Pediatrics International (2011) 53, 454–462
Original Article
ped_3290
doi: 10.1111/j.1442-200X.2010.03290.x
454..462
New insights into the pathogenesis of perinatal hypoxic-ischemic brain injury Brankica Vasiljevic,1 Svjetlana Maglajlic-Djukic,3 Miroslava Gojnic,2 Sanja Stankovic,4 Svetlana Ignjatovic4 and Dragana Lutovac4 Departments of 1Neonatology, and 2Perinatology, Institute of Gynecology and Obstetrics – Clinical Centre of Serbia, 3 Department of Neonatology, University Children’s Hospital, and 4Institute of Biochemistry– Clinical Centre of Serbia, Belgrade, Serbia Abstract
Background: Pathogenesis of perinatal hypoxic-ischemic brain injury (HIE) is complex. In this study, we examined the role of neuroinflammation, oxidative stress and growth factors in perinatal hypoxic-ischemic brain damage. Methods: Ninety neonates (>32 weeks’ gestation) with perinatal HIE were enrolled prospectively. Perinatal HIE was categorized into three stages according to the Sarnat and Sarnat clinical scoring system and changes seen on amplitude integrated electroencephalography. Cerebrospinal fluid (CSF) for interleukin-6 (IL-6) and glutathione peroxidase analysis was taken in the first 48 h of life and subsequent CSF for neuron-specific enolase (NSE) and vascular endothelial growth factor (VEGF) analysis 72 h after birth. Neurodevelopmental outcome was assessed at 12 months of corrected gestational age using the Denver Developmental Screening Test. Results: Concentrations of NSE in CSF correlated with severity of HIE (P < 0.0001) and corresponded well with subsequent neurodevelopmental outcome. Concentrations of IL-6 in CSF were markedly increased in neonates with severe HIE (P < 0.0001) and those with subsequent neurological sequels, but were normal in the majority of neonates with mild and moderate HIE. Glutathione peroxidase activity in CSF was significant with the stage of HIE (P < 0.0001) and gestational age (P < 0.0001) and corresponded well with subsequent neurodevelopmental outcome. Advanced stage of HIE was associated with increased concentrations of VEGF in CSF (P < 0.0001). Neurological outcomes at 12 months of age correlated best with CSF level of NSE (P < 0.001) and IL-6 (P < 0.001). Conclusion: Our results suggest that neuroinflammation plays a principal role in perinatal hypoxic-ischemic brain damage and we postulate that oxidative stress and upregulation of VEGF might be important contributing factors in the pathogenesis of hypoxic-ischemic brain injury, particularly in preterm neonates.
Key words
glutathione peroxidase, hypoxic-ischemic encephalopathy, interleukin-6, neonates, neuron-specific enolase.
Hypoxic-ischemic encephalopathy (HIE) after perinatal asphyxia is a common cause of neonatal morbidity and mortality and neurological disability among survivors.1–3 Each year 1.2 million neonates die and about 1 million infants have permanent neurological disability caused by HIE.4 The pathogenesis of perinatal hypoxic-ischemic brain damage is complex, involves impaired blood–brain barrier permeability, energy failure, loss of cell ion homeostasis, acidosis, increased intracellular calcium, excitotoxicity, free radical mediated toxicity, growth factor deficiency or upregulation and activation inflammatory cascade in the immature brain.5 Free radicals are highly reactive molecules generated predominantly during cellular respiration and normal metabolism.
Correspondence: Brankica Vasiljevic, MD, MSc, Institute of Gynecology and Obstetrics-Clinical Centre of Serbia, Department of Neonatology, 26 Višegradska Street, 11 000 Belgrade, Serbia. Email:
[email protected] Received 3 May 2010; revised 25 September 2010; accepted 27 October 2010.
© 2011 The Authors Pediatrics International © 2011 Japan Pediatric Society
Imbalance between cellular production of free radicals and the ability of cells to defend against them is referred to as oxidative stress. Glutathione peroxidase (GPX) is the principal antioxidant enzyme that protects the cells against intracellular radicals and peroxides coming from the respiratory chain or other metabolic pathways. A number of studies have documented that HIE is associated with increased production of free radicals in animal models. Direct measurement of free radicals in biological samples is difficult because they are extremely reactive and have a short half-life. Therefore, particularly in human studies, indirect approaches have been used to demonstrate free radical production during cerebral ischemia by measuring the products of free radical reaction with other molecules, such as lipids, proteins, and DNA, and the level or activity of antioxidant molecules. There is increasing evidence supporting an association between perinatal inflammatory response and the development of cerebral palsy in early childhood.6 Several experimental animal models indicate that inflammation is involved in the pathogenesis of ischemic brain injury.7 Inflammatory reaction triggered by
Perinatal hypoxic-ischemic brain injury hypoxia-ischemia in the brain consists of a large influx of leukocytes, including polymorphonuclear cells followed by monocytes, astrocytes and microglia activation that require the expression of specific adhesion molecules and chemotactic factors.8 Experimental models suggest that a cytokine network orchestrates in irreversible hypoxic-ischemic brain damage. IL-6 is a pleiotropic cytokine with both proinflammatory and anti-inflammatory effects. Increased expression of IL-6 in the ischemic brain has been found in adult animal models, and one study has shown increased intrathecal levels of IL-6 in adult patients with stroke.9 Hypoxia induces activation of hypoxia-inducible transcription factors (HIF-1, HIF-2), which in turn modulate expression of hypoxically regulated genes, such as those encoding vascular endothelial growth factor (VEGF) and erythropoietin (EPO).10 VEGF confers neuroprotection and promotes neurogenesis and cerebral angiogenesis, but the biological role of VEGF in the ischemic brain remains unknown. Studies from human and experimental strokes indicate that angiogenesis is present in the focal ischemic brain. Angiogenesis may promote ischemic brain plasticity and thereby improve functional neurological outcome. VEGF may have direct effects on neuronal plasticity by stimulating axonal outgrowth and inhibits glutamate and N-methyl-D-aspartate toxicity, promotes neurogenesis through the proliferation and differentiation of neuronal precursors, perhaps by the release of brain-derived neurotrophic factor from endothelial cells. Disruption of the blood–brain barrier (BBB) and early upregulation of VEGF increases BBB leakage edema formation and bleeding and further impairs brain perfusion and tissue oxygenation.11 Neuron-specific enolase (NSE) was originally described by Moore and McGregor in 1965. NSE is intracytoplasmic glycolytic enzyme in neurons. Enolase has five isoenzymes. Those containing the gamma subunit, predominantly found in neurons of the central and peripheral nervous system, are called NSE.12 The major distinctive feature of NSE compared with other enolases is its high degree of stability. In this study, we examined the role of neuroinflammation, oxidative stress, and deficiency or overproduction of growth factors in perinatal hypoxic-ischemic brain damage. An understanding of the mechanisms of perinatal hypoxic–ischemic brain damage is essential to the design of effective neuroprotective interventions.
Methods The our prospective study was performed from January 2007 to January 2009 and was conducted in accordance with the Declaration of Helsinki and was approved by the Ethical Committee for Medical Research of the Medical Faculty at the University of Belgrade. During the study period, 90 neonates (gestational age >32 weeks) with perinatal HIE were admitted to the neonatal intensive care unit at the Institute of Gynecology and Obstetrics– Clinical Centre of Serbia in Belgrade. Our institute serves as a referral center for high-risk pregnancies, with delivery numbers of 7000–7500 per year. Written consent was obtained from all
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parents. Perinatal HIE was diagnosed if fetal distress (meconium staining of liquor or abnormal fetal heart rate), immediate neonatal depression (Apgar score 26 at 5 min and/or necessary intubations in delivery room), metabolic acidosis (pH < 7.20, base deficit 310 mmol/l and lactate >3 mmol/l in arterial cord blood) and early neonatal encephalopathy (within first 24 h of life) were presented. All of the neonates were resuscitated as per the 2005 guidelines of the Newborn Resuscitation Program of the American Academy of Pediatrics and the American Heart Association.13 Complete obstetrical history and physical examinations were obtained on admission. Perinatal HIE was categorized into three stages according to the Sarnat and Sarnat clinical scoring system and changes seen on amplitude-integrated electroencephalography (aEEG), which was recorded during the first 3 days of life using a cerebral function monitor, CFM Olympic 6000 (Olympic Biomedical, Seattle, WA, USA). Mild HIE (HIE stage I) was defined as an altered level of consciousness that included irritability with periods of jitteriness, slight abnormal muscle tone, exaggerated Moro and absence of autonomic dysfunction and with normal continuous aEEG patterns (upper margin of the trace >10 mV and the lower margin >5 mV) and without seizures. Moderate HIE (HIE stage II) was defined as somnolence, decreased activity, hypotonia, weak primitive reflexes, constricted pupils, bradycardia or periodic breathing and with early seizures and moderately abnormal aEEG patterns (upper margin of the trace >10 mV and the lower margin
