Zdrav Vestn 2008; 77: II-51–8
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HYPOXIC-ISCHEMIC BRAIN INJURY IN THE NEONATAL PERIOD – CURRENT CONCEPTS, NOVEL DIAGNOSTIC APPROACHES AND NEUROPROTECTIVE STRATEGIES*
HIPOKSIČNO-ISHEMIČNA OKVARA MOŽGANOV V NEONATALNEM OBDOBJU – SODOBNI POGLEDI, NOVE DIAGNOSTIČNE METODE IN NEVROPROTEKTIVNI
UKREPI
Metka Derganc,1 Damjan Osredkar,2
1 Department of Pediatric Surgery and Intensive Care, University Medical Centre Ljubljana, Zaloška 7,
1525 Ljubljana, Slovenia 2 Department of Pediatric Neurology, University Children’s Hospital, University Medical Centre
Ljubljana, Vrazov trg 1, 1525 Ljubljana, Slovenia
Abstract
Key words
In the presenting paper, we describe mechanisms of brain injury following a hypoxic-ischemic event in the neonatal period. Neuronal death occurs in two major phases, the primary neuronal cell loss at the time of the insult and the delayed neuronal cell loss, occurring about 6 hours – 4 days after the injury. We describe different cellular mechanisms responsible for the neuronal death. The main patterns of brain injury that can be readily recognized with the newer neuroimaging techniques are dependent on the gestational age of the newborn. In order to apply novel neuroprotective treatments to the newborns with hypoxic-ischemic encephalopathy (HIE), the newborns at risk have to be identified as early as possible. Among the most useful diagnostic methods are amplitude-integrated EEG, new markers of brain lesions and different modalities of magnetic-resonance imaging. During resuscitation of neonates with HIE the importance of prevention of hyperoxia, and, during intensive care, of hypocapnia and hyperglycemia is stressed. In the treatment of newborns with HIE hypothermia, by means of both selective head cooling or whole body hypothermia, reduced the risk of death and disability according to three multicenter randomized controlled studies. It is therefore recommended for treatment of HIE in the newborn. One of the potentially beneficial effects of therapeutic hypothermia is also widening of the therapeutic window for intervention with other neuroprotective regimens. Among these, treatments with erythropoietin or minocycline seem to be clinically promising.
hypoxia-ischemia; newborn; neuroprotection; hypothermia; amplitude-integrated electro-encephalography
Izvleček
V pričujočem prispevku sva opisala mehanizme možganske okvare po hipoksično-ishemičnemu dogodku v neonatalnem obdobju. Odmiranje nevronov se odvija v dveh korakih, kot prvotna izguba nevronov v času hipoksično-ishemičnega dogodka in kot zakasnela izguba nevronov, ki sledi 6 ur – 4 dni po prvotnemu dogodku. Opisala sva različne celične mehanizme, ki so povezani z odmiranjem nevronov. Glavni vzorci možganske okvare, ki jih lahko spremljamo s sodobnimi slikovnimi preiskavami, so odvisni od gesta-cijske starosti novorojenčkov. Da bi lahko novorojenčkom s hipoksično-ishemično encefa-lopatijo (HIE) pomagali z nevroprotektivnimi zdravljenji, je potrebno ogrožene novorojenčke odkrivati čim bolj zgodaj. Med najuporabnejšimi diagnostičnimi metodami, ki jih danes uporabljamo v ta namen, sodijo amplitudno-povprečena elektroencefalografija,
Corresponence / Dopisovanje:
Metka Derganc, Department of Pediatric Surgery and Intensive Care, University Medical Centre Ljubljana, Zaloška cesta 7, 1525 Ljubljana, Slovenia
* The authors acknowledge the financial support from the state budget by the Slovenian Research Agency (project No. Z3-9060-0312-06).
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novi biokemični kazalci možganske okvare in različne tehnike magnetno-resonančnega slikanje glave. Opisala sva pomen preprečevanja hiperoksije med oživljanjem novorojenčkov s HIE in preprečevanja hipokapnije ter hiperglikemije v obdobju intenzivnega zdravljenja le-teh. Tri multicentrične raziskave kažejo, da je zdravljenje novrojenčkov s HIE s hipotermijo, tako s selektivnim ohlajanjem glave, kot z ohlajanjem celega telesa, povezano z manjšim tveganjem za smrt in možgansko okvaro, zato je takšno zdravljenje priporočljivo. Eden od možnih koristnih učinkov terapevtske hipotermije je tudi podaljšanje časovnega okna, v katerem je mogoče novorjenčke zdraviti še z drugimi nevro-protektivnimi ukrepi. Med temi klinično največ obetata zdravljenji z eritropoetinom in minociklinom.
Ključne besede    hipoksija-ishemija; novorojenec; nevroprotekcija; hipotermija; amplitudno-povprečena elektroencefalografija
Introduction
Hypoxic-ischemic cerebral injury that occurs during the perinatal period is one of the most commonly recognized causes of severe, long-term neurological deficits in children.1 Hypoxic-ischemic encephalo-pathy (HIE) of the newborn occurs with the incidence of 1–4/1000.2 Between 20 % and 50 % of newborn infants affected by perinatal brain injury die during the newborn period, and 25–60 % of the survivors suffer from permanent neurodevelopmental handicaps, including cerebral palsy, seizures, mental retardation, and learning disabilities.2–4 Hypoxic-ischemic brain injury occurs at or near the time of birth and may be amenable to post-natal neuroprotective inter-ventions.5 The aim of this article is to review current concepts of perinatal hypoxic-ischemic brain injury, novel diagnostic methods and neuroprotective strategies.
Mechanisms of brain injury following hypoxic-ischemic event
Following a hypoxic-ischemic insult, it appears that neuronal death occurs in two major phases, the primary neuronal cell loss at the time of the insult and the delayed neuronal cell loss.6 Neuroimaging studies have shown, that brain injury following a hypoxic-ischemic event evolves over days, if not
weeks.7
Primary cell loss is related to cellular hypoxia, which leads to exhaustion of high-energy metabolism (primary energy failure) and cellular depolarization. During primary energy failure, studies suggest that there are three closely interrelated mechanisms involved in the death of neurons. Firstly, depolarization due to hypoxia causes an influx of sodium and a lesser efflux of potassium with passive chloride entry along the electrochemical gradient. This leads to cell swelling and, if sufficiently severe, cell lysis.8 Secondly, intracellular calcium accumulation occurs due to both excessive entry of calcium due to failure of ion channels and of calcium removal by the sodium-calcium pump.9 Thirdly, extracellular glutamate accumulation (excitotoxicity) due to failure of energy-dependent re-uptake and excessive release is also a key mechanism stimulating intracellular calcium
accumulation through the N-methyl-D-aspartate (NMDA)-receptor-channel complex.10 Further cell membrane damage may occur due to the action of free radicals in the immediate reperfusion phase.11 However, many neurons do not die during the primary phase of neuronal death. Rather, a cascade of pathologic processes is triggered and leads to further loss of neurons, starting some hours later and extending over several days.
This secondary loss of neurons is termed secondary or delayed neuronal death. This phase may be associated with hyperexcitability and cytotoxic edema from about 6 hours – 4 days after the injury, as found in a study on fetal sheep.12 In this study, 15 chronically instrumented fetal sheep following transient cerebral ischemia were studied to estimate changes in extracellular space. The peak of the secondary edema was found at 28 ± 6 hours after the insult. In the human infant, the severity of the secondary energy failure is correlated with adverse neurodevelopmental outcome at 1 and 4 years.13 The mechanisms involved in delayed neuronal death include excitotoxicity,14 cytotoxic actions of activated microglia, mitochondrial
failure,15 NO synthesis,16 exposure to free radicals,17 inflammation,18 and apoptosis.19 Recent data suggest
that apoptosis plays a prominent role in the evolution of hypoxic-ischemic injury in the neonatal brain and may be more important than necrosis after injury.20 A prominent degree of neuronal injury has been also associated with the development of seizures and changes in cerebral blood volume and flow in the near-term fetal sheep model of asphyctic brain injury.15, 16 Clinically, it is this delayed phase of neuronal injury that is amenable to potential intervention(s). Factors such as the severity, pattern and type of insult, as well as the gestational age and metabolic status, including temperature, of the infant are crucial determinants of the neuropathology of hypoxic-ischemic brain injury in the newborn.4 Advanced methods of neuroimaging, such as magnetic resonance imaging (MRI), magnetic resonance spectroscopy, and diffusion-weighted MRI, have identified patterns of damage after ischemic insult to the newborn brain. In a study of 104 children with evidence of bilateral hypoxic-ischemic brain damage, at least three different patterns were observed with the use of MRI.21 These patterns are dependent on the gestational age
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of the infant, because certain neuron groups exhibit age-specific vulnerability. Periventricular leukomala-cia was observed in premature infants with a history of subacute or chronic hypoxia and ischemia. Lesions in the basal ganglia and thalamus occurred in full-term babies who had profound asphyxia. Multicystic changes were seen in a minority of infants who had severe encephalopathy but only a mild hypoxic-ischemic event; this group may include babies who had underlying fetal infections or metabolic disorders that had eluded diagnosis. These data suggest that injury is related to the gestational age at the time of the insult, although the severity or chronicity of the insult may be a better indicator of eventual outcome.
Similar insults to the neonatal brain will manifest themselves differently in different babies in terms of the injury, as observed on imaging studies such as MRI, and in terms of neurodevelopmental outcome. Such variability has also been observed in animal models and appears to be genetically based.22 Certain polymorphisms may increase the risk for many complex diseases.23, 24 However, susceptibility factors for neonatal brain injury have yet to be identified clearly.
Until the last decade, management strategies have largely been supportive and not targeted toward the processes of ongoing injury.25 However, novel exciting strategies aimed at preventing ongoing injury are being clinically evaluated and offer an opportunity for neuroprotection, if brain injury is diagnosed and treated early enough.
Novel diagnostic approaches to newborns with hypoxic-ischemic encephalopathy
Amplitude-integrated electroencephalography
Among newer diagnostic methods used in newborns with HIE is amplitude-integrated electroencephalo-graphy (aEEG). It was designed in the late 1960s by Maynard, but not until it was used to study newborns with HIE in 1980s did it gain widespread clinical use.26, 27 The aEEG has been also shown to be of use for selection of newborns for neuroprotective therapies, such as hypothermia, within the first hours after birth.28 Depending on the severity of the HIE, different background patterns can be observed on aEEG tracings: continuous normal voltage (CNV), discontinuous normal voltage (DNV), burst suppression (BS), continuous low voltage (CLV), and isoelectric or flat trace (FT). Epileptiform activity can also be detected with aEEG as a single seizure (SS), repetitive seizures (RS), or status epilepticus (SE). The presence or absence of sleep-wake cycles can also be determined with the use of aEEG.29, 30 The correlation between the background pattern on aEEG and the grade of HIE was found to be consistent.31, 32 The trends of background patterns on aEEG tracings and the time interval between birth and appearance of sleep-wake cycles have been found to be of predictive value.33, 34 With the use of aEEG it is possible to detect and treat
clinically silent epileptiform discharges, which significantly improves the outcome of these newborns.35 Monitoring of newborns with aEEG has been found to be of clinical value also in newborns with non-neurological life-threatening conditions demanding intensive treatment with extracorporeal membrane oxygenation (ECMO).36 In the study of Pappas et al., all of the newborns who showed continuous normal voltage on aEEG tracings throughout the treatment had a normal neurological outcome. In the Department of Pediatric Surgery and Intensive Care, University Medical Centre Ljubljana, we have been using aEEG for monitoring of newborns with HIE since 2001.30 Our findings are in accordance with the findings of other authors. We have seen good correlation between aEEG and standard EEG recordings when comparing background activity, while the correlation in assessing epileptiform activity was less obvious. In full-term newborns without severe hypoxic-ischemic encephalopathy who had other neurological conditions we have found aEEG to be of value for detection of epileptiform activity. Newer digital aEEG monitors, as opposed to the single channel analog aEEG devices (such as Lec-tromed cerebral function monitor), offer 2-channel aEEG tracings (one aEEG channel for each hemisphere) with simultaneous standard EEG tracings (in our department we use the BrainZ monitor). In a recent study using such devices, good predictive value has been found for aEEG findings combined with clinical examination and magnetic resonance investigation of the head.37
Biochemical markers of hypoxic-ischemic brain injury of the newborn
Among the earliest biochemical markers of brain hypoxia in the CSF and/or plasma studied were lactate which accumulates in hypoxic cells due to glycolysis and hypoxanthine which is formed by ATP breakdown.38, 39 Although several studies found a correlation between the degree of HIE and concentrations of both markers, their long term prognostic value was poor.40 Among enzymes released from the cells during brain hypoxia, the first studied were LDH with isoenzymes, creatine-kinase (particularly its brain-specific isoenzyme CK-BB) and adenylate kinase.41 Again, their concentrations were correlated with early clinical signs of HIE, but only CK-BB in the CSF had a prognostic value for further development in some studies while others could not confirm it.42 Other markers of brain hypoxia in CSF and/or plasma were as follows: monoamine neurotransmit-ters norepinephrine and dopamine;43 neuron-specific enolase;44 components of damaged glial cells, such as acidic glial protein45 and protein S-100;46 hydroperoxide and advanced oxidation protein products;47 nucleated red blood cells and non-protein bound iron,48 and, most recently, activin, a glycoprotein expressed in the central nervous system.49 Again, most of these markers correlated with early clinical signs of HIE whereas they did not have long-term prognostic value.50
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Newer imaging techniques
Bedside ultrasound (US) serial imaging performed with the newer advanced US machines remains an important imaging technique in newborns with HIE.51 Although no study has been performed in which US imaging would be compared with MRI, it is clear from other studies that MRI is invaluable for precise diagnostic evaluation of brain injury. Several studies performed in the latter years have shown importance of MRI in early diagnosis as well as in follow-up studies of newborns with HIE.21, 52, 53 Perinatal lesions are best detected between the first and second week of life. Very early MRI, performed within the first three days of life is of clinical importance when deciding whether to continue with treatment or not in the artificially ventilated newborns, but the MRI changes are subtle.54 Standard T1 weighted MRI performed with a 1–1.5 T or stronger machine is best for evaluation of basal ganglia and posterior end of the capsula interna, while T2 weighted MRI imaging is better for early detection of ischemic lesions and imaging differentiating between white and grey matter.55 Diffusion weighted imaging is best for detection of ischemic lesions within the white matter.54 With the use of MRI in newborns with HIE, it is possible to predict the pattern of the later neurodevelopmental deficit. Venous contrast imaging and arterial angiography can contribute additional diagnostic and prognostic data. Diffusion tensor imaging has an even greater sensitivity for detection of brain lesions. Fractional anisotropy is considerably lowered in white matter after moderate to severe hypoxic-ischemic brain injury. Although neonatal MRI is a field experiencing rapid development in some centers, for many neonatologists this imaging technique is still difficult to perform due to lack of MR machine availability and a relatively complicated procedure for preparation of a newborn undergoing MRI (absence of ferromagnetic material in incubators, respirators, monitors, etc.).
Positron emission tomography (PET) has been used for imaging of brain metabolism in animals and humans with HIE. In a study performed on fetal lambs, global cerebral metabolic rate was significantly lower in lambs subjected to cord occlusion than in controls.56 Although PET has been used in evaluation of adult patients with stroke,57 it’s clinical value in the neonatal period has not been evaluated.
Treatment
Resuscitation of newborns with room air instead of with 100 % oxygen
In the last fifteen years an increasing number of studies have shown that resuscitation with room air instead of with 100 % oxygen poses a considerably smaller oxidative stress on newborns.58 Newborns resuscitated with room air presented spontaneous respirations sooner,59 and those who did not experience hyperoxemia and hypocapnia had a better psychophysical development afterwards.60 Two meta-analysis have been published confirming a smaller
mortality of newborns who were not resuscitated with 100 % oxygen.61 Minimizing oxidative stress is not only expected to protect newborn brain, but also other vital organs. On the basis of the findings from animal and human studies, some authors recommend routine use of resuscitation with room air or at least supplementation of oxygen in lower concentrations.62
The importance of normocapnia and normoglycemia
In 2006 Perlman wrote an extensive overview of different procedures and drugs which can be potentially used in treating newborns with HIE.63 Most new-borns with moderate to severe brain injury have to be artificially ventilated. Normocapnia or slight hypercapnia have to be maintained, while hypocapnia has to be avoided because additional ischemic damage due to vasoconstriction can be expected. The results of animal experiments have shown major morphological changes following hypocapnia during resuscitation, mild hypercapnia has been found to be protective, whereas severe hypercapnia was also found to be related to a poor outcome, possibly because of detrimental effects on circulation.64 Arterial blood pressure should be maintained within normal limits, while hydration should be on the lower limit. However, in a recent meta-analysis, the authors could not find any randomized study to support fluid restriction in HIE of the newborn.65 In newborns with HIE normoglycemia has to be maintained at all times, because hypoglycemia and hyperglycemia both aggravate the brain injury.66, 67 One of the possible mechanisms of cell damage of hyperglycemia is the stimulation of inducible nitric oxide synthase (iNOS) leading to higher concentrations of iNOS, mitochon-drial damage, and intracellular edema.
Drugs with potential neuroprotective effects
Among the first potential drug candidates in the treatment of newborns with HIE was a calcium blocker, nicardipine. Because this drug was related to severe drop in blood pressure, its use was discontinued in
clinical studies.68
Studies on barbiturates were giving contradictory results: while thiopental was not shown to have any effect on the later neurological disability, high doses of phenobarbital (40 mg/kg) immediately after birth were related to a better neurodevelopmental outcome of newborns with HIE.69 However, in another study early administration of phenobarbital immediately after hypoxic-ischemic injury resulted in higher morbidity and mortality of asphyxiated newborns.70 In spite of promising first results with the use of magnesium sulphate (antagonist of glutamate receptors) in treatment of newborns with HIE,71 larger studies did not confirm the clinical benefits of its use.72 Magnesium sulphate treatment did also not show any improvement on aEEG background pattern.73 Animal studies using allopurinol, a xanthine oxidase inhibitor, showed that allopurinol reduces free radical production, preserves the cerebral energy state, reduces brain edema, and improves the brain electri-
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cal activity when given before reperfusion. However, a recent clinical study on newborns with HIE did not show any benefit of allopurinol administration, because treatment started postnatally was too late to reduce the early reperfusion induced free radical surge.74 The authors speculated that allopurinol administration to the fetus with (imminent) hypoxia via the mother during labor might be more effective in reducing free radical induced post-asphyxial brain damage.
Erythropoietin (EPO) is another potential candidate for neuroprotective treatment of newborns with HIE. Its neuroprotective effects are attributed to the fact that EPO lowers intracellular levels of calcium and diminishes glutamate toxicity, apoptosis, inflammation, and has an antioxidative effect.75 Although many animal studies have proven the beneficial effect of EPO administration, clinical data on newborns are still scarce. Besides neuroprotective role, EPO shows also neurogenetic properties, similarly to vascular endo-thelial growth factor (VEGF): they both promote regeneration after hypoxic-ischemic brain injury.76 Animal studies have shown that melatonin is a potent free oxygen radical scavenger.77 Among other potential neuroprotective substances are 2-iminobiotin, es-tradiol, minocycline, and xenon. In a recent randomized clinical study in adults with ischemic stroke minocycline significantly improved outcome compared to placebo.78
Therapeutic hypothermia
The most acclaimed neuroprotective treatment of newborns with HIE to date is early therapeutic hypothermia, starting within 6 hours after hypoxic-ischemic event. Several animal studies have shown beneficial effects of therapeutic hypothermia: lowering of metabolic rate, minimizing the extent of apoptosis, formation of NO and free oxygen radicals, epileptiform activity, cerebral edema, and maintenance of the hematoencephalic barrier.79
Currently there are two approaches to therapeutic hypothermia: head cooling with a specially designed cap (Cool Cap), accompanied with mild hypothermia of the whole body (rectal temperature 34–35 °C), and whole body cooling with a cooling blanket, accompanied by moderate hypothermia of the whole body (rectal temperature 33–34 °C). After early diagnosis of HIE, usually by means of aEEG and clinical signs, hypothermia is initiated within 6 hours after hypoxic-ischemic event and is maintained for the next 48–72 hours. During hypothermia, several parameters have to be strictly monitored: heart rate and function, blood pressure, electrolytes, blood gases, blood glucose, and coagulation factors. Rewarming of the patient has to be graduate and slow, using a warming blanket or warm air.
Several studies have investigated the possible side effects of hypothermia. The results of these studies have shown no major side effects if rectal temperature was maintained between 33–35 °C.80–83 In the year 2005 the results of a smaller multicenter hypothermia study on newborns with severe HIE and of gestatio-
nal age > 35 weeks were published.84 Sixty-five new-borns were randomized within 6 hours after birth and their rectal temperature was maintained at 33.5 °C with a cooling blanket for 48 hours. At one year follow up, 52 % of newborns treated with hypothermia had severe motor disability as opposed to 84 % of newborns that were not cooled, a difference found to be significant. The first large multicentre study published was the »Cool Cap« study,85 which included 234 newborns of gestational age > 36 weeks with acidosis, Apgar scores < 5 at 10 minutes, and clinical and pathological changes on aEEG tracings. Hypothermia was initiated within first 6 hours after birth using a cooling cap, reaching rectal temperatures of 34.5 °C, and was continued for the next 72 hours. At 18 months the treated and non treated groups were compared, but no significant difference in outcome was found. If the results were stratified, a significant difference was found for the less severely asphyxiated newborns in favor of treatment with hypothermia. Severely abnormal motor scores were recorded in 24 % of hypothermia and 64 % of normothermia patients. In the third multicentre study of Shankaran and coworkers,86 208 newborns with gestational age of > 36 weeks, clinical signs and history of HIE were included. Whole body hypothermia was employed for 72 hours, with the rectal temperature being set to 33.5 °C. At 18 months follow-up, their findings demonstrated the safety and effectiveness of whole-body cooling in reducing the risk of death or disability among infants with moderate or severe encephalopathy. Expert groups for neonatal hypothermia support the use of therapeutic hypothermia, but under strict conditions of protocols, that were used in larger studies, extensive follow-up of patients, and contributing data to hypothermia registries.87, 88 In a recent study, not more than 6.4 % of the neonatal units in the United States were found to use therapeutic hypothermia.89 In Slovenia we take part of the nEuro Network Neonatal Hypothermia protocol and registry, founded by Prof. Simbruner in 2001. This protocol was approved by Slovenian Ethical Committee of the Ministry of Health of Republic of Slovenia. The protocol suggests the parents of the patients to be informed, but their informed consent is not mandatory for the initiation of treatment.
Marianne Thoresen, one of the pioneers of therapeutic hypothermia in newborns, suggests that hypothermia should be started immediately after resuscitation (without intermittent warming of the patient) and combining hypothermia with other treatment modalities might prove to be of even greater value. To date, it is also not clear which cooling method, whole body or head cooling is more efficient. In a recent MRI study of newborns with HIE, the methods did not differ in basal ganglia lesions, but there were significantly less cortical lesions in newborns cooled with the cooling cap.90
Conclusions
Until last decade, management strategies of newborns with HIE have been mainly supportive. Three multi-
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centre studies have approved routine clinical use of therapeutic hypothermia, giving the possibility to actively modify the natural history of perinatal brain injury in a favorable way. However, the study protocols have to be strictly followed, the findings should be monitored with registers, and the children should have rigorous follow-up in order to provide best possible care. Other neuroprotective strategies are being investigated, but their clinical value remains to be proven by the future studies.
References
1.  Volpe JJ. Neurology of the newborn. 3rd ed. Philadelphia: WB Saunders; 1995.
2.  Vannucci RC. Hypoxic-ischemic encephalopathy. Am J Perinatol 2000; 17: 113–20.
3.  Berger R, Bender S, Sefkow S, Klingmuller V, Kunzel W, Jensen A. Peri/intraventricular haemorrhage: a cranial ultrasound study on 5286 neonates. Eur J Obstet Gynecol Reprod Biol 1997; 75: 191–203.
4.  Inder TE, Volpe JJ. Mechanisms of perinatal brain injury. Semin Neonatol 2000; 5: 3–16.
5.  Miller SP, Ramaswamy V, Michelson D, et al. Patterns of brain injury in term neonatal encephalopathy. J Pediatr 2005; 146: 453–60.
6.  Gluckman PD, Williams CE. When and why do brain cells die? Dev Med Child Neurol 1992; 34: 1010–4.
7.  McKinstry RC, Miller JH, Snyder AZ, et al. A prospective, longitudinal diffusion tensor imaging study of brain injury in new-borns. Neurology 2002; 59: 824–33.
8.  Williams CE, Mallard C, Tan W, Gluckman PD. Pathophysiology of perinatal asphyxia. Clin Perinatol 1993; 20: 305–25.
9.  du Plessis AJ, Johnston MV. Hypoxic-ischemic brain injury in the newborn. Cellular mechanisms and potential strategies for neuroprotection. Clin Perinatol 1997; 24: 627–54.
10.  Choi DW. Calcium: still center-stage in hypoxic-ischemic neuronal death. Trends Neurosci 1995; 18: 58–60.
11.  Goplerud JM, Mishra OP, Delivoria-Papadopoulos M. Brain cell membrane dysfunction following acute asphyxia in newborn piglets. Biol Neonate 1992; 61: 33–41.
12.  Williams CE, Gunn A, Gluckman PD. Time course of intracellu-lar edema and epileptiform activity following prenatal cerebral ischemia in sheep. Stroke 1991; 22: 516–21.
13.  Roth SC, Baudin J, Cady E, et al. Relation of deranged neonatal cerebral oxidative metabolism with neurodevelopmental outcome and head circumference at 4 years. Dev Med Child Neurol 1997; 39: 718–25.
14.  Tan WK, Williams CE, During MJ, et al. Accumulation of cyto-toxins during the development of seizures and edema after hypoxic-ischemic injury in late gestation fetal sheep. Pediatr Res 1996; 39: 791–7.
15.  Marks KA, Mallard EC, Roberts I, et al. Delayed vasodilation and altered oxygenation after cerebral ischemia in fetal sheep. Pediatr Res 1996; 39: 48–54.
16.  Marks KA, Mallard CE, Roberts I, Williams CE, Gluckman PD, Edwards AD. Nitric oxide synthase inhibition attenuates delayed vasodilation and increases injury after cerebral ischemia in fetal sheep. Pediatr Res 1996; 40: 185–91.
17.  Baud O, Greene AE, Li J, Wang H, Volpe JJ, Rosenberg PA. Glutathione peroxidase-catalase cooperativity is required for resistance to hydrogen peroxide by mature rat oligodendro-cytes. J Neurosci 2004; 24: 1531–40.
18.  Dammann O, Kuban KC, Leviton A. Perinatal infection, fetal inflammatory response, white matter damage, and cognitive limitations in children born preterm. Ment Retard Dev Disabil Res Rev 2002; 8: 46–50.
19.  Beilharz EJ, Williams CE, Dragunow M, Sirimanne ES, Gluckman PD. Mechanisms of delayed cell death following hypoxic-ische-mic injury in the immature rat: evidence for apoptosis during selective neuronal loss. Brain Res Mol Brain Res 1995; 29: 1–14.
Zdrav Vestn 2008; 77: SUPPL II
20.  Hu BR, Liu CL, Ouyang Y, Blomgren K, Siesjo BK. Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation. J Cereb Blood Flow Metab 2000; 20: 1294–300.
21.  Sie LT, van der Knaap MS, Oosting J, de Vries LS, Lafeber HN, Valk J. MR patterns of hypoxic-ischemic brain damage after prenatal, perinatal or postnatal asphyxia. Neuropediatrics 2000; 31: 128–36.
22.  Sheldon RA, Sedik C, Ferriero DM. Strain-related brain injury in neonatal mice subjected to hypoxia-ischemia. Brain Res 1998; 810: 114–22.
23.  Davey Smith G, Ebrahim S. ‘Mendelian randomization’: can genetic epidemiology contribute to understanding environmental determinants of disease? Int J Epidemiol 2003; 32: 1–22.
24.  Hoppe C, Klitz W, Cheng S, et al. Gene interactions and stroke risk in children with sickle cell anemia. Blood 2004; 103: 2391–6.
25.  Vannucci RC, Perlman JM. Interventions for perinatal hypoxic-ischemic encephalopathy. Pediatrics 1997; 100: 1004–14.
26.  Maynard D, Prior PF, Scott DF. Device for continuous monitoring of cerebral activity in resuscitated patients. Br Med J 1969; 4: 545–6.
27.  Viniker DA, Maynard DE, Scott DF. Cerebral function monitor studies in neonates. Clin Electroencephalogr 1984; 15: 185–92.
28.  Groenendaal F, de Vries LS. Selection of babies for intervention after birth asphyxia. Semin Neonatol 2000; 5: 17–32.
29.  de Vries LS, Hellstrom-Westas L. Role of cerebral function monitoring in the newborn. Arch Dis Child Fetal Neonatal Ed 2005; 90: F201–7.
30.  Osredkar D, Paro-Panjan D, Derganc M, Neubauer D. Primerjava med amplitudno povprečenim elektroencefalogramom in standardnim elektroencefalogramom pri novorojenčkih. Slov Pediatr 2002; 9: 56–8.
31.  Hellstrom-Westas L, Rosen I, Svenningsen NW. Predictive value of early continuous amplitude integrated EEG recordings on outcome after severe birth asphyxia in full term infants. Arch Dis Child Fetal Neonatal Ed 1995; 72: F34–8.
32.  al Naqeeb N, Edwards AD, Cowan FM, Azzopardi D. Assessment of neonatal encephalopathy by amplitude-integrated electro-encephalography. Pediatrics 1999; 103: 1263–71.
33.  Toet MC, Hellstrom-Westas L, Groenendaal F, Eken P, de Vries LS. Amplitude integrated EEG 3 and 6 hours after birth in full term neonates with hypoxic-ischaemic encephalopathy. Arch Dis Child Fetal Neonatal Ed 1999; 81: F19–23.
34.  Osredkar D, Toet MC, van Rooij LG, van Huffelen AC, Groenen-daal F, de Vries LS. Sleep-wake cycling on amplitude-integrated electroencephalography in term newborns with hypoxic-ischemic encephalopathy. Pediatrics 2005; 115: 327–32.
35.  Toet MC, Groenendaal F, Osredkar D, van Huffelen AC, de Vries LS. Postneonatal epilepsy following amplitude-integrated EEG-detected neonatal seizures. Pediatr Neurol 2005; 32: 241–7.
36.  Pappas A, Shankaran S, Stockmann PT, Bara R. Changes in amplitude-integrated electroencephalography in neonates treated with extracorporeal membrane oxygenation: a pilot study. J Pediatr 2006; 148: 125–7.
37.  Shah DK, Lavery S, Doyle LW, Wong C, McDougall P, Inder TE. Use of 2-channel bedside electroencephalogram monitoring in term-born encephalopathic infants related to cerebral injury defined by magnetic resonance imaging. Pediatrics 2006; 118: 47–55.
38.  Derganc M, Neubauer D, Vreča I, Zupan I, Grosek Š. Biochemical markers of neonatal brain injury. In: Gennser G, Maršal K, Svenningsen N, Lindstrom K, eds. Fetal and neonatal physiologic measurements III. Malmo: Flenhags; 1989. p. 523–6.
39.  Poulsen JP, Oyasaeter S, Sanderud J, Rognum TO, Saugstad OD. Hypoxanthine, xanthine, and uric acid concentrations in the cerebrospinal fluid, plasma, and urine of hypoxemic pigs. Pediatr Res 1990; 28: 477–81.
40.  Gosar D, Neubauer D, Tretnjak V, Bregant T, Derganc M. Neonatal hypoxia and long-term neuropsychological outcome: a follow-up study into late childhood and adolescence. In: Book of Abstracts of European Academy of Paediatrics. Barcelona: Kenes; 2006: 215.
41.  Vreca I, Derganc M, Grosek S. Adenylate kinase activity in the cerebrospinal fluid of hypoxic newborns. Clin Biochem 1989; 22: 135–9.
Derganc M, Osredkar D. Hypoxic-ischemic brain injury in the neonatal period
II-57
42.  Talvik T, Haldre S, Soot A, Hamarik M, Piirsoo A, Mikelsaar AV. Creatine kinase isoenzyme BB concentrations in cerebrospinal fluid in asphyxiated preterm neonates. Acta Paediatr 1995; 84: 1183–7.
43.  Blennow M, Zeman J, Dahlin I, Lagercrantz H. Monoamine neuro-transmitters and metabolites in the cerebrospinal fluid following perinatal asphyxia. Biol Neonate 1995; 67: 407–13.
44.  Thornberg E, Thiringer K, Hagberg H, Kjellmer I. Neuron specific enolase in asphyxiated newborns: association with encephalopathy and cerebral function monitor trace. Arch Dis Child Fetal Neonatal Ed 1995; 72: F39–42.
45.  Blennow M, Hagberg H, Rosengren L. Glial fibrillary acidic protein in the cerebrospinal fluid: a possible indicator of prognosis in full-term asphyxiated newborn infants? Pediatr Res 1995; 37: 260–4.
46.  Thorngren-Jerneck K, Alling C, Herbst A, Amer-Wahlin I, Marsal K. S100 protein in serum as a prognostic marker for cerebral injury in term newborn infants with hypoxic ischemic ence-phalopathy. Pediatr Res 2004; 55: 406–12.
47.  Buonocore G, Perrone S, Longini M, et al. Oxidative stress in preterm neonates at birth and on the seventh day of life. Pediatr Res 2002; 52: 46–9.
48.  Perrone S, Bracci R, Buonocore G. New biomarkers of fetal-neonatal hypoxic stress. Acta Paediatr Suppl 2002; 91: 135–8.
49.  Florio P, Perrone S, Luisi S, et al. Increased plasma concentrations of activin a predict intraventricular hemorrhage in pre-term newborns. Clin Chem 2006; 52: 1516–21.
50.  Nagdyman N, Grimmer I, Scholz T, Muller C, Obladen M. Predictive value of brain-specific proteins in serum for neuro-developmental outcome after birth asphyxia. Pediatr Res 2003; 54: 270–5.
51.  Daneman A, Epelman M, Blaser S, Jarrin JR. Imaging of the brain in full-term neonates: does sonography still play a role? Pediatr Radiol 2006; 36: 636–46.
52.  Rutherford MA, Pennock JM, Counsell SJ, et al. Abnormal magnetic resonance signal in the internal capsule predicts poor neurodevelopmental outcome in infants with hypoxic-ischemic encephalopathy. Pediatrics 1998; 102: 323–8.
53.  Barnett A, Mercuri E, Rutherford M, et al. Neurological and perceptual-motor outcome at 5–6 years of age in children with neonatal encephalopathy: relationship with neonatal brain MRI. Neuropediatrics 2002; 33: 242–8.
54.  Rutherford M, Srinivasan L, Dyet L, et al. Magnetic resonance imaging in perinatal brain injury: clinical presentation, lesions and outcome. Pediatr Radiol 2006; 36: 582–92.
55.  De Vita E, Bainbridge A, Cheong JL, et al. Magnetic resonance imaging of neonatal encephalopathy at 4.7 tesla: initial experiences. Pediatrics 2006; 118: e1812–21.
56.  Thorngren-Jerneck K, Ley D, Hellstrom-Westas L, et al. Reduced postnatal cerebral glucose metabolism measured by PET after asphyxia in near term fetal lambs. J Neurosci Res 2001; 66: 844–50.
57.  Markus R, Reutens DC, Kazui S, et al. Topography and temporal evolution of hypoxic viable tissue identified by 18F-fluoro-misonidazole positron emission tomography in humans after ischemic stroke. Stroke 2003; 34: 2646–52.
58.  Vento M, Asensi M, Sastre J, Garcia-Sala F, Pallardo FV, Vina J. Resuscitation with room air instead of 100 % oxygen prevents oxidative stress in moderately asphyxiated term neonates. Pediatrics 2001; 107: 642–7.
59.  Saugstad OD, Rootwelt T, Aalen O. Resuscitation of asphyxiated newborn infants with room air or oxygen: an international controlled trial: the Resair 2 study. Pediatrics 1998; 102: e1.
60.  Klinger G, Beyene J, Shah P, Perlman M. Do hyperoxaemia and hypocapnia add to the risk of brain injury after intrapartum asphyxia? Arch Dis Child Fetal Neonatal Ed 2005; 90: F49–52.
61.  Saugstad OD, Ramji S, Vento M. Resuscitation of depressed newborn infants with ambient air or pure oxygen: a meta-analysis. Biol Neonate 2005; 87: 27–34.
62.  The International Liaison Committee on Resuscitation (ILCOR) consensus on science with treatment recommendations for pediatric and neonatal patients: neonatal resuscitation. Pediatrics 2006; 117: e978–88.
63.  Perlman JM. Intervention strategies for neonatal hypoxic-ische-mic cerebral injury. Clin Ther 2006; 28: 1353–65.
64.  Vannucci RC, Vannucci SJ. Perinatal hypoxic-ischemic brain damage: evolution of an animal model. Dev Neurosci 2005; 27: 81–6.
65.  Kecskes Z, Healy G, Jensen A. Fluid restriction for term infants with hypoxic-ischaemic encephalopathy following perinatal asphyxia. Cochrane Database Syst Rev 2005: CD004337.
66.  Efron D, South M, Volpe JJ, Inder T. Cerebral injury in association with profound iatrogenic hyperglycemia in a neonate. Eur J Paediatr Neurol 2003; 7: 167–71.
67. Van den Berghe G, Schoonheydt K, Becx P, Bruyninckx F, Wouters PJ. Insulin therapy protects the central and peripheral nervous system of intensive care patients. Neurology 2005; 64: 1348–53.
68.  Levene MI, Gibson NA, Fenton AC, Papathoma E, Barnett D. The use of a calcium-channel blocker, nicardipine, for severely asphyxiated newborn infants. Dev Med Child Neurol 1990; 32: 567–74.
69.  Hall RT, Hall FK, Daily DK. High-dose phenobarbital therapy in term newborn infants with severe perinatal asphyxia: a randomized, prospective study with three-year follow-up. J Pediatr 1998; 132: 345–8.
70.  Ajayi OA, Oyaniyi OT, Chike-Obi UD. Adverse effects of early phenobarbital administration in term newborns with perinatal asphyxia. Trop Med Int Health 1998; 3: 592–5.
71.  Levene M, Blennow M, Whitelaw A, Hanko E, Fellman V, Hartley R. Acute effects of two different doses of magnesium sulphate in infants with birth asphyxia. Arch Dis Child Fetal Neonatal Ed 1995; 73: F174–7.
72.  Nelson KB, Grether JK. Can magnesium sulfate reduce the risk of cerebral palsy in very low birthweight infants? Pediatrics 1995; 95: 263–9.
73.  Groenendaal F, Rademaker CM, Toet MC, de Vries LS. Effects of magnesium sulphate on amplitude-integrated continuous EEG in asphyxiated term neonates. Acta Paediatr 2002; 91: 1073–7.
74.  Benders MJ, Bos AF, Rademaker CM, et al. Early postnatal allopurinol does not improve short term outcome after severe birth asphyxia. Arch Dis Child Fetal Neonatal Ed 2006; 91: F163–5.
75. Strunk T, Hartel C, Schultz C. Does erythropoietin protect the preterm brain? Arch Dis Child Fetal Neonatal Ed 2004; 89: F364–6.
76.  Ferriero DM. Protecting neurons. Epilepsia 2005; 46 Suppl 7: 45–51.
77.  Tutunculer F, Eskiocak S, Basaran UN, Ekuklu G, Ayvaz S, Va-tansever U. The protective role of melatonin in experimental hypoxic brain damage. Pediatr Int 2005; 47: 434–9.
78.  Lampl Y, Boaz M, Gilad R, et al. Minocycline treatment in acute stroke: an open-label, evaluator-blinded study. Neurology 2007; 69: 1404–10.
79.  Thoresen M, Whitelaw A. Therapeutic hypothermia for hypoxic-ischaemic encephalopathy in the newborn infant. Curr Opin Neurol 2005; 18: 111–6.
80.  Gunn AJ, Gluckman PD, Gunn TR. Selective head cooling in newborn infants after perinatal asphyxia: a safety study. Pediatrics 1998; 102: 885–92.
81.  Simbruner G, Haberl C, Harrison V, Linley L, Willeitner AE. Induced brain hypothermia in asphyxiated human newborn infants: a retrospective chart analysis of physiological and adverse effects. Intensive Care Med 1999; 25: 1111–7.
82. Azzopardi D, Robertson NJ, Cowan FM, Rutherford MA, Rampling M, Edwards AD. Pilot study of treatment with whole body hypothermia for neonatal encephalopathy. Pediatrics 2000; 106: 684–94.
83.  Battin MR, Penrice J, Gunn TR, Gunn AJ. Treatment of term infants with head cooling and mild systemic hypothermia (35.0 degrees C and 34.5 degrees C) after perinatal asphyxia. Pediatrics 2003; 111: 244–51.
84.  Eicher DJ, Wagner CL, Katikaneni LP, et al. Moderate hypothermia in neonatal encephalopathy: efficacy outcomes. Pediatr Neurol 2005; 32: 11–7.
85.  Gluckman PD, Wyatt JS, Azzopardi D, et al. Selective head cooling with mild systemic hypothermia after neonatal ence-phalopathy: multicentre randomised trial. Lancet 2005; 365: 663–70.
86.  Shankaran S, Laptook AR, Ehrenkranz RA, et al. Whole-body hypothermia for neonates with hypoxic-ischemic encephalo-pathy. N Engl J Med 2005; 353: 1574–84.
II-58
Zdrav Vestn 2008; 77: SUPPL II
87.  Higgins RD, Raju TN, Perlman J, et al. Hypothermia and perinatal asphyxia: executive summary of the National Institute of Child Health and Human Development workshop. J Pediatr 2006; 148: 170–5.
88.  Perlman JM. Summary proceedings from the neurology group on hypoxic-ischemic encephalopathy. Pediatrics 2006; 117: S28–33.
89.  Lang TR, Hartman TK, Hintz SR, Colby CE. Hypothermia for the treatment of neonatal ischemic encephalopathy: is the genie out of the bottle? Am J Perinatol 2007; 24: 27–31.
90.  Rutherford MA, Azzopardi D, Whitelaw A, et al. Mild hypothermia and the distribution of cerebral lesions in neonates with hypoxic-ischemic encephalopathy. Pediatrics 2005; 116: 1001–6.
Accepted 2008-02-16