Biochemical Basis of Hypoxic-Ischemic Encephalopathy

Maria Delivoria-Papadopoulos and Peter J. Marro

NeoReviews 2010;11;e184-e193
DOI: 10.1542/neo.11-4-e184

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Article neurology

Biochemical Basis of Hypoxic-Ischemic

Encephalopathy
Maria Delivoria-
Papadopoulos, MD,*
Objectives After completing this article, readers should be able to:
Peter J. Marro, MD† 1. Describe the steps of posthypoxic brain injury.
2. Explain the role of N-methyl-D-aspartate (NMDA) receptors in hypoxia.
3. Delineate the contribution of free radicals to neuronal injury during hypoxia.
Author Disclosure
4. Explain how hypoxia affects calcium influx and modification of apoptotic proteins.
Drs Delivoria-
Papadopoulos and
Marro have disclosed Abstract
no financial Despite improved methods of intrapartum monitoring and advances in neonatal care
and treatment, neonatal hypoxic-ischemic injury continues to produce significant
relationships relevant
morbidity and mortality, often leading to long-term neurologic consequences. Hyp-
to this article. This
oxia creates an imbalance in metabolic demand and cellular energy supply, resulting
commentary does not in the disruption of critical cellular functions and the activation of excitatory neuro-
contain a discussion transmitters. In addition, the structure, function, and modification of cellular pro-
of an unapproved/ cesses, such as the N-methyl-D-aspartate (NMDA) receptor and intracellular calcium
investigative use of a regulation, are affected. Nuclear calcium signals control critical nuclear functions,
including regulation of transcription factors and cell cycle, gene transcription, DNA
commercial
replication, and nuclear envelope breakdown. Nitric oxide synthase and the genera-
product/device.
tion of nitric oxide during hypoxia may contribute significantly to altered cell
function, disruption in calcium homeostasis, and the activation of caspases, leading to
programmed cell death. The biochemical mechanisms involved in hypoxic-ischemic
neuronal injury and death are exceedingly complex and interdependent. This discus-
sion focuses primarily on some of the major cellular and molecular mechanisms of
hypoxic neuronal injury in the newborn brain.

Introduction
Perinatal hypoxia-ischemia is the most common cause of
neurologic disease during the neonatal period. Hypoxic-
Abbreviations ischemic encephalopathy (HIE) is associated with high mor-
tality and morbidity rates, including cerebral palsy, intellec-
Apaf-1: apoptotic protease activation factor
tual disability, and seizures. The incidence of perinatal
ATP: adenosine triphosphate
asphyxia is about 1.0% to 1.5% in most centers and usually is
CaM kinase: calcium/calmodulin kinase
related to gestational age and birthweight. It occurs in 9.0%
CNS: central nervous system
of infants younger than 36 weeks’ gestation and in 0.5% of
CREB: cAMP response element binding
infants older than 36 weeks’ gestation. (1) Perinatal HIE
cyt c: cytochrome C
may develop during the antepartum (20%), intrapartum
HIE: hypoxic-ischemic encephalopathy
(30%), antepartum and intrapartum (35%), or postpartum
ICAD: inhibitor of caspase-activated DNase
(10%) period. (2) HIE develops in the setting of perinatal
IP3: inositol triphosphate
asphyxia, which is a multiorgan system disease and includes
NMDA: N-methyl-D-aspartate
circumstances affecting the cerebral blood flow in the fetus
NNLA: Nw-nitro-l-arginine
and newborn that compromise the supply of oxygen to the
nNOS: neuronal nitric oxide synthase
brain. Assessment and management of these complications is
NO: nitric oxide
an integral part of the treatment of perinatal asphyxia/HIE.
NOS: nitric oxide synthase
(3)
PARP: poly-ADP-ribose-polymerase
A large amount of information has been collected on the

*Professor of Pediatrics and Physiology Emeritus, University of Pennsylvania School of Medicine, Philadelphia, Pa.
†
Division of Neonatology, Barbara Bush Children’s Hospital at Maine Medical Center, Portland, Me.

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 neurology hypoxic-ischemic encephalopathy

fetal cardiovascular and respiratory response to oxygen

limitations, giving rise to a better understanding and
management of neonatal deterioration induced by hyp-
oxia. Besides these physiologic studies, cellular and bio-
chemical mechanisms are being explored increasingly to
investigate the complex and interrelated biochemical
alterations that result in hypoxic brain injury and brain
cell death in the fetus and newborn. (4)(5) It is important
to recognize the factors that may determine the sus-
ceptibility of the developing brain to neonatal and peri-
natal hypoxia. These determinants include the lipid com-
position of the brain cell membrane, the rate of lipid
peroxidation, the presence of antioxidant defenses, the
development and modulation of the excitatory neuro-
transmitter receptors such as the NMDA receptor, and
the intracellular calcium influx mechanisms. In addition
to the developmental status of these cellular compo-
nents, the response of the potential mechanisms to hyp-
oxia determines the fate of the hypoxic brain cell in the
developing brain of the fetus and the newborn.

Steps of Posthypoxic Brain Injury

Energy Breakdown
Under normal conditions, oxidative phosphorylation
and the synthesis of high-energy phosphates such as
adenosine triphosphate (ATP) requires adequate oxygen
supply. Under anaerobic conditions, the metabolic cost
of ATP production is critically increased, leading to a
breakdown of energy balance that depletes brain cells of
high-energy compounds necessary for energy-dependent
metabolic processes in neurons and glial cells.
Excitotoxic Mechanisms (Fig. 1)
The decrease in ATP concentrations leads to cell mem-
brane depolarization and a disruption of voltage-
dependent ion channels, which allows excessive amounts
of calcium to enter the cytosol, initiating the release of
glutamate that activates NMDA receptors. The increased
expression/activation of NMDA receptors further en-
hances cellular calcium influx. The entire mechanism is
accelerated further by the dysfunction of the energy-
dependent reuptake of glutamate both in neurons and
in astrocytes in a vicious cycle. When calcium influx is
unregulated, activation of phospholipases, proteases, and
endonucleases can result in nuclear, organelle, and cell
membrane disruption.
Cell Death (Fig. 2)
Acute or long-term consequences of HIE are related
either to necrosis or to apoptosis of neuronal cells. Ne-
crosis is characterized by passive cell swelling; rapid en-
Figure 1. Effects of hypoxia leading to increased intranuclear
calcium. ATPⴝadenosine triphosphate, NMDAⴝN-methyl-D-
aspartate
ergy loss; generalized disruption of internal homeostasis
leading to eventual lysis of the nucleus, organelles, and
plasma cell membranes; and the release of intracellular
components that induce a local inflammatory response.
The result is edema and injury to neighboring cells. The
inflammatory response triggers expression of the cyto-
kines interleukin-1-beta and tumor necrosis factor-alpha,
which stimulate release of oxygen free radicals from
neutrophils that activate microglial cells.
Necrosis is only one of the mechanisms of cell death
following hypoxia or ischemia to the brain. Programmed
cell death or apoptosis also appears to contribute to cell
death following hypoxia-ischemia, especially cell death
that occurs days to weeks following the insult. (6)(7)
Apoptosis is an active process that requires activation of a
genetic program and specific endonucleolytic digestion
of nuclear DNA. In contrast to necrosis, programmed
cell death is characterized by cell shrinkage, coarse chro-

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neurology hypoxic-ischemic encephalopathy
Figure 2. Pathways of neuronal cell death following initial
energy failure. NMDAⴝN-methyl-D-aspartate. Reprinted
with permission from Marro PJ. The etiology and pharmaco-
logic approach to hypoxic-ischemic encephalopathy in the
newborn. NeoReviews. 2002;3:e99.
matin aggregation with extensive nuclear DNA fragmen-
tation, nuclear pyknosis, and extrusion of membrane-
bound cytoplasmic fragments or apoptotic bodies, but it
is not associated with lysis of the plasma membrane.
(8)(9) Studies in cell culture models have demonstrated
that hypoxia can trigger programmed cell death. (10)
Programmed cell death, as assessed by the cleavage of
genomic DNA, also has been shown to occur in the brain
following focal (6)(11)(12) and global ischemia. (7)(13)
The mechanism by which hypoxia causes DNA fragmen-
tation has been studied extensively but is not well under-
stood.
NMDA Receptors
Glutamate is the primary excitatory amino acid neuro-
transmitter that contributes to a number of essential
developmental processes such as synaptogenesis, synaptic
plasticity, long-term potentiation, learning, and memory
as well as neurodegeneration and hypoxia-induced in-
jury. (14)(15) The physiologic and pathologic effects of
glutamate in the central nervous system (CNS) are me-
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diated through its interaction with specific cell mem-
brane receptors, of which the NMDA, kainate, and
AMPA subtypes are the best characterized. (16)
The NMDA-type glutamate receptor is a predomi-
nant mediator of excitotoxicity in the immature brain
compared with the adult brain due to overexpression of
the receptor in the developing immature CNS. (17)
Within the developmental period, NMDA receptor-
mediated processes may depend not only on the ontog-
eny of the NMDA receptor, but also potential modifica-
tion by intracellular mechanisms.
Structure and Function of the NMDA Receptor
The activity of the NMDA receptor-ion channel complex
is regulated by a number of pharmacologically distinct
binding sites. The NMDA receptor possesses a neuro-
transmitter binding site, or recognition site, that binds
glutamate or NMDA; a coactivator site that binds gly-
cine; a channel site that binds MK-801; a voltage-
dependent magnesium-binding site; a polyamine site; an
ifenprodil site; and an inhibitory divalent cation site that
binds zinc. (17) Ligand binding studies indicate two
distinct binding sites or states associated with the gluta-
mate recognition site, one that preferentially binds ago-
nists and one that preferentially binds antagonists. (18)
The NMDA receptor is associated with a cation-
selective ion channel that gates sodium, potassium, and
calcium ions. In the resting state, when blocked by
magnesium in a voltage-dependent manner, (19)(20)
blockade of the ion channel complex by glutamate or
NMDA is allowed, and agonist-dependent calcium in-
flux occurs. The influx of calcium ions is believed to
initiate biochemical processes responsible for both
NMDA receptor-induced plasticity in the developing
brain and NMDA receptor-mediated excitotoxic cell
death. (15)(21)(22)
Each of the regulatory sites of the NMDA receptor-
ion channel complex is modified during brain develop-
ment, which may play a role in altering the response of
the receptor during development and hypoxia.
Mechanism of NMDA Receptor Modification
During Hypoxia
Brain tissue hypoxia modifies the NMDA receptor rec-
ognition, coactivator, and ion channel sites. A decrease in
the apparent number of NMDA receptors and an in-
crease in receptor affinity for MK-801 were observed in
hypoxic fetal guinea pig and newborn piglet brains.
(23)(24) In these same studies, glutamate- and glycine-
dependent activation of the NMDA receptor was de-
creased and spermine-dependent and basal state receptor

activation were increased during hypoxia. Hypoxia-
induced modification of the recognition, coactivator,
and modulatory sites of the NMDA receptor-ion channel
complex is likely through nitric oxide (NO)-mediated
nitration. In neurons of the CNS, neuronal nitric oxide
synthase (nNOS) is colocalized with the NMDA recep-
tor, (25)(26) thereby favoring nitration of the receptor.
In addition, nNOS activity is decreased by phosphoryla-
tion and increased by dephosphorylation, (27)(28) a
condition likely present during hypoxia. Dephosphory-
lation of the receptor also makes tyrosine sites available
for nitration by peroxynitrite, which is produced by NO
and superoxide radicals, both of which are produced
during hypoxia. Thus, dephosphorylation during hyp-
oxia may facilitate peroxynitrite-mediated nitration of
tyrosine-inhibiting phosphorylation of proteins and al-
teration of the NMDA receptor-ion channel complex.
(29)
In neurons of the CNS, nNOS is activated by calcium
influx through the NMDA receptor-ion channel, but
nNOS is not efficiently stimulated by activation of non-
NMDA receptors that also induce calcium influx. (30)
The synaptic localization of nNOS in the brain may be
mediated by the postsynaptic density protein PSD-95.
Recently, it was demonstrated that nNOS, PSD-95, and
NMDA receptor subunit NR2B from the brain coimmu-
noprecipitate and that the PSD-95 is sufficient to assem-
ble a tight tertiary complex with nNOS and the NR2B
subunit of the NMDA receptor. (31)
In summary, results of these studies indicate that NO
production in the brain is preferentially activated by
calcium influx through the NMDA receptor-ion channel,
that there is a specific structural and functional link
between the NMDA receptor and nNOS, and that the
Table 1. Deleterious Effects of Increased Intracellular Calcium
Action
Activates phospholipases
Activates proteases
Activates nucleases
Activates calcium-ATPase
Enters mitochondrion and uncouples oxidative phosphorylation
Increases neurotransmitter release
Activates protein that transforms xanthine dehydrogenase
to xanthine oxide
Activates nitric oxide synthase
ATP⫽adenosine triphosphate
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neurology hypoxic-ischemic encephalopathy
nitric oxide synthase (NOS) pathway plays a critical role
in the NO-mediated mechanism of hypoxia-induced
modification of the NMDA receptor complex in the
newborn brain.
Free Radicals
Free radicals are molecular species that have unpaired
electrons in the outer orbit with a strong tendency to
initiate chain reactions that result in membrane peroxi-
dation, protein oxidation, nucleic acid oxidation, and cell
damage. Normally, more than 80% of the oxygen con-
sumed by the cell is reduced completely by cytochrome
oxidase to water without production of oxygen free
radicals. The remaining 10% to 20% undergoes other
oxidation reduction reactions in the cytoplasm and mi-
tochondria that produce a superoxide anion radical.
Mechanisms of Free Radical Generation During
Hypoxia
There are a number of potential mechanisms of free
radical generation under hypoxic conditions. During
hypoxia, the increased accumulation of intracellular cal-
cium resulting from excessive activation of NMDA (32)
and non-NMDA receptors is crucial in hypoxia-induced
excitotoxicity. Increased intracellular calcium can initiate
a number of biochemical events that could lead to free
radical generation and cell death (Table 1).
In addition to calcium mediation, other potential
mechanisms of free radical generation during hypoxia
include: 1) reduction of electron transport chain com-
ponents, including ubiquinone (a component that un-
dergoes auto-oxidation to produce free radicals); 2) in-
creased release of ferritin under the conditions of
decreased cellular high-energy compounds; and 3) in-
Result
● Phospholipid hydrolysis and cell membrane injury
● Generation of free radicals
● Cytoskeletal disruption of microtubules
● Proteolysis of other cellular proteins
● Nuclear injury
● Further increase in cytosolic and nuclear calcium
● Consumes ATP at time of energy depletion
● Decreased ATP production
● Activation of glutamate receptors and calcium influx
● Free radical formation
● Generation of nitric oxide
NeoReviews Vol.11 No.4 April 2010 e187
neurology hypoxic-ischemic encephalopathy
creased degradation of ATP during hypoxia, increasing
the substrate for the xanthine oxidase reaction and lead-
ing to increased free radical generation.
Increased free radical generation during hypoxia in
the cerebral cortex of newborn piglets has been docu-
mented directly through spin trapping of free radicals
and measuring the resulting spin adduct signal with
electron spin resonance spectroscopy. The characteristics
of the spin adduct signal identify the free radical species
present in the hypoxic tissue as predominantly an alkoxyl
radical, indicating that free radical-mediated lipid peroxi-
dation is an ongoing event during cerebral hypoxia, a
mechanism of hypoxic neuronal injury. These studies not
only demonstrate increased free radical generation dur-
ing hypoxia in the cerebral cortex of the fetus and the
newborn but reduced hypoxia-induced production of
free radical species due to inhibitors of pathways of free
radical generation.
Nitric Oxide Free Radicals and Neuronal Injury
Three major isoforms of NOS have been identified:
constitutive neuronal, constitutive endothelial, and in-
ducible macrophage isoforms. Following ischemia, NO
produced from neuronal NOS has toxic effects, but NO
produced from endothelial NOS has protective effects in
the brain. (33) Hypoxic brain injury is associated with
the formation of NO. (34)(35) Although NO physiolog-
ically mediates cerebral vasodilation under normal con-
ditions, (36) recent studies suggest that NO, a gaseous
free radical, may react with superoxide anion to form
peroxynitrite and cause neurotoxicity. (37)(38)(39)
NO is reported to cause neuronal damage through
various mechanisms. Studies using the NOS inhibitor
Nw-nitro-l-arginine (NNLA) demonstrated that free
radicals, corresponding to alkoxyl radicals, were induced
by hypoxia but were inhibited by pretreatment with
NNLA. NNLA also inhibited hypoxia-induced genera-
tion of conjugated dienes (products of lipid peroxida-
tion) and preserved Na⫹, K⫹-ATPase activity (an index
of cellular membrane function). These data demon-
strated that NOS generates free radicals during hypoxia
via peroxynitrite production, presumably causing lipid
peroxidation and membrane dysfunction. The produc-
tion of secondarily formed lipid free radicals provides
strong evidence of peroxidative injury. This is particularly
true for alkoxyl radicals, which are generated from lipid
peroxide by either iron or copper ions and can abstract
hydrogen atoms from polyunsaturated fatty acids, lead-
ing to further lipid peroxidation. This suggests that NO
has an in vivo role in the generation of alkoxyl radicals,
leading to free radical-mediated lipid peroxidation.
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The exact molecular mechanism of hypoxic mem-
brane damage is not clear. However, it has been shown
that peroxynitrite (formed by the reaction between su-
peroxide anions and NO) can cause lipid peroxidation in
vitro. (40) Therefore, high concentrations of NO during
hypoxia may result in increased production of peroxyni-
trite, causing lipid peroxidation.
Neuronal Nuclear Calcium Influx
A number of critical nuclear functions, including regula-
tion of transcription factors, cell cycle regulation tran-
scription, DNA replication, and nuclear envelope break-
down, are controlled by intracellular calcium (Table 2).
(41) Furthermore, nuclear calcium signals potentially
control a number of events leading to hypoxia-induced
programmed cell death. Nuclear and cytosolic calcium
signals are regulated differently, and the extranuclear
calcium concentration determines the mode of calcium
entry into the nucleus. Increased intracellular calcium
has been shown to be a primary mediator of activity-
dependent gene transcription under a number of exper-
imental conditions. (42)(43)(44) Several factors may be
involved in calcium-regulated gene expression and tran-
scription, including the site of calcium entry, the ampli-
tude and the spatial properties of the calcium signals, and
the duration of the calcium signal. (45)(46)
Studies have shown that cerebral hypoxia results in
increased nuclear calcium influx in neuronal nuclei of the
cerebral cortex of newborn guinea pigs. (47) The nuclear
calcium influx increased as a function of increased cere-
bral tissue hypoxia. Cerebral hypoxia resulted in in-
creased calcium/calmodulin kinase (CaM kinase) IV ac-
tivity, cAMP response element binding (CREB) protein
phosphorylation, activity of high-affinity Ca⫹⫹-ATPase,
and inositol triphosphate (IP3)-dependent calcium in-
flux in neuronal nuclei of newborn piglets. In addition,
NO donors increased neuronal nuclear calcium influx,
(42) and hypoxia resulted in generation of NO free
radicals and increased high-affinity Ca⫹⫹-ATPase activ-
ity.
During hypoxia, NO-mediated modification of the
Role of Calcium Inside
Table 2.
the Nucleus
● Transcription of genes
● Regulation of the cell cycle
● Replication of DNA
● Breakdown of the nuclear envelope

nuclear membrane high-affinity Ca⫹⫹-ATPase and IP3
receptor is a potential mechanism of increased intranu-
clear calcium that leads to activation of calcium-
dependent nuclear mechanisms and activates cascades of
hypoxic programmed cell death.
Expression and Posttranslational
Modification of Apoptotic Proteins
The Bcl-2 Proteins
The Bcl-2 family of proteins (including Bax and Bcl-
2) control cell proliferation, differentiation, and pro-
grammed cell death during normal brain development.
(48)(49) Bax and Bcl-2 are inducible genes found in the
developing and adult central and peripheral nervous
systems. (50)(51) The antiapoptotic protein Bcl-2 pre-
vents apoptosis by forming a heterodimer with the pro-
apoptotic protein Bax and protects cells from pro-
grammed cell death following hypoxia. (48)(49)
Cerebral hypoxia results in increased expression of
Bax protein in neuronal nuclei of the cerebral cortex of
newborn piglets. (52) Increased Bax protein in the mi-
tochondrial, cytosolic, and neuronal nuclear fractions
indicates increased expression of the protein rather than
its translocation from mitochondria to cytosol during
hypoxia. The expression of antiapoptotic protein Bcl-2
did not increase during hypoxia. Therefore, the ratio of
proapoptotic protein Bax to antiapoptotic protein Bcl-2
increases in all compartments of the cell during hypoxia,
which may lead to activation of the hypoxia-induced
cascade of neuronal death. NOS inhibition prevented the
hypoxia-induced increased expression of proapoptotic
protein Bax, indicating that the hypoxia-induced in-
creased expression of Bax is mediated by NO. (53)
Caspases-3, -8, and -9
Caspases are a unique family of cysteinyl-aspartate pro-
teases that play an important role in the initiation and
execution of apoptosis. (54) They are divided into two
primary classes: Class I caspases have long prodomain
and Class II caspases have short prodomain.
Class I caspases such as 8, 9, and 10 can autocatalyze
their own activation and are activated in the early phase
of apoptosis. These are termed the initiator caspases.
Class II caspases, such as 3, 6, and 7, require cleavage
by another protease and are responsible for the break-
down of cells. These are termed the effector or the
executioner caspases. Upstream caspase activation dur-
ing apoptosis leads to the activation of downstream
caspases in a self-amplifying cascade.
Two pathways of caspase activation have been inves-
tigated: the cell surface death receptor-mediated path-
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neurology hypoxic-ischemic encephalopathy
way and the mitochondria-initiated pathway. The re-
cruitment and cleavage of procaspase-8 to produce the
active form of caspase-8 is a critical biochemical event in
the death receptor-mediated apoptosis. Following its
activation, caspase-8 can activate downstream caspases
by direct cleavage or by indirectly cleaving the proapop-
totic protein and inducing cytochrome c (cyt c) release
from the mitochondria. In the mitochondria-initiated
pathway, caspase activation is triggered by formation
of an oligomeric apoptotic protease activation factor
(Apaf-1)/cyt c complex. The resulting complex formed
by the combination of Apaf-1, cyt c, Bax/Bcl-2, and
procaspase-9 is referred to as the apoptosome, which
leads to recruiting and activating procaspase-9, an up-
stream caspase. Other, less-defined pathways of apopto-
tic caspase activation that also are active in neurons act
predominantly through the caspases-8 or -9. Following
hypoxia in the cerebral cortex of newborn piglets, there is
an increase in protein expression and activity of caspase-
8 and caspase-9 (initiator caspases) as well as the expres-
sion and activity of caspase-3 (executioner caspase).
Nuclear Calcium Influx and Caspase-9 and
Caspase-3 Activation
Increased activity of casapse-9 and caspase-3 during
hypoxia is mediated by nuclear calcium influx. Studies
investigating the role of nuclear calcium influx in caspase
activation during hypoxia were performed in newborn
piglets. (55)(56) The calcium-ATPase inhibitor (cloni-
dine) was administered to block nuclear calcium influx.
Cerebral tissue hypoxia resulted in increased caspase-9
activity, and pretreatment with clonidine prevented this
hypoxia-induced increase in caspase-9 activity. In addi-
tion, hypoxia resulted in increased caspase-3 activity, a
consequence of caspase-9 activation, and pretreatment
with clonidine prevented this increase in caspase-3 activ-
ity. These results demonstrate that hypoxia-induced in-
crease in caspase-3 activity is mediated by nuclear calcium
influx.
NO and Caspase-9 and Caspase-3 Activation
To delineate caspase activation during hypoxia further,
the role of NO derived from neuronal NOS was studied
in newborn piglets, using a relatively selective inhibitor
of nNOS, 7-NINA salt. (44) Following cerebral tissue
hypoxia, an increase in caspase-9 activity was seen, but
pretreatment with the nNOS inhibitor prevented the
hypoxia-induced increase in caspase-9 activity. Cerebral
tissue hypoxia again resulted in increased caspase-3 activ-
ity, a consequence of caspase-9 activation, and pre-
treatment with the nNOS inhibitor prevented this
NeoReviews Vol.11 No.4 April 2010 e189
neurology hypoxic-ischemic encephalopathy
hypoxia-induced increase in caspase-3 activity. These
results demonstrate that the hypoxia-induced increase in
caspase-9 is mediated by nNOS-derived NO.
Caspase-9 can be activated during hypoxia by multi-
ple mechanisms that are dependent on generation of
nNOS-derived NO and neuronal nuclear calcium influx.
NO increases calcium influx in synaptosomes as well as
neuronal nuclei. By increasing nuclear calcium influx,
NO can increase expression of caspase-9 as well as pro-
apoptotic proteins. NO-mediated protein modification
also may alter its activation. Caspase-9 plays a significant
role in the hypoxia-induced programmed cell death in
the newborn brain, and caspase activation during hyp-
oxia in the newborn brain is mediated by both
transcription-dependent and -independent mechanisms.
(57)
DNA Fragmentation
It has been proposed that the cleavage of DNA at its
intranucleosomal linkage region is produced by specific
endonucleases that are dependent on calcium. (58)(59)
Caspase-3, acting as cysteine protease, cleaves and inac-
tivates nuclear enzymes, including poly-ADP-ribose-
polymerase (PARP), a DNA repair enzyme, and inhibitor
of caspase-activated DNase (ICAD). The caspase-
activated DNase then enters the nucleus and cleaves
genomic chromosomal DNA. (60)(61) This nuclear
genomic DNA fragmentation correlates exponentially
with the degree of cerebral tissue hypoxia in newborn
piglets (62) and is characteristic of cellular apoptosis.
Temporal Biochemical Changes
The steps of posthypoxic neuronal injury evolve within
hours (the necrotic process) and days (the apoptotic
process). Temporal biochemical changes and associated
nuclear fragmentation have been assessed in the cerebral
cortex of newborn guinea pigs following hypoxia. Initial
cellular injury may be followed by a failure of cellular
repair mechanisms, leading to further delayed brain in-
jury. Neuronal nuclear calcium influx increases immedi-
ately following hypoxia and remains elevated through
7 days of age. Similarly, nuclear Bax protein expression
increases immediately following hypoxia and remains
elevated through 7 days, but Bcl-2 protein remains sim-
ilar to control during hypoxia. These biphasic temporal
changes may reflect not only the primary hypoxic insult
but also a secondary cellular damage due to recurrent
(continuing) free radical release during the reperfusion–
reoxygenation phase.
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Clinical Implications
Understanding the very complex and interrelated mech-
anisms of cell death after a hypoxic-ischemic result may
serve as background for critical care of the newborn.
Hypoxia at the cellular level results from failure in oxygen
transport from the lung alveolar space to the mitochon-
dria. To prevent neuronal cell death, any insufficiency or
failure in the respiratory and circulatory systems must be
restored in a matter of minutes. Among the hazards in
restoring oxygen supply is medically induced hyperoxia
(high FiO2), which may worsen the neuronal insult by
producing additional oxygen free radicals. An under-
standing of the temporal evolution of the posthypoxic
biochemical disturbances during and following hypoxic-
ischemic insult may offer the opportunity for pharmaco-
logic interventions at key steps of the biochemical events.
American Board of Pediatrics Neonatal-Perinatal
Medicine Content Specification
• Know the incidence, causes and
pathophysiology, including cellular
abnormalities, of acute perinatal asphyxia.
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 neurology hypoxic-ischemic encephalopathy

NeoReviews Quiz
1. Under anaerobic conditions, the production of high-energy phosphates decreases, which leads to cell
membrane depolarization and intracellular calcium influx. Increased intracellular calcium has several
deleterious effects on cell structure and function. Of the following, cytoskeletal disruption of microtubules
is most likely the result of calcium-mediated activation of:
A. Nitric oxide synthase.
B. Nuclease.
C. Phospholipase.
D. Protease.
E. Xanthine oxidase.

2. The two mechanisms of cell death following hypoxia-ischemia of the brain are necrosis and apoptosis.
Whereas necrosis is more immediate in onset after the hypoxic-ischemic injury, apoptosis occurs days to
weeks following the insult. Of the following, the most distinguishing feature of apoptosis is:
A. Cell swelling.
B. Chromatin aggregation.
C. Local cytokine release.
D. Oxygen free radical formation.
E. Plasma membrane lysis.

3. N-methyl-D-aspartate (NMDA) cell membrane receptor is a predominant mediator of excitotoxicity in the

immature developing brain. This receptor has a number of pharmacologically distinct binding sites. Of the
following, the coactivator site of the NMDA receptor is most likely to bind to:
A. Glutamine.
B. Glycine.
C. Magnesium.
D. Mk-801.
E. Zinc.

4. Caspases are a family of cysteinyl-aspartate proteases that play an important role in the initiation and
execution of apoptosis. Caspases are of two classes: class I caspases initiate apoptosis and class II caspases
execute apoptosis. Caspase activation occurs through two pathways: the cell surface death receptor-
mediated pathway and mitochondria-initiated pathway. Of the following, the critical caspase in cell surface
death receptor-mediated apoptosis is:
A. Caspase-3.
B. Caspase-6.
C. Caspase-7.
D. Caspase-8.
E. Caspase-10.

NeoReviews Vol.11 No.4 April 2010 e193

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 Biochemical Basis of Hypoxic-Ischemic Encephalopathy
Maria Delivoria-Papadopoulos and Peter J. Marro
NeoReviews 2010;11;e184-e193
DOI: 10.1542/neo.11-4-e184

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