Neuroprotective Mechanisms of Hypothermia in Brain Ischaemia

REVIEWS
Neuroprotective mechanisms of
hypothermia in brain ischaemia
Midori A.Yenari1,2 and Hyung Soo Han3
Abstract | Cooling can reduce primary injury and prevent secondary injury to the brain after
insults in certain clinical settings and in animal models of brain insult. The mechanisms that
underlie the protective effects of cooling also known as therapeutic hypothermia
are slowly beginning to be understood. Hypothermia influences multiple aspects of brain
physiology in the acute, subacute and chronic stages of ischaemia. It affects pathways
leading to excitotoxicity, apoptosis, inflammation and free radical production, as well as
blood flow, metabolism and bloodbrain barrier integrity. Hypothermia may also influence
neurogenesis, gliogenesis and angiogenesis after injury. It is likely that no single factor can
explain the neuroprotection provided by hypothermia, but understanding its myriad effects
may shed light on important neuroprotective mechanisms.
Apoptosis
Innate, programmed cell death
that is energy-dependent and
leads to nuclear and
cytoplasmic compaction with
characteristic blebbing of the
nucleus. It occurs during
development but also in
disease states.
Necrosis
Acute, uncontrolled cell death
that leads to cell lysis.
Department of Neurology,
University of California, San
Francisco, California
941430248, USA.
2
San Francisco Veterans
Affairs Medical Center, San
Francisco, California 94121,
USA.
3
Department of Physiology,
Kyungpook National
University School of Medicine,
Daegu, 700-422, South
Korea.
Correspondence to M.A.Y.
e-mail: Yenari@alum.mit.edu
doi:10.1038/nrn3174
Published online
22 February 2012
1
The lowering of temperature to preserve tissues is not

a new concept. Cooling organs that are to be used for
transplantation is known to prolong their viability, and
the discovery of well-preserved life forms in cold climates
also points to the protective role of lowered temperatures.
Although the phenomenon that profound temperature
changes can protect the brain from various insults has
been studied for quite some time, a report by Busto and
colleagues1 demonstrated that even small, clinically feasible decreases in body temperature could prevent neuron
death. This finding triggered renewed interest in cooling
or hypothermia as a means of protecting the brain.
Therapeutic hypothermia has been extensively studied
in the laboratory. It is one of the most robust neuroprotectants (that is, it protects neurons against apoptosis and
necrosis) studied to date, and recent clinical studies have
established a role for therapeutic cooling in neuroprotection in some clinical conditions, including anoxic brain
injury due to cardiac arrest 2,3 and hypoxic ischaemic neonatal encephalopathy 4,5. Furthermore, hypothermia has
been widely shown in the laboratory to protect against
experimental stroke, brain and spinal cord trauma,
although this remains to be convincingly shown at the
clinical level. Hypothermia is also an important part of
the practice sometimes referred to as suspended animation, which could be described as the slowing or cessation
of essential metabolic processes without causingdeath6.
Our increasing understanding of the metabolic and
molecular events that occur following brain ischaemia
and related acute neurological insults, such as hypoxia,
trauma and brain haemorrhage (FIG.1), has begun to
shed light on the many facets of and pathways involved

in ischaemic injury and how they are affected by therapeutic hypothermia. Animal models of focal and global
cerebral ischaemia are the most widely studied in terms
of hypothermic neuroprotection. Focal cerebral ischaemia models attempt to create brain injury that resembles stroke, whereas global cerebral ischaemia models
attempt to imitate brain injury resulting from cardiac
arrest. Focal cerebral ischaemia models can be further
divided into models of permanent occlusion, where the
parent vessel remains blocked (which could be said to
represent most clinical strokes), and models of temporary occlusion, where the parent vessel is re-opened
after a period of time. Hypothermia has also been studied in models of brain trauma7 and spinal cord trauma8,
where it had beneficial results, but this area of research
is beyond the scope of this Review.
This Review focuses mostly on animal studies of
brain ischaemia, including both focal and global cerebral ischaemia. These studies have collectively shown
that hypothermia affects nearly every investigated cell
death pathway, including pathways leading to excitotoxicity, apoptosis, inflammation and free radical production, and it is likely that no single factor can explain its
underlying beneficial effect. We first discuss methods
by which cooling is achieved in the laboratory. We then
review findings from animal studies that have begun to
reveal the effects of hypothermia during the acute, subacute and chronic phases of brain injury, and consider the
future prospects of therapeutic hypothermia in terms of
clinical translation and target identification.
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Phases of ischaemic stroke
Acute phase (minuteshours)
Subacute phase (hoursdays)
Chronic phase (weeksmonths)
Blood ow decrease
Apoptosis
Necrotic debris removal
Ionic homeostasis disturbance
Inammation by neutrophils, monocytes

and microglia
Stem cell proliferation,

dierentiation and maturation
Glutamate release increase and

excitotoxicity
Cytokine production
Angiogenesis
Cytotoxic oedema
Vasogenic oedema and intracranial pressure

increase
Intracellular calcium increase
Membrane, mitochondrial and DNA damage

Misfolding of proteins and enzyme
dysfunction
Proteolytic enzyme activation
Gliosis
Reactive oxygen species production

Stimulation of neurogenesis and angiogenesis
Reconnection of lost circuits

Neurovascular remodelling and functional
recovery
Necrosis
Figure 1 | The events involved in the pathogenesis of cerebral ischaemia are classified by their active time. The
events associated with the acute phase initiate and manifest their actions within minutes to hours after the onset of
Nature Reviews | Neuroscience
stroke. These events include the loss of blood flow, loss of ion homeostasis, release of excitotoxic neurotransmitters,
subcellular organelle damage, loss of normal protein structure and function, cell swelling followed by cell lysis, which gives
rise to cytotoxic oedema, and necrosis. Damage to mitochondria can set the stage for the generation of reactive oxygen
species when the occluded vessel is reperfused (reopened), because these mitochondria are no longer able to effectively
neutralize reactive species. Necrotic debris can then give rise to many subacute events, which occur hours to days later
(the subacute phase). Many of these processes are secondary to the initial ischaemic event, such as delayed cell death of
cells (apoptosis) in the periphery of the infarct an area that is exposed to less severe injury. In addition, necrotic debris
can stimulate immune responses and activate proteases. The inflammatory response itself can lead to further reactive
oxygen species generation. Although many subacute events could be considered damaging themselves, some of the
factors generated may be important in setting the stage for processes of recovery and repair, such as neurogenesis and
angiogenesis. In the chronic phase, which starts weeks to months later, many restorative processes occur, such as debris
removal, cell genesis, synaptogenesis and remodelling.
Methodological aspects of brain cooling

Most laboratory studies of hypothermia use small
rodents, although a few use larger mammals including
non-human primates; in rodents, cooling is performed
by applying a cooling blanket or by spraying water or
alcohol on the anaesthetized animals fur. Cooling for
longer durations (that is, for more than 1 day) in awake
and freely moving animals can be accomplished by using
automated misting systems and overheadfans9.
Studies aimed at determining the optimal parameters
to achieve brain protection without causing additional
adverse events have shown that relatively small decreases
in brain temperature are as protective as lower temperatures10,11. Indeed, brain temperatures in the range of
3034C (decreased from normal body temperatures
of 3638C) seem to provide protection that is as robust
as temperatures below 25C. Timing and duration are
also important factors for the effects of cooling, with
early initiation increasing the likelihood of a good outcome. For example, in models of focal cerebral ischaemia, protection is generally only observed if it is initiated
within 12h of ischaemia onset, although it can still be
observed when cooling is delayed by more than 2h,
provided it is maintained for relatively long durations
(2448 h)12. The therapeutic time window seems wider
in models of global cerebral ischaemia12. Importantly,
only a few studies have demonstrated beneficial effects of
cooling in terms of long-term benefit13,14 many studies
in animal models were carried out for relatively short
time periods, with a relatively brief coolingperiod.
Although cooling can be easily achieved in small
rodents, in humans it brings substantial challenges.
Because humans have a larger body mass and because

of the co-morbidities such as diabetes and cardiovascular disease that typically occur in victims of cardiac
arrest and stroke, cooling takes longer and may not be
as well tolerated as in smaller animals12. In addition,
early initiation is not always feasible in clinical settings.
Furthermore, most stroke patients are not anaesthetized
and are not generally comatose. Thus, shivering can
occur during cooling, which not only causes discomfort
but can also inhibit cooling efforts. These methodological aspects of therapeutic cooling raise questions as to
whether the effects of hypothermia observed in the laboratory are durable, and whether findings from studies in
young laboratory animals apply to older adult humans
with various co-morbidities10,15.
Finally, the phenomenon of hibernation in some
mammalian species also deserves some attention, as
hibernation leads to marked reductions in body temperature and slowing of metabolism. The changes that
occur in the brain during hibernation are similar to those
that occur during therapeutic hypothermia. Further,
an understanding of how animals enter and maintain
hibernation may lead to important insights into how
hypothermia should be applied in humans (BOX1).
Acute effects of hypothermia

Effects on metabolism, blood flow and excitoxicity. The
neuroprotective effects of cooling have largely been attributed to the finding that lowered temperatures decrease
the metabolic rate and reduce blood flow in the brain (see
REF.16 for a review). Temperature reductions decrease
brain oxygen consumption and glucose metabolism
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Reperfusion
The period of resumed blood
flow to the tissue after arterial
occlusion.
Hyperaemia
Higher than normal blood flow.
Torpor
A prolonged state of energy
conservation that allows
heterothermic animals to
tolerate limitations in resource
availability that are
encountered in extreme
environments.
by about 5% per degree Celsius17. Hypothermia also

preserves high-energy phosphate compounds, such as
ATP, and maintains tissue pH. By preserving the brains
metabolic stores, cooling can prevent the downstream
consequences of increased lactate production (which is
dependent on anaerobic metabolism) and the development of acidosis. The effect of cooling on cerebral blood
flow is less straightforward. In the absence of injury, cooling decreases cerebral blood flow, whereas under injury
conditions such as stroke, its effects are more complex.
(BOX2). During ischaemia, blood flow is markedly reduced
as a result of vessel occlusion; however, when blood flow is
restored (reperfusion), there is an overshoot of flow (hyperaemia) followed by a gradual decline that occurs over a
period of hours. Hypothermia actually blunts the immediate hyperaemia and prevents the gradual reduction in
cerebral blood flow that follows17. In other types of brain
injury, such as trauma, hypothermia has not always been
shown to affect cerebral bloodflow 17.
Although reports have been inconsistent (see REF. 18
for a review), therapeutic cooling has also been shown to
prevent the accumulation or release of excitotoxic amino
acids such as glutamate. This may be attributable to the
effect of cooling on metabolism, which preserves tissue
ATP levels. ATP is needed to maintain ion gradients, and
when these concentration gradients are disturbed, such as
in the case of ischaemia, calcium influx occurs and leads
to increased extracellular glutamate levels19. Hypothermia
may also prevent the consequences of excitotoxicity by
limiting calcium influx through AMPA channels. The glutamate receptor 2 (GluR2) subunit of the AMPA receptor
is thought to limit calcium influx, and its downregulation
by ischaemia20 may lead to the entry of excess calcium.
Indeed, one study demonstrated that hypothermia attenuates ischaemia-induced downregulation of GluR2 in a
model of global cerebral ischaemia20.
However, several studies in rodents, particularly those
examining mild and moderate hypothermia, have shown
that these acute mechanisms do no fully explain the protective effect of hypothermia. The changes in oxygen and
Box 1 | Hibernation
Hibernation is characterized by a prolonged state of energy conservation that animals
such as arctic ground squirrels and bears routinely undergo in order to withstand
extremely cold environments. In spite of the profound reduction of cerebral blood flow
that ensues, hibernation causes no lasting brain injury and hibernating animals show
relatively increased tolerance to ischaemic insults during and after the hibernation
period119. Several studies have investigated the mechanisms underlying hibernation,
and there seem to be many parallels between the brain changes that occur during
hibernation and the adaptations that the brain undergoes in order to develop
resistance to injury. These overlapping mechanisms include suppression of protein
synthesis, excitotoxicity, inflammatory responses, oxidative stress and activation of cell
death pathways120,121. Small hibernators such as arctic ground squirrels undergo
regulated decreases in core body temperature to near or below freezing during torpor,
whereas core body temperature in larger animals such as bears is far higher during
hibernation. The metabolism of hibernating bears is reduced by 53% from the basal
metabolic rate, even when core body temperature has returned to normothermic levels
towards the end of hibernation122. As metabolic conditions of non-hibernating
mammals under therapeutic hypothermia are similar to those of the hibernating bear,
the therapeutic implications of understanding how animals achieve hibernation may
provide useful insight into how to attain neuroprotection in humans.
glucose consumption and in cerebral blood flow cannot

explain the nonlinear protective effects of cooling 21. That
is, the extent of neuroprotection does not proportionately
increase with temperature decreases. Further, the protective effects of hypothermia cannot simply be explained by
the prevention of ATP loss or reduction in extracellular
glutamate as ATP loss and glutamate accumulation occur
within minutes of ischaemia onset, whereas hypothermia
can be effective even when cooling is initiated well after
these events have occurred12. Furthermore, some studies have shown that hypothermia merely delays many of
these acute events15,22. The debate over whether hypothermia really provides permanent protection or merely
delays the injury processes12 is important and still relevant today. In addition, the finding that cooling is also
protective if it is initiated even hours after ischaemia
onset suggests that hypothermia might have important
effects on injurious processes in the subacute and even
chronic phases of stroke and related insults, as discussed
below (BOX2).
Effects on early molecular events. Hypothermia has also
been shown to affect other acute processes associated
with ischaemia, including the induction of immediate
early gene expression23 and the cellular stress response.
The few studies that examined the effect of cooling on
the stress response specifically the expression of heat
shock proteins, which are upregulated in response to
various cellular stresses are mixed. Some studies
have shown that the expression of 70 kDa inducible heat
shock protein (HSP70) is increased under hypothermic
conditions24,25, and this might be consistent with its neuroprotective properties26. However, other studies have
shown that HSP70 expression is decreased under similar conditions27, and others have shown no influence of
cooling on its expression28. Thus, it is unclear whether
hypothermic neuroprotection is mediated by alterations in the cellular stress response. The significance
of the effect of hypothermia on immediate early gene
expression is also unclear, as no subsequent studies have
been carried out to systematically determine whether
the alteration in gene expression underlies the protective effect of hypothermia, or whether its suppression
represents a global downregulation of transcription and
contributes nothing to the observed protection.
MicroRNAs (miRNAs), a subset of non-coding
RNAs, have been a topic of recent investigationin brain
injury models, including stroke, where their expression
increases as early as 2h after ischaemia onset 29. miRNAs
are thought to play a part in silencing mRNAs, but they
can also regulate a range of signalling pathways29. It is
conceivable that they have an important role in stroke
pathogenesis, and the roles of specific miRNAs are currently under investigation. A recent report in a model
of traumatic brain injury showed that cooling alters the
expression of several miRNAs30. A few miRNAs, including miR874 and miR451, were most strongly affected:
cooling decreased the expression of both miRNAs at 7h,
but miR451 was increased by cooling at 24h compared
to normothermia. Further research is needed to better
define their role in brain injury.
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Subacute effects of hypothermia
The subacute phase of stroke is considered to be the time
during which secondary injury mechanisms occur, and
this is generally considered to be anywhere from 1 to 7
days post-ischaemia. It is during this period that many
reperfusion-related pathways are activated as a result of
the increased generation of reactive oxygen species (ROS)
by injured cells. In addition, inflammatory responses are
activated during this period, along with other cell death
pathways, including those leading to apoptosis. As a result,
the extent of ischaemic injury can widen during the subacute period, and secondary injury such as bloodbrain
barrier (BBB) disruption, oedema formation and haemorrhage can occur. Several studies have now demonstrated
that hypothermia affects multiple cell death and cell survival pathways. Its beneficial effects of suppressing several cell death mechanisms and enhancing cell survival
proteins might explain why hypothermia seems to be the
most robust neuroprotectant studied todate.
Effects on cell death pathways. Hypothermia has been
shown to affect several aspects of apoptotic cell death.
There are two main pathways that lead to apoptosis (FIG.2).
The intrinsic pathway is thought to stem from within
Box2 | Phase-specific effects of hypothermia in cerebral ischaemia

In the acute phase, cooling has beneficial effects on the brain as it preserves metabolic stores and maintains tissue
homeostasis, decreases excitatory amino acid accumulation and alters immediate early gene expression. During the
subacute phase, hypothermia alters apoptotic cell death pathways in such a way as to favour cell survival. It also prevents
bloodbrain barrier (BBB) disruption and its downstream effects, such as oedema and brain haemorrhage. Hypothermia
also inhibits many pro-inflammatory responses, and enhances the expression of trophic factors. In the chronic phase, the
beneficial effects of hypothermia can still be observed, even when cooling was applied during only the acute and early
subacute phase. Weeks to months after cooling (that is, in the chronic phase), the brain seems to have increased precursor
cell generation and differentiation, increased synaptogenesis and improved functional recovery compared to
normothermic brains.
Phase
Effect
Acute phase
Differential alteration of cerebral blood flow

Preservation of energy stores
Reduction of excitatory amino acids
Decrease of lactate production and cellular acidosis
Improvement of brain glucose metabolism
Alteration of immediate early gene expression
Alteration of the cellular stress response
Subacute phase
Prevention of apoptotic death:

decreased BAX expression (pro-apoptotic) and increased BCL-2 expression (anti-apoptotic)
inhibition of caspase-dependent pathway (cytochrome c, APAF1) and non-dependent pathway
(AIF)
inhibition of extrinsic apoptosis pathway
blocking of pro-apoptotic PKC activation and anti-apoptotic PKC degradation
preservation of anti-apoptotic AKT activity and attenuation of pro-apoptotic PTEN action
Inhibition of inflammation:
decreased inflammatory cell infiltration
decreased activation of immune transcription factors
reduced production of free radicals
Reduction of BBB disruption:
preservation of vascular endothelial function
reduction of extracellular protease expression and activity
reduction of brain haemorrhage
Increase of neurotrophins and their receptors
Chronic phase
Enhanced differentiation of precursor cells

Enhanced angiogenesis
Increase of neural and oligodendrocyte precursor cells
Induced neurite outgrowth and increased neuronal connectivity
AIF, apoptosis inducing factor; PKC, protein kinase C; PTEN, phosphatase and tensin homologue.
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Extrinsic pathway
FASL
Intrinsic pathway
FAS
FADD
Intracellular apoptotic signals

Cooling
Procaspase 8
BID
BAX
+
BCL-2
tBID
Caspase 8
PKC
AIF
+
Cytochrome c
Cooling
APAF1
Caspase 3
Caspase 9
Activated
caspases
Cooling
Caspaseindependent
pathway
Cooling
Caspase 3
+ PKC
Apoptosis
Figure 2 | Apoptotic pathways. Apoptosis occurs through three main pathways. The
extrinsic pathway begins outside the cell, with the activation of specific death receptors
Nature
| Neuroscience
on the cell surface. FAS is one of many such death receptors
thatReviews
are activated
by death
ligands. The ligand for FAS is FASL. Signalling from death receptors activates a signalling
complex through a death-inducing signalling complex (DISC). Activation of DISC,
a complex of a death ligand, death receptor and adaptor proteins, such as FASassociated death domain protein (FADD), leads to caspase 8 activation. Activated
caspase 8 initiates the caspase cascade, leading ultimately to cleavage and activation of
effector caspases such as caspase 3. Cleaved caspases eventually cause apoptosis. The
intrinsic pathway is initiated from within the cell in response to death signals such as
DNA damage, a defective cell cycle, detachment from the extracellular matrix, hypoxia,
loss of cell survival factors or other types of severe cellular stress. The intrinsic apoptotic
pathway hinges on the balance of activity between the pro-apoptotic (BCL2
antagonist/killer (BAK), BCL2associated X protein (BAX), BCL2related ovarian killer
protein (BOK), BH3interacting domain death agonist (BID), and so on) and
anti-apoptotic (BCL2, BCLXL, and so on) members of the BCL2 superfamily of
proteins, which are thought to influence mitochondrial signals. Mitochondrial
apoptotic signalling leads to the release of cytochromec from the mitochondrial
intermembrane space to the cytosol, where it forms an apoptosome with apoptotic protease-activating factor 1 (APAF1). The apoptosome activates caspase 9 and proceeds
down the same common pathway as the extrinsic pathway, leading to caspase 3
activation and, eventually, apoptosis. Protein kinase C (PKC) family members can be
either pro-apoptotic (PKC) or anti-apoptotic (PKC). PKC translocates from the
cytosol to the mitochondria to initiate apoptosis, whereas PKC prevents apoptosis.
There is also crosstalk between the intrinsic and extrinsic pathways, as death
receptor-activated caspase 8 can cleave the pro-apoptotic BCL2 family member BID to
a truncated form (tBID), and then can also initiate the intrinsic pathway through the
mitochondria. A third apoptotic pathway has also been described, where apoptosisinducing factor (AIF) is released from the mitochondria and directly leads to apoptosis
without activating caspases. Cooling affects several aspects of apoptotic cell death
pathways (white boxes).
the cell at the level of the mitochondria31, whereas the

extrinsic pathway is triggered via a cell surface receptor 32.
Hypothermia can affect both pathways, but whether
cooling has any effect on neuron survival depends on
whether apoptosis is occurring in a given model or paradigm. Models of moderate ischaemic injury such as
models of global cerebral ischaemia or mild focal cerebral
ischaemia (30minutes or shorter durations of middle
cerebral artery occlusion (MCAO)) lead to predominantly apoptotic cell death, whereas more severe insults
result in predominantly necrotic celldeath.
Cooling can interfere with the intrinsic (or mitochondrial) pathway by changing the expression of
BCL2 family members, reducing cytochromec release
and decreasing caspase activation33. In models of
global cerebral ischaemia, hypothermia leads to reductions in pro-apoptotic BCL2 family members such as
BCL2associated X (BAX) and increases in the antiapoptotic member BCL2. Acting downstream of BCL2
family proteins, protein kinase C (PKC) is a PKC isoform that has been shown to contribute to ischaemic
injury 34, and caspase 3 leads to translocation of PKC
from the cytosol to the mitochondria and to the nucleus
to induce apoptosis35. By contrast, a different isoform,
PKC is anti-apoptotic, and is degraded by caspases.
Interestingly, hypothermia does not seem to alter overall levels of PKC36, but it blocks its translocation to the
mitochondria and the nucleus and stimulates the action
of PKC after ischaemia37.
The extrinsic apoptotic pathway also seems to be activated in ischaemic brain injury. The most widely studied
apoptosis-inducing receptor and ligand in this pathway
are FAS and FASL, respectively. Genetic or pharmacological disruption of this pathway has been shown to improve
outcome from experimental stroke38,39, and hypothermia
seems to suppress the expression of both proteins in
models in which neuroprotection is observed40. In order
for FAS to initiate apoptosis via caspase 8 activation, its
ligand FASL must bind FAS. How FASL binds FAS is
somewhat unclear: many reports indicate that FASL must
be present on the cells surface in order to engage FAS41,
but other reports indicate that FASL must first be cleaved
from the surface by activated matrix metalloproteinases
(MMPs) and solubilized42. Hypothermia seems to prevent this cleavage, as levels of soluble FASL are decreased
in cooled rodent brains, as are levels of several MMPs43
46
. The decreased level of soluble FASL was also associated with decreased caspase 8 activation, which occurs
downstream of FAS activation40.
In models of more severe stroke (MCAO of 2h or
longer), hypothermia does not seem to affect BCL2
family members or caspase activation but prevents
cytochrome c release 47. These observations might
be explained by a third, caspase-independent apoptotic pathway that involves direct cell killing through
the release of apoptosis-inducing factor (AIF) from
mitochondria48. Indeed, in a more severe model of
MCAO, hypothermia reduced apoptotic cell death and
suppressed AIF translocation49.
Other, more recently studied molecules involved in
apoptosis are affected by hypothermia as well. Phosphatase
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and tensin homologue (PTEN) is a tumour suppressor
molecule with pro-apoptotic functions. PTEN deletion
has been shown to prevent ischaemic brain injury 50; however, PTEN phosphorylation, which leads to its deactivation, is normally decreased in brain ischaemia18. Under
conditions of hypothermia in which neuroprotection was
observed, phosphorylated PTEN levels were preserved,
but not under hypothermic conditions that did not result
in neuroprotection36. Thus, the deactivated form of this
pro-apoptotic protein seems to be associated with hypothermic neuroprotection. The mechanisms underlying
this association require further investigation.
Effects on survival pathways. Several neurotrophic
factors in the brain have been studied with regard to
their therapeutic potential in various acute neurological insults. These proteins control synaptic function and plasticity and sustain neuronal cell survival,
morphology and differentiation. In animal models of
brain insult, exogenous administration of one or more
of these factors improved functional neurological outcome without necessarily affecting lesion size. In studies
in which hypothermia had neuroprotective effects following ischaemic brain insult, levels of brain-derived
neurotrophic factor (BDNF)51,52, glial-derived neurotrophic factor (GDNF)53 and neurotrophin54 were
increased in the brain. Further, hypothermia increased
extracellular signal-regulated kinase (ERK) phosphorylation, a downstream element of BDNF signalling52,55.
However, ERK signalling itself does not seem to account
for the protective effect of hypothermia, as pharmacologic inhibition of ERK by U0126 failed to prevent the
benefit of hypothermia56.
Hypothermia also upregulates other survival factors.
As described above, hypothermia upregulates the antiapoptotic protein BCL2 (REFS 57,58) and also promotes
activation of AKT59, a serine/threonine protein kinase that
has multiple roles in glucose metabolism, cell proliferation,
Box3 | Cold shock proteins
Mammalian cells generally respond to cold temperatures by arresting the cell cycle and
inhibiting protein translation and gene transcription. Work in prokaryotic cells has
revealed a class of cold-inducible (or cold shock) proteins that enable bacteria to
withstand decreased temperatures. Two of these genes, cold-inducible RNA-binding
protein (CIRBP) and RNA binding motif protein 3 (RBM3), are specifically induced by
mild hypothermia123. They belong to a highly conserved glycine-rich RNA-binding
protein family and are known to regulate translation by acting as RNA chaperones124.
A few studies have begun to explore their significance in the brain. CIRBP mRNA in the
cortex and hippocampus was found to increase after brain ischaemia in rats, with even
higher increases in ischaemic brains of rats that had been exposed to hypothermia125.
CIRBP levels were also increased in neural stem cell lines cultured under a moderately
low temperature (32C) compared with neural stem cell lines cultured under normal
temperature (37C)98. Even though the capacity of cold shock proteins to increase the
survival of cells (by inhibiting apoptosis) and enhance activation of the extracellular
signal-related kinase signalling pathway has been reported in mammalian cell lines126,
this has not been directly studied in brain injury models. Knockdown of CIRBP by small
interfering RNA in fibroblasts prevented the anti-apoptotic effect of cooling117,
indicating a crucial role for CIRBP in the neuroprotective effect of hypothermia. Future
research on the effects and mechanisms of cold shock proteins in the brain will
contribute to a broader understanding of the effects of therapeutic cooling and lead
to the exploration of cold shock proteins as a therapeutic target.
apoptosis, transcription and cell migration. After phosphorylation by phosphoinositide 3kinase (PI3K), activated
AKT phosphorylates (that is, inactivates) pro-apoptotic
proteins such as glycogen synthase 3 (GSK3) and BCL2
antagonist of cell death (BAD). In a model of ischaemic
brain injury, hypothermia conferred neuroprotection by
maintaining AKT activity, and this protection was lost
when hypothermia was applied in combination with an
AKT inhibitor 59. Thus, although hypothermia has generally been documented to suppress or decrease metabolism and protein expression, it can also upregulate or
maintain proteins involved in cell survival and growth.
The family of cold-shock proteins, which are upregulated
at lower temperatures, has been studied in systems other
than the CNS, but might also be relevant in hypothermiainduced brain protection (BOX3).
Effects on inflammatory mediators. Although the
inflammation that accompanies many acute neurological conditions is thought to contribute to tissue recovery
and repair, studies have shown that it can also exacerbate
acute brain injury 60. The injured brain stimulates innate
immune responses leading to activation of microglia and
circulating leukocytes, and these immune cells can then
release various molecules, including ROS, proteases and
pro-inflammatory cytokines. These molecules can activate more inflammatory cells, leading to a vicious cycle
of cell death and immune activation.
Several animal studies have shown that inhibiting
various aspects of this immune response by brain cooling can have beneficial effects on neurological outcome following brain ischaemia and injury 61 (FIG.2).
Hypothermia indeed affects many aspects of this
immune response. It lowers numbers of neutrophils and
activated microglia in the ischaemic area and reduces
levels of many inflammatory mediators including ROS62
and reactive nitrogen species63, adhesion molecules64,
pro-inflammatory cytokines (such as interleukin1
(IL1), tumour necrosis factor- (TNF) and IL6)59,60
and the chemokines CC-chemokine ligand 2 (CCL2;
also known as MCP1) and C-C motif chemokine 20
(also known as MIP3)65,66. However, anti-inflammatory cytokines such as IL10 and transforming growth
factor- (TGF) are reduced by hypothermia as well67,68,
indicating that hypothermia does not have a purely
anti-inflammatoryeffect.
Hypothermia also suppresses the activation of
nuclear factor-B (NF-B)69, a major transcription factor
that can activate many inflammation-related genes. The
mechanism by which hypothermia suppresses NF-B
activation in inflammatory cells such as microglia and
also in neurons and astrocytes depends on the type
of cerebral insult. In models of focal brain ischaemia,
hypothermia prevented nuclear NF-B translocation
and DNA binding by inhibiting the activity of inhibitor
of NF-B kinase (IKK). IKK is required for the phosphorylation and degradation of NF-B inhibitor (IB),
thereby allowing NF-B to enter the nucleus, where it
can upregulate target genes69,70. In models of global cerebral ischaemia, hypothermia also decreased nuclear
translocation of NF-B, but its regulatory proteins IB
272 | APRIL 2012 | VOLUME 13
REVIEWS
and IKK were unaffected71. Notably, NF-B also regulates genes involved in cell survival and growth72; thus,
the net effect of hypothermia-induced suppression of
NF-B activity is difficult topredict.
Hypothermia also affects the mitogen-activated
protein kinase (MAPK) pathway, another important
enzyme system that regulates inflammation, in a celltype dependent manner. In stimulated cultured microglia, hypothermia suppresses ERK signalling 73, but in
experimental stroke, hypothermia actually activates
ERK in brain endothelial cells. This activation leads to
decreased levels of intercellular adhesion molecule 1
(ICAM1), which is one of many inflammatory factors
regulated by the MAPK signalling pathway and a type
of adhesion molecule involved in attracting circulating
inflammatory cells to theCNS74.
In summary, hypothermia has a largely suppressive
effect on inflammation, and this anti-inflammatory
property might serve as a major protective mechanism
in ischaemic conditions. In a similar way, mild hypothermia reduced inflammatory responses in a model of brain
inflammation where cell death does not occur, and this
suggests that inflammatory responses are temperature
sensitive64.
Effects on the bloodbrain barrier. The restoration
of blood flow after ischaemia can lead to secondary
injuries including oedema and haemorrhage, which
are consequences of BBB disruption. BBB disruption
after stroke or other brain injuries is caused by structural and functional impairment of components of the
neurovascular unit, including tight-junction proteins,
transport proteins, basement membrane, endothelial
cells, astrocytes, pericytes and neurons. Models of brain
ischaemia, trauma and intracerebral haemorrhage have
shown that mild to moderate hypothermia protects the
BBB and prevents oedema formation44,7579. Specifically,
hypothermia prevents the activation of proteases responsible for degrading the extracellular matrix, such as the
MMPs4346,80,81. Activated MMPs have been shown to
degrade several tight-junction proteins that make up
the BBB, leading to oedema formation and brain haemorrhage. Hypothermia reduces the proteolytic activity
of MMPs and the consequent degradation of vascular
basement membrane proteins82 and the extracellular
matrix proteins agrin and laminin82. In addition to suppressing MMPs, hypothermia increases the expression of
endogenous MMP inhibitors, such as metalloproteinase
inhibitor 2 (also known as TIMP2).
In a rat focal cerebral ischaemia model, preservation
of BBB integrity by hypothermia was documented even
5days after the cessation of hypothermia80, demonstrating the potential long-term benefits of hypothermia.
Hypothermia also preserves vascular morphology, as the
distortion of endothelial cells and their separation from
the basement membrane that is associated with ischaemia was attenuated by cooling in a model of experimental stroke82. In addition, it seems to affect other cells that
make up the BBB, such as pericytes, which are increasingly recognized to have an important regulatory role
in BBB integrity. In a model of MCAO, pericytes were
found to migrate away from the vessel wall, leading to

basement membrane disorganization, and moderate
hypothermia preventedthis83.
Hypothermia also attenuates oedema formation by
preserving the brains water balance. Aquaporins are a
family of water channel proteins that control the movement of water across cell membranes. Aquaporin 4 is the
predominant type of aquaporin in the microvasculature
of the CNS and has been observed in the end-foot membrane of astrocytes. Aquaporin 4 expression is increased
in reactive astrocytes in cerebral ischaemic lesions57, and
deleting Aqp4 reduces brain oedema following MCAO84.
Mild hypothermia reduced brain oedema formation by
suppressing aquaporin 4 expression in models of intra
cerebral haemorrhage85 and cardiac arrest 86. In summary, by preserving the structural proteins and cells that
constitute the BBB, and by inhibiting the activation of
damaging proteases and preventing the opening of water
channels, hypothermia prevents secondary brain injury
from brain oedema and haemorrhage.
In addition to water channel regulation, hypothermia modulates molecular transport across the BBB. One
study demonstrated that hypothermia decreased multidrug resistance protein 1 (MDR1)-mediated transport
without affecting passive diffusion and paracellular
transport 87. MDR1 is a type of transport protein that
mobilizes drugs and drug metabolites through transcellular pathways. The finding that hypothermia can affect
drug transport highlights the need for pharmacokinetic
studies: drugs that may be co-administered during cooling should be studied for any temperature-dependent
changes in pharmacokinetics.
By protecting BBB integrity, hypothermia can limit
brain oedema and increases in intracranial pressure.
However, hypothermia may not always lead to BBB
protection and subsequent neuroprotection. Studies in
models of protease-induced brain haemorrhage leading to BBB degradation have shown that hypothermia has inconsistent effects on functional outcome in
these models. Compared to brain ischaemia models,
in which several laboratories have consistently shown
hypothermia-induced neuroprotection, in brain haemorrhage models hypothermia leads to neurological
improvement in some laboratories, whereas others
report no improvement or even a slight worsening of
neurological outcome76,77,88,89. It should be noted that
hypothermia can also inhibit coagulation by decreasing fibrinogen generation and compromising thrombin
generation90. The net result of cooling in this situation
might increase the chance of bleeding 91. Thus, as cooling
could lead to expansion of bleeding, hypothermia may
be a less effective neuroprotectant in brain haemorrhage.
Chronic effects of hypothermia

Chronic effects of hypothermia have been observed
weeks to months after injury and well after cooling has
ceased. Recent work has focused on whether the acute
hypothermic treatment has lasting effects, and whether
hypothermia might affect recovery and repair mechanisms that occur in the brain long after the acute and
subacute phases ofinjury.
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REVIEWS
Nestin
An intermediate filament
protein used as a marker for
CNS stem cells.
Effects on neurogenesis. Recent studies have investigated the brains regenerative capacity following focal
cerebral ischaemia and traumatic brain injury 92. The
influence of therapeutic hypothermia on neurogenesis
and the brains ability to reorganize synaptic connectivity has been studied by a few groups, but is currently far
from clear. After brain insults such as stroke, neurons
in the ischaemic area lose synaptic connectivity and
undergo cell death93. However, it is becoming increasingly recognized that endogenous restorative processes
are also activated after ischaemia, leading to neurogenesis and synaptogenesis92. However, these restorative
processes are incomplete, as evidenced by the persistent disability seen in most stroke and brain trauma victims. Clearly, being able to enhance these endogenous
properties would have clinical benefits. Even though
rodent studies have shown that acute brain insults initiate the proliferation of neural stem cells in the subventricular zone and the hippocampal subgranular zone93,
neurogenesis in the uninjured aged brain is markedly
reduced94. This is relevant as these types of brain insult
occur commonly in old age, whereas gliogenesis is not
affected by age95. Spontaneous recovery through neurogenesis is limited in brain injury, and there is an obvious
need to develop strategies to improve regenerative processes such as proliferation of neuronal precursor cells,
migration of precursor cells to the injury area, differentiation into mature neurons and reconnection between
neurons. Here, we review the influence of hypothermia
on neurological recovery and repair (FIG.3).
Cooling has been shown to differentially affect neurogenesis in uninjured animals. In one study that examined neurogenesis in the developing brain, reduction
of brain temperature to 30C for 21h decreased the
number of proliferating cells in the subgranular zone
of the hippocampus, but not the periventricular zone96.
However, under conditions ofhypoxiaischaemia in
the developing brain, hypothermia to 33C enhanced
the maturation of neural progenitor cells in the striatum and inhibited apoptosis of proliferating neural stem
cells that were already increased by ischaemic stimuli97.
The mechanism of reduced apoptosis seems to be linked
to the cooling-induced upregulation of BCL2 (REF.97).
In a study of cultured neural stem cells, mild hypothermia also inhibited apoptosis, increased the number of
nestin positive cells and inhibited stem cell differentiation into astrocytes98. Adult rodents exposed to forebrain ischaemia and subjected to mild hypothermia had
increased numbers of newborn neurons in the dentate
gyrus compared to animals exposed to ischaemia without cooling 99. By contrast, another study in adult rats
with forebrain ischaemia showed that hypothermia had
no effect on neurogenesis100; however, the duration of
hypothermia in this study was rather short (33C for
45min) and occurred relatively early, either during the
ischaemic period or during the immediate reperfusion
phase. Thus, it is possible that hypothermia may not
have any effect on neurogenesis if it is not applied during a critical time window (or windows) that has yet to
be clearly defined. More research in this area is needed;
in particular to determine the optimal conditions under
which cooling might be expected to positively influence neurogenesis and whether cooling may improve
neurogenesis in aged brains exposed to ischaemia and
related insults.
Effects on gliogenesis and angiogenesis. Oligo
dendrocytes, although less studied than neurons, succumb to brain insults and undergo cell death with a
susceptibility that is similar to neurons, and hypothermia attenuates trauma-induced oligodendrocyte
cell death, demyelination and circuit dysfunction101,102.
Hypothermia (32C) increased the number of oligodendrocyte precursor cells in a primary culture taken from
embryonic mouse brains103. As a result, greater numbers of oligodendrocyte precursor cells that undergo
cell cycle progression were maintained in a less welldifferentiated state. However, an invivo study using a
hypoxia model in preterm fetal sheep demonstrated
that hypothermia (30C) was associated with an overall
reduction in hypoxia-induced loss of immature oligodendrocytes, although it did not prevent the hypoxiainduced reduced proliferation of oligodendrocytes
within the periventricular white matter 104.
Reports of the effects of hypothermia on endo
genous cell genesis in the injured and uninjured brains
are somewhat conflicting. Some reports96,104 indicate
that hypothermia suppresses stem cell proliferation,
whereas many reports indicate the opposite98,99,103, and
some even suggest that cooling promotes progenitor cell
differentiation towards neurogenesis over gliogenesis97,98.
Hypothermia to temperatures lower than 30C seems
to suppress cell proliferation and phase-specific and
nonspecific cell cycle arrest as a result of reduced energy
supply 96,105. However, small temperature decreases seem
to protect against progenitor cell death98,104. Thus, we
speculate that mild hypothermia enables the differentiation of precursor cells while preventing apoptosis, and
that cooling to lower temperatures seems detrimental to
cells and blocks their proliferation.
Astrogliogenesis and angiogenesis are thought to contribute to brain regeneration following brain injury 93,106,
but this has not been studied intensively. Astrocytes
comprise the largest population of cells in the ischaemic
core during the subacute to chronic period after stroke107,
and reactive astrocytes are the main component of the
glial scar. However, glial scar formation in the brain can
obstruct neurite outgrowth and regeneration108,109, and
blocking astrocyte activation and related reactions can
exacerbate inflammation and increase injury responses109.
Thus, enhancement of gliogenesis may do some harm.
How hypothermia affects gliogenesis has not yet been
studied in any depth. Mild hypothermia has been shown
to enhance angiogenesis in focal cerebral ischaemia110,
spinal cord injury111 and traumatic brain injury models112.
Although these angiogenic effects by hypothermia are
presumably beneficial for repair processes, their significance is still uncertain. In fact, a few studies suggest that
angiogenesis may actually be detrimental to brain repair.
For example, one study of acute stroke patients showed
that an early dominance of pro-angiogenic factors
(including platelet-derived growth factors (PDGFs),
274 | APRIL 2012 | VOLUME 13
REVIEWS
Metabolic
acidosis
Necrosis
Excitotoxicity
Cytotoxic oedema
Lactate
production
Na+K+
pump failure
Anaerobic
metabolism
ATP depletion
Low O2
and glucose
EAA release
and receptor
activation
Mitochondrial
dysfunction
FAS
BAX/BCL-2
PKC
Ion imbalance and

calcium inux
Survival pathway
AKT
MAPK
Immediate
early genes
Neutrophil/
macrophage/
microglia activation
Inammatory
cytokines
Intracerebral
haemorrhage
MMP activation
Adhesion molecule
induction
Acute phase
(minuteshours)
Gliosis
Astrocyte
activation
Enzyme activation
and breakdown of
proteins, lipids and
nucleus
ROS/RNS
production
Apoptosis
Neurogenesis
Angiogenesis
Synaptogenesis
Trophic factors
Stress signals
Ischaemic
insult
CytC release
and caspase
activation
Endothelial damage
and BBB dysfunction
Subacute phase
(hoursdays)
Vasogenic
oedema
Chronic phase
(weeksmonths)
Figure 3 | Effects of therapeutic hypothermia on the pathogenesis of cerebral ischaemia. The ischaemic cascade, as
it pertains to events studied in hypothermia paradigms, is outlined with a relative timeline shown at the bottom. Following
ischaemia, acute events such as loss of oxygen and glucose lead to energy loss (that is, loss of ATP) and ion pump failure. The
resulting loss of concentration gradients allows ions to flow down their concentration gradients, leading to cell swelling
(cytotoxic oedema) and to the release of excitatory amino acids (EAAs). Decreased glucoseNature
also causes
the cell
to switch
Reviews
| Neuroscience
from aerobic to anaerobic metabolism, which leads to metabolic acidosis. All of these events will induce acute cell death, or
necrosis. Ischaemia also leads to the upregulation of many immediate early genes and stress signals, which can worsen
tissue injury during the subacute phase of stroke. These genes and signals lead to apoptosis, activation of the inflammatory
response and, subsequently, activation of damaging proteases such as matrix metalloproteinases (MMPs). This can lead to
secondary damage, including brain oedema and haemorrhage. During this phase, factors that set the stage for recovery and
repair are also activated. This includes AKT activation and trophic factor upregulation. During the chronic phase of stroke,
recovery and repair mechanisms predominate, including neurogenesis, angiogenesis and synaptogenesis. Gliosis is also
present and leads to scar formation. Therapeutic hypothermia has been shown to suppress or inhibit processes indicated by
green boxes, whereas yellow boxes show events that are increased or enhanced by hypothermia. Purple boxes indicate events
that are not affected by cooling. BAX, BCL-2-associated X; BBB, bloodbrain barrier; CytC, cytochrome c; MAPK,
mitogen-activated protein kinase; PKC, protein kinase C; ROS/RNS, reactive oxygen species/ reactive nitrogen species.
vascular endothelial growth factors (VEGFs) and their

receptors, stromal cell-derived factor 1 (SDF1) and hepatocyte growth factor (HGF)) was associated with mild
short-term neurological deficits, but that an acute antiangiogenic status (as defined by elevated plasma endostatin levels) also predicted a worse long-term functional
outcome113. Furthermore, pharmacologic stimulation of
angiogenesis using high-dose VEGF impeded recovery
of neurological function in a rat model of global cerebral
ischaemia and caused neuronal damage in uninjured

control brains114.
Effects on neural connectivity. In addition to stem cell
genesis, repair of neuronal connectivity is crucial to functional recovery after stroke. Few studies have examined
the role of hypothermia on neuronal circuit repair. At
the morphological level, neurite and axonal outgrowth
were enhanced by applying deep hypothermia (17C) in
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REVIEWS
Coagulopathies
Abnormal conditions of blood
clotting or blood clot lysis.
Autophagy
The breakdown of a cells own
components by the lysosome.
Anoikis
A form of programmed cell
death that is activated when
cells detach from the
extracellular matrix
organotypic brain slices115,116. A genomic analysis study in

a rat model of traumatic brain injury demonstrated that
mild hypothermia had a significant effect on gene expression. An analysis of hippocampal gene expression profiles
from rats exposed to hypothermia following traumatic
brain injury revealed statistically significant differences
in 133 transcripts compared to injured normothermic
rats. Of these, 57 transcripts were upregulated and 76
were downregulated afterinjury. Those genes belonging
to synapse organization and biogenesis were especially
upregulated in hypothermic animals compared to normothermic animals117. Although the scientific literature is
still scant, current data suggest that overall, hypothermia
supports regenerative processes by enhancing synapse
formation and reorganization.
To summarize, although the full effect of therapeutic
hypothermia in brain repair is unclear, under specific conditions it seems to have a beneficial role in protecting stem
cells, promoting their proliferation and differentiation
and possibly encouraging recovery of neural circuitry.
Conclusions and future directions

Hypothermia is a remarkable phenomenon that preserves tissues and limits injury. It is perhaps the most
robust neuroprotectant studied in the laboratory and has
also been shown to have efficacy in humans. It affects
nearly every metabolic, molecular and cellular event in
cell death to promote tissue preservation (FIG.3). More
recent studies have shown that hypothermia can also
favourably modulate endogenous regenerative and
restorative properties. This multi-faceted aspect of
therapeutic hypothermia may suggest that the goal
of neuroprotection requires multi-target approaches.
It may also be possible to combine hypothermia with
other therapeutic modalities, such as neuroprotectants or thrombolytic agents, to extend the therapeutic
window of the drugs or of hypothermia itself. The use
of hypothermia in combination may lead to the reexamination of the many neuroprotectant drugs that
failed at the clinical level, as many drugs may not have
been studied in the most optimalmanner.
In spite of the robust protective effects of therapeutic hypothermia demonstrated in the laboratory, there
are several obstacles that hamper the application of this
1.
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Acknowledgements
This work was supported by grants from the US National

Institutes of Health (NS40516 to M.Y.), the Veterans Merit
Award (M.Y.), the Korea Healthcare technology R&D Project,
Ministry of Health & Welfare, Republic of Korea (A100870 to
H.S.H.) and the Industrial Strategic technology development
program (10035197 to H.S.H.), which is funded by the
Ministry of Knowledge Economy (MKE), Korea. Grants to M.Y.
were administered by the Northern California Institute for
Research and Education, and supported by resources of the
Veterans Affairs Medical Center, San Francisco, California.
Competing interests statement
The authors declare no competing financial interests.

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REVIEWS

The lowering of temperature to preserve tissues is not

shed light on the many facets of and pathways involved

NATURE REVIEWS | NEUROSCIENCE

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Subacute phase (hoursdays)

Chronic phase (weeksmonths)

Necrotic debris removal

Ionic homeostasis disturbance

Inammation by neutrophils, monocytes

Stem cell proliferation,

Glutamate release increase and

Vasogenic oedema and intracranial pressure

Intracellular calcium increase

Membrane, mitochondrial and DNA damage

Proteolytic enzyme activation

Reactive oxygen species production

Reconnection of lost circuits

Methodological aspects of brain cooling

Because humans have a larger body mass and because

Acute effects of hypothermia

268 | APRIL 2012 | VOLUME 13

by about 5% per degree Celsius17. Hypothermia also

glucose consumption and in cerebral blood flow cannot

NATURE REVIEWS | NEUROSCIENCE

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Box2 | Phase-specific effects of hypothermia in cerebral ischaemia

Differential alteration of cerebral blood flow

Prevention of apoptotic death:

Enhanced differentiation of precursor cells

270 | APRIL 2012 | VOLUME 13

Intracellular apoptotic signals

the cell at the level of the mitochondria31, whereas the

NATURE REVIEWS | NEUROSCIENCE

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272 | APRIL 2012 | VOLUME 13

found to migrate away from the vessel wall, leading to

Chronic effects of hypothermia

NATURE REVIEWS | NEUROSCIENCE

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274 | APRIL 2012 | VOLUME 13

Ion imbalance and

vascular endothelial growth factors (VEGFs) and their

ischaemia and caused neuronal damage in uninjured

NATURE REVIEWS | NEUROSCIENCE

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organotypic brain slices115,116. A genomic analysis study in

Conclusions and future directions

Busto, R. etal. Small differences in intraischemic brain

method in the clinic, including how to achieve effective

A clinical trial of selective head cooling that showed

276 | APRIL 2012 | VOLUME 13

awake and freely moving rodents. J.Neurosci.

35. Raval, A.P. etal. Protein kinase C cleavage initiates

NATURE REVIEWS | NEUROSCIENCE

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97. Xiong, M. etal. Post-ischemic hypothermia promotes

278 | APRIL 2012 | VOLUME 13

114. Manoonkitiwongsa, P.S., Schultz, R.L., McCreery,

This work was supported by grants from the US National

Competing interests statement

The authors declare no competing financial interests.

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