Experimental treatments for hypoxic ischaemic encephalopathy

Early Human Development 86 (2010) 369–377

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Early Human Development j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e a r l h u m d ev

Experimental treatments for hypoxic ischaemic encephalopathy Dorottya Kelen, Nicola J. Robertson ⁎ Neonatology, Institute for Women's Health, University College London, 86-96 Chenies Mews, London WC1E 6HX, United Kingdom

a r t i c l e

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Keywords: Neuroprotection Birth asphyxia Hypoxia–ischaemia Biomarker Magnetic resonance spectroscopy Brain lactate/N acetyl aspartate

a b s t r a c t Hypoxic ischaemic encephalopathy continues to be a significant cause of death and disability worldwide. In the last 1–2 years, therapeutic hypothermia has entered clinical practice in industrialized countries and neuroprotection of the newborn has become a reality. The benefits and safety of cooling under intensive care settings have been shown consistently in trials; therapeutic hypothermia reduces death and neurological impairment at 18 months with a number needed to treat of approximately nine. Unfortunately, around half the infants who receive therapeutic hypothermia still have abnormal outcomes. Recent experimental data suggest that the addition of another agent to cooling may enhance overall protection either additively or synergistically. This review discusses agents such as inhaled xenon, N-acetylcysteine, melatonin, erythropoietin and anticonvulsants. The role of biomarkers to speed up clinical translation is discussed, in particular, the use of the cerebral magnetic resonance spectroscopy lactate/N-acetyl aspartate peak area ratios to provide early prognostic information. Finally, potential future therapies such as regeneration/repair and postconditioning are discussed. © 2010 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . Biomarkers in hypoxic–ischaemic 2.1. Overview of biomarkers . 2.2. MR biomarkers . . . . . 3. Neuroprotective agents . . . . . 3.1. Xenon . . . . . . . . . 3.2. Allopurinol . . . . . . . 3.3. N-acetyl cysteine . . . . 3.4. Melatonin. . . . . . . . 3.5. Erythropoetin . . . . . . 3.6. Alpha-2 agonists . . . . 3.8. Anticonvulsants . . . . . 4. Postconditioning . . . . . . . . 5. Regeneration and repair . . . . 6. Conclusion. . . . . . . . . . . Role of funding source . . . . . . . . References . . . . . . . . . . . . .

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1. Introduction Neonatal encephalopathy remains a significant problem worldwide. An estimated 4 million babies die every year during the

⁎ Corresponding author. Tel.: +44 207 674 6052. E-mail address: [email protected] (N.J. Robertson). 0378-3782/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.earlhumdev.2010.05.011

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neonatal period, and one quarter of these deaths are attributed to neonatal asphyxia [1]. Even in the developed world, neonatal encephalopathy is a common clinical condition affecting approximately 2 per 1000 neonates [2] and accounts for a substantial proportion of admissions to neonatal intensive care; 10–15% of cases will die in the neonatal unit, 10–15% will develop cerebral palsy and up to 40% will have other significant disabilities including blindness, deafness, autism, epilepsy, global developmental delay, and problems with cognition, memory, fine motor skills and behaviour [3–5].

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Within the last decade, therapeutic hypothermia for infants with hypoxic ischaemic encephalopathy has been studied in pre-clinical models [6] and several major randomized clinical trials in the developed world [7]. Despite the clinical heterogeneity of perinatal asphyxia and the use of different cooling methods there are consistent findings that hypothermia reduces the extent of neurological damage and improves survival without disability [8]. Therapeutic hypothermia is now widely offered to moderately and severely asphyxiated infants in countries and centers which participated in the trials [9]. Despite the promising outcome of these trials, the reduction in disability or death at 18 months with therapeutic hypothermia is modest — meta-analyses indicate that the composite adverse outcome reduces from 58% to 47% with cooling [5,8]. Thus approximately half the infants who receive therapeutic hypothermia still have an abnormal outcome and some infants with the most severe injuries may not be rescued [7]. Recent experimental data suggest that hypothermia extends the duration of the therapeutic window [10,11] and that certain drugs given during this time may augment neuroprotection [11–13]. Research is now being focused on pre-clinical studies of drugs, which act synergistically or additively with hypothermia with the hope that combination therapy might reduce the overall number of infants needed to treat to improve intact survival. 2. Biomarkers in hypoxic–ischaemic encephalopathy 2.1. Overview of biomarkers Biomarkers are critically needed in neonatal encephalopathy because the timing of brain injury is heterogeneous and difficult to identify. Biomarkers may have several roles, including: (a) the identification of who is injured, (b) the extent of injury, (c) the timing of injury, (d) identification of the most likely outcomes with and without therapy and (e) speeding up clinical translation of interventions. In a recent systematic review of studies where urine, serum and cerebrospinal fluid biomarkers were taken within the first 96 h after birth in infants with neonatal encephalopathy, four markers were revealed as potentially predictive of abnormal outcomes in the meta-analysis: serum and CSF interleukin-1b, serum interleukin-6, and CSF neuron-specific enolase [14]. None of these biomarkers, however, had been studied extensively enough to warrant routine clinical use and further work is needed for their validation. Speeding up clinical translation is an important aspect to consider in future trials; therapeutic hypothermia for hypoxic ischaemic encephalopathy took some 15 years to translate from pre-clinical large animal studies to an established therapy in the clinic [15]. During this time, experimental studies were performed showing consistent benefit of cooling [6,16] and clinical trials were based on 12–18 month neurodevelopmental outcome data. Thus clinical translation occurred only after these randomized controlled trial (RCT) results were known [8]. Although knowledge of the neurodevelopmental outcome of therapies is critically important, robust and sensitive quantitative biomarkers with properly validated outcome measures at age18 months or later can help with speeding up clinical translation. 2.2. MR biomarkers In clinical practice, magnetic resonance imaging (MRI) is increasingly used in newborn infants after perinatal asphyxia where it is an effective biomarker for both disease and treatment effect [17]. To increase prognostic objectivity, quantitative cerebral biomarkers such as proton (1H) magnetic resonance spectroscopy (MRS) have been utilized [18] and a recent meta-analysis of the prognostic accuracy of MR methods demonstrated that the thalamic 1H MRS Lactate/N-acetyl aspartate (Lac/ NAA) peak area ratio acquired between days 5–14 is a highly sensitive and specific biomarker of long term neurodevelopmental outcome in

infants with neonatal encephalopathy [19] as well as in adult stroke [20]. The 1H MRS lactate peak area metabolite ratios can be used in in vivo preclinical as well as clinical studies; Lac/NAA has been validated as a bridging biomarker and will be used as a surrogate endpoint in phase II clinical trials of neuroprotection. In the UCL piglet perinatal asphyxia model we acquire phosphorus-31 (31P) and 1H MRS at baseline and serially during the 48 h following transient hypoxia–ischaemia; the increase in brain lactate and reduction in NAA over this time correlates with brain tissue injury as described below [21]. The biphasic decrease in nucleotide triphosphate (NTP)/exchangeable phosphate pool (EPP = Pi + PCr + NTP) and reciprocal increase in brain lactate during and following a transient hypoxic–ischaemic insult are shown in Fig. 1. In the late 1980s, studies in infants with birth asphyxia were performed using 31 P MRS and the characteristic pattern of evolving energy failure in the hours and days after birth in neonatal encephalopathy was described [22]. We have recently focused on 1H MRS, in particular brain Lac/NAA. Brain lactate increases during hypoxia–ischaemia as mitochondria are unable to continue nucleotide triphosphate (NTP) synthesis at a rate sufficient to supply the brain. Glycolysis increases to provide additional NTP and lactate concentration increases. Acidosis increases as well as hypoxic depolarization of cells, cytotoxic oedema, excessive intracellular accumulation of calcium and extracellular accumulation of excitatory amino acids. Following reperfusion, clearance of lactate occurs over ∼30 min [23]. A secondary increase in brain lactate may occur as early as 3 h after ischaemia [24,25] due to renewed production in tissues [26] and may persist for considerable periods of time [18,27]. The events leading to the secondary rise in brain lactate are thought to include failure of mitochondrial activity

Fig. 1. Schematic diagram illustrating the biphasic pattern of energy failure associated with a transient hypoxic–ischemic insult visualized using 31P MRS (top) and 1H MRS (bottom) in the UCL piglet model. 31P MRS: nucleotide tri-phosphate (NTP)/ exchangeable phosphate pool (EPP = Pi + PCr + NTP) is shown on the y axis. The change in NTP/EPP during transient hypoxia–ischaemia (HI), resuscitation, the latent phase (period between the recovery from acute HI and the evolution of secondary energy failure (SEF)) and SEF itself are shown. 1H MRS: lactate/N acetyl aspartate (NAA) is shown on the y axis. The change in Lac/NAA during transient HI, resuscitation, latent phase and SEF are shown. During the acute energy depletion, some cells undergo primary cell death, the magnitude of which will depend on the severity and duration of HI. Following perfusion, the initial hypoxia-induced cytotoxic edema and accumulation of excitatory amino acids typically resolve over 30–60 min with apparent recovery of cerebral oxidative metabolism (latent phase). It is thought that the neurotoxic cascade is largely inhibited during the latent phase and that this period provides a “therapeutic window” for therapies such as hypothermia and other agents. Cerebral oxidative metabolism may then secondarily deteriorate 6–15 h later (as SEF). This phase is marked by the onset of seizures, secondary cytotoxic edema, accumulation of cytokines and mitochondrial failure. Mitochondrial failure is a key step leading to delayed cell death.

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leading to a reduced NTP levels, increased glycolysis and a change in redox state. This altered redox state (NADH/NAD+) inhibits pyruvate dehydrogenase activity thus the lactate/pyruvate ratio increases. The increase in ADP drives glycolysis, which is further stimulated by the influence of alkalosis [28] on the pKa of phosphofructokinase. In our model, the rise in brain lactate after hypoxia–ischaemia is associated with cell death [29] and immunohistochemical markers of apoptosis — transferase mediated biotinylated d-UTP nick end-labeling positive (TUNEL+) cells and caspase 3 immunoreactive cells [21]. In other models infiltration of brain microglia 1–3 days after the injury has been associated with brain lactate [30]. 3. Neuroprotective agents

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of perinatal asphyxia by improving cerebral magnetic resonance biomarkers of injury (Lac/NAA) and immunohistochemistry [21]. This supports transition to a clinical trial (TOBYXe; NCT00934700) where the efficacy of the combination of hypothermia and xenon will be tested against cerebral magnetic resonance biomarkers and clinical outcomes. The neuroprotective effects of krypton, argon, neon and helium have been recently investigated in fetal mice cortex cell cultures. The effect of noble gases on cell reducing ability in the absence of OGD was also investigated. Like xenon, neuroprotection was afforded by argon. Krypton and neon had no effect on neuronal injury in vitro whereas helium had detrimental effects on the cells [47]. These data suggest that the cheap and widely available noble gas, argon, may have potential as a neuroprotectant for the future.

3.1. Xenon 3.2. Allopurinol Xenon was discovered by Sir William Ramsay (together with his student Morris Travers) in 1898 while he was Professor of Chemistry at University College London; they found xenon in the residue left over from evaporating components of liquid air. Ramsay estimated the proportion of xenon in the earth's atmosphere as one part in 20 million [31]. Ramsay also discovered krypton and neon and received the Nobel Prize for Chemistry in 1904. Although the noble gas xenon is considered chemically inert, it has surprising biological effects. Since the discovery that xenon has anaesthetic properties in 1951 [32], further research has shown its favourable profile as a safe anaesthetic and more recently as a neuroprotective agent [33]. According to the current level of knowledge, xenon is not teratogenic [34]; indeed it has been seen to reduce isoflurane-induced neuronal death [35,36]. Due to its poor solubility in blood (at a blood/gas distribution coefficient of 0.115) xenon provides very rapid induction and recovery characteristics as an anaesthetic. Xenon is an attractive agent to use in the sick newborn infant because of its apparent cardiovascular stability, [37] myocardial protective properties, [38] previous use in neonates in assessment of cerebral blood flow [39], and the ability to rapidly cross the blood brain barrier [40]. A disadvantage of xenon is its cost and the need for a closed circuit ventilator for delivery and re-use. Xenon is a non-competitive antagonist of the N-methyl-Daspartate (NMDA) subtype of the glutamate receptor [33]. Other actions of xenon include activation of the background two-pore K+ channel, [41] inhibition of the calcium/calmodulin dependent protein kinase II [42], activation of anti-apoptotic effectors Bcl-XL and Bcl-2 [35] and induced expression of hypoxia inducible factor 1α and its downstream effectors erythropoietin and vascular endothelial growth factor, which can interrupt the apoptotic pathway [43]. Xenon is neuroprotective following hypoxia–ischaemia in neonatal rats [12,44] and is effective even when administration is delayed for some hours [45,46]. Data from experimental animals demonstrate a synergy when xenon is administered in combination with mild therapeutic hypothermia [12]. Surprisingly, the combination of both interventions caused an effect, even at low concentrations or mild temperature reductions, while each alone had no effect at all. Remarkably this synergistic interaction still occurred when the administration of hypothermia and xenon occurred asynchronously [45]. Xenon showed attenuation of injury at sub-anesthetic concentrations in vivo and in vitro models of HIE at concentrations of 40% and greater [12]. The neuroprotection observed with xenon correlates with improved neuromotor function at 30 days of age indicating longterm functional protection [12]. It has been suggested that xenon and hypothermic therapy converge on an anti-apoptotic pathway explaining why asynchronous application of the interventions attenuates the injury. We have recently demonstrated that 50% xenon started 2 h after resuscitation from a global hypoxic–ischaemic insult and continued for 24 h augments hypothermic neuroprotection in the piglet model

Free radicals such as hydroperoxide (H2O2), hydroxyl radical (OH), – superoxide radical (O− 2 ), and peroxynitrite (ONOO ) play a significant role in the development of damage after HI. The primary source of superoxide in reperfused reoxygenated tissues appears to be the enzyme xanthine oxidase, released during ischemia by a calciumtriggered proteolytic attack on xanthine dehydrogenase. Allopurinol is a xantine-oxidase inhibitor; in high concentrations allopurinol also scavenges hydroxyl radicals and prevents free radical formation by chelating their catalyst non-protein bound iron. High doses are needed for neuroprotection. Studies in immature rats found that allopurinol treatment 15 min after cerebral hypoxia–ischaemia reduced brain oedema and long term brain damage. In this study, the rat pups received either allopurinol (135 mg/kg subcutaneously) or saline [48]. Preservation of cerebral 31P MRS energetics was found in a study of pre-treatment with allopurinol in neonatal rats, suggesting that neuroprotection may be attributed in part to preservation of energy metabolites [49]. In adult randomised controlled trials, high dose allopurinol (N10 mg/kg) reduces reperfusion injury in patients undergoing coronary bypass surgery [50]; in infants with hypoplastic left heart syndrome who undergo deep hypothermic cardiac arrest, pre-treatment with allopurinol reduces post-operative adverse cardiac and neurological outcomes [51]. The pharmacokinetics of allopurinol in children with birth asphyxia has been investigated [52]. A Cochrane review in 2008 [53] suggested that the available data are not sufficient to determine whether allopurinol has clinically important benefits for newborn infants with hypoxic–ischaemic encephalopathy. The review suggested that larger trials are needed which should assess allopurinol as an adjunct to therapeutic hypothermia in infants with moderate and severe encephalopathy and should be designed to exclude clinically important effects on mortality and adverse long-term neurodevelopmental outcomes. Three trials were included in the meta-analysis of allopurinol for preventing mortality and morbidity in newborn infants with suspected hypoxic–ischemic encephalopathy [54–56]. Meta-analysis did not reveal a statistically significant difference in the risk of death during infancy or incidence of neonatal seizures. Only one trial assessed neurodevelopment in surviving children and did not find a statistically significant effect [54]. As allopurinol acts through its free radical scavenger property it must be administered early, even during delivery; fortunately allopurinol crosses the placenta. In 2009, the Dutch group demonstrated that maternal allopurinol (500 mg) during fetal hypoxia lowers cord blood levels of S-100B in pregnancies in which fetal hypoxia was suspected at N36 weeks gestation [57]; fetal hypoxia was indicated by abnormal/non-reassuring fetal heart rate tracing or fetal scalp pH of b7.20. Following on from this the ALLO-trial has started in the Netherlands [58]; the proposed trial is a randomized double blind placebo controlled multicenter study in pregnant women at term in

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whom the fetus is suspected of intra-uterine hypoxia. Allopurinol 500 mg IV or placebo will be administered antenatally to the pregnant woman when fetal hypoxia is suspected. Fetal distress is being diagnosed by the clinician as an abnormal or non-reassuring fetal heart rate trace, preferably accompanied by either significant ST-wave abnormalities or an abnormal fetal blood scalp sampling (pH b 7.20). Primary outcome measures will be S100B levels and the severity of oxidative stress (measured by isoprostane, neuroprostane, non protein bound iron and hypoxanthine), both measured in umbilical cord blood. 3.3. N-acetyl cysteine N acetyl cysteine (NAC) is an attractive neuroprotective substance as it has low toxicity, is able to cross the placenta and blood brain barrier. Its profile as a glutathione (GSH) precursor, antioxidant, antiapoptotic, and anti-inflammatory agent makes it an interesting substance acting at multiple sites. NAC has been used in the treatment of late acetaminophen-induced acute liver failure and non-acetaminophen-induced acute liver failure in children where it was given as a continuous infusion of 100 mg/kg/24 h. High doses of NAC were well tolerated in this group of patients; NAC was associated with a shorter length of hospital stay, higher incidence of native liver recovery without transplantation, and better survival after transplantation [59]. NAC is used in adults with renal impairment where it plays a role in the prevention of contrast-induced nephropathy after cardiovascular procedures [60]. In adult models of stroke, NAC scavenges oxygen radicals, attenuates reperfusion injury and decreases inflammation and nitric oxide production [61]. NAC has shown promise in models that combine infection with hypoxia–ischaemia and was more neuroprotective than melatonin. In neonatal rats, 200 mg/kg NAC protected the brain from lipopolysaccharide (LPS) sensitized hypoxia–ischaemia. Multiple injections of NAC, started after LPS but before HI, provided remarkable neuroprotection (78% reduction of brain injury) compared with 41% reduction when the first NAC injection was given immediately after HI [62]. Other studies have confirmed the protective effects of LPS evoked degeneration of oligodendrocytes in developing rat brain [63]. Paintlia went on to examine the effects of LPS on preterm labor in pregnant rats and demonstrated that LPSinduced increased inflammation and metabolism of phospholipids at the feto-maternal interface (placenta) is critical for preterm labor and fetal demise during maternal microbial infections and these changes could be blocked by NAC [64]. In a piglet hypoxia-reoxygenation model, NAC (30 mg/kg followed by 20 mg/kg/h started within 5 min of resuscitation and continued for 4 h) reduced cerebral oxidative stress, reduced cerebral lactate accumulation and improved cerebral perfusion [65]. When combined with hypothermia, NAC decreased infarct volume, improved myelin expression and functional outcomes after focal HI injury in 7-day-old rats exposed to 2 h of carotid ligation and hypoxia [13]. Hypothermia was induced after resuscitation and lasted for 2 h; NAC (50 mg/kg) was administered intraperitoneally daily. Synergistic protective effects were seen between hypothermia and NAC. The promising effect of combined therapy was also reported after spinal cord ischaemia; NAC and hypothermia alone had limited neuroprotective effects whereas the combination of NAC and hypothermia was associated with a significant recovery of spinal cord function [66]. 3.4. Melatonin Melatonin (N-acetyl-5-methoxytryptamine) is a remarkable natural neuroprotectant produced from 1-trytophan in the pineal gland, retina and gastrointestinal tract and is known to be a potent free radical scavenger. Melatonin has a direct antioxidant activity as it − scavenges free radicals such as OH, O− as a 2 , H2O2 and ONOO

terminal antioxidant. Additionally melatonin induces antioxidant enzymes including glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and superoxide dismutase. Melatonin readily crosses the blood brain barrier, binds to specific brain receptors and is selectively concentrated in the brain and its subcellular compartments. Melatonin has been shown to be neuroprotective in small animal models. In permanent and transient middle cerebral artery occlusion rat stroke models, a single intraperitoneal injection of melatonin between 5 and 15 mg/kg, 30 min before HI significantly reduced infarct volume at 72 h [67]. In a similar study documenting the benefit and therapeutic window of melatonin given after HI onset, neuroprotection was seen with either single or multiple 5 mg/kg injections up to 2 h after HI [68]. Melatonin has also proven beneficial in a mouse model of ibotenate-induced excitotoxic white matter lesions mimicking periventricular leukomalacia [69]. In adult mice, delayed melatonin treatment reduced both grey and white matter damage and improved neurobehavioural outcome following transient focal cerebral HI; this neuroprotection was associated with reduced oxidative damage suggesting that one of melatonin's major mechanisms of action is mediated through its anti-oxidant and radical scavenging activity [70]. In adult rats, 5 mg/kg melatonin given at the start of reperfusion enhanced electrophysiological and neurobehavioural recovery and reduced cortical and striatal infarct sizes after cerebral ischemia [71]. In adult mice following transient focal cerebral ischemia treatment with melatonin at reperfusion also reduced both gray and white matter damage and improved neurobehavioural outcomes through its antioxidant and radical scavenging activity [72]. Melatonin has been shown to have several neuroprotective actions that include antiinflammatory [73], anti-apoptotic and anti-oxidative effects [74] and preservation of blood brain barrier permeability [74]. In the fetal sheep, 20 mg/kg melatonin starting 10 min after the start of reperfusion and continued for 6 h resulted in an attenuation in the production of 8-isoprostanes following umbilical cord occlusion and a reduction in the number of activated microglia cells and TUNELpositive cells in melatonin treated fetuses, suggesting a protective effect of melatonin [75]. A slower recovery of fetal blood pressure following umbilical cord occlusion was seen with melatonin treated animals, but this was without changes in fetal heart rate, acid base status or mortality [75]. In a recent study melatonin was found to reduce markers of CNS inflammation (microglia, macrophage infiltration) and apoptosis (activated caspase-3, fractin) when administered to the spiny mouse for 7 days at the end of the pregnancy prior to hypoxia– ischaemia and delivery [76]. The potential for maternally administered melatonin to protect the fetal brain from free radical damage was also seen in the fetal sheep [77]. Given that melatonin shows almost no toxicity to humans [78] and possesses multifaceted protective capacity against cerebral ischemia, it is valuable to consider using melatonin in clinical trials. The potential for melatonin to be used as a neuroprotective agent in preterm brain injury has been suggested in rodent models where melatonin prevented white matter myelination defects and promoted oligodendrocyte maturation but not proliferation [79] and prevented learning disorders in brain lesioned newborn mice [80]. A clinical trial of melatonin administration in premature infants is currently running in the UK. The potential benefit of post-insult administration of melatonin combined with therapeutic hypothermia is currently being studied in the UCL piglet model. 3.5. Erythropoetin Erythropoietin is a glycoprotein hormone that controls erythropoiesis. Erythropoietin (Epo) is a cytokine for erythrocyte precursors in the bone marrow and is produced by the peritubular capillary

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endothelial cells in the kidney. The safety of recombinant human Epo (rEPO) has been demonstrated in clinical studies for the prevention and treatment of anaemia of prematurity [81]. Epo is up-regulated in umbilical cord blood from babies who have suffered perinatal asphyxia [82], which may be an endogenous repair mechanism. Several pre-clinical studies have suggested a neuroprotective effect of Epo following hypoxia–ischaemia and reperfusion [83]; in neonatal models systemically administered Epo is neuroprotective [84] with early and high dose treatment (5000U/kg) required to reduce brain injury and promote neurogenesis [85]. There is also data to suggest that multiple doses of Epo are required for sustained neuroprotection and reduced tissue loss [86]. Epo is shown to have neuroprotective effects through different mechanisms such as direct neurotrophic effect, decreased susceptibility to glutamate toxicity, induction of anti-apoptotic factors, decreased inflammation, decreased nitric oxide-mediated injury, direct antioxidant effects, and protective effects on glia. Interestingly the antioxidative effect of Epo may be enhanced by the increased erythropoesis utilizating the free-iron which has accumulated following hypoxia–ischaemia [87]. The dosage for neuroprotection (1000–30,000 U/kg) is above the range used in anaemia; Epo is a relatively large molecule (30.4 kDa) to cross the blood brain barrier. However following high-dose rEpo administration in non-human primates and sheep, neuroprotective concentrations of Epo was found in the CSF by 2–2.5 h after injection [88]. High dose Epo was well tolerated in these studies [88]. A recent randomized controlled study in 167 infants reported that repeated low dose (300 or 500 U/kg) rEpo was safe and resulted in improved neurological outcome in moderate to severe neonatal encephalopathy at 18 months of age [89]. Recombinant Epo was administered every other day for 2 weeks, starting b48 h after birth. Outcomes were not different between the 2 Epo doses. Subgroup analyses indicated that Epo improved long-term outcomes only for infants with moderate HIE and not those with severe HIE. No negative hematopoietic side effects were observed [89]. Pre-clinical studies of the interaction of Epo with hypothermic neuroprotection are required before progression to clinical trials of combined therapy. Interestingly, other neuroprotective agents up-regulate endogenous erythropoietin; for example, xenon up-regulates erythropoietin expression [43] and easily crosses the blood brain barrier providing an alternative mechanism to harness erythropoietin protection in the CNS. Whether higher doses of Epo can improve outcomes without inducing complications is unknown. Concerns over the use of high doses of erythropoietin include possible thrombotic and hematological complications and real safety concerns have arisen in 2 recent adult studies. In a pilot study of out-of-hospital cardiac arrest, patients treated with hypothermia and early high dose Epo (40 000 IU) as soon as possible after resuscitation and every 12 h for the first 48 h were compared with case-matched controls who received therapeutic hypothermia alone [90]. A higher survival rate with none or minimal cerebral sequelae were seen in the Epo treated patients but adverse effects related to vascular thrombosis occurred [90]. In another study of adult stroke patients, a higher death and complication rate was observed in those receiving Epo compared with placebo; complications included intracerebral hemorrhage, brain oedema and thrombotic events [91]. 3.6. Alpha-2 agonists α2 adrenoceptor agonists, including clonidine and dexmedetomidine, have been shown to have neuroprotective potential in animal models of perinatal hypoxia–ischaemia [92–94]. Both agents reduce the size of excitotoxin-induced cortical and white-matter lesions in mouse pups injected intracerebrally with ibotenate [92]; these protective effects were abolished by an a2 antagonist confirming that these agents protect through their activity on the α2 adrenoceptor. Clonidine has

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been shown to improve outcome in preterm fetal sheep when given after the HI insult (started 15 min after a 25 min umbilical cord occlusion and continued for 4 h) [93] — low dose clonidine (10 μg/kg/h) and not the high dose (100 μg/kg/h) was protective. This may relate to the poor α2: α2 α selectivity ratio of clonidine resulting in loss of effect of the drug at the higher dose. Dexmedetomidine is a highly selective agonist of the α 2 adrenoceptor and decreases ischemia induced epinephrine oversecretion [95], increases the expression of adhesion kinase, modulates glutamate release and decreases calcium entry and apoptosis [96]. Dexmedetomidine provides sedation without respiratory depression in the clinic; it has organ protective effects against ischemic and hypoxic injury, including cardioprotection, neuroprotection, and renoprotection. Dexmedetomidine was approved as a sedative agent in intensive care in 1999 in the US. Over the last 15–20 years neuroprotection by dexmedetomidine has been demonstrated in rodent [94,97,98] and rabbit models [99]. Dexmedetomidine, dose-dependently reduces neuronal injury in vitro and in vivo in a neonatal asphyxia rat model [94]. Dexmedetomidine, administered during asphyxia, improves neuromotor function when assessed 30 days later and improved histological markers of brain injury by more than 50% [94]; dexmedetomidine also synergises with xenon adding to its potential to form part of a multimodal neuroprotective strategy [96]. Dexmedetomidine reduces excitotoxin-induced cortical and white-matter lesions [97]. Dexmedetomidine is chemically related to clonidine but has eight times higher affinity for α2 receptors. In preterm fetal sheep low dose clonidine given after the HI insult also improved outcome [93]. Dexmedetomidine reduces pro and increases anti-apoptotic proteins after cerebral ischaemia and reperfusion in rats [100] and reduces isofluraneinduced neuroapoptosis in rats [101]. Furthermore unlike other sedatives, dexmedetomidine itself does not induce apoptosis even at 75 times the sedative dose in neonatal rats [101]. Dexmedetomidine is the most selective α2 adrenoceptor agonist available clinically and has a strong safety profile making it the ideal agent to pursue in clinical trials. Indeed dexmedetomidine has been used safely in both neonatal and paediatric intensive care and pharmacokinetic data is available for dexmedetomidine use from neonatal ages upwards [102]. 3.8. Anticonvulsants Data from both animal and human studies suggest that seizures amplify neonatal hypoxic–ischemic brain damage [103,104]. In a recent study of newborns with HIE with MRS used to assess tissue metabolic integrity, the severity of seizures was independently associated with brain injury [105]. These results provide some support for the hypothesis that effective prevention of neonatal seizures could attenuate brain injury. Anticonvulsant drugs (AEDs) are being studied as possible neuroprotective agents on the basis of the similar cascade of cellular events triggered by seizures and acute hypoxia–ischaemia [106]. Two drugs of particular interest in perinatal asphyxia are topiramate and levetiracetam. Topiramate is an AED drug used safely in children [107]. Topiramate has actions on many cell pathways: it acts through modulation of alphaamino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainite and gamma-aminobutyric acid (GABA) A-activated ion channels and voltage-activated Na+ and Ca++ channels, which may reduce excessive glutamate release after hypoxia-ischaemia [108–110]. Topiramate suppresses the pre-synaptic voltage-sensitive sodium channel of excitatory synapses and enhances GABA-mediated chloride flux and GABA-evoked chloride currents in murine brain neurons and increases seizure threshold [111]. In a neonatal rat stroke model, neither topiramate nor hypothermia alone conferred protection, whereas combined treatment with topiramate and hypothermia improved functional performance and severity of brain injury compared with delayed hypothermia alone [11]. Other studies have shown that topiramate on its own is

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neuroprotective in neonatal rat pups although the post-treatment window appears to be relatively narrow (b2 h) [112]. In another p7 rodent model post-insult topiramate was protective against hypoxic– ischemic white matter injury and decreased subsequent neuromotor deficits [113]. In this study topiramate attenuated AMPA-kainate receptor-mediated cell death and calcium influx, as well as kainateevoked currents in developing oligodendrocytes, similar to the AMPA– kainate receptor antagonist 6-nitro-7-sulfamoylbenzo-(f)quinoxaline2,3-dione (NBQX). Notably, protective doses of NBQX and topiramate did not affect normal maturation and proliferation of oligodendrocytes [113]. Topiramate was shown to protect against white matter injury in a newborn piglet model when given as infusion 1 h after hypoxia– ischaemia followed by maintenance doses for 3 days [114]. Interestingly, topiramate administration did not affect seizure frequency. Two dose regimens were used: a low dose (loading dose of 20 mg/kg and a maintenance dose of 10 mg/kg/day) or a higher dose (loading dose of 50 mg/kg followed by 20 mg/kg/day). Topiramate reduced cell loss in a dose dependent way, however there was concern that increased apoptosis was seen in the white matter of high dose treated animals. Topiramate is an attractive agent to consider for clinical trials, however pre-clinical studies are needed to assess the effect of topiramate on neurodegeneration. One study has suggested that although Topiramte and Levetiracepam alone did not induce cell death in 8 day old rat pups, when both drugs were given in combination with phenytoin, then cell death was increased [115]. In addition further study on the effect of hypothermia on the metabolism of Topiramte is needed as some studies have shown a reduction in both absorption and elimination of topiramate with therapeutic hypothermia [116]. Levetiracetam was approved by the US FDA in 1999; in the last 10 years it has become one of the first-line AEDs and has been evaluated in small uncontrolled studies and case series as an off-label treatment for status epilepticus, for nonepileptic neurological conditions and for some psychiatric conditions. Levetiracetam has not been approved for any indication other than chronic epilepsy. In a recent small pilot study of the treatment of neonatal seizures with levetiracetam, there were no adverse effects and all six patients treated with oral levetiracetam became seizure free within 6 days [117]. In addition to its efficacy at seizure treatment, some studies suggest that levetiracetam is neuroprotective [118]. Levetiracetam was administered by intraperitoneal bolus injections of 5.5, 11, 22 and 44 mg/kg 30 min before occlusion followed by a continuous 24 h infusion of 1.25, 2.6, 5.1 and 10.2 mg/kg per h, respectively. Levetiracetam administration reduced the infarct volume by 33% at the highest dose tested [119]. Although there is a lack of clinical experience with levetiracetam, this agent is worth investigating further as a neuroprotective agent as in contrast to other conventional AEDs such as phenobarbitone and phenytoin, it does not induce cell death in the developing brain even at high dose [120]. 4. Postconditioning The “ischaemic conditioning” phenomenon was first described in 1986 in the heart whereby brief non-lethal episodes of ischaemia and reperfusion in advance of prolonged lethal ischaemia boosted the intrinsic resistance of the myocardium to injury [121]. This was termed ischaemic preconditioning (IPC); such resistance has been reproduced in all species tested including humans and in a variety of organs including the heart, kidney and brain [122]. As perinatal hypoxia–ischaemia is unpredictable, IPC has no credible translational potential in this clinical scenario. It was later found that inducing brief episodes of non-lethal ischaemia and reperfusion to the heart and brain after an episode of sustained lethal ischaemia (termed ischaemic postconditioning (IPostC)) has a protective effect comparable to IPC [123]. IPostC was initially described in the myocardium and consisted of interrupted

reperfusion early in the reperfusion phase following an ischaemic insult [124]. Because it can be implemented after an ischaemic event, it has lent itself to clinical trials in myocardial ischaemia [125] where there is mechanical control of reperfusion. In animal models of stroke, IPostC (performed within 30 min of reperfusion) reduces infarct size in focal [126] and global [127] cerebral ischaemia. IPostC therefore opens up enormous translational potential for neuroprotection of the newborn with perinatal asphyxia especially in the light of 2 recent developments: (1) IPostC is effective in improving brain metabolism, normalising cerebral blood flow and providing long term protection if initiated as late as 3 and 6 h after the index ischaemia in stroke models (delayed IPostC) [128]; (2) remote IPostC (whereby a conditioning stimulus is induced in a remote non-vital organ such as a limb) reduces infarct size if performed immediately at reperfusion and at 3 h after reperfusion [129]; such an intervention constitutes a practical clinical strategy which could be readily applied at the time of resuscitation or in the first few hours after birth in a newborn infant with neonatal encephalopathy due to perinatal hypoxia–ischaemia. IPostC has been described in 2009 as “a novel avenue to protect the brain against stroke” but more studies are needed in different animal models, genders and ages [130]. 5. Regeneration and repair During HI brain injury neurons, glia and endothelial cells are damaged and lose their function or die. Endogenous regeneration mechanisms have been shown to exist in the brain as ischemic brain injury stimulates neural stem cell proliferation and differentiation in cerebral neurogenic areas [131–133]. The capacity of the neonatal brain to respond with enhanced endogenous neurogenesis following neonatal HI may, however, depend on timing and severity of insult. In addition endogenous neurogenesis may only partially restore brain damage after an HI insult. Recent advances in regenerative medicine suggest that stem cell transplantation may improve repair of the damaged brain [134]. Regenerative effects of stem cell transplantation are likely to involve both replacement of damaged cells by exogenous cells as well as improvement of endogenous repair processes by releasing trophic factors [134,135]. It is considered by some that activation of antiapoptotic mechanisms as well as improvement of the trophic milieu necessary for endogenous repair processes may be the most important mechanism underlying the improved functional outcome after stem cell treatment [134]. Several types of stem cells have been used to treat ischemic brain injury in rodent models including neuronal stem cells, mesenchymal stem cells and hematopoietic stem cells. Genetically modified stem cells may be more effective than native unmodified stem cells [134]. The use of engineered stem cells is thought to have an advantage over administration of growth factors due to the fact that stem cells have a long-term effect with only a single intervention. Stem cells keep on secreting the desired growth factor as long as the cell is present, and since stem cells tend to migrate predominantly to the site of injury, production of the desired growth factor occurs in the appropriate location. The therapeutic potential of systemic administration of soluble growth factors is limited by the fact that the plasma half-life of most growth factors is very short and these macromolecules cannot easily pass the blood brain barrier. Stem cell transplantation has the potential to become a future neuroprotective and regenerative therapy for ischemic brain damage, however there are still hurdles to overcome before clinical application of stem cell transplantation can safely be considered. 6. Conclusion Carefully conducted pre-clinical studies are needed to define the best combination of drugs and interventions that will optimize

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