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

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://neoreviews.aappublications.org/cgi/content/full/neoreviews;11/4/e184

NeoReviews is the official journal of the American Academy of Pediatrics. A monthly publication, it has been published continuously since 2000. NeoReviews is owned, published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, 60007. Copyright 2010 by the American Academy of Pediatrics. All rights reserved. Online ISSN: 1526-9906.

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Biochemical Basis of Hypoxic-Ischemic Encephalopathy

Maria DelivoriaPapadopoulos, MD,* Peter J. Marro, MD

Objectives
1. 2. 3. 4.

After completing this article, readers should be able to:

Author Disclosure Drs DelivoriaPapadopoulos and Marro have disclosed no nancial relationships relevant to this article. This commentary does not contain a discussion of an unapproved/ investigative use of a commercial product/device.

Describe the steps of posthypoxic brain injury. Explain the role of N-methyl-D-aspartate (NMDA) receptors in hypoxia. Delineate the contribution of free radicals to neuronal injury during hypoxia. Explain how hypoxia affects calcium inux and modication of apoptotic proteins.

Abstract
Despite improved methods of intrapartum monitoring and advances in neonatal care and treatment, neonatal hypoxic-ischemic injury continues to produce signicant morbidity and mortality, often leading to long-term neurologic consequences. Hypoxia creates an imbalance in metabolic demand and cellular energy supply, resulting in the disruption of critical cellular functions and the activation of excitatory neurotransmitters. In addition, the structure, function, and modication of cellular processes, such as the N-methyl-D-aspartate (NMDA) receptor and intracellular calcium regulation, are affected. Nuclear calcium signals control critical nuclear functions, including regulation of transcription factors and cell cycle, gene transcription, DNA replication, and nuclear envelope breakdown. Nitric oxide synthase and the generation of nitric oxide during hypoxia may contribute signicantly 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 discussion focuses primarily on some of the major cellular and molecular mechanisms of hypoxic neuronal injury in the newborn brain.

Introduction Abbreviations
Apaf-1: ATP: CaM kinase: CNS: CREB: cyt c: HIE: ICAD: IP3: NMDA: NNLA: nNOS: NO: NOS: PARP: apoptotic protease activation factor adenosine triphosphate calcium/calmodulin kinase central nervous system cAMP response element binding cytochrome C hypoxic-ischemic encephalopathy inhibitor of caspase-activated DNase inositol triphosphate N-methyl-D-aspartate Nw-nitro-l-arginine neuronal nitric oxide synthase nitric oxide nitric oxide synthase poly-ADP-ribose-polymerase

Perinatal hypoxia-ischemia is the most common cause of neurologic disease during the neonatal period. Hypoxicischemic encephalopathy (HIE) is associated with high mortality and morbidity rates, including cerebral palsy, intellectual disability, and seizures. The incidence of perinatal asphyxia is about 1.0% to 1.5% in most centers and usually is related to gestational age and birthweight. It occurs in 9.0% of infants younger than 36 weeks gestation and in 0.5% of infants older than 36 weeks gestation. (1) Perinatal HIE may develop during the antepartum (20%), intrapartum (30%), antepartum and intrapartum (35%), or postpartum (10%) period. (2) HIE develops in the setting of perinatal asphyxia, which is a multiorgan system disease and includes circumstances affecting the cerebral blood ow in the fetus and newborn that compromise the supply of oxygen to the brain. Assessment and management of these complications is an integral part of the treatment of perinatal asphyxia/HIE. (3) 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 Childrens Hospital at Maine Medical Center, Portland, Me. e184 NeoReviews Vol.11 No.4 April 2010

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fetal cardiovascular and respiratory response to oxygen limitations, giving rise to a better understanding and management of neonatal deterioration induced by hypoxia. Besides these physiologic studies, cellular and biochemical 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 susceptibility of the developing brain to neonatal and perinatal hypoxia. These determinants include the lipid composition of the brain cell membrane, the rate of lipid peroxidation, the presence of antioxidant defenses, the development and modulation of the excitatory neurotransmitter receptors such as the NMDA receptor, and the intracellular calcium inux mechanisms. In addition to the developmental status of these cellular components, the response of the potential mechanisms to hypoxia 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.

Figure 1. Effects of hypoxia leading to increased intranuclear calcium. ATPadenosine triphosphate, NMDAN-methyl-Daspartate

Excitotoxic Mechanisms (Fig. 1)

The decrease in ATP concentrations leads to cell membrane depolarization and a disruption of voltagedependent 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 enhances cellular calcium inux. The entire mechanism is accelerated further by the dysfunction of the energydependent reuptake of glutamate both in neurons and in astrocytes in a vicious cycle. When calcium inux is unregulated, activation of phospholipases, proteases, and endonucleases can result in nuclear, organelle, and cell membrane disruption. 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 inammatory response. The result is edema and injury to neighboring cells. The inammatory response triggers expression of the cytokines 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 specic endonucleolytic digestion of nuclear DNA. In contrast to necrosis, programmed cell death is characterized by cell shrinkage, coarse chroNeoReviews Vol.11 No.4 April 2010 e185

Cell Death (Fig. 2)

Acute or long-term consequences of HIE are related either to necrosis or to apoptosis of neuronal cells. Necrosis is characterized by passive cell swelling; rapid en-

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diated through its interaction with specic cell membrane receptors, of which the NMDA, kainate, and AMPA subtypes are the best characterized. (16) The NMDA-type glutamate receptor is a predominant 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 receptormediated processes may depend not only on the ontogeny of the NMDA receptor, but also potential modication 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 neurotransmitter binding site, or recognition site, that binds glutamate or NMDA; a coactivator site that binds glycine; a channel site that binds MK-801; a voltagedependent 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 glutamate recognition site, one that preferentially binds agonists and one that preferentially binds antagonists. (18) The NMDA receptor is associated with a cationselective 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 inux occurs. The inux 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 receptorion channel complex is modied during brain development, which may play a role in altering the response of the receptor during development and hypoxia.

Figure 2. Pathways of neuronal cell death following initial energy failure. NMDAN-methyl-D-aspartate. Reprinted with permission from Marro PJ. The etiology and pharmacologic approach to hypoxic-ischemic encephalopathy in the newborn. NeoReviews. 2002;3:e99.

matin aggregation with extensive nuclear DNA fragmentation, nuclear pyknosis, and extrusion of membranebound 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 fragmentation has been studied extensively but is not well understood.

Mechanism of NMDA Receptor Modication During Hypoxia

Brain tissue hypoxia modies the NMDA receptor recognition, coactivator, and ion channel sites. A decrease in the apparent number of NMDA receptors and an increase in receptor afnity for MK-801 were observed in hypoxic fetal guinea pig and newborn piglet brains. (23)(24) In these same studies, glutamate- and glycinedependent activation of the NMDA receptor was decreased and spermine-dependent and basal state receptor

NMDA Receptors
Glutamate is the primary excitatory amino acid neurotransmitter 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 injury. (14)(15) The physiologic and pathologic effects of glutamate in the central nervous system (CNS) are mee186 NeoReviews Vol.11 No.4 April 2010

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activation were increased during hypoxia. Hypoxiainduced modication 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 receptor, (25)(26) thereby favoring nitration of the receptor. In addition, nNOS activity is decreased by phosphorylation and increased by dephosphorylation, (27)(28) a condition likely present during hypoxia. Dephosphorylation 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 hypoxia may facilitate peroxynitrite-mediated nitration of tyrosine-inhibiting phosphorylation of proteins and alteration of the NMDA receptor-ion channel complex. (29) In neurons of the CNS, nNOS is activated by calcium inux through the NMDA receptor-ion channel, but nNOS is not efciently stimulated by activation of nonNMDA receptors that also induce calcium inux. (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 coimmunoprecipitate and that the PSD-95 is sufcient to assemble 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 inux through the NMDA receptor-ion channel, that there is a specic structural and functional link between the NMDA receptor and nNOS, and that the
Table 1.

nitric oxide synthase (NOS) pathway plays a critical role in the NO-mediated mechanism of hypoxia-induced modication 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 peroxidation, protein oxidation, nucleic acid oxidation, and cell damage. Normally, more than 80% of the oxygen consumed 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 mitochondria 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 calcium 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 components, including ubiquinone (a component that undergoes auto-oxidation to produce free radicals); 2) increased release of ferritin under the conditions of decreased cellular high-energy compounds; and 3) in-

Deleterious Effects of Increased Intracellular Calcium

Result

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
ATPadenosine triphosphate

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 inux Free radical formation Generation of nitric oxide

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creased degradation of ATP during hypoxia, increasing the substrate for the xanthine oxidase reaction and leading to increased free radical generation. Increased free radical generation during hypoxia in the cerebral cortex of newborn piglets has been documented 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 peroxidation is an ongoing event during cerebral hypoxia, a mechanism of hypoxic neuronal injury. These studies not only demonstrate increased free radical generation during 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.

The exact molecular mechanism of hypoxic membrane damage is not clear. However, it has been shown that peroxynitrite (formed by the reaction between superoxide anions and NO) can cause lipid peroxidation in vitro. (40) Therefore, high concentrations of NO during hypoxia may result in increased production of peroxynitrite, causing lipid peroxidation.

Neuronal Nuclear Calcium Inux

A number of critical nuclear functions, including regulation of transcription factors, cell cycle regulation transcription, DNA replication, and nuclear envelope breakdown, 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 activitydependent gene transcription under a number of experimental conditions. (42)(43)(44) Several factors may be involved in calcium-regulated gene expression and transcription, including the site of calcium entry, the amplitude 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 inux in neuronal nuclei of the cerebral cortex of newborn guinea pigs. (47) The nuclear calcium inux increased as a function of increased cerebral tissue hypoxia. Cerebral hypoxia resulted in increased calcium/calmodulin kinase (CaM kinase) IV activity, cAMP response element binding (CREB) protein phosphorylation, activity of high-afnity Ca-ATPase, and inositol triphosphate (IP3)-dependent calcium inux in neuronal nuclei of newborn piglets. In addition, NO donors increased neuronal nuclear calcium inux, (42) and hypoxia resulted in generation of NO free radicals and increased high-afnity Ca-ATPase activity. During hypoxia, NO-mediated modication of the

Nitric Oxide Free Radicals and Neuronal Injury

Three major isoforms of NOS have been identied: constitutive neuronal, constitutive endothelial, and inducible 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 physiologically mediates cerebral vasodilation under normal conditions, (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 generation of conjugated dienes (products of lipid peroxidation) and preserved Na, K-ATPase activity (an index of cellular membrane function). These data demonstrated that NOS generates free radicals during hypoxia via peroxynitrite production, presumably causing lipid peroxidation and membrane dysfunction. The production 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, leading 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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Role of Calcium Inside the Nucleus

Table 2.

Transcription of genes Regulation of the cell cycle Replication of DNA Breakdown of the nuclear envelope

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nuclear membrane high-afnity Ca-ATPase and IP3 receptor is a potential mechanism of increased intranuclear calcium that leads to activation of calciumdependent nuclear mechanisms and activates cascades of hypoxic programmed cell death.

Expression and Posttranslational Modication of Apoptotic Proteins

The Bcl-2 Proteins
The Bcl-2 family of proteins (including Bax and Bcl2) control cell proliferation, differentiation, and programmed 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 prevents apoptosis by forming a heterodimer with the proapoptotic protein Bax and protects cells from programmed 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 mitochondrial, 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 increased expression of Bax is mediated by NO. (53)

way and the mitochondria-initiated pathway. The recruitment 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 proapoptotic 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 upstream caspase. Other, less-dened pathways of apoptotic 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 caspase8 and caspase-9 (initiator caspases) as well as the expression and activity of caspase-3 (executioner caspase).

Nuclear Calcium Inux and Caspase-9 and Caspase-3 Activation

Increased activity of casapse-9 and caspase-3 during hypoxia is mediated by nuclear calcium inux. Studies investigating the role of nuclear calcium inux in caspase activation during hypoxia were performed in newborn piglets. (55)(56) The calcium-ATPase inhibitor (clonidine) was administered to block nuclear calcium inux. Cerebral tissue hypoxia resulted in increased caspase-9 activity, and pretreatment with clonidine prevented this hypoxia-induced increase in caspase-9 activity. In addition, hypoxia resulted in increased caspase-3 activity, a consequence of caspase-9 activation, and pretreatment with clonidine prevented this increase in caspase-3 activity. These results demonstrate that hypoxia-induced increase in caspase-3 activity is mediated by nuclear calcium inux.

Caspases-3, -8, and -9

Caspases are a unique family of cysteinyl-aspartate proteases 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 breakdown of cells. These are termed the effector or the executioner caspases. Upstream caspase activation during apoptosis leads to the activation of downstream caspases in a self-amplifying cascade. Two pathways of caspase activation have been investigated: the cell surface death receptor-mediated path-

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 activity, a consequence of caspase-9 activation, and pretreatment with the nNOS inhibitor prevented this
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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 multiple mechanisms that are dependent on generation of nNOS-derived NO and neuronal nuclear calcium inux. NO increases calcium inux in synaptosomes as well as neuronal nuclei. By increasing nuclear calcium inux, NO can increase expression of caspase-9 as well as proapoptotic proteins. NO-mediated protein modication also may alter its activation. Caspase-9 plays a signicant role in the hypoxia-induced programmed cell death in the newborn brain, and caspase activation during hypoxia in the newborn brain is mediated by both transcription-dependent and -independent mechanisms. (57)

Clinical Implications
Understanding the very complex and interrelated mechanisms 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 mitochondria. To prevent neuronal cell death, any insufciency 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 understanding of the temporal evolution of the posthypoxic biochemical disturbances during and following hypoxicischemic insult may offer the opportunity for pharmacologic interventions at key steps of the biochemical events.

DNA Fragmentation
It has been proposed that the cleavage of DNA at its intranucleosomal linkage region is produced by specic endonucleases that are dependent on calcium. (58)(59) Caspase-3, acting as cysteine protease, cleaves and inactivates nuclear enzymes, including poly-ADP-ribosepolymerase (PARP), a DNA repair enzyme, and inhibitor of caspase-activated DNase (ICAD). The caspaseactivated 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.

American Board of Pediatrics Neonatal-Perinatal Medicine Content Specication

Know the incidence, causes and pathophysiology, including cellular abnormalities, of acute perinatal asphyxia.

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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 injury. Neuronal nuclear calcium inux increases immediately 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 similar to control during hypoxia. These biphasic temporal changes may reect 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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NeoReviews Quiz
1. Under anaerobic conditions, the production of high-energy phosphates decreases, which leads to cell membrane depolarization and intracellular calcium inux. 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. B. C. D. E. Nitric oxide synthase. Nuclease. Phospholipase. Protease. 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. B. C. D. E. Cell swelling. Chromatin aggregation. Local cytokine release. Oxygen free radical formation. 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. B. C. D. E. Glutamine. Glycine. Magnesium. Mk-801. 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 receptormediated pathway and mitochondria-initiated pathway. Of the following, the critical caspase in cell surface death receptor-mediated apoptosis is: A. B. C. D. E. Caspase-3. Caspase-6. Caspase-7. Caspase-8. Caspase-10.

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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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