1996). Levels remain low
throughout the latent phase and then secondarily rise many
hours later, in association with delayed seizures and
cytotoxic edema (Tan et al., 1996; Fraser et al., 2008). In
term-equivalent fetal sheep, for example, intense seizures
are seen from approximately 9  2 to 30  3 h after cerebral
ischemia; completely suppressing these large amplitude
seizures with a selective glutamate antagonist was asso-
ciated with very limited reduction in neuronal damage in
more mildly affected regions, but no effect on infarction
of the parasagittal cortex and no improvement in recovery
of EEG activity (Tan et al., 1992). Moreover, combined
antiglutamate treatment and mild whole body hypother-
mia after severe asphyxia did not show additive neuro-
protection in fetal sheep (George et al., 2012).
The noble gas xenon provides competitive binding at
the glycine binding site of the NMDA subtype of gluta-
mate receptor (Dickinson et al., 2007) and so potentially
can attenuate excitotoxicity. Moreover, there is some
evidence that it can activate prosurvival kinases, such
as p-Akt and the antiapoptotic factor Bcl-2, and poten-
tially inhibit opening of the mitochondrial permeability
pore (Lobo et al., 2013). There is animal evidence of
an additive neuroprotective effect when adding xenon
to hypothermia treatment. Xenon and hypothermia
administered together, immediately or as late as 5 h after
HI, in neonatal rats significantly reduced apoptotic cell
death and loss of brain matter while improving long-term
neurologic motor function and coordination (Robertson
et al., 2012). In the newborn piglet, the combination of
xenon with whole body cooling was associated with a
75% reduction in global neuropathology after perinatal
asphyxia (Chakkarapani et al., 2010). In a similar para-
digm, others found that xenon-augmented hypothermia
reduced cerebral MRS abnormalities and cell death
markers in some brain regions compared with no treat-
ment, although the effect was not significant compared
to hypothermia alone (Faulkner et al., 2011). A recent
preliminary randomized controlled study confirmed that
it is feasible to treat infants with xenon during therapeutic
hypothermia, using up to 50% xenon for up to 18 h dur-
ing cooling, with no apparent adverse effects seen at
18 months follow-up (Dingley et al., 2014).
A subsequent study found no improvement in magnetic
resonance spectroscopy and MRI biomarkers of outcome
measured within 15 days of birth, but was limited by rel-
atively low dose and late initiation of xenon therapy
(Azzopardi et al., 2016).
CONCLUSION
Therapeutic hypothermia is now established as standard
care to improve neurologic recovery in infants with mod-
erate to severe HIE. However, current protocols are only
partially effective (Jacobs et al., 2013). Further improve-
ments in neurodevelopmental outcomes are likely to
come from combining hypothermia with endogenous
or exogenous neuroprotective agents. Endogenous neu-
roprotective compounds such as EPO and melatonin are
showing particular promise, with multiple potential ben-
efits and excellent safety records in other settings. Tanta-
lizingly, autologous or external stem cells may have
potential to promote long-term neurorepair through
reducing neural inflammation and promoting release of
trophic factors (Fleiss et al., 2014; van den Heuij et al.,
2017; Bennet et al., 2018).
While awaiting the results of further research, it is
important not to forget that the most effective way to opti-
mize treatment with hypothermia is to start treatment as
soon as possible after resuscitation (Gunn and Thoresen,
2015). EEG recordings and other early biomarkers can
help to identify patients who would benefit from treatment
in such a limited time frame (Bennet et al., 2010).
 NEONATAL ENCEPHALOPATHY AND HYPOXIC–ISCHEMIC ENCEPHALOPATHY
ACKNOWLEDGMENTS
The authors’ work reported in this review has been sup-
ported by grants from the Health Research Council of
New Zealand, Lottery Health Board of New Zealand,
the Auckland Medical Research Foundation, The
Norwegian Research Council, The Wellcome Trust,
Charitable donation through SPARKS UK, and the
Moulton Foundation.
REFERENCES
Alonso-Alconada D, Broad KD, Bainbridge A et al. (2015).
Brain cell death is reduced with cooling by 3.5 degrees
C to 5 degrees C but increased with cooling by 8.5 degrees
C in a piglet asphyxia model. Stroke 46: 275–278.
Azzopardi D, Wyatt JS, Cady EB et al. (1989). Prognosis of
newborn infants with hypoxic–ischemic brain injury
assessed by phosphorus magnetic resonance spectroscopy.
Pediatr Res 25: 445–451.
Azzopardi DV, Strohm B, Edwards AD et al. (2009). Moderate
hypothermia to treat perinatal asphyxial encephalopathy.
N Engl J Med 361: 1349–1358.
Azzopardi D, Strohm B, Marlow N et al. (2014). Effects of
hypothermia for perinatal asphyxia on childhood out-
comes. N Engl J Med 371: 140–149.
Azzopardi D, Robertson NJ, Bainbridge A et al. (2016).
Moderate hypothermia within 6 h of birth plus inhaled
xenon versus moderate hypothermia alone after birth
asphyxia (TOBY-Xe): a proof-of-concept, open-label, ran-
domised controlled trial. Lancet Neurol 15: 145–153.
Basu SK, Kaiser JR, Guffey D et al. (2016). Hypoglycaemia
and hyperglycaemia are associated with unfavourable out-
come in infants with hypoxic ischaemic encephalopathy: a
post hoc analysis of the CoolCap Study. Arch Dis Child
Fetal Neonatal Ed 101: F149–F155.
Bax M, Nelson KB (1993). Birth asphyxia: a statement. World
Federation of Neurology Group. Dev Med Child Neurol 35:
1022–1024.
Beilharz EJ, Williams CE, Dragunow M et al. (1995).
Mechanisms of delayed cell death following hypoxic–
ischemic injury in the immature rat: evidence for apoptosis
during selective neuronal loss. Brain Res Mol Brain Res 29:
1–14.
Bennet L (2017). Sex, drugs and rock and roll: tales from pre-
term fetal life. J Physiol 595: 1865–1881.
Bennet L, Peebles DM, Edwards AD et al. (1998). The cerebral
hemodynamic response to asphyxia and hypoxia in the
near-term fetal sheep as measured by near infrared spec-
troscopy. Pediatr Res 44: 951–957.
Bennet L, Roelfsema V, Pathipati P et al. (2006). Relationship
between evolving epileptiform activity and delayed loss of
mitochondrial activity after asphyxia measured by near-
infrared spectroscopy in preterm fetal sheep. J Physiol
572: 141–154.
Bennet L, Roelfsema V, George S et al. (2007). The effect of
cerebral hypothermia on white and grey matter injury
induced by severe hypoxia in preterm fetal sheep.
J Physiol 578: 491–506.
233
Bennet L, Booth L, Gunn AJ (2010). Potential biomarkers for
hypoxic–ischemic encephalopathy. Semin Fetal Neonatal
Med 15: 253–260.
Bennet L, Dhillon S, Lear CA et al. (2018). Chronic inflamma-
tion and impaired development of the preterm brain.
J Reprod Immunol 125: 45–55.
Blood AB, Hunter CJ, Power GG (2003). Adenosine mediates
decreased cerebral metabolic rate and increased cerebral
blood flow during acute moderate hypoxia in the near-term
fetal sheep. J Physiol 553: 935–945.
Blumberg RM, Cady EB, Wigglesworth JS et al. (1997).
Relation between delayed impairment of cerebral energy
metabolism and infarction following transient focal hyp-
oxia–ischaemia in the developing brain. Exp Brain Res
113: 130–137.
Boddy K, Dawes GS, Fisher R et al. (1974). Foetal respiratory
movements, electrocortical and cardiovascular responses
to hypoxaemia and hypercapnia in sheep. J Physiol 243:
599–618.
Brar HS, Platt LD, DeVore GR et al. (1988). Qualitative
assessment of maternal uterine and fetal umbilical
artery blood flow and resistance in laboring patients
by Doppler velocimetry. Am J Obstet Gynecol 158:
952–956.
Bruno VM, Goldberg MP, Dugan LL et al. (1994).
Neuroprotective effect of hypothermia in cortical cultures
exposed to oxygen-glucose deprivation or excitatory amino
acids. J Neurochem 63: 1398–1406.
Chakkarapani E, Dingley J, Liu X et al. (2010). Xenon
enhances hypothermic neuroprotection in asphyxiated
newborn pigs. Ann Neurol 68: 330–341.
Colbourne F, Corbett D (1994). Delayed and prolonged post-
ischemic hypothermia is neuroprotective in the gerbil.
Brain Res 654: 265–272.
Colbourne F, Corbett D (1995). Delayed postischemic hypo-
thermia: a six month survival study using behavioral and
histological assessments of neuroprotection. J Neurosci
15: 7250–7260.
Colbourne F, Auer RN, Sutherland GR (1998).
Characterization of postischemic behavioral deficits in ger-
bils with and without hypothermic neuroprotection. Brain
Res 803: 69–78.
Colbourne F, Corbett D, Zhao Z et al. (2000). Prolonged but
delayed postischemic hypothermia: a long-term outcome
study in the rat middle cerebral artery occlusion model.
J Cereb Blood Flow Metab 20: 1702–1708.
Conway JM, Walsh BH, Boylan GB et al. (2018). Mild hyp-
oxic ischaemic encephalopathy and long term neurodeve-
lopmental outcome—a systematic review. Early Hum
Dev 120: 80–87.
Cowan F, Rutherford M, Groenendaal F et al. (2003). Origin
and timing of brain lesions in term infants with neonatal
encephalopathy. Lancet 361: 736–742.
Davidson JO, Green CR, Bennet L et al. (2015a). Battle of the
hemichannels—connexins and pannexins in ischemic brain
injury. Int J Dev Neurosci 45: 66–74.
Davidson JO, Wassink G, Yuill CA et al. (2015b). How long is
too long for cerebral cooling after ischemia in fetal sheep?
J Cereb Blood Flow Metab 35: 751–758.
234 A.J. GUNN AND M. THORESEN
Davidson JO, Dean JM, Fraser M et al. (2018a). Perinatal brain
injury: mechanisms and therapeutic approaches. Front
Biosci (Landmark Ed) 23: 2204–2226.
Davidson JO, Draghi V, Whitham S et al. (2018b). How long is
sufficient for optimal neuroprotection with cerebral cool-
ing after ischemia in fetal sheep? J Cereb Blood Flow
Metab 38: 1047–1059.
de Haan HH, van Reempts JL, Vles JS et al. (1993). Effects of
asphyxia on the fetal lamb brain. Am J Obstet Gynecol 169:
1493–1501.
de Haan HH, Gunn AJ, Williams CE et al. (1997a). Brief
repeated umbilical cord occlusions cause sustained cyto-
toxic cerebral edema and focal infarcts in near-term fetal
lambs. Pediatr Res 41: 96–104.
de Haan HH, Gunn AJ, Williams CE et al. (1997b).
Magnesium sulfate therapy during asphyxia in near-term
fetal lambs does not compromise the fetus but does not
reduce cerebral injury. Am J Obstet Gynecol 176: 18–27.
Deng W, Yue Q, Rosenberg PA et al. (2006). Oligodendrocyte
excitotoxicity determined by local glutamate accumulation
and mitochondrial function. J Neurochem 98: 213–222.
Dickinson R, Peterson BK, Banks P et al. (2007). Competitive
inhibition at the glycine site of the N-methyl-D-aspartate
receptor by the anesthetics xenon and isoflurane: evidence
from molecular modeling and electrophysiology.
Anesthesiology 107: 756–767.
Dingley J, Tooley J, Liu X et al. (2014). Xenon ventilation dur-
ing therapeutic hypothermia in neonatal encephalopathy: a
feasibility study. Pediatrics 133: 809–818.
Edwards AD, Yue X, Squier MV et al. (1995). Specific inhi-
bition of apoptosis after cerebral hypoxia–ischaemia by
moderate post-insult hypothermia. Biochem Biophys Res
Commun 217: 1193–1199.
Eklind S, Mallard C, Arvidsson P et al. (2005).
Lipopolysaccharide induces both a primary and a second-
ary phase of sensitization in the developing rat brain.
Pediatr Res 58: 112–116.
Faulkner S, Bainbridge A, Kato T et al. (2011). Xenon aug-
mented hypothermia reduces early lactate/N-
acetylaspartate and cell death in perinatal asphyxia. Ann
Neurol 70: 133–150.
Fisher DJ, Heymann MA, Rudolph AM (1982). Fetal myocar-
dial oxygen and carbohydrate consumption during acutely
induced hypoxemia. Am J Physiol 242: H657–H661.
Fleischer A, Anyaegbunam AA, Schulman H et al. (1987).
Uterine and umbilical artery velocimetry during normal
labor. Am J Obstet Gynecol 157: 40–43.
Fleiss B, Gressens P (2012). Tertiary mechanisms of brain
damage: a new hope for treatment of cerebral palsy?
Lancet Neurol 11: 556–566.
Fleiss B, Guillot PV, Titomanlio L et al. (2014). Stem cell ther-
apy for neonatal brain injury. Clin Perinatol 41: 133–148.
Fraser M, Bennet L, van Zijl PL et al. (2008). Extracellular
amino acids and peroxidation products in the periventricu-
lar white matter during and after cerebral ischemia in pre-
term fetal sheep. J Neurochem 105: 2214–2223.
Galinsky R, Lear CA, Dean JM et al. (2018). Complex inter-
actions between hypoxia–ischemia and inflammation in
preterm brain injury. Dev Med Child Neurol 60: 126–133.
George SA, Barrett RD, Bennet L et al. (2012). Nonadditive
neuroprotection with early glutamate receptor blockade
and delayed hypothermia after asphyxia in preterm fetal
sheep. Stroke 43: 3114–3117.
Gilbert RD, Schroder H, Kawamura T et al. (1985). Heat trans-
fer pathways between fetal lamb and ewe. J Appl Physiol
59: 634–638.
Giussani DA, Spencer JA, Moore PJ et al. (1993). Afferent
and efferent components of the cardiovascular reflex
responses to acute hypoxia in term fetal sheep. J Physiol
461: 431–449.
Glass HC, Shellhaas RA, Wusthoff CJ et al. (2016).
Contemporary profile of seizures in neonates: a prospective
cohort study. J Pediatr 174: 98–103 e101.
Glass HC, Shellhaas RA, Tsuchida TN et al. (2017). Seizures
in preterm neonates: a multicenter observational cohort
study. Pediatr Neurol 72: 19–24.
Gluckman PD, Wyatt JS, Azzopardi D et al. (2005). Selective
head cooling with mild systemic hypothermia after neona-
tal encephalopathy: multicentre randomised trial. Lancet
365: 663–670.
Green LR, Bennet L, Hanson MA (1996). The role of nitric
oxide synthesis in cardiovascular responses to acute hypoxia
in the late gestation sheep fetus. J Physiol 497: 271–277.
Greenwell EA, Wyshak G, Ringer SA et al. (2012).
Intrapartum temperature elevation, epidural use, and
adverse outcome in term infants. Pediatrics 129:
e447–e454.
Guiang 3rd SF, Widness JA, Flanagan KB et al. (1993). The
relationship between fetal arterial oxygen saturation and
heart and skeletal muscle myoglobin concentrations in
the ovine fetus. J Dev Physiol 19: 99–104.
Guillet R, Edwards AD, Thoresen M et al. (2012). Seven- to
eight-year follow-up of the CoolCap trial of head
cooling for neonatal encephalopathy. Pediatr Res 71:
205–209.
Gunn AJ, Bennet L (2009). Fetal hypoxia insults and patterns
of brain injury: insights from animal models. Clin Perinatol
36: 579–593.
Gunn AJ, Thoresen M (2006). Hypothermic neuroprotection.
NeuroRx 3: 154–169.
Gunn AJ, Thoresen M (2015). Animal studies of neonatal
hypothermic neuroprotection have translated well in to
practice. Resuscitation 97: 88–90.
Gunn AJ, Parer JT, Mallard EC et al. (1992). Cerebral histo-
logic and electrocorticographic changes after asphyxia in
fetal sheep. Pediatr Res 31: 486–491.
Gunn AJ, Gunn TR, de Haan HH et al. (1997). Dramatic neu-
ronal rescue with prolonged selective head cooling after
ischemia in fetal lambs. J Clin Invest 99: 248–256.
Gunn AJ, Gunn TR, Gunning MI et al. (1998). Neuroprotection
with prolonged head cooling started before postischemic
seizures in fetal sheep. Pediatrics 102: 1098–1106.
Gunn AJ, Bennet L, Gunning MI et al. (1999). Cerebral hypo-
thermia is not neuroprotective when started after postis-
chemic seizures in fetal sheep. Pediatr Res 46: 274–280.
Hagberg H, Mallard C, Jacobsson B (2005). Role of cytokines
in preterm labour and brain injury. BJOG 112 (Suppl. 1):
16–18.
 NEONATAL ENCEPHALOPATHY AND HYPOXIC–ISCHEMIC ENCEPHALOPATHY
Hagberg H, Mallard C, Ferriero DM et al. (2015). The role of
inflammation in perinatal brain injury. Nat Rev Neurol 11:
192–208.
Harris DL, Weston PJ, Harding JE (2015). Lactate, rather than
ketones, may provide alternative cerebral fuel in hypogly-
caemic newborns. Arch Dis Child Fetal Neonatal Ed 100:
F161–F164.
Huch A, Huch R, Schneider H et al. (1977). Continuous trans-
cutaneous monitoring of fetal oxygen tension during
labour. Br J Obstet Gynaecol 84: 1–39.
Hunter CJ, Bennet L, Power GG et al. (2003). Key neuroprotec-
tive role for endogenous adenosine A1 receptor activation
during asphyxia in the fetal sheep. Stroke 34: 2240–2245.
Ikeda T, Murata Y, Quilligan EJ et al. (1998). Physiologic and
histologic changes in near-term fetal lambs exposed to
asphyxia by partial umbilical cord occlusion. Am
J Obstet Gynecol 178: 24–32.
Impey LW, Greenwood CE, Black RS et al. (2008). The rela-
tionship between intrapartum maternal fever and neonatal
acidosis as risk factors for neonatal encephalopathy. Am
J Obstet Gynecol 198: 49 e41–46.
Jacobs SE, Berg M, Hunt R et al. (2013). Cooling for newborns
with hypoxic ischaemic encephalopathy. Cochrane
Database Syst Rev 1: CD003311.
Janbu T, Nesheim BI (1987). Uterine artery blood velocities
during contractions in pregnancy and labour related to
intrauterine pressure. Br J Obstet Gynaecol 94: 1150–1155.
Jary S, Smit E, Liu X et al. (2015). Less severe cerebral palsy
outcomes in infants treated with therapeutic hypothermia.
Acta Paediatr 104: 1241–1247.
Johnston MV (2005). Excitotoxicity in perinatal brain injury.
Brain Pathol 15: 234–240.
Jonsson M, Agren J, Norden-Lindeberg S et al. (2014).
Neonatal encephalopathy and the association to asphyxia
in labor. Am J Obstet Gynecol 211: 667 e661–668.
Katz M, Lunenfeld E, Meizner I et al. (1987). The effect of the
duration of the second stage of labour on the acid-base state
of the fetus. Br J Obstet Gynaecol 94: 425–430.
Keogh MJ, Drury PP, Bennet L et al. (2012). Limited predic-
tive value of early changes in EEG spectral power for neu-
ral injury after asphyxia in preterm fetal sheep. Pediatr Res
71: 345–353.
Laptook AR, Shankaran S, Tyson JE et al. (2017). Effect of
therapeutic hypothermia initiated after 6 hours of age on
death or disability among newborns with hypoxic-ischemic
encephalopathy: A randomized clinical trial. JAMA 318:
1550–1560.
Lear CA, Wassink G, Westgate JA et al. (2018). The
peripheral chemoreflex—indefatigable guardian of
fetal physiological adaptation to labour. J Physiol 596:
5611–5623.
Lee AC, Kozuki N, Blencowe H et al. (2013). Intrapartum-
related neonatal encephalopathy incidence and impairment
at regional and global levels for 2010 with trends from
1990. Pediatr Res 74: 50–72.
Lemyre B, Ly L, Chau V et al. (2017). Initiation of passive
cooling at referring centre is most predictive of achieving
early therapeutic hypothermia in asphyxiated newborns.
Paediatr Child Health 22: 264–268.
235
Li J, Takeda Y, Hirakawa M (2000). Threshold of ischemic
depolarization for neuronal injury following four-vessel
occlusion in the rat cortex. J Neurosurg Anesthesiol 12:
247–254.
Li H, Gudmundsson S, Olofsson P (2003). Uterine artery blood
flow velocity waveforms during uterine contractions.
Ultrasound Obstet Gynecol 22: 578–585.
Liu X, Jary S, Cowan F et al. (2017). Reduced infancy and
childhood epilepsy following hypothermia-treated neona-
tal encephalopathy. Epilepsia 58: 1902–1911.
Lobo N, Yang B, Rizvi M et al. (2013). Hypothermia and
xenon: novel noble guardians in hypoxic–ischemic enceph-
alopathy? J Neurosci Res 91: 473–478.
Longo LD, Koos BJ, Power GG (1973). Fetal myoglobin:
quantitative determination and importance for oxygena-
tion. Am J Physiol 224: 1032–1036.
Lorek A, Takei Y, Cady EB et al. (1994). Delayed ("second-
ary") cerebral energy failure after acute hypoxia–ischemia
in the newborn piglet: continuous 48-hour studies by phos-
phorus magnetic resonance spectroscopy. Pediatr Res 36:
699–706.
Maitre NL (2015). Neurorehabilitation after neonatal intensive
care: evidence and challenges. Arch Dis Child Fetal
Neonatal Ed 100: F534–F540.
Mallard EC, Gunn AJ, Williams CE et al. (1992). Transient
umbilical cord occlusion causes hippocampal damage in
the fetal sheep. Am J Obstet Gynecol 167: 1423–1430.
Mallard EC, Williams CE, Johnston BM et al. (1995).
Repeated episodes of umbilical cord occlusion in fetal
sheep lead to preferential damage to the striatum and sen-
sitize the heart to further insults. Pediatr Res 37: 707–713.
Martinez-Biarge M, Cheong JL, Diez-Sebastian J et al. (2016).
Risk factors for neonatal arterial ischemic stroke: the impor-
tance of the intrapartum period. J Pediatr 173: 62–68 e61.
McIntosh GH, Baghurst KI, Potter BJ et al. (1979). Foetal
brain development in the sheep. Neuropathol Appl
Neurobiol 5: 103–114.
Miller SP, Ramaswamy V, Michelson D et al. (2005). Patterns
of brain injury in term neonatal encephalopathy. J Pediatr
146: 453–460.
Modanlou H, Yeh SY, Hon EH (1974). Fetal and neonatal
acid–base balance in normal and high-risk pregnancies:
during labor and the first hour of life. Obstet Gynecol 43:
347–353.
Morishima HO, Glaser B, Niemann WH et al. (1975). Increased
uterine activity and fetal deterioration during maternal
hyperthermia. Am J Obstet Gynecol 121: 531–538.
Murray DM, O’Connor CM, Ryan CA et al. (2016). Early EEG
grade and outcome at 5 years after mild neonatal hypoxic
ischemic encephalopathy. Pediatrics 138: e20160659.
(ePub).
Niquet J, Seo DW, Allen SG et al. (2006). Hypoxia in presence
of blockers of excitotoxicity induces a caspase-dependent
neuronal necrosis. Neuroscience 141: 77–86.
Northington FJ, Zelaya ME, O’Riordan DP et al. (2007). Failure
to complete apoptosis following neonatal hypoxia–ischemia
manifests as "continuum" phenotype of cell death and occurs
with multiple manifestations of mitochondrial dysfunction
in rodent forebrain. Neuroscience 149: 822–833.
236 A.J. GUNN AND M. THORESEN
Novak I, Morgan C, Adde L et al. (2017). Early, accurate diag-
nosis and early intervention in cerebral palsy: advances in
diagnosis and treatment. JAMA Pediatr 171: 897–907.
Odd DE, Gunnell D, Lewis G et al. (2011). Long-term impact
of poor birth condition on social and economic outcomes in
early adulthood. Pediatrics 127: e1498–e1504.
Osredkar D, Thoresen M, Maes E et al. (2014). Hypothermia
is not neuroprotective after infection-sensitized neonatal
hypoxic–ischemic brain injury. Resuscitation 85: 567–572.
Parer JT (1998). Effects of fetal asphyxia on brain cell struc-
ture and function: limits of tolerance. Comp Biochem
Physiol A Mol Integr Physiol 119: 711–716.
Peebles DM, Spencer JA, Edwards AD et al. (1994). Relation
between frequency of uterine contractions and human fetal
cerebral oxygen saturation studied during labour by near
infrared spectroscopy. Br J Obstet Gynaecol 101: 44–48.
Peeters LL, Sheldon RE, Jones Jr MD et al. (1979). Blood flow
to fetal organs as a function of arterial oxygen content. Am
J Obstet Gynecol 135: 637–646.
Pitcher JB, Robertson AL, Cockington RA et al. (2009).
Prenatal growth and early postnatal influences on adult
motor cortical excitability. Pediatrics 124: e128–e136.
Ramsey EM (1968). Uteroplacental circulation during labor.
Clin Obstet Gynaecol 11: 78–95.
Rees S, Mallard C, Breen S et al. (1998). Fetal brain injury fol-
lowing prolonged hypoxemia and placental insufficiency: a
review. Comp Biochem Physiol A Mol Integr Physiol 119:
653–660.
Reid SM, Meehan E, McIntyre S et al. (2016). Temporal trends
in cerebral palsy by impairment severity and birth gesta-
tion. Dev Med Child Neurol 58 (Suppl. 2): 25–35.
Reuss ML, Rudolph AM, Heymann MA (1981). Selective dis-
tribution of microspheres injected into the umbilical veins
and inferior venae cavae of fetal sheep. Am J Obstet
Gynecol 141: 427–432.
Robertson CM, Finer NN, Grace MG (1989). School perfor-
mance of survivors of neonatal encephalopathy associated
with birth asphyxia at term. J Pediatr 114: 753–760.
Robertson NJ, Tan S, Groenendaal F et al. (2012). Which neu-
roprotective agents are ready for bench to bedside transla-
tion in the newborn infant? J Pediatr 160: 544–552.e544.
Roelfsema V, Bennet L, George S et al. (2004). The window of
opportunity for cerebral hypothermia and white matter
injury after cerebral ischemia in near-term fetal sheep.
J Cereb Blood Flow Metab 24: 877–886.
Roth SC, Baudin J, Cady E et al. (1997). Relation of deranged
neonatal cerebral oxidative metabolism with neurodeve-
lopmental outcome and head circumference at 4 years.
Dev Med Child Neurol 39: 718–725.
Sabir H, Scull-Brown E, Liu X et al. (2012). Immediate hypo-
thermia is not neuroprotective after severe hypoxia–
ischemia and is deleterious when delayed by 12 hours in
neonatal rats. Stroke 43: 3364–3370.
Sarnat HB, Sarnat MS (1976). Neonatal encephalopathy fol-
lowing fetal distress. A clinical and electroencephalo-
graphic study. Arch Neurol 33: 696–705.
Sato M, Noguchi J, Mashima M et al. (2016). 3D power
Doppler ultrasound assessment of placental perfusion dur-
ing uterine contraction in labor. Placenta 45: 32–36.
Shankaran S, Laptook AR, Ehrenkranz RA et al. (2005).
Whole-body hypothermia for neonates with hypoxic–
ischemic encephalopathy. N Engl J Med 353: 1574–1584.
Shankaran S, Pappas A, McDonald SA et al. (2012). Childhood
outcomes after hypothermia for neonatal encephalopathy.
N Engl J Med 366: 2085–2092.
Shankaran S, Laptook AR, Pappas A et al. (2014). Effect of
depth and duration of cooling on deaths in the NICU among
neonates with hypoxic ischemic encephalopathy: a ran-
domized clinical trial. JAMA 312: 2629–2639.
Shankaran S, Laptook AR, Pappas A et al. (2017). Effect of
depth and duration of cooling on death or disability at age
18 months among neonates with hypoxic–ischemic enceph-
alopathy: a randomized clinical trial. JAMA 318: 57–67.
Sinding M, Peters DA, Frokjaer JB et al. (2016). Reduced pla-
cental oxygenation during subclinical uterine contractions
as assessed by BOLD MRI. Placenta 39: 16–20.
Sirimanne ES, Blumberg RM, Bossano D et al. (1996). The
effect of prolonged modification of cerebral temperature
on outcome after hypoxic–ischemic brain injury in the
infant rat. Pediatr Res 39: 591–597.
Spain JE, Tuuli MG, Macones GA et al. (2015). Risk factors
for serious morbidity in term nonanomalous neonates.
Am J Obstet Gynecol 212: 799 e791–797.
Tan WK, Williams CE, Gunn AJ et al. (1992). Suppression of
postischemic epileptiform activity with MK-801 improves
neural outcome in fetal sheep. Ann Neurol 32: 677–682.
Tan WK, Williams CE, During MJ et al. (1996). Accumulation
of cytotoxins during the development of seizures and
edema after hypoxic–ischemic injury in late gestation fetal
sheep. Pediatr Res 39: 791–797.
Thoresen M, Penrice J, Lorek A et al. (1995). Mild hypother-
mia after severe transient hypoxia–ischemia ameliorates
delayed cerebral energy failure in the newborn piglet.
Pediatr Res 37: 667–670.
Thoresen M, Tooley J, Liu X et al. (2013). Time is brain: start-
ing therapeutic hypothermia within three hours after birth
improves motor outcome in asphyxiated newborns.
Neonatology 104: 228–233.
Thornton JS, Ordidge RJ, Penrice J et al. (1998). Temporal and
anatomical variations of brain water apparent diffusion
coefficient in perinatal cerebral hypoxic–ischemic injury:
relationships to cerebral energy metabolism. Magn Reson
Med 39: 920–927.
Thornton C, Leaw B, Mallard C et al. (2017). Cell death in the
developing brain after hypoxia–ischemia. Front Cell
Neurosci 11: 248.
Tooley JR, Satas S, Porter H et al. (2003). Head cooling with
mild systemic hypothermia in anesthetized piglets is neuro-
protective. Ann Neurol 53: 65–72.
Torvik A (1984). The pathogenesis of watershed infarcts in the
brain. Stroke 15: 221–223.
Tsuji M, Naruse H, Volpe J et al. (1995). Reduction of cyto-
chrome aa3 measured by near-infrared spectroscopy predicts
cerebral energy loss in hypoxic piglets. Pediatr Res 37:
253–259.
Turbow RM, Curran-Everett D, Hay Jr WW et al. (1995).
Cerebral lactate metabolism in near-term fetal sheep. Am
J Physiol 269: R938–R942.
 NEONATAL ENCEPHALOPATHY AND HYPOXIC–ISCHEMIC ENCEPHALOPATHY
van den Heuij LG, Fraser M, Miller SL et al. (2017). Delayed
intranasal infusion of human amnion epithelial cells
improves white matter maturation after asphyxia in preterm
fetal sheep. J Cereb Blood Flow Metab 39 (2): 223–239.
Epub Sept: 271678X17729954.
Vannucci RC (2000). Hypoxic–ischemic encephalopathy. Am
J Perinatol 17: 113–120.
Vannucci RC, Towfighi J, Vannucci SJ (2004). Secondary
energy failure after cerebral hypoxia–ischemia in the
immature rat. J Cereb Blood Flow Metab 24: 1090–1097.
Vesoulis ZA, Liao SM, Rao R et al. (2017). Re-examining the
arterial cord blood gas pH screening criteria in neonatal
encephalopathy. Arch Dis Child Fetal Neonatal Ed 103
(4): F377–F382. Epub Sep 23.
Wassink G, Bennet L, Davidson JO et al. (2013). Pre-existing
hypoxia is associated with greater EEG suppression and
early onset of evolving seizure activity during brief repeated
asphyxia in near-term fetal sheep. PLoS One 8: e73895.
Wassink G, Gunn ER, Drury PP et al. (2014). The mechanisms
and treatment of asphyxial encephalopathy. Front Neurosci
8: 40.
Wassink G, Davidson JO, Lear CA et al. (2018). A working
model for hypothermic neuroprotection. J Physiol 596
(23): 5641–5654.
Welin AK, Sandberg M, Lindblom A et al. (2005). White mat-
ter injury following prolonged free radical formation in the
0.65 gestation fetal sheep brain. Pediatr Res 58: 100–105.
Westgate JA, Gunn AJ, Gunn TR (1999). Antecedents of neo-
natal encephalopathy with fetal acidaemia at term. BJOG
106: 774–782.
Westgate J, Wassink G, Bennet L et al. (2005). Spontaneous
hypoxia in multiple pregnancy is associated with early fetal
decompensation and greater T wave elevation during brief
237
repeated cord occlusion in near-term fetal sheep. Am
J Obstet Gynecol 193: 1526–1533.
Westgate JA, Wibbens B, Bennet L et al. (2007). The intrapar-
tum deceleration in center stage: a physiological approach
to interpretation of fetal heart rate changes in labor. Am
J Obstet Gynecol 197: 236.e1–11.
Wiberg N, Kallen K, Olofsson P (2006). Physiological devel-
opment of a mixed metabolic and respiratory umbilical
cord blood acidemia with advancing gestational age.
Early Hum Dev 82: 583–589.
Wilkening RB, Meschia G (1983). Fetal oxygen uptake, oxy-
genation, and acid–base balance as a function of uterine
blood flow. Am J Physiol 244: H749–H755.
Williams CE, Gunn AJ, Synek B et al. (1990). Delayed sei-
zures occurring with hypoxic–ischemic encephalopathy
in the fetal sheep. Pediatr Res 27: 561–565.
Williams CE, Gunn A, Gluckman PD (1991). Time course
of intracellular edema and epileptiform activity fol-
lowing prenatal cerebral ischemia in sheep. Stroke 22:
516–521.
Winkler M, Rath W (1999). A risk-benefit assessment of oxy-
tocics in obstetric practice. Drug Saf 20: 323–345.
Wood T, Osredkar D, Puchades M et al. (2016).
Treatment temperature and insult severity influence the
neuroprotective effects of therapeutic hypothermia. Sci
Rep 6: 23430.
Wu YW, Mathur AM, Chang T et al. (2016). High-dose eryth-
ropoietin and hypothermia for hypoxic–ischemic encepha-
lopathy: a phase II trial. Pediatrics 137: e20160191.
Wyatt JS, Edwards AD, Azzopardi D et al. (1989). Magnetic
resonance and near infrared spectroscopy for investigation
of perinatal hypoxic–ischaemic brain injury. Arch Dis
Child 64: 953–963.