An Pediatr (Barc). 2018;88(4):228.e1---228.e9

www.analesdepediatria.org

SPECIAL ARTICLE

Oxidative stress in perinatal asphyxiaand hypoxic-ischaemic encephalopathy�

Antonio Nuneza, Isabel Benaventeb, Dorotea Blancoc, Héctor Boixd,Fernando Cabanase, Mercedes Chaffanel f, Belén Fernández-Colomerg,José Ramón Fernández-Lorenzoh, Begona Loureiro i, María Teresa Moral j,Antonio Pavónk, Inés Tofé l, Eva Valverdem, Máximo Ventoa,∗, co-researchers of theclinical trial1

a Hospital Universitario y Politécnico La Fe, Valencia, Spainb Hospital Universitario Puerta del Mar, Cádiz, Spainc Hospital Universitario Gregorio Maranón, Madrid, Spaind Hospital Universitario Vall d’Hebron, Barcelona, Spaine Hospital Universitario Quirónsalud Madrid, Pozuelo de Alarcón, Madrid, Spainf Hospital Regional Universitario Carlos Haya, Málaga, Spaing Hospital General Universitario Asturias, Oviedo, Spainh Complejo Hospitalario Universitario de Vigo, Vigo, Pontevedra, Spaini Hospital Universitario de Cruces, Barakaldo, Vizcaya, Spainj Hospital Universitario 12 de Octubre, Madrid, Spaink Hospital Universitario Virgen del Rocío, Sevilla, Spainl Hospital Universitario Reina Sofía, Córdoba, Spainm Hospital Universitario La Paz, Madrid, Spain

Received 11 April 2017; accepted 17 May 2017Available online 12 March 2018

KEYWORDSAsphyxia;Hypoxic-ischaemicencephalopathy;Oxidative stress;

Abstract Birth asphyxia is one of the principal causes of early neonatal death. In survivorsit may evolve to hypoxic-ischaemic encephalopathy and major long-term neurological mor-bidity. Prolonged and intense asphyxia will lead to energy exhaustion in tissues exclusivelydependent on aerobic metabolism, such as the central nervous system. Energy deficit leads toATP-dependent pumps blockage, with the subsequent loss of neuronal transmembrane poten-

Free radicals;Reactive oxygenspecies

tial. The most sensitive areas of the brain will die due to necrosis. In more resistant areas,neuronal hyper-excitability, massive entrance of ionic calcium, activation of NO-synthase, freeradical generation, and alteration in mitochondrial metabolism will lead to a secondary energy

� Please cite this article as: Nunez A, Benavente I, Blanco D, Boix H, Cabanas F, Chaffanel M, et al. Estrés oxidativo en la asfixia perinatal

y la encefalopatía hipóxico-isquémica. An Pediatr (Barc). 2018;88:228.e1---228.e9.

∗ Corresponding author.E-mail address: maximo.vento@uv.es (M. Vento).

1 Appendix A lists the co-researchers in the clinical trial.

2341-2879/© 2017 Asociacion Espanola de Pediatrıa. Published by Elsevier Espana, S.L.U. All rights reserved.

228.e2 A. Nunez et al.

Newborn;Antioxidants;Hypothermia

failure and programmed neuronal death by means of the activation of the caspase pathways. Athird phase has recently been described that includes persistent inflammation and epigeneticchanges that would lead to a blockage of oligodendrocyte maturation, alteration of neurogene-sis, axonal maturation, and synaptogenesis. In this scenario, oxidative stress plays a critical rolecausing direct damage to the central nervous system and activating metabolic cascades leadingto apoptosis and inflammation. Moderate whole body hypothermia to preserve energy storesand to reduce the formation of oxygen reactive species attenuates the mechanisms that leadto the amplification of cerebral damage upon resuscitation. The combination of hypothermiawith coadjuvant therapies may contribute to improve the prognosis.© 2017 Asociacion Espanola de Pediatrıa. Published by Elsevier Espana, S.L.U. All rightsreserved.

PALABRAS CLAVEAsfixia;Encefalopatíahipóxico-isquémica;Estrés oxidativo;Radicales libres;Especies reactivas deoxígeno;Recién nacido;Antioxidantes;Hipotermia

Estrés oxidativo en la asfixia perinatal y la encefalopatía hipóxico-isquémica

Resumen La asfixia intraparto es una de las causas más frecuentes de muerte neonatalprecoz pero también puede, en los supervivientes, evolucionar a una encefalopatía hipóxico-isquémica responsable de una elevada morbilidad neurológica. La presencia de episodios dehipoxia-isquemia prolongados conduce a un rápido agotamiento energético en los tejidos exclu-sivamente dependientes del metabolismo aeróbico, como el sistema nervioso central. El déficitenergético conlleva una paralización de las bombas ATP-dependientes y subsiguiente pérdidadel potencial neuronal transmembrana. La población neuronal de las regiones más sensibles delSNC mueren por necrosis, mientras que en otras áreas se produce una hiperexcitabilidad neu-ronal con entrada masiva de calcio iónico, activación de NO-sintasa, generación de radicaleslibres que alteran el funcionamiento mitocondrial, provocando un fallo energético secundarioy muerte neuronal por apoptosis. Recientemente se ha propuesto una tercera fase en la quefactores como la inflamación persistente y los cambios epigenéticos causarían un bloqueo de lamaduración de los oligodendrocitos, alteración de la neurogénesis, del crecimiento axonal y dela sinaptogénesis. En este contexto, el estrés oxidativo va a tener un papel protagonista comoresponsable tanto en causar dano directo al SNC como en activar cascadas metabólicas condu-centes a la apoptosis e inflamación. La hipotermia moderada precoz, al preservar las reservasenergéticas y disminuir la formación de especies reactivas de oxígeno, atenuará el dano cere-bral posreanimación. La combinación de la hipotermia con terapias coadyuvantes para modularel estrés oxidativo podría contribuir a mejorar el pronóstico.© 2017 Asociacion Espanola de Pediatrıa. Publicado por Elsevier Espana, S.L.U. Todos los dere-chos reservados.

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o-called oxy-regulator tissues, such as the central ner-ous system (CNS) and myocardium, require high amountsf energy to maintain membrane potentials and thereforeepend on aerobic metabolism, which generates energyuch more efficiently than anaerobic metabolism.1 In theNS, the transmission of action potentials involves ATP-ependent ion pumps, which use a large amount of energy.urthermore, the CNS cannot store energy in forms that cane mobilised rapidly, such as phosphocreatine or glycogen,nd therefore depends on a continuous supply of glucosend oxygen. In consequence, oxygen and glucose depriva-ion lead to a rapid depletion of energy stores and cell deathn a matter of minutes.2---4

Intrapartum asphyxia is characterised by periods ofypoxia/ischaemia during labour that, depending on sever-

ty, may result in death or cause hypoxic-ischaemicncephalopathy (HIE). The mortality due to HIE is of 1 to 8eaths per 1000 live births in developed countries and can be

Ime

s high as 26 deaths per 1000 live births in developing coun-ries. The main challenge for neonatologists is to reduce theorbidity and mortality associated with HIE, since despite

he widespread use of hypothermia, as many as 45% of theseatients still die today, while a high percentage of survivorsevelop significant disabilities.5

The purpose of this review article is to describe theiochemical basis of oxidative metabolism as well as thedjuvant therapies that may help control the production ofree radicals and improve the outcomes of hypothermia.

xidative metabolism2,6---9

erobic metabolism and oxidative phosphorylationFig. 1)

n multicellular organisms, oxygen must be available foritochondrial oxidative phosphorylation to produce the

nergy required to sustain life. Substrates such as glucose,

Oxidative stress in perinatal asphyxia and hypoxic-ischaemic encephalopathy 228.e3

Cytoplasm Glucose (glycolysis)Amino acids (oxidative deamination)

Fatty acids (β oxidation)

Acetyl-coA

Mitochondria

INTERMEMBRANE SPACE

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high-energy electrons, which are transported to the respiratoryin turn is used to resynthesise ATP.

amino acids and fatty acids are converted to acetyl coen-zyme A (acetyl-coA) in the mitochondrial matrix. Acetyl-coAis metabolised by the different components of the Krebscycle. This process releases high-energy electrons that arecarried to the electron transport chain by nicotinamideadenine dinucleotide and flavin adenine dinucleotide. Theelectron transport chain consists of a series of adjacentenzyme complexes (i, iii and iv) capable of pumping protons(H+) to the intermembrane space, thus generating a mem-brane potential ( m). ATP-synthase then allows protons toflow back into the matrix down the electrochemical gradi-ent, which releases energy used to synthesise ATP from ADP.Oxygen accepts the electrons produced by the Krebs cycle,preventing damage to mitochondrial structures. Energy isused so efficiently that 36 ATP molecules are obtained from1 glucose molecule and 106 ATP molecules from 1 palmiticacid molecule.2,6---9

Reactive oxygen species and free radicals (Fig. 2)

Oxygen is reduced by the addition of 4 electrons to itsoutermost shell. Oxygen has a limited reactivity, so it isreduced sequentially (slow reduction). However, its reac-tivity increases exponentially in the presence of transitionmetals like copper or iron (rapid reduction). The abundantpresence of iron in the CNS thus increases the speed of oxida-tive processes, as occurs in HIE. In the sequential process,oxygen is reduced by a single electron to a superoxide anion(•O2

−), by two electrons to hydrogen peroxide (H2O2), or

by three electrons to a hydroxyl radical (•OH). All thesesubstances, which are common products of mitochondrialoxidative phosphorylation, are known as reactive oxygenspecies. However, some of these species, like the superoxide

Obtf

in. The energy is used to generate a membrane potential that

nion, the hydroxyl radical and others like nitric oxide (•NO)r lipid peroxy radicals (LOO•) are also free radicals, that is,pecies with a single unpaired electron in their outer shellhat are therefore highly reactive and interact with nearbyubstrates to obtain the additional electron they require tottain stability, generating a chain reaction that can dam-ge cellular structures. The most prevalent free radical inhe human body is the superoxide anion. Not all reactivepecies are free radicals: for example, hydrogen peroxideH2O2), which acts as a signalling molecule in several physi-logical pathways, can also be a precursor of free radicals,specially in the presence of transition metals (Fe++/Fe+++;u+).6

ources of free radicals

itochondrial activity is the main physiological source ofree radicals. Within the mitochondria, oxygen is fullyeduced and gives rise to water if it reacts with protons (H+).nder metabolic stress conditions (resuscitation, infection,arenteral nutrition, etc.) there is an increased productionf free radicals that can damage mitochondrial structure andinder energy production. Some of the most frequent causesf oxidative stress are the activation of the NADPH oxidaseNOX) system in phagocytes in response to infection, oxygenupplementation or the administration of certain drugs.6,8,9

iological antioxidant systems

xidative stress reflects a disturbance in the balanceetween the production of free radicals and their neu-ralisation. A certain degree of oxidative stress is requiredor proper functioning of enzymatic pathways that require

228.e4 A. Nunez et al.

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he fraction of inspired oxygen used during reoxygenation (Solb

eactive oxygen species for cellular signalling. However, aronounced imbalance leads to tissue damage and acute orhronic disease.9

The antioxidant defence system (ADS) includes enzymatics well as non-enzymatic systems. Antioxidant enzymeseutralise reactive species through chemical reactions.on-enzymatic antioxidant systems include proteins thatind transition metals (transferrin, ceruloplasmin, ferritin),itamins that block lipid peroxidation (A, E and C) andow molecular weight compounds that reduce reactivepecies. Reduced glutathione (GSH) reacts with anotherolecule of GSH to form oxidised glutathione (GSSG), arocess that releases two electrons that can be capturedy free radicals, contributing to their neutralisation. Thenzyme GHS reductase then catalyses the reduction ofSSG to GSH. Some of the key antioxidant enzymes areuperoxide dismutases (SODs), which catalyse the conver-ion of superoxide to hydrogen peroxide, and catalasesCATs) and glutathione peroxidases (GPxs), which convertydrogen peroxide to water and molecular oxygen. Otherystems involve glutaredoxins, thioredoxins, peroxiredoxinsnd haem oxygenases, which degrade the pro-oxidant haemomplex in red blood cells, converting it to biliverdin. TheDS develops late in gestation to prepare the foetus forostnatal oxygenation.6,9

xidative stress, tissue damage, and theirssessment

ree radicals cause changes in proteins, lipids, carbohy-rates, RNA and DNA. There are biomarkers of overalledox status, such as the oxidised-reduced glutathione ratioGSH/GSSG). In situations of oxidative stress, the level of

SSG increases and the ratio decreases, while the oppositeccurs in situations promoting antioxidant activity or reduc-ion, where there is an increase in GSH. Another method ishe measurement of antioxidant enzymes. Thus, increases in

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newborn piglet model of hypoxia/reoxygenation correlated tot al.17).

xidative stress are associated with a proportional increasen protective enzyme activity. Nevertheless, research is cur-ently underway to search for biomarkers derived fromhe oxidation of different cell components that could beetected in samples of biological fluids. In recent years,he use of several biomarkers from different biological fluidsor the detection of oxidative stress has been validated inewborns.10---16

athophysiology of ischaemia-reperfusionnjury

ree radical generation in reoxygenation injury

ypoxia-ischaemia results in depletion of ATP, and thisauses damage that is initially reversible but that cannote repaired if the depletion is prolonged. Oxy-regulator tis-ues, of which the CNS is the prime example, are mostensitive to the lack of oxygen. Studies conducted in the970s proved that the initial damage caused by a period ofypoxia/ischaemia was amplified significantly during reper-usion/reoxygenation. The damage caused by reoxygenationas directly proportional to the duration and intensity ofypoxia-ischaemia and to the oxygen concentration useduring reoxygenation/reperfusion.8 An experimental animalodel of hypoxia-ischaemia in newborn piglets found that

ervous tissue damage and the urine concentration of mark-rs of DNA damage (Fig. 3) were proportional to the FiO2

sed during reoxygenation.17 Research is currently beingonducted on specific markers of tissue hypoxia. Lactate is

reliable marker of the intensity of tissue hypoxia, but doesot reflect its duration. An experimental model in newborniglets demonstrated that the concentrations of metabo-

ites resulting from the oxidation of purines, pyrimidines andhospholipids detected by mass spectrometry can be used toalculate a ‘‘metabolite index’’ that provides a reliable andeproducible measure of the intensity and duration of brain

Oxidative stress in perinatal asphyxia and hypoxic-ischaemic encephalopathy 228.e5

Redox status

ATP

ADP

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Uric acid

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is an accumulation of purines from the degradation of ATP, espconverted to xanthine oxidase, which generates a large amount

hypoxia, which can be used for the purpose of prognosisor to guide decision-making regarding the most appropriatetreatment.18---20

Sources of reactive species inischaemia-reperfusion

There are multiple sources of reactive species duringischaemia-reperfusion (IR), chief of which are the mitochon-drial respiratory chain complex and the xanthine oxidase(XO) system. The NADPH oxidase (NOX) system and NO syn-thase uncoupling are also involved, although their roles areless prominent.8

Under normal conditions, the production of free radicalsis well regulated. Nearly all electrons that flow down theelectron transport chain are captured by oxygen (98%), whileonly 2% produce reactive species. However, under hypoxicconditions, the transfer of electrons in the respiratory chainhalts due to the lack of oxygen, and �m drops. Membranedepolarization inhibits the activity of ATP synthase and ATPproduction stops, leading to the lysis of mitochondria andcell death. During reoxygenation, the �m is re-establishedand the synthesis of ATP resumes. However, the abruptreactivation of the electron flow leads to an excessive pro-duction of reactive species that may aggravate the neuraltissue damage initially caused by apoptosis.2,7,8 Fig. 4 showselectron microscopy images of brain slices from an experi-mental murine model of hypoxia-reperfusion that reveal thestructural disruption, swelling, loss of cristae, membranerupture and finally vacuolation and destruction of mitochon-dria.

In the reoxygenation stage of IR, oxygen reacts with

hypoxanthine and XO, driving a burst of superoxide radi-cals and hydrogen peroxide that in turn give rise to otherreactive species in a chain reaction, amplifying the tissuedamage initially caused by prolonged ischaemia. The activ-

ptts

lly hypoxanthine. During reoxygenation, xanthine reductase iseactive oxygen species, leading to oxidative stress.

ty of XO is also subject to post-translational regulationy the partial pressure of oxygen in tissue, with XO activ-ty increasing with decreasing pressure. This would explainow oxidative stress could begin in the hypoxaemic stagend be amplified during reoxygenation. Recent studies haveemonstrated that XO can produce •NO from nitrites. Theimultaneous generation of superoxide and •NO may resultn the production of peroxynitrite, a powerful nitratinggent.21,22

hases of hypoxic-ischaemic encephalopathy

he course of HIE can be divided into 3 phases (Fig. 5),lthough in reality it is a continuum in which these eventsan overlap. The first phase lasts 6 h and is characterisedy a reduction in blood flow in the foetus, causing sys-emic hypotension and loss of autoregulation of cerebrallood flow. Cerebral ischaemia leads to hypoxia, acidosisnd brain damage as a result of primary energy failure.he cell resorts to anaerobic metabolism to compensateor the decreased oxygen supply, which causes lactic aci-osis, ATP depletion, intracellular accumulation of Na+,a++ and water and inhibition of neurotransmitter reup-ake with secondary excitotoxicity. The massive influx ofa++ leads to activation of lipases and NO synthase, freeadical production, mitochondrial dysfunction and releasef apoptogenic substances to the cytoplasm. The secondhase (6---48 h) is characterised by continued excitotox-city, loss of mitochondrial activity and oxidative stressecondary to changes in membrane potential with a reducedroduction of ATP and an alkaline intracellular environ-ent despite adequate oxygenation. Finally, there is a third

hase that may last days, weeks or even months charac-erised by inflammation and epigenetic changes that leado abnormalities in axonal development, neurogenesis andynaptogenesis.23,24

228.e6 A. Nunez et al.

Effects of reoxygenation on mitochondrial structure (electron microscopy)

Mitochondria: normal structure Mitochondria: structural damage due to oxidative stress

Figure 4 The images show morphological and structural changes including structural disruption, swelling, loss of cristae, mem-brane rupture and finally vacuolation and destruction of cerebral mitochondria in a model where newborn mice were exposed todifferent oxygen concentrations. Courtesy of Isabel Torres-Cuevas (doctoral thesis; supervisor: Máximo Vento).

Phase I: Hypoxia Phase II: Reoxygenation

Prolonged hypoxia Hyperoxia

Hyperpolarization (ΔΨ)

Resumed production of ATPExcessive production of ROS

Oxidative damage and loss of mitochondrial functionRelease of pro-apoptotic factorsActivation of apoptotic pathway

Programmed cell death

Hyper-reactivation of electron flow (respiratory chain)

Halt in electron flow (respiratory chain)

Halt in ATP productionDepolarization (ΔΨ)

Depletion of ATP storesInhibition of ATP-dependent pumps

Accumulation of Ca++ in mitochondrial matrixAccumulation of Na+ in cytoplasm

Release of K+ to extracellular environmentInhibition of neurotransmitter reuptake

ExcitotoxicityCell death

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Figure 5 Phases of hypo

trategies to prevent damage from reactive oxygenpecies

reventing the damage caused by reactive oxygen speciesequires very early intervention, as the damage may havetarted in utero and could be amplified during postna-al resuscitation. Therefore, the treatment protocol shouldnclude interventions that target primary energy failure.

hese could be implemented in the mother during labour ifhere were evidence of foetal distress, or in newborns withlinical or electrophysiological findings suggestive of HIE inhe first minutes of life.25,26

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chaemic encephalopathy.

We proceed to give a brief description of interventionshat address the pathogenic effects of oxidative stress.

ypothermia

ypothermia slows down the metabolic activity of tissuesuch as the CNS, with a corresponding reduction in ionxchange activity and ATP demand. The reduction in mito-

hondrial activity combined with activation of uncouplingroteins (UCPs) significantly decreases production of reac-ive species while preserving the membrane potential andreventing release of apoptogenic proteins. There is an

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Oxidative stress in perinatal asphyxia and hypoxic-ischaemic

extensive body of research on the mechanism of action ofhypothermia and its clinical effectiveness. However, despitethe proven efficacy of hypothermia as a neuroprotectiveintervention in patients with moderate or severe HIE, upto 45% of treated patients, especially those with severeHIE, have unfavourable outcomes. This may change in thevery near future with the use of adjuvant therapies thatare currently being investigated in experiments and clinicaltrials.5,23,25,27

Neonatal resuscitation with low oxygenconcentrations

The use of low oxygen concentrations during resuscitation isassociated with a reduction in oxidative stress during reoxy-genation, mortality, and the incidence of HIE.28 The recentguidelines of the International Liaison Committee on Resus-citation published in 2015 recommend the use of room airin the resuscitation of term newborns.29

Allopurinol or inhibition of xanthine oxidase

Allopurinol is a selective inhibitor of XO, reduces produc-tion of NO from nitrites and acts as a free-iron chelatorand a hydroxyl radical scavenger. These properties make ita suitable candidate for neuroprotection.21,22

A pilot study was conducted in 199830 with administra-tion of 40 mg/kg of allopurinol in asphyxiated newborns. Thestudy found a reduction in oxidative stress and improvedcerebral perfusion and cortical activity in the absence ofallopurinol-related toxicity. However, a subsequent study31

found no improvements in survival or neurodevelopmentaloutcome with administration of allopurinol in the first 4 hpost birth compared to the use of placebo, which the authorsattributed to the delay in its administration after birth.The same group of researchers administered allopurinol topregnant women in labour with signs of foetal hypoxia.32

Newborns who had therapeutic concentrations of allopuri-nol in cord blood had significantly lower plasma levels ofS-100B protein, a marker of brain damage, and non-protein-bound iron, a marker of oxidative stress.32 A follow-up studyof moderately asphyxiated newborns found that the grouptreated with allopurinol in the first hours post birth (<4 h)and a second dose at 12 h scored higher in the WechslerIntelligence Scale for Children (WISC) compared to new-borns that received placebo.33 Recently, the same authorspublished the results of a randomised placebo-controlledmulticentre trial of allopurinol in women in labour withclinical indices of foetal hypoxia prompting immediate deliv-ery, which did not confirm a significant reduction in thelevels of S100B; however, the post hoc analysis revealeda potential benefit in newborns of mothers treated withallopurinol.34

A multicentre study is currently being conducted inEurope (ALBINO trial; EudraCT-2016-000222-19) with par-ticipation of 18 public hospitals in Spain. It is a phase IIIrandomised placebo-controlled trial of allopurinol whose

primary endpoint is death and/or severe neurodevelop-mental impairment. The protocol consists of administrationof a dose of allopurinol to newborns with suspected HIEsecondary to neonatal asphyxia in the first 30 minutes

LEoi

ephalopathy 228.e7

f life, with a second dose administered 12 h after therst in newborns treated with hypothermia. The assess-ent includes metabolomic, neurophysiological and imaging

ests, in addition to clinical followup of participants untilge 24 months.

elatonin

elatonin (N-acetyl-5-methoxytryptamine) is an endoge-ous indolamine with antioxidant, anti-inflammatory andntiapoptotic properties that has shown promising resultsn patients with HIE. Its anti-inflammatory activity is basedn the downregulation of pro-inflammatory molecules andhe suppression of NO production in neural tissue. Mela-onin also blocks the release of pro-apoptotic proteins byitochondria and modulates the activity of GABA and glu-

amate receptors.35 Melatonin used as adjuvant therapy toypothermia in patients with HIE is associated with a reduc-ion in oxidative stress and improvements in survival andeurodevelopmental outcomes,36---38 although these resultsre limited by the small sample sizes. Further studies withdequate statistical power are required to confirm the effec-iveness of melatonin and address additional questions.

onclusions

ypoxic-ischaemic encephalopathy continues to be a seri-us challenge in neonatology. Despite the widespread use ofypothermia, the associated morbidity and mortality con-inue to be high. Reactive oxygen species play a significantole in the pathogenesis of asphyxia leading to HIE. There-ore, a reduction of their production, their neutralisation orhe inhibition of the inflammatory and pro-apoptotic path-ays that they activate are strategies that combined with

nterventions involving other targets (for instance, to reducexcitotoxicity or promote angiogenesis or neurogenesis) canmprove intact survival in newborns with HIE.

unding

his study has been funded by Grant EC11-244 (Institutoe Salud Carlos III; Ministry of Economy, Industry and Com-etitiveness; Spain) awarded to MV and through a contractetween AN and the Maternal and Child Health and Devel-pment Network (SAMID), funded by the 2008---2011 R&D&Iational Programme of the General Vice Directorate of the

nstituto de Salud Carlos III and project RD16/0022/0001 ofhe European Regional Development Fund.

onflicts of interest

he authors have no conflicts of interest to declare.

ppendix A. List of co-researchers

ist of hospitals and co-researchers that participated in theC11-244 clinical trial (Instituto de Salud Carlos III; Ministryf Economy, Industry and Competitiveness; Spain), with reg-stration number EudraCT-2011-005696-17

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Hospital Universitario y Politécnico La Fe, Valencia:ntonio Nunez-Ramiro; Anna Parra-Llorca; Ana García-obles; Ana García-Blanco; María Cernada; Raquel Escrig-ernández; Ana Gimeno-Navarro; Nuria Boronat González.

Hospital Universitario Puerta del Mar, Cadiz: Isabelenavente; Simón Lubián López.

Hospital Regional Universitario Materno Infantil Carlosaya, Malaga: Mercedes Chaffanel; Enrique Salguero; Maríael Mar Serrano Martín; Rafael Maese Heredia.

Hospital Universitario Gregorio Maranón, Madrid: Dorotealanco; María Arriaga.

Hospital Universitario La Paz, Madrid: Eva Valverde;alaika Cordeiro; Adelina Pellicer; Laura Sánchez; Palomaópez Ortego; Maricarmen Bravo.

Hospital Universitario 12 de Octubre, Madrid: Maríaeresa Moral-Pumarega; María del Carmen Pérez-Grande;oelia Ureta Velasco; Rocío Mosqueda Pena; Ana Melgaronis.

Hospital Universitario de Cruces, Barakaldo: Begonaoureiro; Jon López de Heredia; María Jesús Martinez;ristina Iniesta.

Hospital Universitario Vall d’Hebron, Barcelona: Héctoroix; Yolanda Castilla; Cristina Fernández-García.

Complejo Hospitalario Universitario Vigo, Vigo: Juanernández-Lorenzo; Eva González-Colmenero; María Luisaonzález-Durán; Ana Concheiro-Guisán.

Hospital Universitario Central de Asturias: Belén Fernán-ez Colomer; Rosa Patricia Arias Llorente.

Hospital Universitario Virgen del Rocío, Seville: Antonioavón; José Fernando Ferreira López.

Hospital Universitario Reina Sofía, Cordoba: Inés Tofé;ilar Jaraba.

Hospital Universitario Quironsalud, Madrid: Fernandoabanas; M. Angeles Caballero; Manuela López-Azorín.

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