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Journal of Perinatology (2007) 27, S39–S46 r 2007 Nature Publishing Group All rights reserved. 0743-8346/07 $30 www.nature.com/jp ORIGINAL ARTICLE Pathogenesis of hypoxic-ischemic brain

JM Perlman Department of , Weill Cornell Medical College, New York, NY, USA

Defining Accumulating evidence points to an evolving process of brain injury after The definition of birth asphyxia is imprecise. The process occurs intrapartum - that initiates in utero and extends into a during the first and second stages of labor and a fairly consistent recovery period. The processes leading to include or description is that of a condition of impaired which if , and result from the combined effects of cellular energy failure, persistent leads to and . The process is , glutamate release, intracellular Ca2 þ accumulation, generation of identified by fetal acidosis (as measured in umbilical arterial free radicals that serve to disrupt essential components of the cell. Many ), which reflects the degree of anaerobic generated factors including the duration or severity of the insult influence the during periods of hypoxia or increased demand.2 The progression of cellular injury after hypoxia-ischemia. A secondary cerebral umbilical arterial pH that best defines ‘asphyxia’ remains unclear. energy failure occurs from 6 to 48 h after the primary event and involves Initially, asphyxia was defined as a cord umbilical arterial pH less mitochondrial dysfunction secondary to extended reactions from primary than 7.20. With this definition, the incidence of ‘asphyxia’ ranged insults (e.g., calcium influx, excitatory neurotoxicity and oxygen free from 5 to 20%.3 If a pH less than 7.10 was used, the incidence ranges radicals) as well as the release of circulatory and endogenous inflammatory from 2 to 8%.4 Accumulating evidence suggests that an umbilical cells/mediators that also contribute to ongoing brain injury. Strategies arterial pH less than7.00 reflects degrees of acidosis often referred to aimed at neuroprotection need to be comprehensive targeting the pathways as pathologic or severe fetal acidemia, where the risk of adverse leading to eventual mitochondrial injury. neurologic sequelae is increased.5,6 A cord pH less than7.0 Journal of Perinatology (2007) 27, S39–S46. doi:10.1038/sj.jp.7211716 complicates approximately 0.3% of all deliveries.5,7 Even with this Keywords: hypoxia-ischemia; apoptosis; necrosis; mitochondria; degree of acidosis, the likelihood of brain injury is low. Thus the glutamate; calcium; reperfusion majority of infants, that is, greater than 60%, exhibit no difficulties in the delivery room, are triaged to the regular newborn nursery, and exhibit an uncomplicated neonatal course in almost all cases.7 Even when infants with severe fetal acidemia are admitted to intensive care (usually because of respiratory difficulties) about 80 to 90% Introduction exhibit a benign neurologic course8–10 and it is only a small Impaired cerebral blood flow (CBF) is the predominant percentage that progress to moderate to severe , that pathogenetic mechanism underlying most of the neuropathology is approximately 10% of all infants delivered in the presence of severe attributed to intrapartum hypoxia-ischemia.1 This disruption in fetal acidemia.7,10 This resilience of the brain is in part based on the CBF is most likely to occur as a consequence of interruption in ability of the fetus to adapt to the interruption of placental blood flow placental blood flow and gas exchange that may be acute and/or to preserve cerebral and oxygen delivery. intermittent. The fetus adapts in multiple ways including both circulatory and noncirculatory responses to preserve cerebral Cerebral responses to interruption of placental blood perfusion and thus oxygen delivery. However, when the disruption flow is severe and/or the adaptive mechanisms fail, neuronal Biochemical ensues. Brain injury may occur in two phases, during the acute The cerebral circulation is exquisitely sensitive to changes in PaO2 insult and during a recovery period following circulatory and PaCO2. Thus, interruption of placental blood flow with restoration termed . This article will deal with hypoxia and hypercarbia both independently increase CBF.11–13 interruption of placental blood flow, the fetal adaptive responses to In addition, the vascular responses to both partial of this state, and the mechanisms contributing to acute and delayed carbondioxide (PaCO2) and of oxygen (PaO2) vary perinatal hypoxic-ischemic brain injury. widely amongst brain regions. For example, cerebral white matter Correspondence: Dr JM Perlman, Division of Newborn Medicine, Department of Pediatrics, as compared to brainstem and cortical structures has limited 14 Weill Cornell Medical College, 525 East 68th Street, N-506, New York, NY 10021, USA. vasodilatory responses to both PaCO2 and hypoxia. Pathogenesis of hypoxic-ischemic brain injury JM Perlman S40

Autoregulation In the usual state CBF remains constant over a wide range of changes in systemic mean arterial , a term referred to as autoregulation.15 The fetal curve differs from the adult curve in two ways. First, the curve is narrower, particularly at the upper limit, in the less mature animal, and second the normal mean arterial blood pressure in the less mature animal is only marginally above the lower limit of the curve.16,17 Autoregulation may be disrupted with hypoxia, hypo/hypercarbia or acidosis.14,18–21 Under such conditions, the cerebral circulation becomes pressure passive and directly reflects systemic blood pressure changes. The importance of impaired CBF autoregulation, even after modest asphyxia in the genesis of ischemic cerebral injury is supported by animal and experimental studies.14,22 The implications are enormous, namely a decrease in blood pressure Figure 1 Schematic depicting the circulatory responses to interruption will result in a decrease in CBF, thus markedly increasing the of placental blood flow. Note the increased blood flow to brain, vulnerability for neuronal injury, particularly within the border and adrenal glands at the expense of blood flow to the less vital organs. zone regions of the brain such as parasagittal cortex or periventricular white matter.14 have been associated with subsequent normal neurologic development.38

Fetal adaptation to interruption of placental blood flow Noncirculatory factors contributing to neuronal Circulatory responses preservation The important circulatory manifestations of interruption of Additional factors considered potentially important in preserving placental blood flow have been well categorized in experimental neuronal integrity with asphyxia include biologic alterations that studies. These include: (1) a redistribution of cardiac output to are maturation dependent. Some examples include: (1) decreasing preserve blood flow to the more vital organs (i.e., brain, rate of brain metabolism during early development that results in a myocardium, adrenal gland) with reduced flow to less vital organs slower depletion of high-energy compounds during hypoxia- (i.e., , intestine, muscle); (2) loss of cerebral vascular ischemia in the fetus as compared with the term infant or autoregulation resulting in a pressure passive circulation as adult;39,40 (2) the use of alternate energy substrate, the neonatal described above; and (3) eventual diminution in cardiac output brain having the capacity to use lactate and for with resultant , and ultimately a critical decrease in energy production;41,42 (3) the relative resistance of the fetal and cerebral perfusion and oxygen delivery (Figure 1).23–28 The neonatal myocardium to hypoxia ischemia;43,44 and (4) the mechanisms involved in the redistribution of blood flow include potential protective role of fetal , that is, it has been peripheral , which is triggered by a carotid calculated that if the PO were to decrease to below a value of chemoreflex, endocrine factors and then maintained or 2 approximately 3 torr, and if the venous PO were to decrease to subsequently modified by endocrine and local components.29,30 2 10 torr (a value that may be found in the cerebral venous blood In the experimental model, with initial arterial hypoxemia, fetal with asphyxia), the infant would have more oxygen available for vascular resistance can decrease by at least 50% to maintain CBF brain uptake with a fetal rather than with an adult dissociation with a minimal decrease in oxygen delivery.31–33 Critical to this curve.45 Recent data suggest a potential important role for ischemic state is a normal or elevated mean arterial blood pressure. preconditioning.46 Perhaps as a consequence of some or all of the However, with persistent hypoxemia, and eventual hypotension, above-mentioned factors, even when interruption of placental blood cerebral vascular resistance cannot decrease further, resulting in a flow, that is asphyxia is prolonged or severe, most newborn infants marked reduction in CBF.34 The critical ischemic threshold for recover with minimal or no neurologic sequelae. neuronal necrosis in developing brain remains unclear. Clearly, postnatal observations indicate that this is a very complex issue. In adults, CBF thresholds have been identified; below a critical threshold functional disturbances (electroencephalographic Mechanisms of neuronal cell death following slowing) occur,35 and below an even lower CBF threshold ion hypoxia-ischemia pump failure occurs.36,37 Yet, values in both preterm and term The mechanism of neuronal cell death in animals and humans infants below the threshold associated with pump failure in adults after hypoxia-ischemia includes neuronal necrosis and apoptosis.47

Journal of Perinatology Pathogenesis of hypoxic-ischemic brain injury JM Perlman S41

Necrosis is a passive process of cell swelling, with disruption of handle multiple oxidation reactions that can yield highly toxic cytoplasmic organelles, loss of membrane integrity, eventual lysis oxygen free radicals under conditions of (Figure 2). of neuronal cells and activation of an inflammatory process. In Mitochondria are key regulators in the process of cell death via contrast, apoptosis is an active process distinguished from necrosis release of the numerous proapoptotic such as cytochrome by the presence of cell shrinkage, nuclear pyknosis, chromatin c, caspase-2 and -9, and AIF from the intermembrane space.57–59 condensation and genomic fragmentation, events that occur in the These key elements of apoptosis, that is caspase-3, caspase-12 and absence of an inflammatory response.48 Two parallel pathways lead BAX, are upregulated in the immature brain.52,59 Caspases are to chromatin processing during apoptosis. One an intrinsic cysteine proteases that orchestrate in a proteolytic cascade, pathway that involves activation of caspase-3, cleavage of inhibitor targeting key homeostatic and structural proteins, leading to DNA of caspase-activated DNAse with subsequent activation of caspase- fragmentation and cell death. The release of caspase-3 has recently activated DNAse which in turn produces oligonucleosomal DNA been categorized in a series of experiments. Thus, during the fragmentation and advanced chromatin condensation.49 The second primary phase of mitochondrial dysfunction there is limited pathway involves apoptosis-inducible factor (AIF) that is caspase activation of caspase-3 whereas secondary damage is associated independent and results in DNA fragmentation and chromatin with neuronal loss and a striking elevation in caspase-3.60 The condensation.50 These two processes of neuronal death can be found pathways leading to caspase activation have not been fully after hypoxia-ischemia in both animals and humans.14,51 elucidated but there are data suggesting that proapoptotic proteins Several factors appear to be important in determining the mode (cytochrome c and apoptosis-inducing factor) are released from of death. The first relates to the intensity of the initial insult with mitochondria. Cytochrome c interacts with APAF-1 (apoptosis severe injury resulting in necrosis and milder insults resulting in protease-activating factor 1) and caspase-9, leading to further apoptosis.48 The second relates to the impact of age on cell death activation of downstream caspases.61 Activation of caspase-3 is mechanisms.52 Thus, there is a developmental downregulation of much more pronounced in the immature than in the adult brain caspase-3 as well as other elements involved in caspase-3 in response to various hypoxic-ischemic insults62,63 with activity activation, that is Bcl-2–associated X that occurs in increasing progressively between 1 and 24 hour of reperfusion.63 tandem with a reduction with physiologic Caspase inhibition reduces brain injury after neonatal HI, seen in the mature brain. Cytochrome c a critical component of the supporting a critical role in HI injury and a potential avenue for respiratory chain that generates triphosphate (ATP) in neuroprotection.63 AIF, which acts independently of caspases, the inner mitochondrial membrane increases during brain translocates to the nucleus and induces chromatin condensation development.52,53 In addition, there is a relative downregulation of and DNA fragmentation.50,62 AIF levels do not change during AIF with maturation.52 Calpains, which are cysteine proteases development, which is in contrast to the increase in the apoptosis- located within the cytoplasm, are activated by intracellular calcium related intermembrane mitochondrial proteins cytochrome c and elevation and have been implicated in excitotoxic neuronal injury.54 caspase. AIF is released earlier from mitochondria shortly after Again, there is a maturational difference with caspain activity hypoxia-ischemia and precedes that of cytochrome c.59 peaking 3 hour after hypoxia-ischemia in immature brain whereas in the mature brain it peaks later, that is at 72 hour and beyond.52 Clearly, because the mechanisms of cell death that is necrosis versus apoptosis differ, strategies to minimize in an affected infant after hypoxia-ischemia likely will have to include interventions that target both processes.

Importance of the mitochondria to the processes of cell death Mitochondria appear to play a central role in determining the fate of cells subjected to hypoxia-ischemia.55 Mitochondria are major buffers of intracellular calcium ions and can become overloaded by cytoplasmic Ca2 þ flooding secondary to opening of 2 þ N-methyl-D-aspartate (NMDA) and voltage-sensitive Ca channels. Diminished mitochondrial function can lead to decreased energy production to maintain membrane ion gradients, potentially Figure 2 Flow diagram depicting potential biochemical mechanisms perpetuating a vicious cycle of membrane depolarization and of hypoxic-ischemic cerebral injury. ATP, ; 56 NMDA receptor channel opening. In addition, mitochondria NMDA, N-methyl-D-aspartate; NOS, synthase.

Journal of Perinatology Pathogenesis of hypoxic-ischemic brain injury JM Perlman S42

Mechanisms contributing to neuronal necrosis/apoptosis peroxidation, and NO neurotoxicity disrupt essential components of following hypoxia-ischemia the cell, resulting in death.1,14,56,65,66 Many factors, including the Brain injury likely occurs in two phases in most cases, that is duration or severity of the insult, influence the progression of during the acute insult and secondarily during the recovery period cellular injury after hypoxia-ischemia. following circulatory restoration, termed reperfusion injury (Figure 3). Delayed (secondary) brain damage Following , which occurs in the delivery room, Acute injury cerebral perfusion and oxygenation is restored. During this recovery At the cellular level, the reduction in CBF and oxygen delivery to a phase, the of phosphorus metabolites and the critical level initiates a cascade of deleterious biochemical events. intracellular pH return to baseline. However, a process of cerebral Thus, oxygen depletion results in a switch to anaerobic metabolism energy failure recurs from 6 to 48 h later in a second phase of an energy-inefficient state with a rapid depletion of high-energy injury. This phase, is characterized by a decrease in the ratio of phosphate reserves, including ATP, accumulation of lactic acid; phosphocreatine/inorganic phosphate, an unchanged intracellular and an inability to maintain cellular functions.64 These changes pH, and stable cardio respiratory status, contributes to further brain 64,67 result in disruption of transcellular ion pumping, with the injury. In the human infant, the severity of the second energy subsequent intracellular accumulation of sodium, calcium, and failure is correlated with adverse neurodevelopmental outcome at 1 68 water. As a result of the membrane depolarization there is a release and 4 years. The mechanisms of secondary energy failure may of excitatory neurotransmitters, specifically glutamate, from axon involve mitochondrial dysfunction secondary to extended reactions terminals. Glutamate then activates specific cell surface receptors, from primary insults (e.g., calcium influx, excitatory neurotoxicity, resulting in a further influx of Na þ and Ca2 þ into postsynaptic oxygen free radicals or NO formation). Evidence suggests that . Within the cytoplasm, free fatty acids accumulate circulatory and endogenous inflammatory cells/mediators also 66,68,69 secondary to increased membrane phospholipid turnover. The fatty contribute to ongoing brain injury. acids undergo peroxidation by oxygen free radicals that arise from reductive processes within mitochondria and as well as from Specific mechanisms of injury by-products in the synthesis of prostaglandins, xanthine and uric Accumulation of cytosolic calcium acid (Figure 3). Ca2 þ ions accumulate within the cytoplasm as Calcium an intracellular secondary messenger is necessary for a consequence of increased cellular influx as well as decreased numerous cellular reactions. Under physiologic conditions, efflux across the plasma membrane associated with release from cytosolic Ca2 þ is strictly regulated at a very low mitochondria and endoplasmic reticulum. In selected neurons, the intracellular concentration. During hypoxia-ischemia, there is an intracellular calcium induces the production of nitric oxide (NO), increase in Ca2 þ influx into neuronal cells caused in part by a free that diffuses to adjacent cells susceptible to NO stimulation of the N-methyl d-aspartate receptors of the agonist- toxicity. The combined effects of cellular energy failure, acidosis, operated Ca2 þ channel by glutamate. In an opposite direction, glutamate release, intracellular Ca2 þ accumulation, Ca2 þ efflux across the plasma membrane is interrupted by the energy failure. In addition, Ca2 þ is also released into cytoplasm from the mitochondria by the Na þ H þ -dependent antiport system and from the endoplasmic reticulum by inositol triphosphate derived from the plasma membrane. This results in increased intracellular Ca2 þ , which interferes with many enzymatic reactions, including activation of lipases, proteases, endonucleases, and phospholipases, and the formation of oxygen free radicals as by-products of prostaglandin synthesis. Overall, the accumulation of cytosolic Ca2 þ after hypoxia-ischemia has major detrimental effects on neuronal cells leading to irreversible brain damage.14,56

Excitatory neurotransmitter release Glutamic acid is a major excitatory within the brain. The action of glutamate is mediated by a number of receptor subtypes, with the NMDA receptor predominating in the developing brain, and is increased in areas of active development (e.g., Figure 3 Flow diagram illustrating the two-stage process of brain striatum or ). Within the NMDA receptor site, there injury with acute hypoxia-ischemia. are regions of agonist activity (glutamate, glycine) as well as

Journal of Perinatology Pathogenesis of hypoxic-ischemic brain injury JM Perlman S43 regions of antagonist activity (Mg2 þ or phencyclidine .72 Furthermore, a low dose of bacterial endotoxin (PCP)).14,56,65 Glutamate metabolism has been shown to be administered 4 h before hypoxia-ischemia in rats induces cerebral tightly coupled to metabolism within neurons. During infarction in response to short periods of hypoxia-ischemia that hypoxia-ischemia, glutamate accumulates within the synaptic cleft, usually cause little or no injury. This endotoxin-induced secondary to increased release from axon terminals as well as sensitization of the immature brain occurs independent of CBF impaired reuptake at presynaptic nerve endings. The excessive changes and/or and is associated with an altered glutamate acting on the receptor sites facilitates the intracellular expression of CD14 mRNA.73 Inflammatory may have a entry of Ca2 þ via the NMDA receptor-mediated channel and direct toxic effect via increased production of inducible NOS, promotes the biochemical cascade mentioned above, leading to cyclooxygenase, and free radical release, or indirectly via induction neuronal death.14,56,66 of glial cells to produce neurotoxic factors such as excitatory amino acids. The important role of cytokines in perpetuating excitotoxic Formation of free radicals injury is suggested from experimental observations in which À À Oxygen free radicals (O2 ,H2O2,OH ) are produced within the administration of -1 endogenous receptor antagonists cytoplasm and mitochondria under physiologic conditions. The free and neutralizing antibody are associated with a reduction in radicals are destroyed rapidly by endogenous antioxidants excitotoxic and ischemic injury in mice.74,75 However, cytokines (e.g., dismutase and catalase) and scavengers that potentiate brain damage after ischemia also appear to play a (e.g., a-tocopherol, ascorbic acid and glutathione).14,66,69 During beneficial neurotrophic effect. TNF knockout mice were shown to and following hypoxia-ischemia, the generation of oxygen free have increased neuronal cell degeneration after ischemia and radicals is increased in excess of the protective capability of the cell. excitotoxic injury, suggesting a critical role for endogenous TNF in Two important sources of oxygen free radicals are the by-products regulating the cellular responses to brain injury.76 In addition, of xanthine (derived from the breakdown of ATP) and administration of exogenous interleukin-6 following ischemia prostaglandin synthesis (derived from the breakdown of free fatty produced by middle occlusion also results in marked acids) (Figure 2). Oxygen free radicals contribute to tissue injury neuroprotection.77 Thus, inflammatory cytokines appear to exert by attacking the polyunsaturated fatty acid component of the both beneficial and deleterious effects after ischemia. This dual cellular membrane, resulting in membrane fragmentation and cell effect will likely complicate the task of developing targeted death.14,56,66 The developing brain appears to be extremely interventions against the inflammatory response. vulnerable to oxidative damage in part because of the high concentrations of unsaturated fatty acids, a high rate of oxygen Summary consumption and relatively low concentrations of antioxidants.14 The processes leading to neuronal cell death are initiated during Another source of free radical formation is NO, which is formed an acute event with a loss of high-energy phosphate compounds, during the conversion of L-arginine into L-citrulline by NO intracellular acidosis followed by excessive extracellular glutamate synthase (NOS). NOS is strongly activated during hypoxia-ischemia accumulation within the extracellular milieu, intracellular and with reperfusion, and a large amount of NO is produced for accumulation of calcium, free radical generation as well as extended periods. When combined with superoxide, NO generates a activation of inflammatory mediators that may extend well beyond potent radical, , which activates lipid peroxidation. In the initial process. addition, NO enhances glutamate release.14,66,69 An additional pathway of free radical induced injury is via the Translating basic science into clinical practice release of iron. Physiologically, iron is maintained in a nontoxic Under most circumstances infants at highest risk for evolving to ferric state and is bound to proteins (, transferrin). During brain injury secondary to intrapartum hypoxia-ischemia are hypoxia-ischemia, free ferric iron is released from these proteins initially identified in the immediate neonatal period starting as and reacts with peroxides to generate potent hydroxyl radicals. In early as in the delivery room and extending through the first six addition, free ferric iron is reduced to a ferrous form, which further postnatal hours.78 Given the complex pathways of pathogenesis, it contributes to free radical injury.14,69 is unreasonable to expect that one intervention will suffice for all infants. Rather, any intervention is likely to vary depending on Inflammatory mediators factors such as the severity or duration of the process. Currently, Inflammatory mediators appear to play a critical role in the induced modest likely reflecting its multiple actions pathogenesis of hypoxic-ischemic brain injury.70 Expression of on the pathways mentioned above, offers the greatest promise, interleukin (IL)-1-b and tumor necrosis factor (TNF)-a messenger particularly in infants who present with moderate encephalopathy RNA (mRNA) has been demonstrated within 1 to 4 h after and without early .79,80 However, for infants with severe hypoxia-ischemia,71 along with the induction of a and b encephalopathy and/or early seizures, this effect is less chemokines followed by neutrophil invasion of the area of favorable.79,80 The latter observations likely reflect a pathway of

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