A.J. Gunn* and M. Thoresen†
*Dept of Physiology, The University of Auckland, New Zealand;†University of Bristol, St Michaels Hospital,
Child Health, level D, Southwell Street BS2 8EG, Bristol, UK
Summary: The possibility that hypothermia during or after
resuscitation from asphyxia at birth, or cardiac arrest in adults,
might reduce evolving damage has tantalized clinicians for a
very long time. It is now known that severe hypoxia-ischemia
may not necessarily cause immediate cell death, but can pre-
cipitate a complex biochemical cascade leading to the delayed
neuronal loss. Clinically and experimentally, the key phases of
injury include a latent phase after reperfusion, with initial re-
covery of cerebral energy metabolism but EEG suppression,
followed by a secondary phase characterized by accumulation
of cytotoxins, seizures, cytotoxic edema, and failure of cerebral
oxidative metabolism starting 6 to 15 h post insult. Although
many of the secondary processes can be injurious, they appear
to be primarily epiphenomena of the ‘execution’ phase of cell
death. Studies designed around this conceptual framework have
shown that moderate cerebral hypothermia initiated as early as
possible before the onset of secondary deterioration, and con-
tinued for a sufficient duration in relation to the severity of the
cerebral injury, has been associated with potent, long-lasting
neuroprotection in both adult and perinatal species. Two large
controlled trials, one of head cooling with mild hypothermia,
and one of moderate whole body cooling have demonstrated
that post resuscitation cooling is generally safe in intensive
care, and reduces death or disability at 18 months of age after
neonatal encephalopathy. These studies, however, show that
only a subset of babies seemed to benefit. The challenge for the
future is to find ways of improving the effectiveness of
treatment. Key Words: Hypothermia, induced, hypoxic-isch-
emic encephalopathy, hypoxia.
Moderate to severe hypoxic-ischemic encephalopathy
(HIE) continues to be an important cause of acute neu-
rologic injury at birth, occurring in 2 to 3 cases per 1000
term live births.1The possibility that mild cooling might
improve recovery from HIE is a ‘dream revisited’. John
Floyer suggested over 300 years ago that it might be
beneficial for babies to get a little cold after birth.2As
recently noted,3it was observed in antiquity that exposed
babies could remain viable for prolonged periods, and
packing in ice and snow was advocated for the wound-
ed.4Napoleon’s Surgeon General, Baron Larrey,5re-
ported that injured soldiers died more rapidly if they
were kept warm by being put closer to a fire.
Modern clinical interest in hypothermia began in the
1930s and 1940s with reports of successful resuscitation
of hypothermic drowning victims, even after prolonged
periods of asphyxia. In the 1940s Temple Fay reported
treating patients with severe head injury and intracere-
bral aneurysms with hypothermia induced by cold baths
and by opening the windows in winter.6Experimentally,
moderate hypothermia improved neurological recovery
in dogs exposed to focal brain ischemia and injury.7,8
Similarly, hypothermia in perinatal rodents greatly ex-
tended the ‘time to last gasp’ during hypoxia and im-
proved subsequent functional outcome.9
In retrospect, these experimental studies addressed the
effect of cooling during severe hypoxia, which has now
been extensively proven to be associated with potent,
dose-related, long-lasting neuroprotection.10The real
clinical issue, of course, was and is whether cooling after
resuscitation from asphyxial injury is beneficial. Al-
though the studies described above had not addressed
this question, these and similar findings led to several
uncontrolled case series in the 1950s and 1960s in which
infants not breathing spontaneously at five minutes after
birth were immersed in cold water until respiration re-
sumed and then allowed to spontaneously rewarm.11–14
Subsequently at least one study suggested that cooling
could be combined with positive pressure ventilation.15
Although outcomes after cooling at birth were said to be
Correspondence to: Dr Alistair Jan Gunn, Depts of Physiology and
Paediatrics, Faculty of Medical and Health Sciences, The University of
Auckland Private Bag 92019, Auckland, New Zealand Fax: (9)
3737497. E-mail: email@example.com
NeuroRx?: The Journal of the American Society for Experimental NeuroTherapeutics
Vol. 3, 154–169, April 2006 © The American Society for Experimental NeuroTherapeutics, Inc.
better than for historical controls,14this experimental
treatment was overtaken by the recognition that even
mild hypothermia is associated with increased oxygen
requirements and more importantly, greater mortality in
premature newborns (?1500 g),16and the disappointing
outcomes of delayed induced cooling after near-drown-
The present review will describe subsequent experi-
mental work that provided a theoretical basis for treat-
ment after severe hypoxia-ischemia, examine recent clin-
ical studies that demonstrate that delayed hypothermia
can improve recovery in infants with acute HIE, and then
highlight unanswered issues surrounding the clinical use
PATHOPHYSIOLOGICAL PHASES OF
Delayed failure of oxidative metabolism
The seminal observation derived from both experi-
mental and clinical observations was that HIE is not a
single ‘event’ but is rather an evolving process. Although
neurons may die during the actual ischemic or asphyxial
event (primary cell death), many neurons initially re-
cover at least partially from the primary insult, only to
die many hours, or even days later (secondary or delayed
cell death). Using magnetic resonance spectroscopy,
Wyatt and co-workers showed that infants with evidence
of moderate to severe asphyxia often have normal cere-
bral oxidative metabolism shortly after birth, but many
then went on to develop delayed energy failure 6 to 15
hours later.18,19Those infants who did not show even
transient recovery had a very high mortality. In survi-
vors, the degree of secondary energy failure after 24 to
48 hours was closely associated with neurodevelopmen-
tal outcome at 18 months and 4 years of age.20An
identical pattern of initial recovery of cerebral oxidative
metabolism followed by secondary energy failure was
seen after hypoxia-ischemia in the piglet21and again
closely correlated with the severity of cell death in the
cortical area that was examined.22
It is this delay that has raised the tantalizing possibility
that asphyxial cell death could be prevented even if
treatment could not start until well after reperfusion.
Characterizing the phases of injury
Subsequent studies have described the phases associ-
ated with evolving neural injury in more detail as illus-
trated by figure 1. The hypoxia-ischemia event is the
primary phase of cell injury. During this phase there is
rapid depletion of high energy metabolites, leading to
progressive hypoxic depolarization of cells, with severe
cytotoxic edema (cell swelling), excessive intracellular
accumulation of calcium, and extracellular accumulation
of excitatory amino acids (EAAs) due to both failure of
reuptake and excessive release.23Following return of
cerebral circulation and / or oxygenation, after the end of
the insult, the initial hypoxia-induced cytotoxic edema
and accumulation of EAAs typically resolve over ap-
proximately 30 to 60 minutes,24,25with at least partial
recovery of cerebral oxidative metabolism, in a ‘latent’
phase. However, cerebral oxidative metabolism may
then secondarily deteriorate many hours later (approxi-
mately 6 to 15 h), in a phase that may extend over many
days.19,21At term equivalent, this secondary deteriora-
tion is often marked by the onset of seizures (figure
2),26,27secondary cytotoxic edema,24accumulation of
excitotoxins,23failure of cerebral mitochondrial activi-
ty21and ultimately, cell death.27,28
Surprisingly, although there are extensive data describ-
ing the timing and development of the delayed failure of
mitochondrial oxidative metabolism after acute insults,
its precise pathogenic significance remains highly con-
troversial. For example, there is a close correlation be-
tween histological loss of the key mitochondrial cyto-
chromes and neuronal loss,29–31and between the timing
of loss of cytochrome activity after severe anoxia in the
cat and subsequent delayed onset of neurological deteri-
oration.32Taken with in vitro evidence that the increase
in intracellular calcium levels during hypoxia/reoxygen-
ation triggers subsequent delayed functional impairment
and morphological disintegration of mitochondria,33
these data support the concept that mitochondrial failure
is a key step leading to cell death. Others, however, have
reported that secondary energy failure is directly corre-
Timing of Pathological Events After Hypoxia
Timing of Pathological Events After Hypoxia- -IschemiaIschemia
1 hour6 hours 5 days
FIG. 1. Flow diagram illustrating the relationship between the
phases of cerebral injury after a severe reversible hypoxic-isch-
emic insult. During reperfusion after the insult, there is a period
of approximately 30 to 60 minutes during which cellular energy
metabolism is restored, with progressive resolution of the acute
cell swelling secondary to hypoxic-depolarization. This is fol-
lowed by a latent phase. During this phase the intracytoplasmic
components of apoptosis are activated, and the early inflamma-
tory reaction is initiated, with induction of cytokines. This may be
followed by secondary deterioration leading to ultimate delayed
neuronal death after 3 days. As indicated by the bar, treatment
with cerebral hypothermia needs to be initiated in the latent
phase before the onset of secondary deterioration, and then
continued for over 48 hours for long lasting neuroprotection.
NeuroRx?, Vol. 3, No. 2, 2006
lated with the loss of neuronal markers at different
times,34implying that it is no more than a reflection of
final cell death.
Regardless of the precise sequence of events, this con-
sistent pattern of delayed deterioration, across species
and a variety of experimental models and clinical obser-
vations suggests that the effectiveness of any treatment
must be highly dependent on the timing of initiation and
continuation. As discussed in detail below experimental
studies of hypothermia have confirmed this hypothesis.
Mechanisms of delayed cell death
The precise events which initiate the cascade leading
to delayed cell death after hypoxia-ischemia (HI) are still
incompletely understood, but are undoubtedly multifac-
torial. It is likely partly related to excessive entry of
calcium into cells both during and after hypoxia-isch-
emia,35loss of trophic support from growth factors,36
induction of free radicals during hypoxia and early reper-
fusion,37,38and/or a secondary inflammatory, reaction,39
which may act through activation of cell surface death
receptors,40or synthesis of down-stream mediators of
cell death such as nitric oxide synthase and reactive
Cell death may involve necrotic and apoptotic events.
Necrosis is usually defined by loss of plasma membrane
integrity associated with a random pattern of DNA deg-
radation, whereas apoptosis is defined morphologically
by a microscopic picture of condensation of chromatin
(i.e. a dark shrunken nucleus) with ‘homogenization’
(loss) of the reticular formation. Ultimately the cells
break down into small, neatly ‘packaged’ fragments.44
By analogy with the active process of developmental loss
of excess cells (including neurons), it was suggested that
an apoptotic morphology reflected active or ‘pro-
grammed’ cell death.28,45,46
Post-hypoxic apoptosis can be triggered by the mech-
anisms discussed above, including glutamate receptor
excitotoxicity and consequent intracellular calcium accu-
mulation,47–49inflammation,50and oxidative stress.51
The intracytoplasmic stage of apoptosis involves alter-
ations in the ratio of various intracellular factors such as
the proto-oncogene Bcl-2, which inhibits apoptosis, and
-12 3 12 2448 7296 120
0 15 30 45 60
Secondary cytotoxic edema
FIG. 2. Data from the near-term fetal sheep, illustrating pathophysiological events after a 30 minute episode of severe ischemia (shown
by the vertical black arrow). After reperfusion, there is near-normalization of oxidative cerebral energy metabolism in the mitochondria
as shown by recovery of the acute rise in impedance (a measure of cytotoxic edema) but depressed electroencephalogram (EEG)
activity. From approximately 6 h after reperfusion there is a rapid onset of intense delayed seizures and cytotoxic edema, which begin
to resolve from 36 h. The profound suppression of the EEG after resolution of seizures correlates strongly with loss of cortical neurons.27
GUNN AND THORESEN156
NeuroRx?, Vol. 3, No. 2, 2006
Bax, which promotes apoptosis,52and activation of cys-
teine proteases (caspases).53The final, irreversible exe-
cution phase of apoptosis is intranuclear, involving en-
donuclease mediated DNA fragmentation.54In contrast,
necrosis was suggested to reflect biophysical damage to
the cell (cell membrane instability, ion shifts etc), par-
ticularly lysis in the primary phase.28,55Both patterns are
clearly described in infants dying after perinatal as-
Recently, it has become clear that post-hypoxic cell
death 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
the primary insult.58–60Consistent with this, there is
evidence that mitochondrial calcium overload is a critical
event in both apoptotic and necrotic cell death.48Gener-
ally apoptosis-like or programmed cell death seems to be
more important in the developing brain than in
Regardless of the precise pattern of delayed death, the
concept remains an important one, since if neuronal and
glia cell death is an active response (preprogrammed or
functionally mediated by secondary mechanisms such as
cytotoxin exposure), then it should logically be possible
to interrupt these events.
THE ‘PHARMACODYNAMICS’ OF
The timing and duration of treatment is critical
Cooling the brain for a few hours can be modestly
protective but is exquisitely dependent on the timing
after the end of the hypoxia-ischemia. For example, mild
hypothermia (decreasing temperature by 2 to 4°C) for
one to 3 hours after 15 minutes of reversible ischemia or
global hypoxia in the piglet, significantly improved re-
covery and reduced neuronal loss 3 days later.67,68Sim-
ilar data have been reported in the neonatal rat.69–71
However, protection seems to be lost if the initiation of
brief hypothermia is delayed by as little as 15 to 45
minutes after the primary insult.72–74The observations
discussed above, namely, that secondary deterioration
continues for days after injury, suggest that hypothermia
would be more effective if it was maintained for a rela-
tively longer period.
Subsequent studies have strongly supported this pro-
posal. For example, in unanesthetized infant rats sub-
jected to moderate hypoxia-ischemia, mild hypothermia
(2 to 3°C decrease in brain temperature) for 72 hours
from the end of hypoxia prevented cortical infarction,
while 6 hours of cooling had an intermediate effect.70In
the same model, however, a greater reduction in body
temperature, of 5°C, for 6 hours, starting immediately
after the insult, gave significant neuroprotection both
after 1 and 6 weeks survival as well as neurobehavioral
improvement.75Similarly, in anesthetized piglets ex-
posed to either hypoxia with bilateral carotid ligation or
to hypoxia with hypotension, either 12 hours of mild
whole body hypothermia (35oC) or 24 hours of head
cooling with mild systemic hypothermia started imme-
diately after hypoxia prevented delayed energy failure,76
reduced neuronal loss55,77and suppressed post-hypoxic
In practice, however, such early initiation of cooling is
either impossible or clinically untestable, because most
infants requiring resuscitation do not present with HIE at
birth and many do not go on to develop HIE later. By the
time that HIE can be reliably diagnosed, hours may have
gone by. Thus, given that in practice treatment must start
some time after birth, it was critical to determine just
how late cooling could be started, and yet remain signif-
icantly protective. There is as yet no specific marker for
when evolving cell death becomes irreversible. Empiri-
cally though, a range of experimental studies strongly
suggest that the latent phase before secondary energy
failure is established represents the realistic window of
opportunity for intervention.78
For example, in the near-term fetal sheep, moderate
hypothermia induced 90 minutes after reperfusion, in the
early latent phase, and continued until 72 hours after
ischemia, prevented secondary cytotoxic edema, and im-
proved electroencephalographic recovery.27There was a
concomitant substantial reduction in parasagittal cortical
infarction and improvement in neuronal loss scores in all
regions. When the start of hypothermia was delayed until
just before the onset of secondary seizures in this para-
digm (5.5 hours after reperfusion) partial neuroprotection
was seen (Figure 3).79With further delay until after
seizures were established (8.5 hours after reperfusion),
there was no electrophysiological or overall histological
protection with cooling (Figure 3).80
Similarly to the studies of early cooling, neuroprotec-
tion with delayed cooling requires relatively prolonged
periods of cooling, typically longer than 12 hours. Cool-
ing was continued for 3 days in the fetal sheep studies
because pilot studies demonstrated intense rebound sei-
zure activity and increased cell loss if cooling was
stopped after less than 24 to 48 h. In contrast, even very
rapid spontaneous rewarming after 3 days of cooling was
associated with only minor, transient epileptiform activ-
ity.81These results are consistent with the report from
Colbourne and colleagues in the adult gerbil that when
the delay after cerebral ischemia before initiating a 24
hour period of cooling was increased from 1 to 4 hours,
neuroprotection in the CA1 region of the hippocampus
after six months recovery fell from 70 to 12%.82This
chronic loss could be prevented by extending the dura-
tion of moderate (32 to 34°C ) hypothermia to 48 hours
or more, even when the start of cooling was delayed until
6 hours after reperfusion.83,84
HYPOTHERMIC NEUROPROTECTION 157
NeuroRx?, Vol. 3, No. 2, 2006
How much should we cool?
There is an obvious potential trade-off between the
adverse systemic effects of cooling, which increase
markedly below a core temperature of approximately
34°C,85and cerebral benefits. Supporting this logic, in
the adult dog, deep hypothermia (to a rectal temperature
of 15°C) after cardiac arrest was detrimental, whereas
mild hypothermia (34 to 36°C), from 10 minutes until 12
hours after cardiac arrest was beneficial.86Overall, sub-
sequent studies suggest that a reduction in cerebral tem-
perature to between 32 and 34°C is required for effective
neuronal rescue. In fetal sheep cooled from 90 minutes
after ischemia, substantial neuroprotection was seen only
in fetuses in whom there was a sustained decrease in the
extradural temperature to less than 34°C (normal tem-
perature in the fetal sheep is 39.5°C).27Further, in the
adult gerbil, cooling from the normal rectal temperature
of 37°C down to 32°C was associated with greater be-
havioral and histological neuroprotection than 34°C.87
Although we do not know the optimal degree of cerebral
cooling in newborns, the first controlled trials of hypo-
thermia after cardiac arrest in adults strongly support this
target range, with improved neurological outcome in pa-
tients cooled to between 32 and 34°C.88,89
Is neuroprotection maintained long-term?
There have been reports that hypothermia only de-
layed, rather than prevented, neuronal degeneration after
global ischemia in the adult rat90–92and after relatively
mild hypoxia-ischemia in the 7 day old rat.93The most
likely explanation is that the duration and / or degree of
hypothermia may have been inadequate as suggested by
the finding that cooling by 5 °C for 6 hours75or 72 hours
of very mild cooling in infant rats were associated with
long-term improvement after carotid occlusion and hyp-
oxia.70Subsequent studies both in the 7 day rat and in
adult species have confirmed that a sufficiently pro-
longed phase of moderate cooling can be associated with
persistent behavioral and histological protection for
many weeks and months.75,83,84,87,94–96Broadly, these
studies tend to suggest that the later cooling is started, the
more prolonged the treatment needs to be for neuropro-
How does it work?
The precise mechanisms of hypothermic neuroprotec-
tion are still unclear. Although, pragmatically, this may
not matter too much to clinicians, it is critical to efforts
to develop more effective combination treatments.
Broadly, it is now well established that cooling sup-
Early (90 min) Cooling
Delayed (5.5 h) Cooling
Post Seizure (8.5 h) Cooling
Neuronal Loss Score
FIG. 3. The effect of cerebral cooling in the fetal sheep started at different times after reperfusion and continued until 72 h, on neuronal
loss after 5 days recovery from 30 minutes of cerebral ischemia.27,79,80Compared with sham cooling (n?13) cooling started 90 minutes
after reperfusion (n?7) was protective, whereas cooling started shortly after the start of the secondary phase (8.5 hours after reperfusion,
n?5) was not. Cooling started just before the end of the latent phase (5.5 hours after reperfusion, n?11) was partially protective. Only
cooled fetuses in which the extradural temperature was successfully maintained below 34°C have been included. DG: dentate gyrus of
the hippocampus. CA1/2: cornu ammonis fields 1 and 2 of the hippocampus. *p?0.005 compared with sham-cooled (control) fetuses;
data are Mean ? SEM.
GUNN AND THORESEN 158
NeuroRx?, Vol. 3, No. 2, 2006
presses many of the pathways leading to delayed cell
death. As well as reducing cellular metabolic de-
mands,98,99hypothermia reduces excessive accumulation
of cytotoxins such as glutamate and oxygen free radicals,
suppresses the post-ischemic inflammatory reaction and
inhibits the intracellular pathways leading to pro-
grammed (i.e., apoptosis-like) cell death.
Suppression of excitotoxins and free radicals. The
combination of hypoxic depolarization and EAA accu-
mulation are key factors in the initiation of neuronal
injury in the primary phase, during hypoxia-ischemia.
Hypothermia produces a graded reduction in cerebral
metabolism of about 5% for every degree of temperature
reduction,98which delays the onset of anoxic cell depo-
larization. However, the protective effects of hypother-
mia even in this phase are not simply due to reduced
metabolism, since cooling improves outcome even when
the absolute duration of depolarization is controlled.100
Cooling potently reduces post-depolarization release of
many toxins including EAAs in both adults,101and new-
borns,102as well as free radicals.103Similarly, cooling
started during reperfusion reduces levels of extracellular
EAAs and NO production in the piglet.102
However these mechanisms rapidly resolve during the
latent phase of recovery from HI, and thus cannot readily
account for the protective effects of delayed cooling.
Intracellular pathways in the latent phase. The ef-
fects of hypothermia on pathways distal to cell mem-
brane ion channels are likely to be more important. For
example, intra-insult hypothermia did not prevent intra-
cellular accumulation of calcium during cardiac arrest in
vivo,104or during glutamate exposure in vitro.105In con-
trast, in vitro neuronal degeneration was prevented by
cooling initiated after the excitotoxins had been washed
out.105Thus, the ability of hypothermia to reduce release
of excitotoxins does not appear to be central to its post-
insult neuroprotective effects, rather these data suggest
that the critical effect of hypothermia is to block the
intracellular consequences of excitotoxin exposure.
Suppression of apoptosis/ programmed cell death?
There is increasing evidence to suggest that hypothermia
may have a particular role in suppressing the evolution of
programmed cell death. Recent clinical studies have
shown that apoptosis is a major contributor to post-
asphyxial cell death in the developing brain.56,57Studies
using morphological criteria have had mixed outcomes.
In the piglet, hypothermia started after severe hypoxia-
ischemia was reported to reduce apoptotic cell death, but
not necrotic cell death,55with similar results reported
after injury in rats.106,107However, in the adult rat, de-
layed post-ischemic cell death prevented by hypothermia
had a necrotic appearance on detailed electron micro-
scopic criteria.108However, it is now clear that apoptotic
mechanisms can be involved even in ‘necrotic’ cell
death. Although multiple pathways are likely to be in-
volved in such post-ischemic apoptosis, caspase-3, one
of the family of cysteine proteases, is reported to play a
crucial role.109–111Protection with post-ischemic hypo-
thermia in the near-term fetal sheep was closely linked
with suppression of activated caspase (Figure 4).112
These data are consistent with in vitro studies of hypo-
thermia after severe hypoxia in developing rat neurons.
Strikingly, whereas in that model preconditioning acti-
vates a program that stimulated the expression of anti-
apoptotic gene products and regulatory components of
the cell cycle, hypothermia did not trigger active pro-
cesses, but depressed cell activity and abolished hypoxia-
associated protein synthesis.113
Suppression of inflammatory second messengers.
Brain injury leads to induction of the inflammatory cas-
cade with increased release of cytokines and interleukins
(IL).42These compounds are believed to exacerbate de-
layed injury, whether by direct neurotoxicity and induc-
tion of apoptosis42or by promoting stimulation of cap-
illary endothelial cell proinflammatory responses and
leukocyte adhesion and infiltration into the ischemic
brain.43There is good evidence that cooling can suppress
this inflammatory reaction. In vitro, hypothermia po-
Isolectin B4 (Cells/Field)
ShamIschemia 2h Cool6h Cool
Caspase 3 (asp 175)
(total intragyral cells)
Sham Ischemia2h Cool6h Cool
FIG. 4. Delayed head cooling is associated with suppression of both apoptotic and inflammatory processes, as shown by reduced
numbers of activated microglia (isolectin B4 positive cells, left panel) and by reduced numbers of cells expressing activated caspase 3
(right panel) in subcortical white matter five days after 30 minutes of cerebral ischemia in near-term fetal sheep.112Suppression was
greater when cooling was initiated 2 h after the start of the ischemic insult than when it was delayed until 6 h.112
NeuroRx?, Vol. 3, No. 2, 2006
tently inhibits proliferation, superoxide and NO produc-
tion by cultured microglia.114In the adult rat, hypother-
mia suppresses the post-traumatic release of IL-1?,115
and the accumulation of polymorphonuclear leuko-
cytes.116Similarly, hypothermia delays neutrophil accu-
mulation and microglial activation following transient
ischemia (Figure 4).112,117Thus, these data suggest that
the hypothermic protection against post-ischemic neuro-
nal damage might be, in part, due to suppression of
Excitotoxity after hypoxia-ischemia. Classically,
cell death due to abnormal glutamate receptor activation
(excitotoxicity) has been related to pathologically ele-
vated levels of extracellular glutamate, as occurs during
hypoxia-ischemia. Following reperfusion, we and others
have shown that glutamate levels rapidly return to con-
trol values,23,25and thus naively we might predict that
excitotoxicity should not be important after reperfusion.
However, more recent data show that pathological hy-
perexcitability of glutamate receptors can continue for
many hours following hypoxia-ischemia,118–121and
treatment with the specific, noncompetitive NMDA an-
tagonist MK-801 blocks this activity and improves neu-
ronal outcome.122,123It is still unknown whether hypo-
thermia affects this abnormal post-hypoxic receptor
function, however, brain cooling can effectively inhibit
epileptiform activity,77,124,125raising the possibility that
this may well be a significant potential mechanism of
action in the latent phase.
Summary of clinical implications
The experimental studies discussed above suggest that
a prolonged duration of moderate cerebral hypothermia
might be able to improve long-term outcome if started as
soon as possible, within approximately 6 hours of hy-
poxic-ischemic injury. Based on these extremely encour-
aging data, clinical trials were undertaken.
CLINICAL TRIALS OF HYPOTHERMIA
Phase I and II studies
A number of small controlled trials of head cooling
with mild hypothermia126–129and of whole body cool-
ing130in asphyxiated newborns have now been reported,
in addition to several case series.131–133Although none
of these studies were powered or designed to evaluate
neurological outcome, there is some suggestion of im-
proved outcomes.127,134,135For example, in a controlled
study of head cooling among infants with early stage 2 or
3 encephalopathy, mild systemic hypothermia was asso-
ciated with a trend to reduced cerebral palsy in survivors
(odds ratio 0.46 [0.08, 2.56] vs normothermia).127A
retrospective study of whole body cooling to between 32
and 34°C in 10 infants found a significant reduction of
major neurologic abnormalities and abnormal MRI find-
ings at follow-up compared with 11 historical con-
trols.134A larger randomized pilot study of head cooling
with mild hypothermia compared to normothermia found
a significant reduction in neuron specific enolase (NSE)
levels in cerebral spinal fluid with cooling but only a
small increase in normal developmental outcome at 6
months of age in 18 of 23 cooled patients (78.3 %)
compared with 19 of 27 (70.4 %) normothermic in-
Finally, a large phase II randomized clinical trial of 65
infants has been reported, in which body cooling to a
rectal temperature of 33 °C for 48 hours was initiated
within 6 hours of birth. In contrast with the previous
studies, the deeper central cooling in this study was
associated with some adverse effects although these were
clinically manageable, including longer dependence on
inotropic agents, prolongation of prothrombin times, and
lower platelet counts with more patients requiring
plasma and platelet transfusions.136The combined out-
come of death or severe motor scores was significantly
lower in the hypothermia group (52%) than the normo-
thermia group (84%) (P ? 0.019).137Severely abnormal
motor scores were recorded in 64% of normothermia
patients and in 24% of hypothermia patients.
Phase III studies
The first large multicenter randomized controlled trial
of hypothermia for HIE was the CoolCap trial.138,139In
this study of term infants with moderate to severe HIE,
head cooling with mild systemic hypothermia, defined as
a rectal temperature of 34-35 °C (n?116), or conven-
tional care (n?118), death or disability at 18 months was
reduced in infants with less severe electroencephalo-
graphic changes at trial entry (n?172, odds ratio 0.42;
95% CI 0.22-0.80, p?0.009).138In contrast, however,
there was no benefit in those with the combination of
seizures and profound suppression of the amplitude in-
tegrated electroencephalogram (aEEG) recording before
cooling was started. The improvement was primarily due
to a reduction in motor disability, with a more than 50
percent reduction in severe neuromotor disability in sur-
vivors and improved continuous BSID-II scores. In con-
trast there was no change in early neonatal mortality (27
cooled vs. 26 control cases), with a small apparent re-
duction in late mortality (9 vs. 16 cases respectively).
Although this is not a large category, the difference in
late events is intriguing since the great majority of late
deaths in both groups were related to complications of
These data suggest that cooling can safely improve
survival without severe neurodevelopmental disability in
infants with less severe aEEG changes. The only consis-
tent minor adverse effects were scalp edema under the
cap, which resolved rapidly before or after removal of
the cap, transient hyperglycemia from 4 to 24 hours
GUNN AND THORESEN 160
NeuroRx?, Vol. 3, No. 2, 2006
5.4?3.1 mmol/L, at 4 hours, p?0.001), and sinus bra-
dycardia (which is a well known, essentially normal
response during hypothermia140) that did not require
treatment. Conversely, there was an apparent reduction
in the incidence of elevated liver enzymes in the cooled
group (38% of cooled infants vs 53% of controls,
In a second large multi-center trial, this time of whole
body cooling, Shankaran and colleagues enrolled 208
infants who met specific clinical and laboratory criteria
suggesting exposure to severe perinatal hypoxia and had
moderate or severe HIE on neurological examination by
trained examiners.141Infants in the experimental group
(n?102) were placed on a cooling blanket and cooled to
a rectal temperature of 33.5?0.5 °C for 72 hours. After
18 months of follow up, the incidence of death and/or
moderate-to-severe disability was significantly reduced
in the cooled infants (45%) vs the normothermic group
(62%, relative risk 0.72; 95 % CI, 0.55-0.93). There was
no difference for death or disability alone.
Other ongoing trials
A number of trials are still in progress, including the
Total Body Cooling trial (TOBY) in England, the Neo-
Network trial in central Europe and the Infant Cooling
Evaluation (ICE) trial in Western Australia. The TOBY
trial’s inclusion criteria are identical to those of the Cool-
Cap trial. This trial should allow direct comparison of
total body vs head cooling in patients meeting the same
inclusion criteria. The NeoNetwork trial is a whole body
cooling trial using a target rectal temperature of 33.5° for
72 h, with similar entry criteria to the TOBY trial using
a cooling blanket. The ICE trial aims to enroll infants
from a wide geographic region, using a simplified, prag-
matic protocol. A target rectal temperature of 33o–34oC
is achieved by turning off the ambient heating systems
and by applying “Hot-Cold” gel packs (cooled to 10oC)
around the infant’s head and chest. In a preliminary
report on 26 infants with HIE who were randomized to
normothermia or to systemic hypothermia, Inder and
colleagues reported that the hypothermia group had less
cortical gray matter signal abnormality on magnetic res-
onance imaging (MRI) (1/12 vs 7/14 infants in the nor-
mothermic group; P ? .036), but had similar numbers of
basal ganglia lesions, which raises the possibility of se-
lective regional benefits from treatment with hypother-
mia.142Interestingly, Rutherford and colleagues have
also reported a reduced incidence of severe cortical le-
sions in infants treated with head cooling.143However, in
that report, both head and whole body cooling were
associated with a decrease in basal ganglia and thalamic
lesions that was significant in infants with moderate
aEEG changes but not in those with severe aEEG find-
Taken together, the remarkably similar effect sizes in
two independent, well controlled studies strongly suggest
that induced hypothermia is beneficial. In many ways, as
often happens, these studies have actually raised many
more questions than they answered. In particular, the
multicenter trials reported to date make it clear that neu-
roprotection with hypothermia as currently used is only
partial, such that many patients die of neural injury or
survive with disability despite hypothermia.138,141This
issue is not limited to neonatal HIE, since trials of cool-
ing for neurological recovery after adult comatose car-
diac arrest have suggested a similar limitation.88,89Both
the two neonatal and the adult cardiac arrest trials have
suggested that the number needed to treat is approxi-
mately 6, meaning that 6 patients need to receive the
intervention for one to have a positive outcome. In this
section, we will focus on issues relating to the practical
use (and abuse) of therapeutic hypothermia.
How late is really too late?
The real clinical window of opportunity for treatment,
with hypothermia, or any other putative therapy, is sim-
ply not clear. It may well be both longer and shorter than
suggested by experimental work. It is important to ap-
preciate some of the limitations of the experimental stud-
ies. Crucially they used very carefully standardized in-
sults, occurring at a precisely known time. In contrast,
the precipitating insult in neonatal encephalopathy is a
well defined event, such as placental abruption that is
terminated at birth, in only approximately 25% of cas-
es.144In other cases the preceding insult seems to evolve
over hours during labor, and in at least some cases,
perhaps 10% of the total, the infant seems to have been
compromised even before labor started.144,145Thus, it
seems very likely that the effective window of opportu-
nity to treat HIE will, in some cases, be somewhat less
than suggested experimentally. The clinical trials were
unable to address this issue, as too few infants were able
to start treatment early after birth; just 12% of infants
started treatment before 4 h in the CoolCap trial for
Equally, we must also recognize that the speed of
evolution of delayed cell death is a function of severity
of injury. Milder insults are associated with much more
delayed neuronal loss.28,146Thus, whereas the most se-
vere insults may need treatment essentially at the time of
resuscitation, or be untreatable, it remains possible that
more mildly affected infants, such infants who have
long-term learning difficulties but no handicap, could
benefit from cooling even after 6 h. This does not imply
of course that such delay is anyway desirable or accept-
able, merely that if unavoidable, it might, in a narrowly
defined subset of children, still have some benefit.
NeuroRx?, Vol. 3, No. 2, 2006
Who should be treated, or: How bad is too bad?
Following on from this issue, it is possible that one
reason for the limited response in the two multicenter
trials may have been that the trials recruited many infants
who were ‘untreatable’. The foundation studies of sec-
ondary energy failure in children with HIE showed that
some infants with apparent hypoxic-ischemic encepha-
lopathy did not show any initial recovery of cerebral
oxidative metabolism, and have extremely poor out-
comes, typically death.18This finding should not be
over-interpreted; the number of MRS studies that could
be performed was limited, and so brief recovery of en-
ergy metabolism could have been missed. Further, al-
though such cases may never be treatable, many infants
with moderate to severe hypoxic-ischemic encephalopa-
thy do show initial, transient recovery of cerebral oxida-
Nonetheless, ideally, we would like to identify the
potentially treatable cases in advance, to avoid offering
false hope, and to target treatment more effectively. Clin-
ical evaluation of the severity of HIE, using criteria
modified from Sarnat and Sarnat, was highly predictive
of the risk of death or disability in both trials. Despite
this, strikingly, and contrary to the authors’ and many
others’ original expectations at the time that the trials
were developed, the relative improvement was similar
both for infants with moderate (Stage II) and severe
(Stage III) HIE.139,141Thus despite its prognostic reli-
ability, clinical evaluation does not seem to distinguish
between ‘treatable’ and ‘untreatable’ cases.
In contrast, the CoolCap trial suggested that EEG mon-
itoring could identify a subgroup of infants with pro-
found suppression of amplitude and onset of seizures at
the time of randomization who did not respond.138Al-
though these findings are extremely suggestive, it is im-
portant to take in to account several potential limitations
of these findings. The EEG recruitment criteria were
designed primarily to exclude cases of mild HIE (who
have a known normal outcome) rather than to distinguish
‘treatable’ from ‘untreatable’ infants, and the EEG inter-
pretation of a 20 min compressed trace was performed by
site investigators, of whom few had previous experience
in assessing aEEG, not experts. Pragmatically, and per-
haps most importantly, this is the first and to date only
study to examine this question.
Thus, the authors believe that it is premature to judge
this issue. The Sarnat and Sarnat score for example was
developed many decades ago,147and is based on assess-
ment of infants who are more than 24 hours old, before
the therapeutic era. Focused clinical and animal studies
are now needed to investigate whether there may be
components of clinical examination, biochemical tests or
of EEG recordings that might be more predictive of the
timing (as opposed to severity) of HIE and of the re-
sponse to cooling.
Is head or whole body cooling better?
In order to provide adequate neuroprotection with min-
imal risk of systemic adverse effects in sick, unstable
neonates, ideally only the brain would be cooled. Al-
though this has been demonstrated experimentally using
cardiac bypass procedures,148it is clearly impractical in
routine practice. Pragmatically, partially selective cere-
bral cooling can be obtained using a cooling cap applied
to the scalp, while the body is warmed by some method
such as an overhead heater to limit the degree of systemic
hypothermia.126,128,149A mild (?34 to 35°C) degree of
systemic hypothermia is still desirable during head cool-
ing; firstly to reduce the steepness of the intracerebral
gradient which develops during true selective head cool-
ing,150avoiding excessively cold cap temperatures which
might cause scalp injury or exacerbate local scalp ede-
ma,128and to provide at least some cooling of deep
cerebral structures such as the brain stem. This approach
has recently been demonstrated in the piglet to be asso-
ciated with a substantial (median, 5.3°C), sustained de-
crease in deep intracerebral temperature at the level of
the basal ganglia compared with the rectal tempera-
ture.151,152Figure 5 shows examples of changes in re-
gional brain temperatures in piglets, either during mod-
erate whole body cooling, to a rectal temperature of
34.5°C, or head cooling combined with moderate central
hypothermia to 34.5°C. During whole body cooling there
was less than 0.6°C difference between the warmest
(basal ganglia) and the coldest parts of the brain (the
cortex). In contrast, during head cooling there was an
approximately 6°C gradient between the superficial and
deep brain. Nevertheless, in the 1.5 kg term piglet, which
has a smaller head relative to body size than the human,
it was possible to use this cooling cap to maintain the
deep part of the brain a mean of 3.4 °C colder than rectal
temperature for more than 24 h.152A gradient of over
6°C was achieved in a subsequent study,153suggesting
that the small premature head could be selectively
Overall, these data suggest that partially selective cool-
ing of the head is likely to be feasible. Consistent with
this, in asphyxiated newborns, although direct tempera-
ture measurements are not yet generally feasible, head
cooling has been shown to increase the gradient between
nasopharyngeal and rectal temperature by approximately
1oC.126However, it is not possible to tell from the recent
trials whether it is more or less effective than whole body
cooling. Intriguingly, recent short-term recovery studies
in the piglet do suggest that the optimal degree of cooling
is greater in the cortex than in the basal ganglia.154
Supporting this experimental observation, in a recent
case series, head cooling but not whole body cooling
seemed to be associated with a reduction in the incidence
of severe cortical lesions,143as examined by MRI. If this
is correct, we would predict that the long-term followup
GUNN AND THORESEN 162
NeuroRx?, Vol. 3, No. 2, 2006
of the CoolCap study may show a greater effect on
How long should cooling be continued?
Based on the experimental data discussed earlier, all of
the recent clinical studies have continued cooling for 48
to 72 h. There is robust evidence from adult rodent
models that cooling for 48 h is better than for 24 h83,84;
indeed the authors have observed rebound deterioration
after 24 h in the fetal sheep (unpublished). However, it is
unclear whether cooling for 72 h is ‘better’ than 48 h.
Equally, there are experimental and clinical data report-
ing rebound seizure activity after rewarming from 72 h
of cooling,81,155and thus it remains possible that cooling
for 4 or 5 days might provide further benefit.
Alternatively there is some evidence that rapid re-
warming can impair recovery, with transient uncoupling
of cerebral circulation and metabolism,156and in adult
rats can exacerbate traumatic axonal injury and impair
cerebrovascular responsiveness, compared with slow re-
warming.157,158There are no systematic data from large
animals. The clinical studies of therapeutic hypothermia
have empirically chosen to rewarm infants at no more
than 0.5°C per h,128,130,132however, it remains possible
that rewarming still more slowly might be beneficial.
Further, there is some evidence that worsening of intra-
cranial pressure during rewarming in adult patients with
head injury may be able to be avoided by an extremely
slow rewarming schedule, although it is still not known
whether this improves long-term outcomes. This is an
area where significant further experimental work in large
species would be helpful.
How much should we cool?
This is one area where the clinical studies did not
closely follow the experimental evidence. As previously
reviewed, in general the evidence is that five degrees of
cooling (approximately a rectal temperature of 32 °C) is
better than a reduction of three degrees (equivalent to 34
°C).159The trials of whole body cooling however have
used a target rectal temperature of around 33.5°C. This
suggests that at present we are using the upper half of the
ideal range, and that deeper cooling, at least for a time,
might allow still greater cerebral protection. The reason
for this discrepancy is of course that the clinicians de-
signing the studies were most concerned not to cause
side effects in these highly unstable infants. As reviewed
next, mild cooling in an intensive care environment has
been impressively safe. Only large clinical trials can
inform us whether this would also be true of deeper
Just how safe is cooling in infants with HIE?
While the above studies have suggested that mild hy-
pothermia is generally safe they have also highlighted
the importance of understanding the physiological im-
pact of hypothermia.85It is important to appreciate that
although for example, there was no increase in the rate of
complications such as infection in the newborn studies,
this likely reflects in large part the design of these trials,
which included both routine screening and treatment for
possible infection.138,141Hypothermia has profound anti-
inflammatory effects and in older adults seems to in-
crease the risk of infective complications such as pneu-
monia and bacteremia,85,160and thus this potential risk
needs to continue to be carefully monitored in clinical
use. Similarly, as noted above, although no increase in
hemorrhagic complications was reported in either of the
two phase III trials,138,141Eicher et al have reported such
an increase in association with still lower body temper-
atures.136It is unclear at this time whether this is a
specific concern with cooling to 33°C and lower, or
simply a chance finding. It is reassuring that in piglet
studies where the cortex was cooled significantly (less
than 30 degrees) no hemorrhagic changes were seen in
One consistent metabolic effect associated with hypo-
thermia was transient mild hyperglycemia, both in
adult88and infant trials,138with no increase in the rate of
Whole body cooling
0 720 1440
-3600 360 720
1080 1440 1800
FIG. 5. Examples of cooling in term piglets showing that during whole body cooling the deep brain temperature closely approximates
rectal temperature (Left). In contrast, during head cooling it is possible to maintain deep brain structures at significantly lower
temperature than the rectal temperature. This method of cooling may further limit the side effects associated with systemic hypothermia
and be feasible for premature infants.153
NeuroRx?, Vol. 3, No. 2, 2006
hypoglycemia. A similar transient initial rise in glucose
concentrations has been observed in the piglet and near-
term fetal sheep,27,161In the piglet, as cooling was con-
tinued increased glucose administration became neces-
sary to maintain normal levels.161It is probable that the
initial rise in glucose levels reflects hypothermia-induced
Cardiovascular effects include a significant increase in
blood pressure at initiation of cooling, both experimen-
tally79and clinically.131This response is mediated by
rapid peripheral vasoconstriction, i.e. centralization of
blood flow.162Further, hypothermia slows the atrial
pacemaker and intracardiac conduction. Consequently,
hypothermia to less than approximately 35.5°C is asso-
ciated with mild but sustained sinus bradycardia, how-
ever this has not required treatment.128,130,138,141This
linear relation between heart rate and core temperature
likely partly reflects the increased metabolic need with
increasing temperature. Electrocardiograms done in in-
fants with sustained heart rates of ?90 bpm confirmed
that some show markedly prolonged QT duration above
the 98thpercentile corrected for age and heart rate, with-
out arrhythmia. These changes resolve with rewarm-
ing.163Although such prolonged QT in the absence of
ventricular arrhythmia may be safe, close monitoring is
clearly essential and other therapies which lengthen the
QT interval (such as macrolide antibiotics) should be
With what should cooling be given?
One potential way of improving the effectiveness of
treatment would be to combine cooling with another
agent. There is increasing evidence that hypothermia can
markedly augment the effects of drug therapy. For ex-
ample, post-ischemic hypothermia attenuated neurobe-
havioral deficits in adult rats when combined with de-
layed NMDA receptor antagonist treatment, more than
either treatment alone,164and synergistically enhanced
the protective effects of MK-801 during hypoxia-isch-
emia in the neonatal rat.165,166Similarly, in the neonatal
rodent, a combination of xenon and hypothermia admin-
istered 4 hours after hypoxic-ischemic injury provided
synergistic neuroprotection up to 30 days after the in-
sult.167Xenon is known to be an antagonist of the N-
methyl-D-aspartate subtype of the glutamate receptor,
and thus, this finding also supports this general approach.
Further, it is of interest that in the piglet model of global
hypoxia-ischemia neuroprotection and improved neuro-
logical function were seen only when the subjects were
anaesthetized during the hypothermic period, suggesting
a potentially important interaction between hypothermia
and sedation / anesthesia.168
A simple increase in protection is not the only useful
outcome from combination treatment. For example, in
the rat, repeated intraperitoneal doses of a glutamate
antagonist substantially delayed the eventual develop-
ment of neuronal loss in the hippocampus.169It is un-
known whether more prolonged treatment might have
had permanent protection, however, it suggests that this
or similar agents might be used to prolong the window of
opportunity for other therapies. Consistent with this pos-
tulate, Liu and colleagues have recently reported that
injection of a single dose of an AMPA/KA receptor
antagonist, Topiramate, after hypoxia-ischemia in the
neonatal rat was not protective by itself, but that this
treatment significantly extended the window of opportu-
nity for protection with a short (3 h) interval of hypo-
thermia.170Conversely, in the adult rat, brief, mild hy-
pothermia which was not significantly protective in its
own right, markedly increased the window of opportu-
nity for treatment with the anti-apoptotic agent insulin-
like growth factor 1 after hypoxia-ischemia.171
MK-801 has been one of the most extensively inves-
tigated glutamate antagonists. However, this drug and
others, have numerous, clinically unacceptable side ef-
fects. Treatment can cause hallucinations, sedation and
learning and memory deficits since it indiscriminately
blocks physiologic effects at the NMDA receptor at neu-
roprotective doses.172NMDA receptor blockade impairs
induction of long-term potentiation (LTP) which is im-
portant for memory formation.173In addition, MK-801
causes acute but reversible neuronal vacuolization when
systemically administered at neuroprotective doses in
adult rodents.174Even more concerning, in the develop-
ing brain NMDA antagonists such as MK-801 have long
lasting effects on neuronal circuits,175can trigger wide-
spread apoptotic neurodegeneration in the developing
brain176and indeed while MK-801 after head injury pro-
tected against primary necrotic damage it actually in-
creased severity of secondary apoptotic damage.177
Thus, at present, despite the considerable promise of
this approach, much more basic investigation is required
to identify the most effective and clinically acceptable
agent for use in combination therapy with hypothermia.
There is now nearly overwhelming clinical and exper-
imental evidence that moderate cerebral cooling after
cardiac arrest and in infants with acute hypoxic-ischemic
encephalopathy can improve medium-term neurological
recovery in at least some infants. The long-term effects
of hypothermia, at school age and later, are not yet
known, but we can be cautiously optimistic, while await-
ing the results of the studies which are still in progress.
The key therapeutic requirements for neuroprotection are
that hypothermia is initiated as soon as possible in the
latent phase, prior to secondary deterioration, and con-
tinued for a sufficient period in relation to the evolution
GUNN AND THORESEN 164
NeuroRx?, Vol. 3, No. 2, 2006
of delayed encephalopathic processes, typically 48 hours
The studies to date show that although hypothermia is
an important advance, as currently applied it is not a
‘magic bullet’. Only approximately 15% have better out-
come after cooling group as compared to those treated
with standard care. Many important clinical questions,
which we may summarize as: when, how deep, how
long, to whom, by what method and combined with
what, will need to be answered before we can really
know how best to use hypothermia.
Acknowledgments: The authors’ work reported in this re-
view has been supported by grants from the Health Research
Council of New Zealand, Lottery Health Board of New Zea-
land, the Auckland Medical Research Foundation, The Norwe-
gian Research Council, Laerdal Foundation for Acute Medi-
cine, The Wellcome Trust and SPARKS (UK).
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