mechanisms of birth asphyxia and a novel resuscitation

Mechanisms of Birth Asphyxia and a Novel Resuscitation Strategy

Mohamed Helmy

Department of Biosciences Faculty of Biological and Environmental Sciences

University of Helsinki and

Finnish Graduate School of Neuroscience

Academic Dissertation

To be presented for public examination with the permission of

the Faculty of Biological and Environmental Sciences of the University of Helsinki in Telkänpönttö (lecture hall 2402), Biocenter 3, Viikinkaari 1, Helsinki,

on Friday the 4th of April 2014, at 12 o’clock noon.

Helsinki, 2014

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Supervised by Professor Kai Kaila Department of Biosciences University of Helsinki, Finland and Professor Juha Voipio Department of Biosciences University of Helsinki, Finland Thesis advisory committee Professor Turkka Kirjavainen Department of Paediatrics University of Helsinki Central Hospital, Finland and Professor Heikki Tanila A. I. Virtanen Institute University of Eastern Finland, Finland Reviewed by Professor Robert D. White Memorial Hospital, Indiana Director, Regional Newborn Program United States and Docent Mikko Airavaara, PhD Institute of Biotechnology University of Helsinki, Finland Opponent Professor Lena Hellström-Westas Department of Women's and Children's Health, Pediatrics Uppsala Universitet, Sweden Custos Professor Kristian Donner Department of Biosciences University of Helsinki, Finland

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Cover picture by Niina Markkanen. ISBN 978-952-10-9773-7 ISSN 1799-7372 This book was published in the series: Dissertationes Biocentri Viikki Universitatis Helsingiensis Unigrafia Oy Helsinki, 2014

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Contents List of original publications (vi) Abstract (vii) Abbreviations (ix) 1 Introduction (1) 2 Review of the literature (3)

2.1 The neonatal brain (3) 2.2 Birth asphyxia in the human newborn (3)

2.2.1 Epidemiology (3) 2.2.2 Etiology (4)

2.3 Diagnosis and features of birth asphyxia (4) 2.3.1 Pathophysiology of birth asphyxia (4) 2.3.2 Acidosis at birth and other clinical parameters (9) 2.3.3 Neonatal encephalopathy, multi-organ failure and associated

conditions (10) 2.3.4 Seizures and electroencephalography in the neonate (11) 2.3.5 Patterns of brain injury (12) 2.3.6 Brain imaging (13) 2.3.7 Cerebrovascular response in birth asphyxia (14)

2.4 pH regulation and neuronal function in the neonatal brain (15) 2.4.1 Regulation of pH in the neonate (15) 2.4.2 Regulation of intracellular pH (17) 2.4.3 Acid-base transporters in the blood-brain barrier (19) 2.4.4 The Na/H exchanger isoform 1 and 2 (20) 2.4.5 Modulation of neuronal activity by pH (21) 2.4.6 Chemoreception (25)

2.5 Current management protocols for birth asphyxia (25) 2.5.1 Resuscitation (25) 2.5.2 Use of drugs to suppress neonatal seizures (26) 2.5.3 Hypothermia (26)

2.6 Animal models of birth asphyxia (28) 2.7 Outcome after birth asphyxia (29)

2.7.1 Psychosocial and economic burden of disease (29)

Contents

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2.7.2 Behavioral and neurologic outcome after birth asphyxia (30)

3 Aims of the study (32) 4 Methods (33)

4.1 Novel rodent model of birth asphyxia and therapeutic strategy based on alteration of respiratory conditions (33)

4.2 EEG and behavioral assessment of seizures (33) 4.3 In vivo pH measurement using pH-sensitive microelectrodes (35) 4.4 In vivo neuronal pH measurement using

two-photon microscopy (36) 4.5 Intracerebral measurement of O2 partial pressure (36) 4.6 Assessment of BBB permeability (37) 4.7 Blood parameters (37) 4.8 Behavioral and neurologic outcome after experimental birth

asphyxia (37) 4.9 Statistical analyses (37)

5. Results and discussion (39) 5.1 Large seizure burden after asphyxia but not after hypoxia or

hypercapnia (39) 5.2 Brain alkalosis triggers seizures (40) 5.3 Postasphyxia seizures are not associated with reduced brain

oxygenation (41) 5.4 The BBB generates a pH gradient between the brain and the rest of

the body (41) 5.5 BBB integrity is not altered by asphyxia (41) 5.6 Blocking NHE1 and NHE2 in BBB abolishes brain alkalosis and

seizures (42) 5.7 GRN alters the course of recovery (44) 5.8 Behavioral and neurologic outcome after asphyxia and GRN (45) 5.9 Translational implications of the study (46)

6 Conclusion (48) Acknowledgements (50) References (51)

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List of original publications

I. Helmy, M., Tolner, E.A., Vanhatalo, S., Voipio, J., and Kaila, K., 2011. Brain alkalosis causes birth asphyxia seizures, suggesting therapeutic strategy. Annals of Neurology. 69(11), 493-500.

II. Helmy, M., Ruusuvuori, E., Watkins, P.V., Voipio, J., Kanold, P.O., and Kaila, K., 2012. Acid extrusion via blood-brain barrier causes brain alkalosis and seizures after neonatal asphyxia. Brain. 135(3), 3311-3319.

III. Helmy, M., Alafuzoff, A., Piepponen, P., Kaila, K., 2013. Graded restoration of normocapnia improves outcome after birth asphyxia. Manuscript.

The novel rodent model of birth asphyxia and therapeutic strategy based on graded restoration of normocapnia are the original ideas of Kai Kaila. Research was designed by Kai Kaila, Juha Voipio, and the author. Manuscripts were written by the author with Kai Kaila, with input from co-authors. All the work was done by the author unless stated otherwise.

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Abstract

Birth asphyxia is a major cause of infant and childhood death, disability and neurodevelopmental delay worldwide. During birth, impairment of respiration is reflected in elevated levels of CO2 and diminished levels of O2 in the neonate. The fundamental presentation and diagnostic criterion of birth asphyxia is severe acidosis, most commonly measured in umbilical blood. Resuscitation is associated with normalization of blood pH values and arterial blood gases. Within hours of a moderate or severe asphyxic insult during birth, severe seizures are triggered. In the present study, asphyxic conditions during birth are modeled as an induced hypoxia and hypercapnia in postnatal day 6 rat pups. Respiratory conditions are altered so that pups breathe 20 % CO2 with either 9 % O2 or 4 % O2 (N2 balanced) for 60 or 45 minutes, respectively. Brain extracellular and intraneuronal pH became rapidly acidotic during asphyxic conditions. After experimental asphyxia, immediate restoration of normoxia and normocapnia was associated with a large seizure burden. Seizures in the postasphyxia period were tightly correlated with a recovery and alkaline overshoot in brain pH. Enhanced acid extrusion from the brain was attributed to increased Na/H exchange across the blood-brain barrier. Pharmacologic blockade of Na/H exchange in the blood-brain barrier with amiloride or its analog abolished brain alkalosis and seizures. These findings suggest that a brain-confined alkalosis is generated by Na/H exchangers in the blood-brain barrier when normocapnic conditions are immediately restored after experimental birth asphyxia. A putative therapeutic strategy was tested, where the CO2 level of inhaled air in the postasphyxic period was reduced in steps, so that normocapnic conditions are gradually restored. This graded restoration of normocapnia was achieved by exposing the pup to 10 % CO2 in air for 30 minutes, followed by 5 % CO2 in air for a further 30 minutes, and finally with room air. A dramatic attenuation of brain alkalosis and seizures was induced by graded restoration of normocapnia. Immediate restoration of normocapnia after asphyxia was associated with adverse outcome in juvenile and adult rats, manifest as compromised sensorimotor coordination, altered emotional reactivity to acute stress, diminished inhibition of fear-motivated behavior, impaired memory and learning, abnormal social interaction, and increased

Abstract

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seizure susceptibility. Graded restoration of normocapnia after asphyxia was associated with significant and favorable improvement of outcome, such that behavioral deficits were rescued, and seizure threshold was not significantly different from control animals. The findings of the this study suggest a central role for Na/H exchange in the blood-brain barrier in mediating the postasphyxia brain alkalosis as measured in the present study as well as in human babies. Importantly, the findings also suggest a putative therapeutic strategy in which recovery from acidosis during neonatal resuscitation is controlled through a graded restoration of normocapnia.

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Abbreviations

[Ca2+] calcium concentration [Ca2+]i intracellular free calcium concentration [H+] hydrogen ion concentration [K+]o extracellular potassium concentration [Na+] sodium concentration βCO2 bicarbonate dependent buffering capacity βi non-bicarbonate dependent buffering capacity AE anion exchange aEEG amplitude-integrated electroencephalography ANOVA analysis of variance ATP adenosine triphosphate BBB blood-brain barrier CBF cerebral blood flow CSF cerebrospinal fluid CT computed tomography DC direct current DNA deoxyribonucleic acid EEG electroencephalography ECl equilibrium potential of chloride EHCO3 equilibrium potential of bicarbonate EGABA reversal potential of current gated by γ-aminobutyric acid type

A receptors GABAA γ-aminobutyric acid type A receptor GRN graded restoration of normocapnia IQ Intelligence Quotient MIA N-methyl-isobutyl-amiloride MCT monocarboxylate cotransporter NBC Na/HCO3 cotransporter NCBE Na-coupled Cl/HCO3 exchanger NDCBE Na-driven Cl/HCO3 exchanger NHE Na/H exchanger NMDA N-methyl-D-aspartate

Abbreviations

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NO nitric oxide NOS nitric oxide synthase PaO2 partial arterial pressure of oxygen PaCO2 partial arterial pressure of carbon dioxide PCr/Pi phosphocreatine/inorganic phosphate PMCA plasma membrane Ca2+ ATPase PVL periventricular leukomalacia S-N-K Student-Newman-Keuls

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1 Introduction

Birth asphyxia is the most common cause of neonatal seizures, but what triggers those seizures is unknown (Evans and Levene, 1998). The disease is associated with a wide spectrum of neurodevelopmental and neurological disorders and disability in life (Cornette and Levene, 2008). A severe episode of birth asphyxia is associated with conditions that seriously impair an individual’s integration into society as an independent adult (Lawn et al., 2009). Furthermore, it is becoming increasingly apparent that more subtle cognitive and affective deficiencies are major and permanent consequences of birth asphyxia, and are in most need of prevention (Marlow et al., 2005; de Haan et al., 2006; van Handel et al., 2007). The nature of the disease, as one afflicting the newborn during a critical time window, and its sequelae, means that huge difficulties are placed on the individual and the family who have to find ways to cope with this tragedy on a daily basis (Soria et al., 2007). The direct costs and burden of disease imposed on societies around the world are alarming (Lawn et al., 2005; Mangiaterra et al., 2006); in England alone, preventing 10 % of birth asphyxia cases would save about £20 million per year (Millburn, 2000). The indirect costs imposed on health, educational, and labor systems in particular are probably vast (Kurinczuk et al., 2010; Saugstad, 2011). The available management options for birth asphyxia are inadequate and rational therapies are urgently needed (Johnston et al., 2011; Seshia et al., 2011; Tagin et al., 2012). An important factor influencing translational research in birth asphyxia is the physiological and clinical relevance of animal models in which hypotheses addressing the mechanism of disease can be tested (Painter, 1995; Roohey et al., 1997; Yager, 2004). The most commonly used rodent model, the Vannucci model (Rice et al., 1981), is a modified Levine preparation of unilateral carotid artery ligation followed by hypoxia (Levine, 1960). The Vannucci model does not reflect systemic pH changes occurring in the neonate after birth asphyxia, namely, there is no profound systemic acidosis following the insult (Vannucci and Vannucci, 1997). Furthermore, the experimental insult is biphasic (Rice et al., 1981), meaning that formulating hypotheses within the model in the context of resuscitation is not possible.

Introduction

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How do rapid pH changes during birth asphyxia and resuscitation contribute to the pathophysiology of the disease? Given the profound effect of pH on neuronal excitability (Ruusuvuori and Kaila, 2014), what are the consequences of rapid pH changes after birth asphyxia on brain function? If present, are pathological pH alterations of brain function in the neonatal period associated with long-lasting effects on neurodevelopmental outcome? In the present study, a novel rodent model of birth asphyxia is developed which investigates the immediate pathological effects of asphyxia on neonatal acid-base physiology and brain excitability, and the associated outcome in later life. Based on our work, we are offering an alternative and therapeutic approach to neonatal resuscitation.

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2 Review of the literature

2.1 The neonatal brain

Studying the developing brain has aroused our fascination and curiosity (Hamburger, 1988; Buzsáki, 2006; Lagercrantz and Changeux, 2010). Research on the development of the visual system in mammals clearly indicate that refinement of neuronal connections during early development is dependent on electrical activity (Wiesel and Hubel, 1963; Penn and Shatz, 2010). The neonatal electroencephalogram (EEG) is characterized by distinct patterns of spontaneous and evoked activity (Vanhatalo and Kaila, 2006). Spontaneous or endogenous electrical activity in the developing brain is believed to play a central role in the formation of neuronal networks (Khazipov and Buzsáki, 2010). Neonatal seizures represent an injurious phenomenon during this highly critical time (Holmes and Ben-Ari, 2001). Abnormal brain activity and function is reflected in the neonatal EEG, and is correlated with poor neurodevelopmental outcome (Hellström-Westas, 2012). There are no age-specific therapies for neonatal seizures (Jensen, 2009). Coherent management of neonatal seizures requires directly addressing the underlying pathophysiology (ibid). pH has profound effects on neuronal excitability, but how rapid changes in systemic pH occurring during birth might contribute to the pathophysiology of neonatal seizures is not known. What the neonatal brain experiences during this time will affect the individual for the rest of his or her life. The price of not paying close attention to the unique nature of neonatal brain disorders is one we cannot afford to pay (Gerhardt, 2008).

2.2 Birth asphyxia in the human newborn

2.2.1 Epidemiology

The incidence of birth asphyxia ranges between 2 and 30 in 1000 live births (Dilenge et al., 2001; Golubnitschaja et al., 2011). The incidence is much greater in developing countries due to the quality of prenatal and obstetric care (ibid). The burden of morbidity of birth asphyxia has been estimated to be 42 million disability adjusted life years - the number of years lost due to ill-health, disability or early death (Lawn et al., 2011). Worldwide, birth asphyxia

Birth asphyxia in the human newborn

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accounts for 23 % of neonatal deaths, and 8 % of childhood (< 5 years) deaths (Bryce et al., 2005).

2.2.2 Etiology

Asphyxia is combined hypercapnia and hypoxia. Birth asphyxia is impaired respiratory gas exchange between mother and infant during labor (Cowan et al., 2003). Perinatal asphyxia and birth asphyxia are synonymous: both refer to the period not only immediately before birth and throughout labor, but also birth itself and the immediate postpartum period, generally thought of as the period of stabilization right after birth. Neonatal encephalopathy is the neurological syndrome following birth asphyxia (Scher, 2006). The clinical grade of neonatal encephalopathy is given by Sarnat and Sarnat (1976). In the present study, the term hypoxic-ischemic encephalopathy is used only in reference to the clinical staging of neonatal encephalopathy in section 2.3.3. Causes can be placental in origin, such as premature separation of the placenta or contracture of the uterus, or umbilical, such as cord compression (Berger and Garnier, 1999). Other causes include maternal hypo- or hypertension, malpresentation during birth, and other problems associated with complicated labor (Gieron-Korthals and Colon, 2005). Three important features indicative of intrapartum asphyxia are: (i) signs of fetal distress, such as meconium stained amniotic fluid; (ii) depression at birth; and (iii) overt neurological syndrome (neonatal encephalopathy) in the postpartum period (Volpe, 2008).

2.3 Diagnosis and features of birth asphyxia

2.3.1 Pathophysiology of birth asphyxia

The mammalian birth process is a time of unique physiological changes that adapt the neonate to extra-uterine life (Lagercrantz and Slotkin, 1986; Winberg, 2005). Some degree of asphyxia, as reflected in neonatal blood-gas analysis, is expected after normal labor (James et al., 1958). If labor is complicated, the asphyxia can be severe and prolonged. A multitude of pathophysiological responses occur during and after such an asphyxic insult during birth. The need for novel experimental models to drive research aiming to elucidate mechanisms underlying the pathophysiology of birth asphyxia has been emphasized (Erecinska et al., 2004; Silverstein and Jensen, 2007). Primary energy failure of birth asphyxia is initiated by severe hypoxia and hypercapnia as follows:

Diagnosis and features of birth asphyxia

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Reduced partial arterial pressure of oxygen (PaO2) impairs oxidative phosphorylation, thus depriving the brain of high energy phosphate compounds, namely adenosine triphosphate (ATP). In the neonatal rat brain, hypoxia is associated with upregulation of glycolysis (Vannucci and Vannucci, 1980). Clinical studies indicate that brain glycolysis is increased in babies during birth asphyxia, and is interpreted as a regulatory mechanism to preserve brain function (Khong et al., 2003; Shah et al., 2004; Perlman, 2006). Systemic blood flow is shunted preferentially to central organs, and glucose influx into the brain is increased, as shown in fetal primates (Behrman et al., 1970). Despite these changes to facilitate glycolysis, anaerobic respiration cannot efficiently meet the energy demands of the brain, and this is reflected in a diminishing ratio of phosphocreatine to inorganic phosphate (PCr/Pi) and increasing lactate, as shown in neonatal dogs and piglets (Mayevsky et al., 1988; Nioka et al., 1991; Penrice et al., 1997). In piglets, when PCr/Pi falls below a critical, age-dependent threshold, activity of Na-K ATPase is impaired (Digiacomo et al., 1992). This impairment of the Na-K ATPase in the presence of deranged cerebral oxidative metabolism in both animal models and babies (Wyatt et al., 1989a) underlies much of the pathophysiology of hypoxia in primary energy failure. Ionic gradients across the cell membrane cannot be effectively maintained, there is an efflux of K+, and membrane potential depolarizes (Hansen, 1985). The downstream effects of membrane depolarization, leading to cell damage and death (Northington et al., 2011), are: (i) a massive influx of Ca2+ into the cell; (ii) Na+ and Cl- enter the cell, causing cytotoxic cell edema (Siesjö, 1981); and (iii) excitatory neurotransmitters, namely glutamate, are released. The role of raised intracellular free Ca2+ concentration ([Ca2+]i) and extracellular glutamate will be discussed below. Elevation of partial arterial pressure of carbon dioxide (PaCO2) shifts the

equilibrium of the CO2/HCO3- buffer system, which dramatically lowers pH.

Tissue acidosis alters: (i) brain activity, discussed in section 2.3.4; (ii) cerebral blood flow (CBF), discussed in section 2.3.7; (iii) key enzymes in energy production, namely, lowering pH reduces affinity of phosphofructokinase for fructose-6-phosphate, thus inhibiting glycolysis (Trivedi and Danforth, 1966). Here, it is important to emphasize that depletion of ATP and altered levels of glycolytic intermediates are to be expected after normal labor as shown in neonatal rat pups delivered spontaneously and vaginally - in comparison, pups delivered by cesarean section showed minimal alteration of metabolic status (Vannucci and Duffy, 1974).


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Reperfusion injury in the brain takes place after the acute period of birth asphyxia (Hope et al., 1984). Delayed, progressive decreases in ATP and PCr/Pi have been measured in babies and animal models, and reach a nadir within 2 – 3 days (Azzopardi et al., 1989; Penrice et al., 1997; Vannucci et al., 2004). This was accompanied by an increase in lactate (ibid). Laboratory and clinical research aim to establish how we may intervene in the progression of reperfusion injury, so as to ameliorate the course of disease towards a more favorable outcome (Hope et al., 1984; Hope et al., 1987; Taylor et al., 1999). Calcium overload and excitoxicity. Increased [Ca2+]i of brain cells is a major contributor to the pathophysiology of severe birth asphyxia (Silver and Erecinska, 1990). As the cell is energy-depleted and the membrane depolarized, intracellular Ca2+ buffering capacity is rapidly overwhelmed (Siesjö et al., 1989; Puka-Sundvall et al., 2000). Ca2+ enters the cell through voltage-gated channels and N-methyl-D-aspartate (NMDA) receptors, down its concentration gradient. Furthermore, intracellular calcium concentration [Ca2+]i increases due to release of Ca2+ from mitochondria by the Na/H- and Ca/H-dependent antiport systems, and from the endoplasmic reticulum by inositol triphosphate signaling cascades (Bernardi, 1999). The downstream deleterious effects of raised [Ca2+]i include (Orrenius et al., 2003): (i) perturbation of cytoskeletal organization; (ii) impaired mitochondrial function; (iii) activation and phosphorylation of endonucleases, proteases including calpains (Annunziato et al., 2007), phospholipases, and the liberation of free fatty acids, all of which have ever-widening effects that alter membrane, receptor, and vascular bed function; and (iv) altered gene expression and protein synthesis. Apoptotic programs can be initiated by the following: (i) during the secondary wave of energy depletion and raised [Ca2+]i, mitochondrial membrane derangement triggers pro-apoptotic signaling, caspases, and calpains; (ii) free radical damage of DNA; and (iii) stress-induced alteration of gene induction and transcription, notably ‘immediate early genes’ or proto-oncogenes (Gubits et al., 1993; Taylor et al., 1999; Hagberg et al., 2009; Northington et al., 2011). Major pathophysiological cascades triggered by Ca2+ overload will be discussed below. The pivotal role of Ca2+ in regulation of cell structure, function and fate is intricately linked with H+ concentration, transport and buffering. The relationship between Ca2+ and pH will be discussed in sections 2.4.1 and 2.4.2. To the best of my knowledge, there is no data on the dynamics of brain extracellular [Ca2+] in the course and progression of birth asphyxia pathophysiology.


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Excitotoxicity refers to neuronal death due to excessive stimulation by glutamate (Olney, 1986). Membrane depolarization, energy depletion, and raised [Ca2+]i increase glutamate release, and impair glutamate uptake (Johnston, 2001). Excitotoxicity plays a crucial role in acute cell death and damage during and after focal ischemia, but not during or directly after (asphyxia-related) global ischemia (Cherici et al., 1991; Hossmann, 1994; Berger and Garnier, 1999). Indeed, in vivo, glutamate concentration in the brain of fetal sheep and newborn piglets was highest several hours after a hypoxic insult (Thoresen et al., 1997; Henderson et al., 1998). Disturbed signaling in the brain, involving excessive stimulation by glutamate, manifests as epileptiform activity during a secondary wave of energy and metabolic derangement (Williams et al., 1991; Tan et al., 1992). During asphyxia, severe acidosis effectively silences brain electrical activity; the interaction between neuronal excitability and pH is discussed in section 2.4.5. Free radical production is tightly linked with reventilation, and a major feature of reperfusion injury (Pourcyrous et al., 1993). Several sources of free radicals increase oxidative stress, while scavenging capacity is reduced (Todd et al., 2011). Free radicals peroxidize polyunsaturated fatty acids, compromise membrane function, damage DNA, and activate pro-apoptotic pathways (Wood and Youle, 1994; Buonocore et al., 2010; Johnston et al., 2011). A key source of free radicals are mitochondria. Leak of oxygen radicals and a steeper cytosolic-to-mitochondrial [Ca2+] gradient together alter mitochondrial membrane integrity and permeability, thereby depolarizing it (Nicotera and Leist, 1997). Collapse of the mitochondrial membrane potential triggers cascades of events which include release of pro-apoptotic proteins and liberation of components of the electron transport chain, powerful predictors of cell fate (Hagberg et al., 2009; Niatsetskaya et al., 2012). Mitochondrial dysfunction during secondary energy failure was correlated with epileptic activity and histopathology in piglets (Thoresen et al., 1996). Other sources of free radicals are (also) Ca2+-dependent and include nitric oxide synthase, cyclooxygenase, lipoxygenase, xanthine oxidase, catecholamine release, and leucocyte activation (Traystman et al., 1991; Johnston et al., 2011). Inflammation during reperfusion injury contributes to brain dysfunction and damage (Alvarez-Diaz et al., 2007). Microglial activation within hours of the insult is associated with release of excitatory amino acids, reactive oxygen and nitrogen species, and cytokines, most notable among which are interleukin-1β, tumor necrosis factor-α, and interferon-γ (Ivacko et al., 1996; Hagberg and


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Mallard, 2005; Hansen-Pupp et al., 2005; Wikström et al., 2008). Oligodendrocytes and white matter are particularly vulnerable to inflammatory injury (Alvarez-Diaz et al., 2007). Infection, often in the shape of chorioamnitis in babies or lipopolysaccharide in animal models, greatly exacerbates brain inflammation after birth asphyxia (Hagberg et al., 2012; Kasdorf and Perlman, 2013). Effect of pH. Alkaline pH induces cell death through unknown mechanisms (Majima et al., 1998). It has been deduced that when alkalosis increases both ionotropic glutamate receptor-and voltage sensitive calcium channel-mediated Ca2+ entry into neurons (see section 2.4.5), the downstream deleterious effects of raised intracellular [Ca2+] are exacerbated (Kristián and Siesjö, 1998; Yao and Haddad, 2004). It has been suggested that Ca2+-mediated effects leading to cell death, such as activation of phospholipases, proteases, and endonucleases, are antagonized by increased intracellular [H+], while reduced [H+] accelerates these processes (Kubohara and Okamoto, 1994; Trump and Berezesky, 1995). In several types of cells, recovery from acidosis per se and alkalosis induce mitochondrial dysfunction (Lemasters et al., 1997; Honda et al., 2005). Reperfusion injury after birth asphyxia in babies was associated with an alkaline brain pH (Azzopardi et al., 1989). Increasing severity and duration of an alkaline brain pH measured within two weeks after an asphyxic insult during birth was correlated with more severe brain damage weeks to months after birth, and negative neurodevelopmental outcome at one year of age; the most severely affected infants had the most alkaline brain pH (Robertson et al., 1999; Robertson et al., 2002). Brain cell death was increased in the presence of brain alkalosis after experimental hypoxia-ischemia in neonatal mice (Kendall et al., 2006). Brain alkalosis was measured in adult patients days to weeks after stroke, and was correlated with neurologic damage (Syrota et al., 1985; Levine et al., 1987; Welch et al., 1990; Hugg et al., 1992). Experimental ischemia in adult rats was associated with brain alkalosis, which was speculated to worsen cerebral tissue injury (Mabe et al., 1983; Yoshida et al., 1985; Chopp et al., 1990a; Chopp et al., 1990b).


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2.3.2 Acidosis at birth and other clinical parameters

The single most important criterion for the diagnosis of birth asphyxia is profound acidosis measured in umbilical blood, pH < 7.00 or base deficit of at least 12 mmol.l-1 (MacLennan, 1999; Poland and Freeman, 2002; Hankins and Speer, 2003). Acidosis at birth has been shown to be a powerful predictor (but not necessarily an immediate cause) of negative outcome (Low et al., 1983; van den Berg et al., 1996; Sehdev et al., 1997; Ross and Gala, 2002). One study found that a base deficit > 16 mmol.l-1 measured in any one blood gases analysis within 4 hours of birth, administration of chest compressions for more than one minute, and delayed onset of respiration for 30 minutes or more, together predict with 93 % probability severe adverse outcome (Shah et al., 2006). Another study identified umbilical cord pH and first blood pH and HCO3

- analysis as a primary predictors of neurological outcome (Ambalavanan et al., 2006). Other clinically significant parameters measured in babies include: (i) Blood glucose varied greatly in the first 6 hours of life after birth asphyxia, despite consistent management protocols (Nadeem et al., 2011). Hypoglycemia was associated with brain injury and worse outcome (Salhab et al., 2004; Nadeem et al., 2011); (ii) Hyperammonemia has been reported in infants with severe birth asphyxia, and is associated with CNS irritability, hyperthermia, hypertension, and heart oscillations (Goldberg et al., 1977); (iii) Erythropoietin synthesis is selectively increased in the CNS after birth asphyxia, and exogenously administered erythropoietin does not cross the BBB (Juul et al., 1999); (iv) Markers of disturbed energy metabolism in high-risk babies include elevated levels of lactate in cerebrospinal fluid (CSF) (Mathew et al., 1980), and increased urinary lactate:creatinine ratio (Huang et al., 1999a); (v) Excitatory amino acids namely glutamate and aspartate measured in CSF of postasphyxia neonates show elevated levels (Riikonen et al., 1992; Hagberg et al., 1993); (vi) Inflammatory markers including interleukin-6 and tumor necrosis factor-α are increased in the plasma and CSF of postasphyxia babies (Silveira and Procianoy, 2003); (vii) Free radicals and markers of oxidative stress in CSF of high-risk infants include elevated levels of hypoxanthine, ascorbic acid (a pro-oxidant in neonates), non-protein-bound iron, and in plasma the oxidized form of coenzyme Q-10 (Saugstad and Gluck, 1982; Silvers et al., 1994; Hara et al., 1999; Ogihara et al., 2003); (viii) Brain-specific markers in CSF include increased levels of neurofilament protein, glial


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fibrillary acidic protein, protein S-100, neuron-specific enolase, and brain-derived neurotrophic factor (Blennow et al., 2001; Douglas-Escobar and Weiss, 2012).

2.3.3 Neonatal encephalopathy, multi-organ failure and associated conditions

Neonatal encephalopathy is diagnosed in the presence of altered level of arousal, muscle tone, and seizures (Scher, 2006). Very often the term ‘hypoxic-ischemic encephalopathy’ is used in the literature to denote birth asphyxia and/or the clinical staging of the disease, as given by Sarnat and Sarnat (1976). Regardless, the grade of encephalopathy is considered mild if the baby is ‘jittery’ and ‘hyperexcitable’, with exaggerated reflexes, sympathetic over-reactivity, and change in muscle tone within 6 hours; moderate in the presence of lethargy, hypotonia, and diminished reflexes, with or without seizures; or severe if the baby is flaccid with obtundation, shows brainstem dysfunction apparent as apnea and/or oculomotor and pupillary disturbances, autonomic dysfunction, increased intracranial pressure, and has seizures (Sarnat and Sarnat, 1976; Amiel-Tison and Ellison, 1986). Management of moderate and severe encephalopathy involves the use of therapeutic hypothermia, antiepileptic drugs when seizures are present, and assisted ventilation as indicated (Yates et al., 2012). On top of the neurological syndrome, the condition is often exacerbated by dysfunction of other body organs. Multi-organ failure occurs in 60 to 80 % of babies born with birth asphyxia (Perlman et al., 1989; Martin-Ancel et al., 1995). A common complication of birth asphyxia is renal impairment due to increased renal artery resistance and acute tubular necrosis (Akinbi et al., 1994; Andreoli, 2004), and, when present, oliguria is associated with poor outcome (Perlman and Tack, 1988). Gastrointestinal complications include increased mesenteric artery resistance (Akinbi et al., 1994), increased risk of necrotizing enterocolitis (Caplan et al., 1994), and abnormal liver function (Zanardo et al., 1985). Disseminated intravascular coagulation is a serious complication and may cause severe intracranial hemorrhage (Chessells and Wigglesworth, 1971). Syndrome of inappropriate antidiuretic hormone secretion may occur (Speer et al., 1984). Cardiac output may be compromised in the postasphyxic period, causing decreased stroke volume and systemic hypotension (Perlman et al., 1989).


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2.3.4 Seizures and electroencephalography in the neonate

The life-time incidence of seizures is highest during the neonatal period (Cowan, 2002). The single most important cause of neonatal seizures is birth asphyxia (Evans and Levene, 1998). The susceptibility of the neonatal brain to seizures is often explained in terms of an innate ‘imbalance’ between excitation and inhibition (Chapman et al., 2012), but this concept has been criticized (Loscher et al., 2013). The neonatal brain shows distinctive patterns of neuronal activity which are necessary for development (Hellström-Westas and Rosén, 2005; Vanhatalo and Kaila, 2006; Sieben et al., 2013). Seizures represent an injurious phenomenon during this critical period (Holmes and Ben-Ari, 2001). It has been inferred that seizures after birth asphyxia disrupt activity-dependent formation of connections during early development (Penn and Shatz, 1999). Several clinical studies have shown that birth asphyxia seizures are associated with negative neurodevelopmental outcome (Brunquell et al., 2002; Miller et al., 2002; Hellström-Westas and Rosén, 2005; Tekgul et al., 2006; Ronen et al., 2007; Glass et al., 2009; Kontio et al., 2013). Recurrent seizures in infancy are associated with enhanced susceptibility to seizures in later life (Isaeva et al., 2010) and altered myelination (Gospe, 1998). Prognosis after birth asphyxia seizures includes symptomatic epilepsy, and, therefore, its co-morbidities (McBride et al., 2000). Animal models have shown that neonatal seizures are associated with epileptogenesis (Thibeault-Eybalin et al., 2009). Recurrent flurothyl seizures in immature rats were associated with decreased seizure threshold, behavioral deficiencies, and altered neural connectivity in adolescence (Huang et al., 1999b; Liu et al., 1999). Electroconvulsive seizures in immature rats alter myelination and synapse formation (Jorgensen et al., 1980; Elfving et al., 2008). In rodent models, seizure threshold was decreased in adolescent or adult life after neonatal hypoxia, anoxia, or hypoxia-ischemia (Shimomura and Ohta, 1988; Jensen et al., 1992; Holmes et al., 1998; Huang et al., 1999b; Holmes, 2004; Isaeva et al., 2010; Rakhade et al., 2011). Seizure activity on the electroencephalogram (EEG) can be defined as “…rhythmic and stereotyped activity, with a minimum duration of 10 seconds, with a clear beginning and end and an evolution in morphology and frequency…” (Shany and Berger, 2011). Burst suppression pattern is “…background with bursts of high amplitude delta and theta activity with intermixed or superimposed sharp waves and spikes lasting 1 – 10 s alternating


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with periods of marked background attenuation (voltage consistently below 5 µV)…”, and is often associated with grave outcome (Sinclair et al., 1999). Seizures start 3 to 11 hours after birth asphyxia (Perlman and Risser, 1996; Ekert et al., 1997; Toet et al., 1999). Clinical detection of seizures grossly underestimates electrographic seizure activity (Murray et al., 2008; Shah et al., 2012). The importance of routine monitoring of brain electrical activity in the neonatal intensive care unit cannot be overstated (de Vries and Hellström-Westas, 2005; Vanhatalo and Kaila, 2006). Continuous monitoring of brain activity is vital for immediate diagnostic information and to guide management; parameters obtained include the presence of seizures, degree of encephalopathy, progression of disease, and data for antiepileptic drug management (Shellhaas et al., 2011). Furthermore, neonatal EEG after birth asphyxia is of high prognostic value, allowing for prediction of outcome (Holmes et al., 1982; Hellström-Westas and Rosén, 2005; Tsuchida, 2013). Amplitude-integrated EEG (aEEG) provides a wealth of information on brain functional integrity (Hellström-Westas et al., 2006b) and is predictive of neurodevelopmental outcome (Spitzmiller et al., 2008; Kontio et al., 2013). The relative ease with which aEEG can be applied and interpreted makes it a valuable tool in the management and study of birth asphyxia (Hellström-Westas and Rosén, 2006).

2.3.5 Patterns of brain injury

Primary brain swelling occurs in 22 % of asphyxiated babies, and reaches a maximum after 29 to 72 hours (Clancy et al., 1988; Lupton et al., 1988). Mannitol, which is sometimes used to reduce brain edema, is of no benefit in the treatment of birth asphyxia (Adhikari et al., 1990). After birth asphyxia “…cerebral edema is probably a marker that severe brain damage has occurred rather than a mechanism by which damage is caused…” (Whitelaw and Thoresen, 2002). The pattern of brain injury varies with gestational age, severity of the insult, and intervention, with overlap in the distribution and type of injury (Myers, 1972; Johnston et al., 2002). The most common injury is selective neuronal necrosis, which is of four main types (Miller and Ferriero, 2009): (i) a severe and prolonged insult in the term and preterm infant is associated with diffuse neuronal necrosis (ibid); (ii) a moderate to severe insult in the term infant is associated with cerebral cortical-deep nuclear (especially basal ganglia and thalamus) neuronal necrosis (ibid); (iii) deep nuclear-brain stem neuronal


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necrosis is seen with abrupt and severe insults in the term infant (ibid); (iv) pontosubicular and cerebellar patterns occur in preterm infants (ibid). Periventricular leuokomalacia (PVL) is white matter injury of brain regions surrounding the lateral ventricles. It affects preterm infants (Volpe, 2009). PVL occurs as an ‘amalgam’, where neuronal and axonal injury is present in addition to white matter damage, forming an ‘encephalopathy of the premature’ (ibid). The two components of PVL are focal, which is necrosis of periventricular white matter and loss of cellular elements (and is either cystic in less than 5 % of cases, or non-cystic) and diffuse, which is astrogliosis and microgliosis. Neuronal and axonal injury occurs in thalamus, basal ganglia, cerebral cortex, brain stem, and cerebellum. PVL occurrence in the premature infant peaks when subplate neurons (Kostovic et al., 1989), which are selectively vulnerable to asphyxia, are at a maximum (McQuillen and Ferriero, 2004). Parasagittal cerebral injury is necrosis of the cortex and underlying white matter of that region (Van Houten et al., 1996).

2.3.6 Brain imaging

The diagnostic value of brain imaging techniques are limited by their degree of invasiveness, local availability, required expertise, and need for transportation of an ill baby from the neonatal intensive care unit (O'shea et al., 2005; Mathur et al., 2008). Magnetic resonance imaging (especially diffusion-weighted) is the imaging approach of choice for both diagnostic and prognostic purposes, and provides data on all types of brain injury mentioned in section 2.3.5 (Lawrence and Inder, 2008). Early measurements within 3 days of life show subtle abnormalities (Rutherford et al., 2006). Interpretation of patterns of brain injury in serial measurements over the first 3 weeks of life can predict neurodevelopmental outcome (ibid). Computed tomography is useful in the absence of magnetic resonance imaging, and can provide some diagnostic data when images are compared over weeks (Barkovich and Raybaud, 2012). Magnetic resonance spectroscopy of phosphorus and H+, is the most sensitive imaging technique of disturbed neonatal brain function in the immediate postasphyxic period; parameters of cerebral function which can be measured include brain phosphocreatine, adenosine triphosphate, lactate, choline, glutamate, and pH (Ashwal et al., 2013). Ultrasonography is portable and cheap, but is false-negative in more than half of cases, and is of value only in the presence of cystic-PVL (which is rare), or concomitant hemorrhagic


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necrosis or other brain lesions (Barkovich and Raybaud, 2012). Near infrared spectroscopy is also portable and can be used to monitor disease progression; the data provided on cerebral oxygenation and hemodynamics is of diagnostic and prognostic value especially when combined with other modalities (Wyatt et al., 1989a).

2.3.7 Cerebrovascular response in birth asphyxia

Cerebral blood flow (CBF) is the ratio of cerebral perfusion pressure to cerebrovascular resistance. Autoregulation of CBF refers to the ability of the cerebral arteries to adjust their level of resistance as systemic blood pressure fluctuates to maintain CBF within an established range (Paulson et al., 1990). With the onset of asphyxia in fetal primate and sheep, cardiac output was redistributed preferentially to the brain and adrenals (Behrman et al., 1970; Hernandez-Andrade et al., 2005). Hypoxia, hypercapnia, and acidosis initially increase CBF in rat (Johannsson and Siesjö, 1975). When asphyxia was prolonged in fetal sheep, CBF eventually fell due to systemic hypotension, secondary to decreased cardiac output, and due to loss of autoregulation (Lou et al., 1979). In newborn piglets and lambs, termination of an induced hypoxia or ischemia was associated with an initial, short-lived ‘reactive’ hyperemia; CBF subsequently returned to baseline (Rosenberg, 1988; Leffler et al., 1989; Marks et al., 1996). Hours or days later, perinatal CBF may show a ‘delayed’ hyperemia in babies and fetal sheep (Marks et al., 1996; Meek et al., 1998). The responsiveness of cerebral vasculature to changes in PaCO2, or CO2 reactivity, has marked effects on perinatal CBF (Purves and James, 1969). CO2 reactivity is reduced following birth asphyxia in the term infant (Wyatt et al., 1989b). In a neonatal rat model of hypoxia-ischemia, mild hypercapnia when applied during the insult preserved CBF, high-energy phosphate reserves, inhibited glutamate secretion, and was associated with attenuated neuropathologic damage (Vannucci et al., 1995; Vannucci et al., 1997). Conversely, hypocapnia-induced ischemia has been shown to be associated with decreased high-energy phosphates and brain cell death in piglets (Fritz et al., 2003). Hypocapnia in preterm infants has been associated with intraventricular hemorrhage and periventricular leukomalacia (Greisen and Vannucci, 2001; Fabres et al., 2007).

pH regulation and neuronal function in the neonatal brain

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2.4 pH regulation and neuronal function in the neonatal brain

2.4.1 Regulation of pH in the neonate

Buffering capacity is the ability of a solution to resist changes in pH resulting from a change in acid or base concentration (Roos and Boron, 1981; Chesler, 1990). In other words, buffers oppose large and rapid changes in [H+]. Because of the major role of the CO2/HCO3

- buffer system, buffers can be classified into two groups, either bicarbonate dependent or bicarbonate independent. The most important physiological buffer in mammals is the bicarbonate buffer system: CO2 + H2O ⇔ H+ + HCO3

-. This is because: (i) CO2 is soluble, diffuses easily across cell membranes, and its reversible hydration to hydrogen and bicarbonate ions is effectively catalyzed by carbonic anhydrases (Maren, 1967); (ii) CO2 is excreted by the lungs, making the system an open one, and thus capable of quick and powerful buffering responses to endogenous pH changes; and (iii) because [HCO3

-] is high, the buffer system can take high [H+] loads without saturation (Voipio, 1998). The total buffering capacity is the sum of bicarbonate dependent (βCO2) and non-bicarbonate dependent, intrinsic (βi) buffers. Intracellular βi arises from phosphates and the histidine groups of proteins, which cannot cross the plasma membrane and so form a closed buffer system within the cell (Roos and Boron, 1981). In an ideal open system βCO2(open) = 2.3.[HCO3

-] (Michaelis, 1922; van Slyke, 1922); however, instantaneous equilibration is never achieved, and βCO2 has been shown to be less than the theoretical maximum (Chesler, 1998). In a closed system, bicarbonate dependent buffering capacity is greatly diminished to values that at physiological pH levels are much smaller than the maximum βCO2(closed) ≈ 0.58.[HCO3

-] that is seen when pH is ~ 6.3, the acid dissociation constant of the buffer (Roos and Boron, 1981; Chesler, 1998). During birth, asphyxia imposes a significant acid load on the neonate, with a consequent fall in pH, indicating that the buffer systems have been overwhelmed (James et al., 1958). As will be discussed below, hypercapnia is seen as increase in PaCO2, which increases [H+], while hypoxia-induced anaerobic metabolism produces acid equivalents which titrate HCO3

-; the combined result is respiratory and metabolic acidosis seen in systemic pH (Lutz, 1992; Tan and Campbell, 2008). In the fetus, the system is only open in the presence of efficient feto-maternal circulation and gaseous exchange; in other words, a working placenta.


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Perinatally, the system is a closed one insofar as neonatal and maternal circulation have dissociated, or are in the process of dissociating, and the first breath has not yet been taken (Wood et al., 1973). Indeed, the time during which the bicarbonate system is closed, and its buffering capacity diminished, is directly reflected in the neonate’s acid-base status; a longer period of normal labor is associated with lower umbilical cord blood pH (Nicolaides et al., 1989; Dickinson et al., 1992; Weiner et al., 1992). Simultaneous with this transient incapacitation of the bicarbonate buffer system and increase in PaCO2, hypoxia-induced anaerobic glycolysis and cellular metabolism increase H+ which titrate HCO3

- (Lutz, 1992). After birth, the neonatal lungs remove the acid load incurred during birth as expired CO2, and this is reflected in a gradual ‘normalization’ of PaCO2 and systemic pH over a period of about two hours (Brouillette and Waxman, 1997; Armstrong and Stenson, 2007). Pulmonary functional capacity is directly correlated with its efficacy to lower PaCO2 (Thibeaul et al., 1968). It is important to note that the breathing movements are set to, and maintained in, motion by PaCO2 (see section 2.4.6). The second organ contributing to acid-base balance in the mammal is the kidney. The kidneys excrete H+ as NH4

+ and H2PO4- and reabsorb HCO3

-. Severe acidosis has a negative effect on the function of the developing kidneys (Andreoli, 2004), and may lead to its failure (see section 2.3.3). To the best of my knowledge, there is no data on the direct contribution of the kidneys to pH compensatory mechanisms in the perinatal period; however, it is thought not to be large (Moretti and Papoff, 2012). Indirectly, kidney and lung functions influence one another through nervous and humoral mediators, and this bidirectional relationship must be considered in the management of acid-base disorders in the neonate (Gehlbach and Schmidt, 2004; Koyner and Murray, 2008). Physiological buffers normally operating under a closed system include proteins and phosphates. Hemoglobin and albumin are rich in histidine, making them effective H+ buffers, since the imidazole ring of histidine confers upon these proteins an acid dissociation constant within the physiological pH range. Albumin also binds calcium in a strictly pH-dependent manner, whereby acidemia increases plasma ionized [Ca2+] (van Slyke et al., 1928). In the normal neonate, plasma ionized [Ca2+] decreases in the first day, then increases over the ensuing three days (Bagnoli et al., 1985). Postasphyxia, plasma


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ionized [Ca2+] is lower than normal two days after birth (Ilves et al., 2000; Yoneda et al., 2005), an important factor whose pH-dependence is overlooked in the management of the neonate (Foghandersen et al., 1983).

2.4.2 Regulation of intracellular pH

Buffering capacity is the ability of a solution to resist changes in pH resulting from a change in acid or base concentration. The total intracellular buffering capacity is the sum of bicarbonate dependent (βCO2) and non-bicarbonate dependent, intrinsic (βi) buffers. βi arises from phosphates and the histidine groups of proteins, which cannot cross the plasma membrane and so form a closed buffer system within the cell (Roos and Boron, 1981). In an ideal open system βCO2 = 2.3.[HCO3

-] (Michaelis, 1922; van Slyke, 1922); however, instantaneous equilibration is never achieved, and βCO2 has been shown to be less than the theoretical maximum (Chesler, 1998). Carbonic anhydrases are an important rate-limiting factor (Huang et al., 1995). The membrane-bound carbonic anhydrase isoforms IV and XIV have their catalytic site located in the extracellular space, while carbonic anhydrase isoforms II and VII are active intraneuronally (Ruusuvuori et al., 2013). Neuronal isoforms of carbonic anhydrases are expressed in an age- and cell region-specific manner (ibid). Passive movement of H+ down a concentration gradient across a cell membrane depolarized to -60 mV and with an extracellular pH of 7.3 would drive intracellular pH towards 6.3 (Ruusuvuori and Kaila, 2014). However, intraneuronal pH is around 7.1, indicating that: (i) thermodynamic forces tend to favor the passive movement of H+ into the cell; and (ii) acid is actively extruded from the cell (Bevensee and Boron, 1998). The fact that acid is also loaded into the cell is of great significance; acid loaders allow for quick responses to rapid and local alkaline loads (or acid deficits), contribute to a more stable intraneuronal pH, and their plasmalemmal localization establishes pH microdomains (Ruusuvuori and Kaila, 2014). Indeed, the presence of membrane proteins which can act as acid extruders or loaders and their specific expression in the subcellular domain argues for a conserved role of H+ as modulators of intercellular brain signaling (ibid; see also section 2.4.5). The main mechanisms underlying movement of acid-base equivalents across the cell membrane are: (1) Na/H exchanger (NHE): NHEs are electroneutral exchangers mediating extrusion of H+, and are fueled by the inward Na+ gradient established by the Na-K ATPase (Orlowski and Grinstein, 1997). There are at least nine isoforms


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of the NHE expressed in the brain (Orlowski and Grinstein, 2004; Schwede et al., 2013). These NHE isoforms, 1 to 9, vary greatly in terms of tissue-expression, subcellular distribution, and sensitivity to amiloride (Casey et al., 2010). The NHE isoforms 1 and 2 are the most abundant in the blood-brain barrier (Lam et al., 2009), and are discussed in section 2.4.4. In addition to antagonizing acidification of intracellular pH, NHE activity contributes to the maintenance of cell volume, facilitates cell adhesion, proliferation, and migration, and is subject to regulation by hormones, mitogens, and physical stimuli such as hyperosmolarity (Casey et al., 2010). (2) Na-driven Cl/HCO3 exchanger (NDCBE): NDCBE is heavily expressed in multiple brain regions (Grichtchenko et al., 2001). HCO3

- is carried into the cell and the stoichiometry of HCO3

- to Na+ is 2:1. The other member of this group described, the Na-coupled Cl/HCO3 exchanger (NCBE), also acts as an acid extruder; its Cl--dependence is debated (Jacobs et al., 2008). NDCBE and NCBE are expressed early in prenatal life in mice (Hubner et al., 2004; Jacobs et al., 2008). (3) Na/HCO3 cotransporters (NBC): NBCs are diversely expressed in the brain, and are acid extruders and acid loaders (Boron, 2004). As acid extruders, electrogenic and electroneutral NBCs carry Na+ and HCO3

- into the cell in a 1:2 and 1:1 stoichiometry, respectively (ibid). As acid loaders, electrogenic NBCs carry Na+ and HCO3

- out of the cell in a 1:2 or 1:3 stoichiometry (Bevensee et al., 1997a; Bevensee et al., 1997b; Boron, 2004). (4) Anion exchangers (AE): AEs mediate electroneutral exchange of monovalent anions, mainly Cl- and HCO3

- (Alper, 2009). AEs mediate the uptake of Cl- and removal of HCO3

- and so act as acid loaders (Kopito et al., 1989). AE isoforms are heterogeneously inhibited in a pH- and cell tonicity-dependent manner, and are their dysfunction has been implicated in seizure disorders (Alper, 2009). (5) Plasma membrane Ca/H ATPase (PMCA): PMCA isoforms mediate an efflux of Ca2+ and an influx of H+ (Schwiening et al., 1993; Thomas, 2009). The role of PMCAs in neuronal pH regulation is not fully understood (Carafoli and Stauffer, 1994; Jensen et al., 2007). PMCA activity contributes to the alkalinization of the extracellular (synaptic) space associated with synchronous neuronal firing (Paalasmaa and Kaila, 1996; Makani and Chesler, 2010). (6) Monocarboxylate cotransporters (MCTs): Predominantly lactate anions, but also pyruvate and ketone bodies are moved into our out of brain cells in


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association with a H+ by the MCTs (Hertz and Dienel, 2005). In the neonate, acid-base status cannot be predicted from lactate concentration (Deshpande and Platt, 1997). (7) Vacuolar type proton ATPase: This enzyme has been suggested to pump H+ out of brain cells (Pappas and Ransom, 1994; Jouhou et al., 2007); however, in view of the small contribution to acid extrusion attributed to this mechanism, its normal function is unclear (Chesler, 2003). The enzyme is present in organellar membranes and creates the H+ motive force which energizes synaptic vesicles (Casey et al., 2010). (8) A significant acid loading HCO3

- conductance is imposed on neurons by γ-aminobutyric acid type A (GABAA) and glycine receptors (Kaila and Voipio, 1987). (9) Basal permeability of neutral forms of weak acids and bases (Voipio, 1998). It is evident that pH of brain cells is intricately controlled by an impressive array of cellular machinery. Extra- and intracellular neurophysiological, metabolic and ultra-structural functions and responses are strictly regulated by intracellular pH (Srivastava et al., 2007). The unique role of pH in modulating brain function in terms of neuronal excitability is discussed in section 2.4.5.

2.4.3 Acid-base transporters in the blood-brain barrier

The major site of blood-brain interaction is the endothelial cell lining the walls of brain capillaries (Abbott et al., 2010). While fetal blood-brain barrier (BBB) permeability can be classified as leaky, and later in gestation intermediate, postnatal BBB is tight (Jones et al., 1992). Indeed, the neonatal rat and mouse BBB physical, transport, and metabolic functions are excellently developed, as the BBB is capable of maintaining brain interstitial homeostasis even in the face of osmotic stress; furthermore the neonatal rat and mouse BBB has high electrical resistance (ibid). The neonatal BBB is a dynamic system regulating brain interstitial content, and can alter transporter activity in response to a variety of physiological and pathological stimuli, peptides, and hormones released both locally and into the general circulation (ibid). Brain extracellular pH is more acidic than blood pH (Somjen, 2004). The healthy BBB therefore maintains a H+ concentration gradient between brain and blood. A prominent feature of the BBB is that it rapidly responds to respiratory and metabolic acid-base disturbances to regulate brain H+ and ion homeostasis (Marshall and Nims, 1940; Nattie, 1998).


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Among the various types of animal epithelia, the remarkably high electrical resistance of the BBB confers upon it unique solute-transport properties (Bradbury, 1985; Crone, 1986). The dynamics of PaCO2/pH alterations across the BBB can be illustrated by modeling the BBB as a direct current (DC) voltage source and a resistor in series (Voipio et al., 2003). The brain is at one potential, and the body is at another. The BBB is exclusively responsible for increasing brain potential with respect to body during hypercapnia (Voipio and Kaila, 2000; Nita et al., 2004). Hyperventilation-induced alkalosis is reflected in shifts of the opposite polarity (ibid). This indicates that the potential gradient across the BBB is highly sensitive to changes in pCO2. A multitude of channels, carriers, and receptors in the BBB have been described (Abbott, 2013). Brain acidification is mediated largely by Cl/HCO3 transporters, and brain alkalinization by NHEs (Nattie, 1998). The most important isoforms responsible for alkalinizing the brain after an imposed acid load are NHE1 and NHE2 (Orlowski and Grinstein, 2004; Lam et al., 2009; Mokgokong et al., 2013). In brain capillaries, NHE1 and NHE2 are located on the luminal side of endothelial cells (Crone, 1986; Goldstein et al., 1986; Lam et al., 2009; Redzic, 2011; Benarroch, 2012). NHE1 and NHE2 in the BBB have been shown to be most susceptible (compared to other NHE isoforms) to inhibition by amiloride and its analogues (NHE1 > NHE2 > other NHE isoforms), allowing for experimental interventions that address its function (Orlowski and Grinstein, 1997; Sipos and Brem, 2000; Masereel et al., 2003).

2.4.4 The Na/H exchanger isoform 1 and 2

NHE1 and NHE2 are allosterically activated by small decreases in intracellular pH (Orlowski and Grinstein, 1997). These exchangers are dependent on the trans-plasmalemmal Na+ gradient to induce H+ efflux (ibid). It is important to note that while NHE1 and NHE2 activity is increased by low intracellular pH, physiological levels of ATP are required for full functional expression, even though ATP is not directly consumed (ibid). The set-point of NHEs is the intracellular pH at which the anitporter is activated (Bianchini and Pouyssegur, 1994). Various kinds of stimuli modulate NHE function through altering its set-point (Orlowski and Grinstein, 2004; Pedersen et al., 2006). A prominent and remarkable feature of NHE1 is the susceptibility of its set-point to modulation by a wide variety of factors and stimuli under physiological and pathological conditions (Sardet et al., 1991; Noel and Pouyssegur, 1995; Orlowski and Grinstein, 1997; Pedersen et al.,


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2006). For instance, several studies have shown that mitogenic factors alter the NHE1 set-point so that exchanger activity is increased until intracellular pH has acquired a new value 0.15 – 0.3 pH units above control conditions; exchanger activity returns to near-quiescence when intracellular pH attains the new set-point value (Aronson et al., 1982; Moolenaar et al., 1983; Paris and Pouyssegur, 1984; Grinstein et al., 1985). Indeed, chronic activation of NHE1 through altering its set-point has been implicated in a variety of pathophysiological disturbances (Pedersen and Cala, 2004; Xue and Haddad, 2009). Increased NHE1 and NHE2 activity has been associated with brain injury in at least nine animal models of hypoxia and/or ischemia, and blocking their action was shown to ameliorate brain damage (Horikawa et al., 2001; Kitayama et al., 2001; Pilitsis et al., 2001; Suzuki et al., 2002; Kendall et al., 2006; Hom et al., 2007; Hwang et al., 2008; Park et al., 2010; Cengiz et al., 2011). However, it is important to note that: (i) the mechanism of NHE-dependent brain damage is unknown; (ii) a crucial factor to achieve neuroprotection in all these studies is the pre-treatment with NHE blockers; and (iii) in addition to blocking NHEs, amiloride and its analogs also block Na+ channels (Annunziato et al., 2009), acid sensing ion channels (Xiong et al., 2008), and alter [Ca2+]i through their effect on intracellular [Na+], and consequently, trans-membranous Na/Ca exchange (Kaila and Vaughan-Jones, 1987). Translational research involving NHEs is an exciting field with promising potential (Xue and Haddad, 2009; Neuwelt et al., 2011).

2.4.5 Modulation of neuronal activity by pH

The evidence presented in the preceding sections outlines the elaborate mechanisms through which pH is buffered and regulated inside and outside the cell, and across pH compartments in the body, the most conspicuous of which in the neonate is the brain (Sacher, 1968). The unique modulatory role of pH in brain signaling is underscored by the very low concentrations of free H+: in the physiological pH range (6.5 to 8) 10 to 300 nM. H+-sensitive targets in the brain are the imidazole rings of histidine in membrane proteins, capable of binding and unbinding H+ in the physiological pH range (Ruusuvuori and Kaila, 2014). Brain signaling is modulated by pH when this interaction with H+ alters the probability of conformational change of membrane proteins and thus their function.


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In general, neuronal excitability is suppressed by acidosis and enhanced by alkalosis (Somjen and Tombaugh, 1998; Ruusuvuori and Kaila, 2014). A notable exception are chemosensitive neurons controlling breathing, discussed in section 2.4.6. Extracellular pH. In several preparations extracellular acidification diminishes calcium and sodium voltage-gated channel conductance, and shifts voltage dependence and steady-state inactivation to more positive levels, while alkalosis has opposite effects (Hille et al., 1975; Ohmori and Yoshii, 1977; Begenisich and Danko, 1983; Iijima et al., 1986; Zhou and Jones, 1996). In acutely isolated relay neurons of the ventrobasal thalamic complex of rat, low threshold Ca2+ currents were more sensitive to changes in extracellular pH than high-threshold Ca2+ currents, both being depressed by intracellular acidification, and enhanced by intracellular alkalinization (Shah et al., 2001). The effect of H+ on calcium channel conductance are probably mediated via a direct site of interaction within the channel pore (Chen et al., 1996; Zhou and Jones, 1996). In cultured fetal rat hippocampal neurons, rises in [Ca2+]i evoked by increasing [K+]o were inhibited by extracellular acidification and enhanced by extracellular alkalinization (Church et al., 1998). In goldfish horizontal cells, extracellular acidification inhibited currents mediated by inward rectifier K+ channels, Ca2+ channels, ionotropic glutamate receptors, and hemichannels (Jonz and Barnes, 2007). Acid-sensing ion channels, preferentially permeable to Na+ but also to Ca2+, K+, Li+ and H+, are widely distributed in the brain and are activated by a fall in extracellular pH (Waldmann et al., 1997). Widely distributed in the central nervous system, 2-pore domain K+ channels mediate leak currents and are sensitive to changes in extracellular pH (Lesage and Lazdunski, 2000; Goldstein et al., 2001; Patel and Honore, 2001; Talley et al., 2003). In rat cerebellar granule neurons it was shown that extracellular H+ reversibly and non-competitively inhibited the NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and kainate receptor-mediated current by acting on an extracellular site (Traynelis and Cullcandy, 1991). H+ decreased NMDA channel opening frequency and the relative proportion of longer bursts (Traynelis and Cullcandy, 1990). In mouse hippocampal slices, extracellular alkalinization increased NMDA receptor-mediated responses (Makani and Chesler, 2007). The effect of extracellular pH on NMDA receptors is mediated through allosteric binding of H+ to NR2 subunits (Low et al., 2000; Williams et


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al., 2003; Banke et al., 2005; Dravid et al., 2007; Erreger et al., 2007; Sobolevsky et al., 2007; Gielen et al., 2008; Gielen et al., 2009). H+ inhibited AMPA receptor-mediated currents and enhanced the apparent rate and extent of AMPA receptor desensitization (Ihle and Patneau, 2000; Lei et al., 2001). Experiments in catfish horizontal cells suggest that histidine groups on the external face of AMPA and kainate receptor channels allow for extracellular H+ modulation of receptor function (Christensen and Hida, 1990). Kainate receptor subunits are differentially sensitive to changes in extracellular pH (Mott et al., 2003). Modulation of ionotropic glutamate receptor function by extracellular H+ (Traynelis et al., 2010) has been interpreted as an intrinsic mechanism acting to regulate Ca2+ influx and glutamatergic signaling, and which is protective during hypoxic and/or ischemic conditions (Giffard et al., 1990; Tang et al., 1990; Kaku et al., 1993). In acutely isolated pyramidal neurons from area CA1 of rat hippocampus, extracellular alkalinization suppressed GABAA receptor-mediated conductance (Pasternack et al., 1996). In presynaptic nerve endings (enriched in synaptosomes) isolated from rat brain corpora striata, extra- but not intracellular acidification inhibited dopamine release by blocking voltage-gated Ca2+ channels (Drapeau and Nachshen, 1988). In rat hippocampal slices, increasing partial pressure of CO2 increased extracellular adenosine and inhibited excitatory synaptic transmission (Dulla et al., 2005). Intracellular pH. High-threshold Ca2+ currents in rat CA1 neurons were depressed by intracellular acidification, and enhanced by intracellular alkalinization (Tombaugh and Somjen, 1997). Those Ca2+ channels are differentially sensitive to modulation by cytosolic pH (ibid). Similar results were obtained in catfish horizontal cells (Takahashi et al., 1993), chick dorsal root ganglion neurons (Mironov and Lux, 1991) and chick sensory neurons (Kiss and Korn, 1999). In rat hippocampal slices, transient changes in excitatory postsynaptic potentials seen in response to changes in partial pressure of CO2 were mediated by intracellular pH (Lee et al., 1996). In primary cultures of neonatal rat hippocampal neurons, recovery from intracellular acidification was associated with a delayed but massive increase in the frequency of miniature excitatory postsynaptic currents and rise in [Ca2+]i (Trudeau et al., 1999). In catfish horizontal cells, application of glutamate acidified cells and suppressed high-threshold Ca2+ currents (Dixon et al., 1993).


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In synaptoneurosomes prepared from P8 rat forebrain, the metabotropic glutamate receptor, or its transduction pathway, was inhibited by intracellular acidification (Vignes et al., 1996). In cultured hippocampal neurons of mice, intracellular acidification decreased spontaneous glutamate release in the CA1 pyramidal area (Sinning et al., 2011). In amphibian and teleost blastomeres, intracellular acidification rapidly and reversibly inhibited gap junctional conductance (Spray et al., 1981). Intracellular alkalinization of mouse horizontal cells greatly increased gap junctional conductance (Palacios-Prado et al., 2009). In catfish horizontal cells, an anomalous type inwardly-rectifying K+ current was enhanced by intracellular alkalinization, but acidification had little effect (Takahashi and Copenhagen, 1995). 2-pore domain K+ channels mediating a weak inward rectifier current are depressed by intracellular acidification (Lesage and Lazdunski, 2000). pH and neuronal activity. Neuronal activity itself alters pH. Channel or transporter-mediated fluxes of acid-base equivalents generate opposite pH changes on the two sides of the membrane (Chesler, 2003). These channels and transporters are heterogeneously localized and expressed on the subcellular level, and across neuronal populations. Their activity is associated with robust, spatially- and temporally-restricted pH shifts (Ballanyi and Kaila, 1998; Kaila and Chesler, 1998). Carbonic anhydrases play a central role in the regulation of brain signaling by pH (Chesler, 2003; Ruusuvuori and Kaila, 2014). Neuronal activity produces and consumes metabolic acid equivalents (Ballanyi and Kaila, 1998; Zhan et al., 1998). Implications. The diverse and largely synergistic effects of pH shifts on excitability suggest an evolutionary origin for H+ as a modulator of inter- and intracellular brain signaling (Ruusuvuori and Kaila, 2014). The neonatal brain is intrinsically and exquisitely sensitive to small changes in pH (Ruusuvuori et al., 2010). In particular, neonatal brain activity is exceptionally prone to pH changes occurring in response to hypo- or hyperventilation; i.e. changes in PaCO2. Hyperventilation-induced alkalosis precipitates petite-mal seizures (Guaranha et al., 2005). The profound seizure-promoting effect of alkalosis, specifically in the pediatric domain, was explored in laboratory and clinical studies, where it was shown that fever-associated hyperventilation induces febrile seizures of infancy (Schuchmann et al., 2006; Schuchmann et al., 2009; Schuchmann et al., 2011). pH is pivotal in the modulation of brain excitability,


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making it an ideal target for therapeutic interventions in epileptic disorders (Pavlov et al., 2013).

2.4.6. Chemoreception

The multiple signals/multiple targets theory of central chemoreception (Putnam et al., 2004) is a modification of the multiple factors theory (Gray, 1946). Neurons controlling breathing movements located in the brainstem respond to increased pCO2 and the associated extra- and intracellular fall in pH by increasing their firing rate (Putnam et al., 2004). The chemosensitive response to acidic stimuli is mediated by numerous ion channels (ibid). Experiments in the ventral respiratory group of the isolated brainstem-spinal cord of neonatal rats suggest that extracellular H+ is the primary stimulating factor in central chemosensitivity (Voipio and Ballanyi, 1997). Respiratory rhythm in the neonate is probably generated by the ventral respiratory group in response to increased pCO2 and decreased pH (Suzue, 1984; Hilaire and Duron, 1999). Elevation of PaCO2 is necessary to initiate breathing (Cook et al., 1963; Kuipers et al., 1997; Mortola, 2001). Afferent signals from chemoreceptors in the carotid body are enhanced by hypoxia, and their sensitivity to O2 is greater in the immature rat than in the adult animal (Hilaire and Duron, 1999).

2.5 Current management protocols for birth asphyxia

2.5.1 Resuscitation

Recommendations for neonatal resuscitation are provided, for instance, by the Neonatal Resuscitation Program prepared by the American Heart Association (Kattwinkel et al., 2010). Briefly, the baby is quickly assessed in terms of gestational age, whether or not it is breathing, and muscle tone. Preliminary steps include providing warmth, drying, clearing the airway, and stimulating. Further assessments are based on heart rate and pulse oximetry. Interventions include positive-pressure ventilation, chest compressions, endotracheal intubation, and intravenous adrenaline. The protocol recommends that resuscitation should begin with room air, and 100 % O2 should be used only after prolonged hypoxemia or bradycardia. Resuscitation with 100 % O2 has been shown to depress ventilation and is associated with adverse outcome in babies and animal models (Feet et al., 1998; Saugstad et al., 1998; Hellström-Westas et al., 2006a; Markus et al., 2007; Sabir et al., 2012). Implementation

Current management protocols for birth asphyxia

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of the Neonatal Resuscitation Program in developing countries has been particularly challenging (Ersdal et al., 2012).

2.5.2 Use of drugs to suppress neonatal seizures

The first drug of choice to be administered after detection of neonatal seizures is usually phenobarbital (Booth and Evans, 2004). Phenobarbital enhances postsynaptic GABA responses (Macdonald and Barker, 1979) through an allosteric binding at the GABAA receptor channel (Leeb-Lundberg et al., 1980). Phenobarbital also blocks glutamate release and responses, and inhibits voltage-activated sodium and calcium channels (Loscher and Rogawski, 2012). Phenobarbital inhibits only 43 % of birth asphyxia seizures (Painter et al., 1999). Phenytoin, lorazepam, or midazolam are usually added as a second-line drug, or are administered as a third-line drug (Booth and Evans, 2004). Phenobarbital plus phenytoin inhibit about 60 % of seizures (Painter et al., 1999). If no effect is seen, lidocaine, clonazepam, or pyridoxine is administered (van Rooij et al., 2010). Evidence on which to refine protocols for treatment of neonatal seizures with antiepileptic agents is lacking (Hellström-Westas et al., 1995; van Rooij et al., 2013). Antiepileptic agents may cause electroclinical uncoupling or dissociation of seizures, leading to the false impression that seizures have been suppressed, and therefore underlining the need for EEG monitoring (Scher et al., 2003). Commonly used drugs are known to increase neuronal apoptosis (Jensen, 2009), and alter brain activity in babies (Malk et al., 2013). Studies from the laboratory and the clinic have shown that extended treatment with antiepileptic drugs during development, foremost among which is phenobarbital, is associated with negative and long-lasting effects on cognition, behavior, and motor function (Farwell et al., 1990; Marsh et al., 2006; Cross, 2010). Indications for mechanical ventilation include frequent and refractory convulsions, and administration of large doses of anticonvulsant(s) (Levene and Senha, 2002). Given the poor efficacy and harmful side-effects of antiepileptic agents, it is not surprising that the need for adjuvant or alternative therapies for neonatal seizures has been very frequently emphasized in the literature.

2.5.3 Hypothermia

Controlled cooling of the head or body is currently the mainstay of management for birth asphyxia (Higgins et al., 2006). Protocols vary, but

Current management protocols for birth asphyxia

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generally therapy should be initiated within 6 hours of birth, and core temperature is kept between 32.5 to 34.5 oC for 72 hours (ibid). Studies attribute the neuroprotective mechanisms of hypothermia to reduction or inhibition of cerebral metabolism, glutamate release, nitric oxide production, apoptosis, leukotriene production, and preservation of endogenous antioxidants and protein synthesis (Shalak and Perlman, 2004). It has been shown that hypothermia significantly lowers blood pH and base excess in a piglet model (Kerenyi et al., 2012). The question of how hypothermia-induced changes in systemic pH might contribute to a possible therapeutic effect is a very interesting one that needs to be addressed in future work. Some data show significant improvement in outcome with hypothermia after severe birth asphyxia, while other show benefit only after moderate insults (Tagin et al., 2012). In the Total Body Hypothermia for Neonatal Encephalopathy Trial, or TOBY, 44 % of infants who received hypothermia had improved secondary outcome in 5 of 12 tests of motor and cognitive function at 18 months, compared to 28 % of non-cooled infants, but no significant difference between the two groups was found in primary outcome, defined as death or severe neurodevelopmental disability (Azzopardi et al., 2009). More recently, similar data have been reported by others (Groenendaal et al., 2013). In a follow-up study of children 6 to 7 years old, no significant difference in primary outcome, defined as death or IQ below 70, was found between the two groups (Shankaran et al., 2012). Continuous aEEG monitoring of postasphyxia babies showed that therapeutic hypothermia did not decrease the frequency of seizures (Yap et al., 2009). A systematic review showed that in developing countries (China, India, South Africa, Uganda, and Malaysia), hypothermia was of no benefit compared to no cooling (Pauliah et al., 2013). Clinical studies in developing countries show that interventions not requiring advanced technology, such as cheap resuscitation apparatus (Richards-Kortum and Oden, 2013), educating parents and providing toys (Carlo et al., 2013) or nurturing an enriched environment (Sale et al., 2009), when applied in developing countries significantly improve outcome. In conclusion, as “…the outcome of birth asphyxia is devastating, work should continue to find adjuvant therapy to hypothermia…” (Tagin et al., 2012), and this is a judgment shared by others (Johnston et al., 2011).

Animal models of birth asphyxia

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2.6 Animal models of birth asphyxia

Important considerations in models of birth asphyxia include the following: (i) the animal species; (ii) the maturity of the animal; (iii) the nature of the insult (type, severity, timing); (iv) the translational (clinical) relevance of the pathophysiology under study; (v) short and long term outcome after neonatal insult, including patterns of brain injury and tests of sensorimotor coordination, memory, cognition, behavior, and seizure threshold; and (vi) implications for therapy (Thorngren-Jerneck et al., 2001; Northington, 2006). Cross-species calibration of maturity are defined by neurodevelopmental milestones, namely the time of: (i) the developmental profile of molecular markers such as carbonic anhydrase isoform VII and the K+-Cl- cotransporter 2 (Blaesse et al., 2009; Ruusuvuori and Kaila, 2014) (ii) neurogenesis, cell proliferation and migration; (iii) synaptogenesis and neurotransmitter development; (iv) gliogenesis, oligodendrocyte maturation and myelination; (v) subplate development; and (vi) appearance of age-dependent behavioral phenotypes (Clancy et al., 2001; Erecinska et al., 2004; Yager and Ashwal, 2009; Semple et al., 2013). The majority of birth asphyxia studies are conducted in rodent models (Yager and Ashwal, 2009) and the most commonly used model was introduced by Vannucci (Rice et al., 1981) as a modification of the Levine hypoxia-ischemia preparation in adult rats (Levine, 1960). Briefly, a P7 rat pup is anaesthetized and the left common carotid artery is ligated. The pup is returned to the nest for 1- 2 hours. The pup is then placed in 8 % O2 for a variable period of time, up to 3 hours (Rice et al., 1981; Welsh et al., 1982). In this model, systemic pH does not change from the control value (Vannucci and Vannucci, 1997). Other rat models of birth asphyxia involve ligation of both carotid arteries (Schwartz et al., 1992), temporary ligation of the umbilical cord (Sakata et al., 2000), or exposing animals to anoxia or hypoxia, where inspired O2 is maintained at 0, 2, 3 or 4 % for one hour (Jensen et al., 1991). The Vannucci model has been adapted to mice; a similar protocol is carried out in P7 animals but hypoxia is maintained up to 90 minutes (Ditelberg et al., 1996; Sheldon et al., 1998). The importance of investigating long-term neurobehavioral outcome after experimental birth asphyxia has been emphasized (Northington, 2006). The rat is highly suited to such research, but only a small proportion of studies, about 20 %, have addressed outcome in later life in rodent models after experimental birth asphyxia (Roohey et al., 1997).

Animal models of birth asphyxia

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Primate models have elaborated our understanding of the etiology of birth asphyxia (Myers, 1979), but ethical constraints, cost, and accessibility are obviously prohibitive for most research ventures. Fetal and newborn piglet, sheep, and less commonly dog models have been immensely valuable in investigating the pathophysiology of birth asphyxia during and shortly after an insult, partly due to their size (Painter, 1995). A model of cerebral palsy has been produced in the rabbit (Derrick et al., 2007). The availability of different models which can be adapted and in which specific research hypotheses can be formulated is an advantage for the birth asphyxia research community (Painter, 1995; Northington, 2006). Recommendations for model refinement highlight a model’s suitability for testing possible therapeutic interventions, and the associated functional outcome (Yager, 2004; Northington, 2006).

2.7 Outcome after birth asphyxia

2.7.1 Psychosocial and economic burden of disease

Research into novel treatments for birth asphyxia has been set as a top priority if the Millennium Development Goals are to be achieved (Lawn et al., 2011). Despite this, and the obviously huge social and economic burden imposed by the disease, birth asphyxia research is hampered by political inertia (Saugstad, 2011). Common outcomes after birth asphyxia, such as cognitive impairment and epilepsy, have a strong and pervasive negative influence, not only on the quality of life the child will lead, but also on the parents, the large majority of which will report anxiousness, depression, and other problems of coping (Soria et al., 2007). Primarily the health and educational systems are faced with costly and enormous challenges in dealing acutely and chronically with birth asphyxia (Darmstadt et al., 2005). Though obviously still a problem, birth asphyxia incidence in developed countries has been reduced through effective prenatal and obstetric care (Thornberg et al., 1995). The dramatic effectiveness of simple and low technology therapeutic interventions, such as fostering a family-oriented approach in the neonatal intensive care unit (Miceli et al., 2000; Kleberg et al., 2007; Gerhardt, 2008), and close attention to patient privacy in the immediate environment (White, 2003; White, 2011), has been demonstrated. Because resources are limited in developing countries, therapies and algorithms specifically designed for such an environment are necessary (Co et al., 2007; Richards-Kortum and Oden, 2013). Birth asphyxia is

Outcome after birth asphyxia

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complicated at its origin and in its progression by strained services or outright poverty, emphasizing the need for cost-effective improvement of neonatal care (Knippenberg et al., 2005). The evidence speaks for the enormity of birth asphyxia as a serious social problem, leading to repeated calls for action (Martines et al., 2005; Lawn et al., 2006).

2.7.2 Behavioral and neurologic outcome after birth asphyxia

The spectrum of poor neurodevelopmental outcome after birth asphyxia is large (de Vries and Jongmans, 2010). Over a third of babies surviving moderate and severe birth asphyxia have identifiable cerebral palsy at 2 years of age, often presenting as dyskinesia or spastic quadriplegia, with or without other impairments such as mental retardation, blindness, and epilepsy (McBride et al., 2000; Brunquell et al., 2002; Ronen et al., 2007; Nunes et al., 2008; Odd and Whitelaw, 2013). However, adverse outcome after birth asphyxia is most often not associated with motor disability (van Handel et al., 2007; Lindström et al., 2008). Teenagers with a diagnosis of asphyxia at birth are much more likely to perform poorly in tests of intellectual development and to suffer epilepsy compared to peers with no perinatal insult (Rantakallio et al., 1987). Other than in tests of intelligence, cognitive dysfunction may manifest as problematic school performance, social maladjustment, and behavioral difficulties (Marlow et al., 2005; Lindström et al., 2006). Verbal processing and memory can be negatively affected (Gadian et al., 2000; Steinman et al., 2009), and vision and hearing can be impaired (Robertson and Finer, 1993; Mercuri et al., 1997). Neuropsychiatric disorders more prevalent among survivors of birth asphyxia include attention deficit hyperactivity disorder, schizophrenia, and autism (de Haan et al., 2006; van Handel et al., 2007). In rats, neonatal hypoxia/ischemia, hypoxia, or anoxia was associated with the following in adolescent or adult life: (i) poor performance in spatial memory and learning tasks (Young et al., 1986; Boksa et al., 1995; Buwalda et al., 1995; Chen et al., 1995; Iuvone et al., 1996; DellAnna et al., 1997; Holmes et al., 1998; Balduini et al., 2000; Van de Berg et al., 2000; Ikeda et al., 2001; Weitzdoerfer et al., 2002; Arteni et al., 2003; Holmes, 2004); (ii) locomotive hyperactivity and impaired attention (Hershkowitz et al., 1983; Speiser et al., 1983; Shimomura and Ohta, 1988; Buwalda et al., 1995; Iuvone et al., 1996; Nyakas et al., 1996; DellAnna et al., 1997) (iii) neophobia (Laviola et al., 2004); (iv) social withdrawal (Laviola et al., 2004; Weitzdoerfer et al., 2004); (v) diminished inhibition of fear-motivated behavior (Lai et al., 2011);

Outcome after birth asphyxia

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(vi) decreased seizure threshold (Shimomura and Ohta, 1988; Jensen et al., 1992; Holmes et al., 1998; Huang et al., 1999b; Ikeda et al., 2001; Holmes, 2004; Isaeva et al., 2010; Rakhade et al., 2011); and (vii) mild deficits in sensorimotor coordination, increase in muscle tone, or subtle gait abnormalities (Young et al., 1986; Strata et al., 2004).

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3 Aims of the Study

(1) To develop a rodent model of birth asphyxia based on combined hypercapnia and hypoxia. (2) Using the novel model, to examine how changes in systemic pH after birth asphyxia are reflected in brain pH. (3) Given the findings of the experiments addressing aims (1) and (2), the third aim was to investigate the mechanisms underlying seizure generation in the postasphyxia period. (4) To develop a novel therapeutic strategy based on graded restoration of normocapnia, which could be implemented in the clinic. (5) To assess neurobehavioral and neurologic outcome in later life associated with immediate vs. graded restoration of normocapnia.

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4 Methods

4.1 Novel rodent model of birth asphyxia and therapeutic strategy based on alteration of respiratory conditions

The present rodent model and therapeutic strategy are the original ideas of Kai Kaila. The novel rodent model of birth asphyxia consists of altering the air inhaled by a spontaneously breathing postnatal day 6 (P6) rat pup so that CO2 is increased and O2 is decreased. CO2 is increased to 20 % and O2 is concomitantly decreased to either 9 % or 4 % (see Figure 1, next page). In the 9/20 asphyxia model CO2 is increased to 20 % and O2 decreased to 9 % for 60 minutes. In the 4/20 asphyxia model CO2 is increased to 20 % and O2 decreased to 4 % for 45 minutes. Control animals are isolated for equivalent periods of time. The ambient temperature is 35 oC. To investigate the effects of hypercapnia alone or hypoxia alone, animals were exposed to gas mixtures of one or the other component of asphyxia, namely: 20 % CO2 (hypercapnia for 60 minutes), 9 % O2 (9 % hypoxia for 60 minutes), and 4 % O2 (4 % hypoxia for 45 minutes). The novel therapeutic strategy for birth asphyxia consists of gradually restoring normocapnic conditions after the insult: graded restoration of normocapnia (GRN). Immediately after asphyxia pups were made to breathe 10 % CO2 in normoxia for 30 minutes, followed by 5 % CO2 in normoxia for a further 30 minutes. GRN applied after 9/20 asphyxia is denoted by 9-GRN, and after 4/20 asphyxia by 4-GRN as shown in Figure 1.

4.2 EEG and behavioral assessment of seizures

Seizures in neonatal rat pups are associated with a wide range of behavioral manifestations (Haas et al., 1990). The most violent expression of neonatal seizures is loss of righting reflex (LRR), a form of tonic-clonic seizures (Racine, 1972) and as such is given the highest score in a Racine scale modified for neonatal animals (Moshe and Albala, 1983; Veliskova, 2006).

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Figure 1. Schematic illustration of the model, putative therapeutic strategy, and experimental paradigms. (A) Alteration of respiratory conditions. Changes in ambient inhaled CO2 (red) and/or O2 (black) are represented as lines. (A1) Control condition and paradigms where only CO2 or only O2 is altered. (A2) Birth asphyxia model. (A3) Graded restoration of normocapnia (GRN). (B) Experimental paradigms. GRN: graded restoration of normocapnia; pCO2: partial pressure of CO2; pO2: partial pressure of O2.

Methods

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The seizure burden was quantified as the cumulative number of LRRs during a 2 hour observation period after the termination of asphyxia. Each incidence of LRR was given a score of 1. The score was averaged per pup, followed by calculation of cumulative seizure burden with data points given at 1 minute intervals. In some experiments, animals were injected with amiloride or its analogue N-methyl-isobutyl-amiloride (MIA) intraperitoneally in a dose of 2.5 mg/kg body weight 30 minutes prior to the onset of asphyxia (Kendall et al., 2006). EEG seizures in the birth asphyxia model were investigated using epidural silver ball electrodes implanted under isoflurane anesthesia over parietal cortex, with ground and reference over cerebellum (Schuchmann et al., 2006). EEG was measured in the freely moving animal. Recordings were made using a differential amplifier with a bandwidth of 0.07 Hz to 2 kHz. The sampling rate was 5 kHz.

4.3 In vivo pH measurement using pH-sensitive microelectrodes

Brain extracellular pH (pHo) and body pH were measured during and after asphyxia, with and without prior administration of MIA, and GRN. Under isoflurane anesthesia, the scalp of an animal was removed, and 2 burr holes were made over parietal cortex. A silver-silver chloride ground was placed subcutaneously over the scapula. The body pH and reference electrodes were placed under the loose skin of the dorsum. After recovery from anesthesia, brain pHo was measured under experimental conditions using pH-sensitive microelectrodes. In some experiments, body pH was measured using pH-sensitive electrodes, with and without pretreatment with MIA. pH-sensitive microelectrodes were prepared and calibrated as described by Voipio and Kaila with some modifications (Voipio and Kaila, 1993). Tip diameters were 4-8 µm for the ion-sensitive electrodes and 2-4 µm for the reference electrodes. The pH microelectrodes had a resistance of 8 to 12 GΩ (in calibration solution and brain tissue in a head-fixed configuration). The reference electrodes were filled with physiological saline. The pH-sensitive microelectrode and reference electrode were each placed into a burr hole to a depth of 500 µm. Body pH electrodes were made from polyvinyl chloride tubes using the same membrane and backfilling solutions as those of pH microelectrodes. The reference electrode was filled with 0.5 % agar in 0.9 %

Methods

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NaCl. The trans-BBB potential was obtained as the difference of the reference electrodes in the brain and the body (Held et al., 1964; Woody et al., 1970); see Voipio et al. (2003). 4.4 In vivo neuronal pH measurement using 2-photon microscopy

Intraneuronal pH changes in asphyxia and GRN were measured using two-photon microscopy by loading the neurons with an acetoxymethyl ester dye (Bandyopadhyay et al., 2010). The dual-excitation ratiometric pH dye 20,70-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein (BCECF) was used (Ruusuvuori et al., 2004). To the best of my knowledge these are the first experiments measuring intraneuronal pH using BCECF in vivo. A 3 mm craniotomy over parietal cortex was carefully made under isoflurane anesthesia. When moved to the two-photon microscope, light anesthesia (0.7 to 0.9 % isoflurane) was maintained in the gas mixtures or air to which the pup was exposed. Cortical layer 2/3 cells at a depth of 250 to 300 µm were imaged. Data were collected for the course of 4 min, with excitation at 825 nm (F825), and for 1 min at 770 nm (F770). Images were acquired using a 570 nm dichroic mirror. Averaged frames were analyzed using custom software written by Paul Watkins (Department of Biology, University of Maryland). The averaged data collected at 825 nm excitation was divided by the averaged data collected at 770 nm to give the ratiometric intraneuronal pH measurement of one 5 min period (F825/F770). Calibration of the BCECF signal was made in P0 rat pup brain slices using the high K+/nigericin method (Eisner et al., 1989). The linear slope obtained from in vitro calibrations is expected to faithfully represent in vivo ΔpHi changes, and the data is presented as such, rather than absolute pH values.

4.5 Intracerebral measurement of O2 partial pressure

Intracerebral partial pressure of oxygen (pO2) was measured in asphyxia using modified Clark-type polarographic oxygen microelectrodes connected to a high-impedance picoamperometer (Offenhauser et al., 2005). Calibrations were made in saline warmed to 35 oC and equilibrated with 100 % O2, air, or 100 % N2.

Methods

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4.6 Assessment of BBB permeability

BBB permeability in the asphyxia group was assessed using 10 % sodium fluorescein (Lenzser et al., 2005) which was injected intraperitoneally 30 minutes after termination of asphyxia, 4 ml/kg body weight. Brains were collected 30 minutes after sodium fluorescein administration. Animals isolated for equivalent periods of time without asphyxia served as controls. BBB damage was induced in positive controls with an intra-cardiac injection of mannitol. The procedure was set up by myself; subsequently, brain homogenization, homogenate preparation, and fluorometry were done by Merle Kampura.

4.7 Blood parameters

A clinical device, GEM 4000 Instrumentation Laboratory, (Beneteau-Burnat et al., 2008) was used to measure blood pH, calcium, partial pressure of oxygen (pO2), partial pressure of carbon dioxide (pCO2), and lactate in asphyxia, GRN, and control animals. Arteriovenous blood was collected by rapid decapitation of the rat pups.

4.8 Behavioral and neurologic outcome after experimental birth asphyxia

Outcome after asphyxia and GRN was investigated using a comprehensive battery of tests for sensorimotor and higher cognitive function. At P7 righting reflex and negative geotaxis were tested; at P14 chimney and horizontal wire tests; at P30 grid-walking, wire-mesh ascending, vertical screen, traversing a square bridge, jumping down, and the rotarod tests; at P34-42 social interaction (done by Petteri Piepponen), inhibitory (passive) avoidance, and performance in the Morris water-maze. Finally, seizure threshold was assessed at P50-55 using the timed intravenous pentylenetetrazol (PTZ) infusion test (Pollack and Shen, 1985).

4.9 Statistical analyses

Brain pHo, correlation to seizures, and trans-BBB potential: in Study I, unpaired t test was used to compare means. The correlation between ΔpH and normalized seizure burden was calculated using Spearman rank order correlation coefficient, with P based on a 2-tailed test in accordance with the

Methods

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hypothesis of alkalosis-promoted seizures. Values are presented as mean ± standard error of the mean. In Study II, values of pH or trans-BBB potential were compared using the Mann-Whitney U-test (two tailed). Data are shown as mean ± standard deviation. Intraneuronal pH: pH at the end of the two experimental paradigms was compared using Student’s two-tailed unpaired t-test. Data are presented as mean ± standard deviation (Study II). Assessment of BBB permeability: brain content of sodium fluorescein was compared between the experimental paradigms using Mann-Whitney U-test (two-tailed). Data are shown as mean ± standard deviation (Study II). Blood parameters: these analyses were done by Titta Kotilainen. For details, see Study I and its Supporting Information Table 1. Behavioral assessment of seizures: these analyses were done by Petteri Piepponen. In Study III, groups were compared with Kruskal-Wallis non-parametric ANOVA combined with Mann-Whitney U-test. The criterion for significance was P < 0.001. Values are presented as mean ± standard error of the mean. Behavioral and neurologic outcome after experimental birth asphyxia: these analyses were done by Petteri Piepponen. Data obtained from behavioral tests were analyzed using an one-way analysis of variance (ANOVA) combined with Student-Newman-Keuls (S-N-K) post-hoc test. The criterion for significance was p < 0.05. Values are presented as mean ± standard error of the mean.

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5 Results and Discussion

5.1 A large seizure burden is generated after asphyxia

Animals died during alteration of respiratory conditions only during 4/20 asphyxia. Mortality was c.a. 31 % of all rat pups; no mortality was seen during the recovery phase. The mortality during all other paradigms was nil. Although LRR is the most violent expression of neonatal seizures in rodents and hence is given the highest score in a modified Racine scale (Racine, 1972; Moshe and Albala, 1983; Veliskova, 2006), it is not equivalent to the electroencephalographic seizure burden, which would include less intense seizure activity. Assessment of LRR occurrence is, however, a reliable tool for comparison of seizure burden between the distinct experimental paradigms in this work. The cumulative seizure burden in the two hours of observation after 9 % hypoxia and 4 % hypoxia was 0 and 1.4 LRR/pup, respectively (Study III, Figure 1; see also Study I, Figure 1). For the present work, an important part of the model was that the levels of hypoxia used were not sufficient to trigger severe seizures. After hypercapnia, the score was 1.8 LRR/pup. In striking contrast, the cumulative seizure burden after 9/20 asphyxia and 4/20 asphyxia was very dramatically increased, 14.5 and 31.4 LRR/pup, respectively. Pronounced EEG seizures were measured after 9/20 asphyxia. Seizures were of duration 1 to 5 seconds, EEG seizure frequency 12 to 14 Hz, and amplitude 50 to 200 µV (Study I, Figure 1). In P6 rat pups over the two-hour observation period after alteration of respiratory conditions, cumulative seizure burden after all of 9 % hypoxia for 60 minutes, 4 % hypoxia for 45 minutes, and 20 % hypercapnia for 60 minutes was negligible, while a huge seizure burden was incurred after 9/20 asphyxia for 60 minutes and 4/20 asphyxia for 45 minutes. Hypoxia per se and hypercapnia per se were insufficient instigators of persistent alteration in brain excitability, whereas recovery from asphyxia (their combined presence) was associated with a large and progressive seizure burden.

Results and Discussion

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5.2 Brain alkalosis triggers seizures

Baseline brain pHo measured with pH-sensitive microelectrodes was 7.19 ± 0.01 (Study I, Figure 1; see also Study II Figure 2). During 9/20 asphyxia, brain pHo became rapidly acidotic, attaining a minimum of 6.77 ± 0.01 pH, followed by a small recovery towards the end of the asphyxic conditions. Following termination of asphyxic conditions, as the pup breathed air, brain pHo increased at a rate of 0.031 pH units/min during the first 5 minutes and 0.0054 pH units/min during the final 115 minutes of the recording period. At the end of the 2 hour observation period, brain pHo was remarkably alkaline at 7.60 ± 0.01. Intraneuronal pH changes matched brain pHo changes in terms of direction during and after 9/20 asphyxia. Changes in fluorescence measured with two-photon microscopy (F825/F770) indicated that intraneuronal pH decreased by 0.30 ± 0.08 pH units during asphyxia (Study II, Figure 1). Recovery from asphyxia was associated with an alkalosis, where intraneuronal pH at the end of the two hour observation period had plateaued at 0.27 ± 0.12 pH units above baseline. These results indicate that during asphyxia a large acid load is imposed on the brain, and after asphyxia acid-equivalents are removed from the brain; both from the intracellular and extracellular compartments. When asphyxia is terminated, brain pH not only recovers to pre-exposure levels, but overshoots, showing a progressive increase throughout the two hours of observation. In light of the evidence presented in section 2.4.5, these dramatic pH changes will have profound effects on brain activity. During 9/20 asphyxia- or hypercapnia-induced acidosis, the baseline activity in the EEG of P6 pups was strongly suppressed (Study I, Figure 1B), with the peak-to-peak amplitude falling from 10 to 15 µV under control conditions to 5 to 7 µV. After asphyxia, the pronounced brain alkalosis was associated with EEG seizures and a large, accumulating seizure burden. Indeed, postasphyxia seizure burden was robustly and steeply dependent on brain pHo (Study I, Figure 3). The dependence of postasphyxia seizure burden on brain pHo shows a correlation of 0.99 (P < 0.0001, Study I, Figure 3). Brief exposure to 5 % CO2 in air during post-asphyxia seizures suppressed them. The present data clearly indicate that brain pH is a major factor determining the postasphyxia seizure burden. A key finding of this study is that inhibiting brain


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alkalosis through pharmacological or respiratory means blocks postasphyxia seizures; these results and their implications are discussed below.

5.3 Postasphyxia seizures are not associated with reduced brain oxygenation

Cerebral pO2 is an index of neonatal cerebral blood flow (Austin et al., 2003). Brain alkalosis may induce cerebral vasoconstriction, thereby promoting hypoxic seizures (Apkon and Boron, 1995; Weinand et al., 1995). To examine this possibility intracerebral pO2 was measured in the 9/20 asphyxia paradigm (Study I, Figure 4). pO2 under control conditions was 10 ± 1.18 mmHg, and fell by 5 to 9 mmHg during asphyxia. Recovery was associated with a transient increase in brain oxygenation. pO2 measurements did not indicate any degree of hypoxia at any time during recovery. These findings are not in conflict with the reactive hyperemia measured after hypoxia or ischemia in piglet and lamb models (see section 2.3.7).

5.4 The BBB generates a pH gradient between the brain and the rest of the body

Body pH changes measured with pH-sensitive electrodes were strikingly different to brain pH changes. During 9/20 asphyxia body pH decreased from 7.42 ± 0.03 to 7.19 ± 0.03 (Study II, Figure 2). After asphyxia body pH recovered to a level that was identical to pre-exposure baseline (7.42 ± 0.01). Unlike brain pH, body pH did not show an alkaline overshoot. In fact, in the ten minutes immediately after asphyxia a small acid transient was measured in body pH, exactly when brain pH was recovering at maximum rate (four out of six recordings). Two hours after asphyxia brain pHo and intraneuronal pH were pathologically alkalotic, while body pH was the same as pre-asphyxia baseline. The structure capable of generating pH gradients of the described dynamics and magnitude between the brain and the body is the BBB. If this is the case then an experimental intervention which inhibits acid extrusion by the BBB during recovery should inhibit brain pHo alkaline overshoot and postasphyxia seizures.

5.5 BBB integrity is not altered by asphyxia

Altering acid-base balance in the mature mammal with an intact BBB is reflected in trans-BBB potential changes (see section 2.4.3). During 9/20 asphyxia brain potential had increased relative to body potential by +4.08 ±


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0.24 mV (Study II, Figure 2). Two hours after asphyxia the trans-BBB potential shift was -3.08 ± 0.37mV. As in the adult human (Voipio et al., 2003), brain potential of P6 rat pups was increased with respect to body potential in association with brain acidosis and decreased with alkalosis. In the present work, the measured shifts in trans-BBB potential, and the smooth trace correlating with pH, are consistent with a structurally intact BBB capable of active regulation and response, and this method of verification is more accurate than methods measuring tracer extravasation into the brain (Kang et al., 2013). Nevertheless, to exclude alteration of BBB permeability in the present model, sodium fluorescein extravasation into the brain was measured (Study II, Figure 2C). No significant difference was found in brain fluorescein after asphyxia when compared with control (128.1 ± 38.7 and 129.9 ± 33.1 ng sodium fluorescein/mg brain tissue, respectively, P = 0.9). Mannitol disruption of BBB in positive controls increased sodium fluorescein extravasation by about four-fold to 549.8 ± 99.1 ng sodium fluorescein/mg brain tissue (P = 0.02).

5.6 Blocking NHE1 and NHE2 in BBB abolishes brain alkalosis and seizures

The data presented thus far suggest that postasphyxia seizures are triggered by brain alkalosis brought about by a pathophysiological increase in acid extrusion from the brain by the BBB. The most likely candidate in the BBB responsible for removal of an acid load imposed on the brain is the NHE, isoforms 1 and 2 (Orlowski and Grinstein, 2004; Lam et al., 2009; Mokgokong et al., 2013). Of the described NHE isoforms, NHE1 is set apart by its ubiquitousness, its susceptibility to modulation by stress, and its particularly pronounced inhibition by amiloride and MIA. Being on the luminal side of endothelial cells makes NHE1 and NHE2 directly accessible to pharmacologic inhibitors present in systemic circulation (Crone, 1986; Goldstein et al., 1986; Lam et al., 2009; Redzic, 2011; Benarroch, 2012). Key findings in the present study following pharmacologic blockade of NHE1 and NHE2 activity in the BBB by pretreatment with amiloride or MIA are: (i) postasphyxia seizure burden was very remarkably reduced in the presence of MIA by 88 %, and similar results were obtained with amiloride (P < 0.0002 compared to saline control, Study II, Figure 3B); (i) MIA abolished overshoot alkalosis of brain pHo (P = 0.004 compared to saline control, Study II, Figure


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2B); (iii) the trans-BBB potential in the presence of MIA two hours after asphyxia was 0 ± 0.1 mV (P = 0.004 compared to saline control, Study II, Figure 2B), indicating that MIA abolished both the steady decrease of the trans-BBB potential and the brain pHo alkaline overshoot in parallel; and (iv) in contrast to postasphyxia animals not treated with MIA, no transient acid load was measured in body pH during the steep brain pHo recovery from acidosis (immediately after asphyxia) in the presence of MIA (Study II, Figure 2B). Can any of our findings be explained by amiloride or MIA action on neuronal NHE? This is unlikely for two reasons. First, MIA and especially amiloride penetrate the BBB very poorly (Sipos and Brem, 2000; Fisher, 2002; Liu et al., 2010). Second, in P7 brain slice preparations bath application of MIA (10 µM) had little effect on intraneuronal steady state pH and recovery from induced acidosis in the presence or absence of CO2/HCO3

-, indicating that Na/H exchange in neonatal cortical layer 2/3 pyramidal neurons is insensitive to amiloride derivatives (Study II, Supplementary Data). This work was done by Eva Ruusuvuori (see Study II, Supplementary Data). The most parsimonious explanation for the experimental effects of amiloride derivatives on postasphyxia brain alkalosis, seizures, trans-BBB potential, and body pH is that the NHE is actively extruding acid from the brain and into the body in a pathologically up-regulated manner after asphyxia. Blocking NHE1 and NHE2 activity thus prohibits the generation of postasphyxia brain alkalosis, and this is reflected in a trans-BBB potential identical to baseline, control conditions. In the absence of a BBB-generated brain alkalosis, seizures are virtually abolished. The transient acid load measured in body pH after asphyxia, most likely induced by active acid removal from the brain, the volume of which constitutes a major fraction of the total neonatal body (Sacher, 1968), is abrogated by blocking NHE1 and NHE2. A central role for NHE1 and NHE2 in the BBB in the pathophysiology of birth asphyxia as described here sits comfortably with what we already know about the exchanger (see sections 2.4.3 and 2.4.4). A wide variety of stimuli and signaling factors present or released during asphyxia might act on the NHE set-point, thus lowering its threshold of activation and up-regulating acid extrusion beyond physiologically acceptable brain pH (Aronson et al., 1982; Moolenaar et al., 1983; Paris and Pouyssegur, 1984; Grinstein et al., 1985; Sardet et al., 1991; Bianchini and Pouyssegur, 1994; Noel and Pouyssegur, 1995; Orlowski and Grinstein, 1997; Orlowski and Grinstein, 2004; Pedersen and Cala, 2004; Pedersen et al., 2006; Xue and Haddad, 2009).


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It is important to note that a neuroprotective effect of NHE blockers in experimental studies was observed if they were administered before or immediately after an insult (Horikawa et al., 2001; Kitayama et al., 2001; Pilitsis et al., 2001; Suzuki et al., 2002; Kendall et al., 2006; Hom et al., 2007; Hwang et al., 2008; Park et al., 2010; Cengiz et al., 2011). Therefore, it is probable that an asphyxia-induced upregulation of NHE activity in the BBB is mediated by chronic alteration of its set-point during or shortly after asphyxia. This has serious clinical implications, namely that if amiloride or other more specific NHE blockers were to be administered to postasphyxia babies as a possible therapeutic drug, timing the administration becomes a critical and limiting factor (Kendall et al., 2006).

5.7 GRN alters the course of recovery

GRN consists of resuscitating the neonate with gas mixtures that contain an atmospheric level of O2, but a higher level of CO2 which is then progressively withdrawn. In the present model, after 9/20 asphyxia or 4/20 asphyxia, normoxia was restored immediately but normocapnia was restored gradually, so that the pup inhaled 10 % CO2 in air for thirty minutes, 5 % CO2 in air for a further thirty minutes, and finally, room air (see Figure 1, page 30). An exciting finding of the present study was that in the animals treated with GRN after 9/20 asphyxia and 4/20 asphyxia (9-GRN and 4-GRN respectively), seizure burden was greatly reduced. Indeed, cumulative seizure burden after asphyxia was reduced by a remarkable 85 % by the simple operation of GRN in both the 9 % and 4 % models (2.2. and 4.4 LRR/pup after 9-GRN and 4-GRN respectively, P < 0.0001 for 9/20 asphyxia vs. 9-GRN and 4/20 asphyxia vs. 4-GRN, Study III, Figure 1). Recovery-associated brain pHo and intraneuronal pH shifts towards the alkaline were greatly attenuated when GRN was applied. Brain pHo in 9-GRN increased at slower rates compared to 9/20 asphyxia. At the end of the observational period brain pHo was 7.34 ± 0.02, significantly less than brain pHo two hours after 9/20 asphyxia which, to reiterate from section 5.2, was 7.60 ± 0.01 (P < 0.0001, Study I, Figure 2). Similarly, while intraneuronal pH had increased by 0.27 ± 0.12 above baseline two hours after 9/20 asphyxia, intraneuronal pH in the 9-GRN paradigm at the same time point was increased by only 0.03 ± 0.1 (P < 0.0001, Study II, Figure 1). Asphyxia was associated with a large and progressive seizure burden that was tightly correlated with progressive brain


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alkalinization after asphyxia, and GRN effectively aborted this process by suppressing sudden changes in brain pH (Study I, Figure 3). These data raise two related postulates. The first is that brain alkalosis after birth asphyxia and consequent seizures are likely to be triggered by a fast restoration of normocapnia. In view of this, the current neonatal resuscitation protocols, which do not directly address how quickly and in what manner normocapnia is to be restored, appear dangerous. Thus, the question of how to clinically manage fast shifts in PaCO2 known to occur after birth asphyxia becomes, indeed, crucial (Engle et al., 1999). The second postulate is that if normocapnia is only gradually restored after birth asphyxia, in other words if GRN is applied, the downstream deleterious effects of brain alkalosis and seizures can be avoided.

5.8 Behavioral and neurologic outcome after asphyxia and GRN

A high seizure burden after birth asphyxia is known to be associated with poor neurodevelopmental outcome (see sections 2.3.4 and 2.7.2). In Study III a representative set of behavioral tests was engaged to see if the greatly diminished seizure burden following GRN would be associated with improved outcome in the present model of birth asphyxia, and thereafter, justify further investigation and clinical trials. The main findings (Study III, Figures 2 and 3) were: (i) a large seizure burden in the postasphyxia period was associated with negative neurodevelopmental outcome, manifest as compromised sensorimotor coordination, altered emotional reactivity to acute stress, diminished inhibition of fear-motivated behavior, impaired memory and learning, abnormal social interaction, and increased seizure susceptibility; (ii) these negative effects on behavior were more severe after 4/20 asphyxia, compared to 9/20 asphyxia; and (iii) in line with our previous observation that GRN after birth asphyxia dramatically reduced the postasphyxia seizure burden, we found that GRN also resulted in life-long and vastly improved neurodevelopmental outcome, virtually abolishing the deficits that followed an immediate restoration of normocapnia. To the best of my knowledge, GRN is the only maneuver after an asphyxic insult which results in a striking reduction in seizure susceptibility in adult life (Study III, Figure 2F).


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These findings are encouraging. The significance is that weaning a neonate after intrapartum asphyxia into a normocapnic state, rather than immediately establishing normocapnia, attenuates brain alkalosis and seizures, with favorable long-term consequences on development.

5.9 Translational implications of the study

The present model of birth asphyxia was designed to emulate the clinical condition and to investigate the impact of respiratory management on the pathophysiology and outcome of birth asphyxia. The findings have translational implications of direct importance to neonatal resuscitation paradigms. To facilitate comparisons between the present observations and clinical data, we examined blood pH after 9/20 asphyxia and 9-GRN (Study I, Figure 5). Not surprisingly, blood samples collected immediately after experimental birth asphyxia indicated profound acidemia and hypercapnia, mimicking the clinical condition (Armstrong and Stenson, 2007). Interestingly, a large decrease in blood ionized [Ca2+] after asphyxia was measured, a phenomenon known to occur in postasphyxia babies (Ilves et al., 2000). This drop in blood ionized [Ca2+] was suppressed by GRN, during the presence of increased concentration of H+ which compete with Ca2+ for shared binding sites (van Slyke et al., 1928). Importantly, GRN significantly decelerated both the sudden, large drop in blood partial pressure of CO2, and the rapid recovery from acidemia measured after birth asphyxia. It has recently been suggested that elevated levels of partial pressure of CO2 may be beneficial for neonatal vitality after birth asphyxia (Kro et al., 2013). Conversely, severe hypocapnia in the postasphyxia period was associated with adverse outcome (Klinger et al., 2005). The present study argues for a robust association between immediate restoration of normocapnia and pronounced brain alkalosis, seizure promotion, and poor neurodevelopmental outcome. Consistent with the present results, magnetic resonance spectroscopy following severe human birth asphyxia has shown an abnormally high brain intracellular pH (Robertson et al., 2002). A high magnitude and prolonged duration of alkalosis are predictive of poor neurodevelopmental outcome (Robertson et al., 1999). The elevated intraneuronal and extracellular brain pH in the present study as well as the elevated intracellular pH in the newborn human brain after birth asphyxia


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(Robertson et al., 2002) persist beyond the time of normalization of the postasphyxia acid–base parameters in the blood (Brouillette and Waxman, 1997). Previous work from our laboratory has shown that brain alkalosis is a mechanistic trigger of EEG and behavioral seizures in experimental and clinical infantile febrile seizures (Schuchmann et al., 2006; Schuchmann et al., 2009; Schuchmann et al., 2011). A therapeutic strategy was proposed for suppression of febrile seizures, where the percentage of inhaled CO2 is increased to ameliorate hyperventilation-induced brain alkalosis (Schuchmann et al., 2006). In another study by us, 5 % inhaled CO2 effectively suppressed seizures in human patients and macaque monkeys (Tolner et al., 2011). The present data suggest a putative therapeutic strategy to ameliorate the fast, alkalosis-promoting loss of CO2 that takes place during recovery from asphyxia. It is important to note that while these three lines of study into disorders of brain excitability explored by the Laboratory of Neurobiology incorporate the powerful effects of CO2 on brain function (Lennox et al., 1936; Pollock, 1949; Woodbury and Karler, 1960) into putative therapeutic strategies, the mechanisms underlying the disorders, and the rationale of utilizing CO2 in a particular therapy, are different. As a resuscitation strategy, GRN addresses the underlying pathophysiology of birth asphyxia during its critical window of opportunity. The decision to treat a baby for birth asphyxia with hypothermia, can potentially be taken within a minute of life (Kattwinkel et al., 2010). It is therefore feasible to incorporate GRN into current neonatal resuscitation protocols. In practice, GRN can be easily applied. If the baby is breathing spontaneously, inhaled air can be altered using a mask or hood. If the baby is mechanically ventilated, normocapnia can be gradually restored by gas mixtures in the ventilator input, controlling ventilator settings, or by a combination of both. Controlling ventilator settings to allow for ‘permissive hypercapnia’ is a lung-protective strategy (Ambalavanan and Carlo, 2001). Individualization of therapy is a recognized and highly recommended concept in neonatal management (White, 2011) and GRN is easily amenable to individualization.

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6 Conclusion

Since Little first described the disease state more than 150 years ago (Little, 1861), birth asphyxia has remained defiant, unyielding to modern medical management (Shalak and Perlman, 2004). Current neonatal resuscitation protocols are oriented towards achieving specific O2 saturations at certain, short, time intervals (Kattwinkel et al., 2010). Comprehensive recommendations on how to manage CO2 during this highly critical period are conspicuously absent (Wikström et al., 2011). This is a major flaw, particularly given the central role of the CO2/HCO3

- buffer system in pH homeostasis, and that birth asphyxia is a disease that presents, fundamentally, as an acid-base disturbance (Georgieva et al., 2013). The present study offers a novel model, in which the pathophysiology of birth asphyxia can be investigated, and a putative therapeutic strategy which directly addresses postasphyxia brain pH changes. The main findings of the present study are: (i) exposure to combined hypoxia and hypercapnia, in other words asphyxia, leads to pronounced seizures; (ii) seizure burden is dependent on rapid brain alkalinization in the postasphyxic period; (iii) postasphyxia brain alkalosis and the associated seizures are attributable to an enhanced efflux of acid equivalents from the brain that is suppressed by blocking Na/H exchange located in the blood–brain barrier; (iv) experimental birth asphyxia is associated with adverse neurodevelopmental outcome; and (v) brain alkalosis and seizures can be dramatically attenuated by controlling the rate of restoration of normocapnic conditions using a graded decrease in inhaled CO2, and such a maneuver is associated with a large and favorable improvement in neurodevelopmental outcome. Antiepileptic drugs do not control more than half of neonatal seizures and have been shown to have serious, life-long consequences on development (Painter et al., 1999; Cross, 2010). An increasing amount of evidence cautions against drug interventional strategies prescribed in neonatal resuscitation (Iacovidou et al., 2012). Disappointing results with hypothermia have prompted calls for rational therapies to be used in the management of birth asphyxia (Johnston et al., 2011). Furthermore, hypothermia is expensive and technically challenging to setup and maintain, and a hospital or center will be limited by the number of devices available. Algorithms and apparatus that can be adapted to the limited

Conclusion

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resources in developing countries, where birth asphyxia is most prevalent, are urgently needed (Hofmeyr et al., 2009). The findings of the present study raise the important and worrying question of whether standard resuscitation protocols where normocapnia is established rapidly in fact promote seizures. The putative therapeutic strategy, graded restoration of normocapnia, establishes normocapnia gradually. It is likely to be safe, effective, and easily tailored to an individual neonate. Birth asphyxia imposes an immense burden on individuals, families, and societies, and repeated calls for action have been made (Martines et al., 2005; Lawn et al., 2006; Lawn et al., 2009). The potentially profound and far-reaching impact of graded restoration of normocapnia as a therapy for birth asphyxia seizures, its simplicity, validity, and low cost, and the encouragingly favorable outcome in our model, are compelling reasons to pursue clinical research.

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Acknowledgements The study was carried out at the Laboratory of Neurobiology, Department of Biosciences, University of Helsinki, Finland. Two-photon microscopy was carried out at the University of Maryland, College Park, USA. Kai and Juha for teaching me and sharing with me their enthusiasm. No amount of thanks will suffice to express my gratitude for your mentoring. Professor Letten Saugstad for encouraging me, and the Letten Foundation for financially supporting my study. Eva for always being there. Katri for hovering over me. Else for teaching me. Sinikka for her expertise. Petteri for very constructive discussions. Merle for her good-humored assistance. Sissi and Chrisse for being cooperative. Patrick and Paul for being nothing less than super-cool when I visited. Nikolai for neatly solving technical issues. Miklós for his insights. Alex, Ramil, and Stas for practical tips. Henri, Aleksander, and everyone else for support and friendship. Waugh for sharing with me his fascination with all things living and how they go about doing it – may your soul rest in peace you old grouch. Pyne for the words. Gill for not panicking. Sabine for laughing with me through thick and thin. Ahmed and Mahmoud, for egging me on, and for providing me with the opportunity to play uncle. Kata, Aksu, and the gang for warmly embracing me into the madhouse. Darsh and Lotta for living hockey. Jenna for sushi and all that came with it. Katarina for the music. Sarah for courage. Mother. Father. Wish you were here.

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ORIGINAL ARTICLE

Brain Alkalosis Causes Birth AsphyxiaSeizures, Suggesting Therapeutic Strategy

Mohamed M. Helmy, MD,1 Else A. Tolner, PhD,1 Sampsa Vanhatalo, MD, PhD,2

Juha Voipio, PhD,1 and Kai Kaila, PhD1,3

Objective: The mechanisms whereby birth asphyxia leads to generation of seizures remain unidentified. To study thepossible role of brain pH changes, we used a rodent model that mimics the alterations in systemic CO2 and O2

levels during and after intrapartum birth asphyxia.Methods: Neonatal rat pups were exposed for 1 hour to hypercapnia (20% CO2 in the inhaled gas), hypoxia (9%O2), or both (asphyxic conditions). CO2 levels of 10% and 5% were used for graded restoration of normocapnia.Seizures were characterized behaviorally and utilizing intracranial electroencephalography. Brain pH and oxygen weremeasured with intracortical microelectrodes, and blood pH, ionized calcium, carbon dioxide, oxygen, and lactatewith a clinical device. The impact of the postexposure changes in brain pH on seizure burden was assessed during 2hours after restoration of normoxia and normocapnia. N-methyl-isobutyl-amiloride, an inhibitor of Naþ/Hþ exchange,was given intraperitoneally.Results: Whereas hypercapnia or hypoxia alone did not result in an appreciable postexposure seizure burden,recovery from asphyxic conditions was followed by a large seizure burden that was tightly paralleled by a rise inbrain pH, but no change in brain oxygenation. By graded restoration of normocapnia after asphyxia, the alkalineshift in brain pH and the seizure burden were strongly suppressed. The seizures were virtually blocked bypreapplication of N-methyl-isobutyl-amiloride.Interpretation: Our data indicate that brain alkalosis after recovery from birth asphyxia plays a key role in thetriggering of seizures. We question the current practice of rapid restoration of normocapnia in the immediatepostasphyxic period, and suggest a novel therapeutic strategy based on graded restoration of normocapnia.

ANN NEUROL 2011;69:493–500

Treatment of seizures is a priority in hypoxic-ischemic

encephalopathy of birth asphyxia,1,2 but neonatal

seizures are notoriously difficult to suppress. Current

anticonvulsant medication is largely ineffective,3 and it is

therefore necessary to seek approaches tailored to neona-

tal physiology and pathophysiology. Asphyxia implies a

local or global decrease in O2 and an accumulation of

CO2 and other end products of energy metabolism, such

as lactate. Intrapartum asphyxia is a persistence of this

abnormal state, exposing the fetus or newborn to hypoxia

and hypercapnia with significant acidosis.4 Profound

metabolic acidosis (pH < 7.00) is a defining characteris-

tic of perinatal asphyxia.5

An unexpected observation based on magnetic reso-

nance spectroscopy in human postasphyxic neonatal

encephalopathy (NE) was that, after a delay of 8 to 24

hours from birth, brain intracellular pH (pHi) was more

alkaline than under control conditions.6 A subsequent

study showed that the alkaline pHi level after NE can

persist for 2 weeks, and that a high magnitude and pro-

longed duration of the alkalosis are predictive for poor

clinical outcome.7 These are interesting observations,

because (1) it is generally known that pH plays a key

role in modulating neuronal survival after trauma (where

acidosis is generally protective8–10) and (2) the excitabil-

ity of brain tissue is known to be highly sensitive to

changes in pH, whereby acidosis suppresses neuronal ac-

tivity, whereas alkalosis has an opposite effect.11–15

Resuscitation or spontaneous recovery of an

asphyxiated neonate after breathing of normal (or

View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.22223

Received Mar 3, 2010, and in revised form Jul 12, 2010. Accepted for publication Aug 6, 2010.

Address correspondence to Dr Kaila, Department of Biosciences, P.O. BOX 65 (Viikinkaari 1), 00014 University of Helsinki, Helsinki, Finland.

E-mail: [email protected]

From the 1Department of Biosciences, University of Helsinki; 2Department of Clinical Neurophysiology, Children’s Hospital, Helsinki University Central

Hospital; 3Neuroscience Center, University of Helsinki, Helsinki, Finland..

Additional Supporting Information may be found in the online version of this article.

VC 2011 American Neurological Association 493

oxygen-enriched) air leads to abrupt changes in the acid-

base balance at the systemic level, as is clearly seen in

measurements of blood gas parameters.16 However, it is

not known whether the increase in brain pH that takes

place during recovery from asphyxia plays a role in the

triggering of seizures. Addressing this question would not

only shed light on the basic mechanisms of generation of

postasphyxic neonatal seizures, but would also provide

further insight into putative therapeutic strategies based

on controlling brain pH.17

Unraveling the mechanistic links among brain

trauma, pH changes, and induction of seizures is a chal-

lenging task for experimental work, calling for suitable

experimental models. In the present study, we developed

a novel animal model of birth asphyxia that is not based

on structural damage of the brain and its vasculature.

Instead, our model relies on exposing postnatal day (P) 6

rats18 to gas mixtures containing various levels of CO2

and O2. Brain pH is measured using an intracortical ion-

sensitive microelectrode. We show here that, after induc-

ing asphyxic conditions with a simultaneous decrease in

O2 (to 9% in the inhaled gas) and increase in CO2

(20%), a fast restoration of normocapnia leads to a pro-

gressive brain alkalosis that is paralleled by pronounced

seizure activity, as monitored behaviorally and with intra-

cranial electroencephalographic (EEG) recordings.

Achieving normocapnia after asphyxia in a graded man-

ner by using 10% and 5% CO2 suppresses the alkaline

shift in brain pH and, strikingly, leads to a virtual abol-

ishment of seizures. These data suggest that (1) an

increase in brain pH plays a causal role in the generation

of acute postasphyxic neonatal seizures, and (2) to allevi-

ate seizure burden in the clinic, normocapnic conditions

should be established in a graded manner during sponta-

neous recovery or resuscitation.

Materials and Methods

Animals and Experimental ConditionsMale Wistar rat pups aged P6 (where P0 refers to the day of

birth) were used. All experiments were approved by the Local

Animal Ethics Committee of Helsinki University and the

National Animal Ethics Committee in Finland. Four groups of

pups (plus controls breathing room air only) were studied.

None of the animals died during the experiments. The total

number of animals used was 159. Three groups were exposed

for 60 minutes to altered ambient levels of CO2 and O2 at

constant experimental temperature (35�C), with appropriate

adjustments of N2 to keep the partial pressure of CO2, O2, and

N2 at 100% of atmospheric pressure, as follows (see inset in

Fig 1A): (1) hypoxia was induced by a decrease of O2 to 9%,

(2) hypercapnia by an elevation of CO2 to 20%, and (3) as-

phyxia by a simultaneous increase in CO2 (20%) and decrease

in O2 (9%). After the 60 minute change in ambient gas con-

centrations, the above groups were immediately re-exposed to

room air. In addition, a fourth experimental group was studied,

which experienced the asphyxic conditions as described above,

but re-establishment of normocapnia was made in a graded

manner by lowering the CO2 levels first from 20% to 10% for

30 minutes, then to 5% for a further 30 minutes, and finally

to virtually zero (room air).

In some experiments, N-methyl-isobutyl-amiloride

(Sigma-Aldrich, St Louis, MO; 2.5mg/kg) was injected intra-

peritoneally 30 minutes before asphyxia.19

The mean weight of the pups was 15.2g, and none of

them lost >0.1g during the experiments.

Behavioral Analysis of SeizuresLoss of righting reflex (LRR) occurs in seizures classified as

tonic-clonic.20 After pilot experiments and comparisons

between seizure scorings by experienced observers, we found

that LRR was the most robust and reproducible indicator for a

quantitative analysis of the total seizure burden. Although LRR

is the most violent expression of neonatal seizures in rodents

FIGURE 1: A high seizure burden is induced after asphyxicconditions on abrupt but not graded establishment ofnormocapnia. (A) Rat pups were exposed for 60 minutes to(a) hypoxia (n 5 6), (b) hypercapnia (n 5 8), or (c) asphyxicconditions (n 5 6) followed by immediate return tonormocapnic and normoxic conditions. In d, re-establishmentof normocapnia (n 5 6) was made in a graded manner (seeinset for experimental paradigms; t 5 0 min indicates the endof the 60-minute exposure to 20% CO2 and/or 9% O2). Notesuppression of the large postasphyxic seizure burden basedon the number of loss of righting reflexes (LRR) by gradedestablishment of normocapnia (c vs d). (B) Specimenrecording (paradigm c) shows the suppression of backgroundelectroencephalographic (EEG) activity by asphyxia and acharacteristic electrographic seizure associated with LRRs.EEG was recorded epidurally over parietal cortex. The breaksin the recording are 40 and 130 min, respectively.

ANNALS of Neurology

494 Volume 69, No. 3

and hence is given the highest score in a modified Racine sca-

lem20,21 it is not equivalent to the electroencephalographic sei-

zure burden, which would include less intense seizure activity.

It is, however, a reliable tool for comparison of seizure burden

between distinct experimental paradigms. The seizure burden

was quantified, from video recordings in an experiment blind

manner, as the cumulative number of LRRs during the 2-hour

observation period after the termination of the 60-minute expo-

sure to altered respiratory conditions (see above). Each inci-

dence of LRR was given a score of 1. The score was averaged

per pup, followed by calculation of cumulative seizure burden

with data points given at 1-minute intervals. Seizure quantifica-

tions based on LRRs were done in uninstrumented animals.

AnesthesiaAnesthesia was induced in an acrylic box with isoflurane (3-4% 1-

chloro-2,2,2-trifluoroethyl difluoromethyl ether, isoflurane, Baxter

Medical, Deerfield, IL), and maintained at 1.5 to 3% with a mask

using an Univentor 400 Anesthesia Unit (Agnthos, Stockholm,

Sweden). During the operation, the pup was kept on a Supertech

Thermal Pad set at 35�C on a Cunningham Mouse/Neonate Rat

Sterotaxic Adaptor (Harvard Apparatus, Holliston, MA).

Intracranial EEG RecordingsElectrodes were prepared as before17 with some modifications;

epidural EEG was measured from parietal cortex in freely mov-

ing rat pups with simultaneous video recording. Alternating

current recordings were made using a differential amplifier with

a bandwidth of 0.07Hz to 2kHz. The sampling rate was 5kHz.

A digital-to-analogue converter (DAC) board with 16-bit reso-

lution (National Instruments, Austin, TX) and acquisition soft-

ware based on LabView were used.

pH-Sensitive MicroelectrodespH-sensitive microelectrodes were prepared and calibrated as

described by Voipio and Kaila.22 Tip diameters were 4-8lm for

the ion-sensitive electrodes and 2-4lm for the reference electro-

des. The microelectrodes had a resistance of 8 to 12GO (in cal-

ibration solution and brain tissue in a head-fixed configuration)

and a slope of 55 to 60mV per pH unit. Electrometer ampli-

fiers had an input bias current <10fA. Two burr holes were

made, the first 1.6 to 2.0mm posterior to bregma and 1.6 to

2.0mm lateral to the midline, and the second 1.6mm posterior

and lateral to the first. The pH-sensitive electrode was inserted

through the anterior burr hole to a depth of about 500lm, and

the reference through the posterior burr hole to the same depth.

A silver-silver chloride ground electrode was inserted subcutane-

ously above the scapula. DAC-board and acquisition software

were the same as with the EEG recordings. Unpaired t test was

used to compare means. Values are given as mean 6 standard

error of the mean in the text and figures. Recordings were

started 15 minutes after recovery from anesthesia. The correla-

tion between DpH and normalized seizure burden was calcu-

lated using Spearman rank order correlation coefficient, with p

based on a 2-tailed test in accordance with the hypothesis of

alkalosis-promoted seizures.

Oxygen MicroelectrodesModified Clark-type polarographic oxygen microelectrodes23,24

(10lm, Unisense A/S, Aarhus, Denmark) were connected to a

high-impedance picoamperometer (Unisense A/S) and calibrated

in salines warmed to 35�C and equilibrated with 100% O2, air,

and 100% N2. They were left in degassed water for 20 to 30

minutes before insertion into the brain to a depth of 500lm.

Blood AnalysisGEM 400025 (Instrumentation Laboratory, Bedford, MA) was used

in trunk blood analyses of pH, calcium, partial pressure of oxygen

(pO2), partial pressure of carbon dioxide (pCO2), and lactate. One

hundred microliters of arteriovenous blood was collected by rapid

decapitation of the rat pups at 0, 30, 60, and 120 minutes. Statisti-

cal analysis was performed with R software26 using the NLME27

package. A model object was calculated using a linear model, and

an analysis of variance table was extracted. In the case of interaction

between treatment and time, the treatment effect was analyzed sepa-

rately for each time point. Comparisons between individual pairs of

treatments were done if the overall significance of the treatments

yielded p < 0.05. The GREGMISC26 package was used to fit these

contrasts to a model not including the interaction. p values from

multiple contrasts were adjusted using Holm’s procedure. For

details, see Supporting Information Table 1.

Results

Postasphyxia Seizures Are Induced during FastEstablishment of Normocapnic ConditionsExposing the rat pups to the various gas mixtures for 60

minutes did not produce marked behavioral effects, apart

from brief agitation on initiation of exposure. Seizures

were never seen under these conditions. However, there

were striking differences among the various groups in the

responses that took place after the termination of the

exposures. The start of this 2-hour recovery period is

defined as time zero in the figures.

Figure 1A shows the cumulative seizure burden for

all the paradigms. Control pups as well as pups exposed

to hypoxia (trace a, n ¼ 6) scored zero at all times.

Notably, immediately after ending the hypoxic conditions

(0 minutes), blood lactate levels increased from 1.0 60.06 to 4.8 6 0.8mM (n ¼ 6 for both groups; p <

0.01), demonstrating that the decrease in ambient O2

from room-air level (around 21%) to 9% was sufficient

to shift the energy metabolism of the P6 pups toward a

direction where anaerobic glycolysis plays a significant

role, which is a hallmark of asphyxia.28

The response seen after abrupt cessation of hyper-

capnia (trace b, n ¼ 8) yielded an average cumulative sei-

zure burden score of 1.4 LRRs/pup. In contrast, pups

exposed to asphyxic conditions (combined hypercapnia

and hypoxia) followed by immediate restoration of nor-

mocapnia (trace c, n ¼ 6) showed a very high seizure

Helmy et al: Birth Asphyxia Seizures

March 2011 495

burden, totaling 14.5 LRRs/pup. These results indicate

that the recovery from asphyxic conditions strongly acti-

vates mechanisms that promote seizures.

Based on the above data, we hypothesized that the

pronounced and progressive seizure burden seen after the

establishment of control conditions in the asphyxic group

might be caused by a fast increase in brain pH. Such an

effect on acid-base regulation is expected to take place after

the removal of excess ambient CO2 (20%) in combination

with the removal of the metabolic acid load (ie, production

of lactate) that was observed in pups exposed to 9% O2

(see above). Hence, we introduced yet another experimen-

tal paradigm, where the asphyxic conditions were followed

by a graded restoration of the ambient CO2 level from

20% first to 10% (for 30 minutes) and thereafter to 5%

(for 30 minutes), followed by room air (60 minutes). In

this paradigm with graded restoration of normocapnia, a

dramatic decrease (85%) in the cumulative postasphyxia

seizure burden was observed (trace d, n ¼ 6; a total of 2.2

LRRs/pup) when compared with the large seizure burden

after abrupt restoration of normocapnia (trace c).

To gain more insight into the seizures, we made ep-

idural EEG recordings from pups subjected to hypercap-

nia or asphyxia. In line with the extensive evidence dem-

onstrating that an acidosis suppresses neuronal

excitability (see Discussion), the baseline activity in the

EEG of pups exposed to hypercapnic or asphyxic condi-

tions was strongly suppressed (see Fig 1B), with the

peak-to-peak amplitude falling from 10 to 15lV under

control conditions to 5 to 7lV during hypercapnia or as-

phyxia (n ¼ 6). In agreement with the behavioral data,

no electrographic seizures were detected in the 2 of 2

pups recorded by EEG after exposure to hypercapnia. In

contrast to this, in 4 of 4 pups with abrupt restoration

of normocapnia after asphyxia, pronounced electro-

graphic seizures developed, with a duration of 1 to 5 sec-

onds, frequency of 12 to 14Hz, and amplitude of 50 to

200lV. Electrographic seizures were invariably detected

during LRRs, except for those obvious cases where EEG

recordings were dominated by movement artifacts

Postasphyxia Seizures Are Triggeredby Brain AlkalosisThe above data strongly suggest that postasphyxia seiz-

ures are triggered by a brain alkalosis, which would also

explain the differences in seizure burden after abrupt

FIGURE 2: A large alkalosis takes place in the brain after asphyxic conditions after abrupt (left panels) but not graded (rightpanels) establishment of normocapnia. (A) Experimental paradigms. (B) Specimen recordings of intracortical pH. (C) Averageddata for intracortical pH in experiments with abrupt (n 5 4) and graded (n 5 6) re-establishment of normocapnia. Bars indicate6 standard error of the mean. The time scale in C holds for A and B as well.

ANNALS of Neurology


versus graded restoration of normocapnia. To directly test

this idea, we performed measurements of intracortical

pH using Hþ-sensitive microelectrodes.

In the experimental group where asphyxic conditions

were followed by an abrupt establishment of normocapnia

(n ¼ 6; Fig 2), the pre-exposure baseline pH was 7.19 60.01. During asphyxic conditions, intracortical pH became

rapidly acidotic, attaining a minimum of 6.77 6 0.01, fol-

lowed by a small recovery toward the end of the asphyxic

conditions. Establishing the normoxic/normocapnic condi-

tions induced a fast increase in the intracortical pH at a

rate of 0.031/min during the first 5 minutes and 0.0054/

min during the final 115 minutes.

In the experimental group where asphyxia was fol-

lowed by graded restoration of normocapnia (n ¼ 4; see

Fig 2), the mean pre-exposure baseline pH was virtually

identical to the above group, 7.20 6 0.02. A key obser-

vation was that graded restoration of normocapnia

greatly attenuated both the rate and amplitude of the

postasphyxic alkalinization. Mean pH increased at a rate

that was much smaller than what was seen with the ab-

rupt restoration procedure, with an initial rate of 0.017/

min and at 0.0036/min during the final 115 minutes. At

the end of the observational period, brain pH was much

more alkaline after the abrupt restoration paradigm (7.60

6 0.01) when compared with graded restoration (7.34

6 0.02; p < 0.0001).

The robust correlation between postasphyxic brain

pH changes and seizure burden that is obvious in the

above data is illustrated in Figure 3, where mean values

of seizure burden (compare with Fig 1) are plotted as a

function of deviations of brain pH from its control level

(dotted lines in Fig 2C). The dependence of seizure bur-

den on brain pH shows a correlation of 0.96 (p <0.0001) and 0.99 (p < 0.0001) after graded and abrupt

establishment of normocapnia, respectively. As a whole,

these data clearly show that the magnitude of the alkaline

shifts in the 2 experimental groups is a major factor

determining the postasphyxia seizure burden. This con-

clusion gained further support from experiments that

showed that LRRs evoked in the abrupt-restoration para-

digm were terminated, and seizures were never observed,

after application of 5% CO2 in air (n ¼ 6). Further-

more, N-methyl-isobutyl-amiloride given intraperitone-

ally 30 minutes before asphyxia21 led to strong suppres-

sion (by 83%) of the cumulative seizure burden, to

2.5 LRRs/pup (n ¼ 4).

Postasphyxia Seizures Are Not Triggered byReduced Brain OxygenationTo rule out the possibility that the alkalosis induced con-

striction of brain microvessels,29 thereby leading to

hypoxic seizures,30 we measured intracortical pO2 (Fig 4;

n¼6). Under control conditions, the mean value was 10

6 1.18mmHg and, during asphyxia, pO2 fell by 5 to

9mmHg. Changes in pO2 relative to control levels are

shown in Figure 4B. After asphyxia (with abrupt restora-

tion of normocapnia), a transient elevation in pO2 was

observed that is most likely attributable to a delay

induced by readjustment of ventilation after the exposure

to low pO2 and elevated pCO2.31,32

Changes in Brain pH Are Paralleled by Changesin Blood ParametersTo facilitate comparisons between the present observa-

tions and clinical data, we examined blood pH in the

FIGURE 3: Postasphyxia seizure burden shows a steep androbust dependence on changes in brain pH. The data aretaken from time points within 0 to 120 minutes shown inFigs 1A and 2C. Black squares and red circles in the scatterplots represent values measured during abrupt and gradedrestoration of normocapnia, respectively.

FIGURE 4: Asphyxia leads to a fast fall in brain pO2, andrecovery is associated with an initial transient increaseabove control level. (A) Specimen recording of intracorticalpO2. (B) Averaged data for intracortical changes in pO2(DpO2) in the experimental paradigm with abruptrestoration of normocapnia (n 5 6).


March 2011 497

abrupt and graded restoration paradigms. The blood and

brain pH data are plotted in Figure 5. Both the maxi-

mum acidosis (end of asphyxia) and alkalosis have signif-

icantly smaller amplitudes in the blood as compared to

the brain. The maximum acidosis observed in blood im-

mediately after asphyxia (0 minutes), 7.25 6 0.02, is sig-

nificantly lower than control pH, 7.42 6 0.02, (p <0.0001), and the maximum alkalosis, 7.49 6 0.02 at 60

minutes, is significantly higher than control pH, (p ¼0.0028). At 120 minutes, blood pH was 7.45 6 0.02,

which is significantly higher than normal, (p ¼ 0.0214).

However, it is important to note that the patterns of the

pH changes in the brain and blood are qualitatively

similar.

Statistical analysis of the blood parameters showed

that treatment, time, and the interaction between treatment

and time undergo statistically significant variation, except

for pO2, where interaction yielded nonsignificant results,

but treatment results were significant. The large decrease in

blood ionized calcium due to abrupt restoration of normo-

capnia, which is also seen in asphyxiated babies,33 was sup-

pressed by graded restoration of normocapnia (see Fig 5).

In a manner consistent with the blood pH changes, pCO2

was higher during the graded restoration of normocapnia

paradigm. Abrupt restoration of normocapnia resulted in a

prolonged postasphyxic elevation in pO2. Detailed data

with statistical comparisons among data points are pro-

vided in Supporting Information Table 1.

Discussion

Brain pH changes are known to be intimately involved in

the modulation of neuronal excitability and in the control

of cell death following trauma.10–14,28,34 The present rat

pup model of neonatal birth asphyxia was designed to elu-

cidate the role of the alkaline pH change in triggering seiz-

ures, in a manner where the immediate, confounding

effects of neuronal damage on excitability are excluded.

Our main observations include that (1) moderate hypoxic

(9% O2) or hypercapnic (20% CO2) conditions do not

lead to seizure generation. However, (2) exposure to com-

bined hypoxia and hypercapnia (which mimics asphyxia)

leads to pronounced postasphyxic seizures that show a

steep dependence on brain pH, but not on brain pO2.

Indeed, the most important observations in the present

FIGURE 5: Changes in brain pH and blood parameters (pH,ionized calcium, pCO2, and pO2) during and after asphyxia inthe paradigms with abrupt and graded restoration ofnormocapnia. From top to bottom: Brain pH changes inducedby asphyxia followed by abrupt establishment of nor-mocapnia are paralleled by much smaller pH changes inarteriovenous blood samples. Data points show brain (dottedlines with green and red circles for abrupt and gradedrestoration of normocapnia, respectively) and blood (black,green, and red squares for control group, abrupt, and gradedrestoration of normocapnia, respectively) pH values. Barsindicate 6 standard error of the mean. The large decrease inblood ionized calcium (iCalcium) due to abrupt restoration ofnormocapnia (green squares) is abolished by gradedrestoration of normocapnia (red squares). Graded restorationof normocapnia causes a gradual decrease in blood pCO2levels. Abrupt restoration of normocapnia causes aprolonged postasphyxic elevation in blood pO2. n 5 4 to 9pups for each paradigm and time point.

~

ANNALS of Neurology


study were that (3) the seizure burden was strictly related

to postasphyxic brain alkalosis and (4) this alkalosis could

be dramatically attenuated (by 85%) simply by controlling

the rate of restoration of normocapnic conditions using a

graded decrease in ambient CO2.

The conclusion that the postasphyxic seizures were

directly caused by cortical pH recovery and subsequent

alkalosis is supported by (1) the much higher seizure bur-

den seen on abrupt versus graded restoration of normo-

capnia (see Fig 3); (2) the rapid termination of seizures

observed when applying 5% CO2 as such; and (3) the

dramatic reduction in seizures by the Naþ/Hþ exchange

inhibitor N-methyl-isobutyl amiloride.19 Further support

comes from our previous work,17 where injection of so-

dium bicarbonate in neonatal rat pups caused a rise in

brain pH that was accompanied by seizure activity.

Our work shows that graded restoration of normo-

capnia greatly reduces abnormal neuronal activity, and a

therapy that causes a significant reduction in seizure bur-

den is expected to improve outcome after birth asphyxia.

Extrapolating from our present results suggests that after

birth asphyxia in human neonates, the high CO2 level

that is invariably observed in blood samples should not

be corrected in an aggressive manner to establish fast

normocapnia, because a pronounced alkaline rebound

and consequent triggering of seizures is likely to occur.

In light of the present data, it would be advisable to es-

tablish normocapnia in a graded manner. It is widely

known that profound and acute changes in the arterial

CO2 partial pressure and acid-base balance occur in the

presence of fetal acidemia, but the rationale behind cur-

rent protocols of rapid correction is unclear.35 Moreover,

brain alkalosis is associated with neuropathological out-

come in human neonates,6,7 a conclusion that gains sup-

port from work on piglets.36 Another benefit of graded

restoration of CO2 in neonates and especially in preterm

infants is that hypercapnia has been shown to protect

ventilated newborns’ lungs from damage, probably by

allowing the use of a low tidal volume strategy.37

Optimum neuroprotection after NE may requirethe use of more than one therapy,21 such as hypother-mia38 and application of xenon.39 Interestingly, in P6rats after hypoxia-ischemia, under both normo- andhypothermic conditions, xenon was shown to cause CO2

retention and a decrease in pH that is probably involvedin the observed reduction of brain injury.40

Regarding the molecular mechanisms of the delayed

alkalosis seen after birth asphyxia, there is substantial evi-

dence that stimulation of acid extrusion by the Naþ/Hþ

exchanger plays a key role.19,41,42 Interestingly, the inhibitor

of the Naþ/Hþ exchanger, N-methyl-isobutyl-amiloride, has

been shown to ameliorate brain injury when applied before

hypoxic-ischemic conditions in neonatal mice.19 The brain

is a multicompartment system and, under in vivo condi-

tions, systemic regulation of pH by respiration and kidney-

based mechanisms has a significant role in shaping the

extracellular and intracellular pH.43 Hence, the most parsi-

monious way to explain the brain alkaloses in the present

and previous studies6,7 is to postulate a net loss of acid from

brain tissue that elevates both intra- and extracellular pH.44

In agreement with such a unifying explanation, we found

that applying N-methyl-isobutyl-amiloride before19 the start

of the experimental asphyxia led to a dramatic decrease in

the cumulative seizure burden.

In summary, our results show that birth asphyxia can

lead to a severe alkalosis of brain tissue and subsequent

seizures in the absence of experimentally induced brain

lesion. The data also suggest a putative therapeutic strategy

to reduce the fast, alkalosis-promoting loss of CO2 that

takes place during recovery from asphyxia. An analogous

therapeutic strategy was proposed for suppression of febrile

seizures, where brain alkalosis may play a crucial role.17 In

practice, an easy way to block the postasphyxic seizures

would be to apply air with CO2 in declining steps during

the initial phase of recovery, which is readily achieved

using a mask or hood for ventilatory gas delivery. Obvi-

ously, such a graded restoration of normocapnia does not

exclude a prompt restoration of normoxia.

Acknowledgment

This work was supported by grants from the Letten

Foundation (MH), the Academy of Finland

(KK,EAT,SV), the Sigrid Juselius Foundation (KK, JV,

SV), and the Jane and Aatos Erkko Foundation (KK).

K.K. is a member of the Finnish Center of Excellence in

Molecular and Integrative Neuroscience Research.

We thank Drs S. Andersson, M. Metsaranta, and

E. Ruusuvuori for stimulating discussions, and Dr T.

Kotilainen for assistance in statistical analyses.

Potential Conflicts of Interest

Nothing to report.

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Supplementary Table 1. Title: Blood parameters.

Values sharing the same letter within a row are not significantly different (p > 0.05).

For all the parameters, treatment df = 2, time df = 3 and interaction df = 5.

BRAINA JOURNAL OF NEUROLOGY

Acid extrusion via blood–brain barrier causes brainalkalosis and seizures after neonatal asphyxiaMohamed M. Helmy,1,2 Eva Ruusuvuori,1,2 Paul V. Watkins,3 Juha Voipio,1 Patrick O. Kanold3,4

and Kai Kaila1,2

1 Department of Biosciences, University of Helsinki, Helsinki, Finland

2 Neuroscience Centre, University of Helsinki, Helsinki, Finland

3 Department of Biology, University of Maryland, College Park, MD 20742, USA

4 Institute for Systems Research, University of Maryland, College Park, MD 20742, USA

Correspondence to: Kai Kaila,

Department of Biosciences,

University of Helsinki, P.O. Box 65 (Viikinkaari 1),

00014 Helsinki, Finland.

E-mail: [email protected].

Birth asphyxia is often associated with a high seizure burden that is predictive of poor neurodevelopmental outcome.

The mechanisms underlying birth asphyxia seizures are unknown. Using an animal model of birth asphyxia based on 6-day-old

rat pups, we have recently shown that the seizure burden is linked to an increase in brain extracellular pH that consists of the

recovery from the asphyxia-induced acidosis, and of a subsequent plateau level well above normal extracellular pH. In the present

study, two-photon imaging of intracellular pH in neocortical neurons in vivo showed that pH changes also underwent a biphasic

acid–alkaline response, resulting in an alkaline plateau level. The mean alkaline overshoot was strongly suppressed by a graded

restoration of normocapnia after asphyxia. The parallel post-asphyxia increase in extra- and intracellular pH levels indicated a net

loss of acid equivalents from brain tissue that was not attributable to a disruption of the blood–brain barrier, as demonstrated by a

lack of increased sodium fluorescein extravasation into the brain, and by the electrophysiological characteristics of the blood–brain

barrier. Indeed, electrode recordings of pH in the brain and trunk demonstrated a net efflux of acid equivalents from the brain across

the blood–brain barrier, which was abolished by the Na/H exchange inhibitor, N-methyl-isobutyl amiloride. Pharmacological

inhibition of Na/H exchange also suppressed the seizure activity associated with the brain-specific alkalosis. Our findings

show that the post-asphyxia seizures are attributable to an enhanced Na/H exchange-dependent net extrusion of acid equivalents

across the blood–brain barrier and to consequent brain alkalosis. These results suggest targeting of blood–brain barrier-mediated

pH regulation as a novel approach in the prevention and therapy of neonatal seizures.

Keywords: birth asphyxia; neonatal seizures; resuscitation; pH; Na/H exchange

Abbreviations: BCECF = 20,70-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein; MIA = N-methyl-isobutyl-amiloride;VBBB = blood-brain barrier potential

IntroductionSeizures are common in the newborn baby and most frequently

occur as a result of birth asphyxia. Increased morbidity andmortality,

brain injury and poor neurodevelopmental outcome have been

associated with a higher seizure burden (McBride et al., 2000;

Lingwood et al., 2003; Haynes et al., 2005; Silverstein and Jensen,

2007; Lindstrom et al., 2008; Bjorkman et al., 2010; Stolp et al.,

2012). Directly addressing neonatal seizures is therefore a priority,

but current drugs are largely ineffective and may have serious side

doi:10.1093/brain/aws257 Brain 2012: 135; 3311–3319 | 3311

Received January 30, 2012. Revised August 4, 2012. Accepted August 5, 2012� The Author (2012). Published by Oxford University Press on behalf of the Guarantors of BrainThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-

commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected].

effects on the neonatal brain (Rennie and Boylan, 2003; Evans et al.,

2007; Rennie and Boylan, 2007). The molecular and cellular mech-

anisms underlying birth-asphyxia seizures are unknown, but it is ob-

vious that information on the seizure-triggering mechanisms is of

crucial importance in the design of novel therapeutic strategies.

A profound acidosis (blood pH 7.00 or lower) at the time of

birth is an essential criterion in the diagnosis of perinatal asphyxia

(Poland and Freeman, 2002). However, birth asphyxia seizures do

not coincide with the maximal blood acidosis but are typically first

observed during the recovery period after a delay that usually

ranges in human babies from 2 to 16h (Shalak and Perlman,

2004). A wide range of observations have shown that changes

in extra- and intracellular pH exert a strong modulatory effect on

brain excitability under normal and pathophysiological conditions,

whereby an alkalosis enhances excitability while an acidosis has an

opposite effect (Siesjo, 1985; Chesler, 1990; Kaku et al., 1993;

Kaila, 1994; Casey et al., 2010; Enyedi and Czirjak, 2010;

Ruusuvuori et al., 2010). This underscores the crucial role of

brain pH regulation in seizure generation (Schuchmann et al.,

2006, 2011; Ziemann et al., 2008; Tolner et al., 2011).

Using an animal model of birth asphyxia based on 6-day-old rat

pups, we recently showed that the post-asphyxia seizure burden

was tightly linked to an increase in brain extracellular pH during

the recovery from the asphyxia-induced acidosis and its subsequent

alkaline overshoot, which reached values that were well above the

normal brain pH level (Helmy et al., 2011). Several lines of evidence

showed that this alkaline shift had a direct, causal role in seizure

generation. Moreover, the post-asphyxia alkalosis was dramatically

reduced by restoring the CO2 levels down to normal in a slow,

controlled manner. Accordingly, this ‘graded restoration of normo-

capnia’ virtually abolished the seizures, suggesting a novel thera-

peutic manoeuvre for suppression of birth-asphyxia seizures.

It is not known how post-asphyxia brain alkalosis is caused. In the

present study we used two-photon imaging of intracellular pH in

neocortical neurons in vivo, which revealed an intraneuronal alkalosis

after asphyxia, with a time course and sensitivity to graded restor-

ation of normocapnia that were similar to what we described before

for extracellular alkalosis (Helmy et al., 2011). The post-asphyxia

brain alkalosis is generated in the absence of a rise of pH in the

trunk and blood. Thus, the rise in both extracellular and intracellular

pH recorded in the brain implies a net loss of acid equivalents and is

attributable to net extrusion of acid across the blood–brain barrier.

This conclusion gained direct support from measurements of the

pH gradient across the blood–brain barrier. The post-asphyxia

increase in blood–brain barrier acid extrusion was inhibited by

systemically administered pharmacological blockers of Na/H

exchange, which notably, also abolished post-asphyxia seizures.

Materials and methods

In vivo measurement ofintraneuronal pHMale Sprague-Dawley rat pups, postnatal Day 6–7 (where Day 0

refers to the day of birth) were used. All experiments were approved

by the University of Maryland Institutional Animal Care and Use

Committee. Animals were anaesthetized during surgery with isoflurane.

A small, bloodless craniotomy (3mm in diameter) was made above

parietal cortex. A small tear was made in the dura, and the exposed

brain was covered with physiological saline, described in Stosiek et al.

(2003). For in vivo imaging, anaesthesia was maintained with 0.7–

0.9% isoflurane in air or in special gas mixtures (as described later)

applied through a funnel loosely placed on the muzzle. Body tempera-

ture was maintained by a heating pad at 35�C throughout the

experiments. The pups were first exposed for 60min to 20% CO2

and 9% O2 (asphyxia). In one of the two groups, the pups breathed

air after asphyxia. In the other group, the experimental asphyxia was

followed by an immediate restoration of normoxia, but with a graded

re-establishment of normocapnia (Helmy et al., 2011), by lowering the

CO2 levels first from 20 to 10% (with appropriate adjustments of N2)

for 30min, then to 5% for a further 30min and finally to virtually zero

or room air (graded restoration of normocapnia).

Acetoxymethyl ester dye solution was prepared by dissolving 50 mgof 20,70-bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein (BCECF-

AM) in 4 ml of 20% pluronic acid in dimethyl sulphoxide (DMSO,

Invitrogen) and diluted (1:8) with physiological saline (Stosiek et al.,

2003). Glass pipettes (2–4 mm tip diameter) were pulled (Sutter P2000)

and loaded with dye solution. For bulk loading of cortical neurons,

pipettes were gradually advanced to a depth of �500 mm into the

parietal cortex under two-photon scanning mode, and the dye solution

was pressure injected as described by Bandyopadhyay et al. (2010).

After loading, the pipette was withdrawn from the cortex, and the

craniotomy was covered with warm agarose and a glass coverslip.

Imaging was done using a two-photon laser-scanning microscope

(Ultima IV, Prairie Technologies) with a Spectra Physics Mai Tai

Deep See Ti-Sapphire mode-locked femto-second laser. Layer 2/3

cells were imaged using a �20 water-immersion objective lens

(LUMPlanFI/IR Olympus 0.95NA) at a depth of 250–350 mm from

the surface of the cortex. Twelve frames per minute were acquired

and averaged; data were collected over the course of 4min, with

excitation at 825 nm (F825), and over 1min at 770 nm (F770) at a

higher light intensity to maximize signal-to-noise ratio. Images were

acquired using a 570 nm dichroic filter. Averaged frames were ana-

lysed using a custom software written in MATLAB. The averaged data

collected at 825 nm excitation was divided by the averaged data col-

lected at 770 nm to give the ratiometric intraneuronal pH measure-

ment of one 5min period (F825/F770).

Calibration of the BCECF signal was done in slice preparation.

Sprague-Dawley rat pups were anesthetized with isoflurane and

decapitated. Equipment and solutions used to prepare 200-mm thick

brain slices were described in Zhao et al. (2009). All physiological

solutions were equilibrated with carbogen (5% CO2, 95% O2) and

kept at 35�C. BCECF-AM (50mg) was dissolved in 4 ml of 20% pluro-

nic acid in DMSO and then diluted in physiological solution to a final

concentration of 5 mM. Slices were placed into the dye solution and

allowed to load for 20min. A slice was placed under the two-photon

microscope and first perfused with physiological solution, followed by

high K+/nigericin (Eisner et al., 1989) solutions with a of pH 7.4, 7.2,

7.0 and 6.8, equilibrated with carbogen. Superficial cells were imaged

using the paradigm described earlier. The calibration curve was linear

over the pH range tested. Although the linear slope is expected to

translate faithfully from the in vitro calibration to in vivo measure-

ments, this is not necessarily true for absolute pH values. Therefore,

the data on intracellular pH have been given as changes from the

baseline (see Fig. 1).

pH at the end of the two experimental paradigms was compared

using Student’s two-tailed unpaired t-test. Data are presented as

mean � SD.

3312 | Brain 2012: 135; 3311–3319 M. M. Helmy et al.

In vivo measurement of body and brainpH and blood–brain barrier potentialIn these and subsequent experiments, male Wistar rat pups (Day 6)

were used with approval by the National Animal Ethics Committee in

Finland. The in vivo intracranial pH measurements with H+-sensitive

microelectrodes were done as described previously (Helmy et al.,

2011). For simultaneous body pH measurements, small incisions were

made in the loose skin of the pup’s dorsum. Two 4 cm lengths of

polyvinyl chloride tube (outer diameter 0.6mm, inner diameter

0.2mm) were prepared as pH-sensitive and reference electrodes and

inserted into the incisions. The pH-sensitive body electrodes had the

same polyvinyl chloride-gelled membrane and backfilling solutions as

the intracranial pH microelectrodes (Voipio and Kaila, 1993), with a

H+-sensitive membrane column length of �1mm. The reference elec-

trode was filled with 0.5% agar in 0.9% NaCl.

Brain extracellular pH and body pH were measured in asphyxic con-

ditions (20% CO2, 9% O2 for 1 h) and for 2 h afterwards. N-methyl-

isobutyl-amiloride (MIA; Sigma-Aldrich) was given 2.5mg/kg

bodyweight intraperitoneally 30min before asphyxia (Kendall et al.,

2006; Helmy et al., 2011). The potential difference across the

blood–brain barrier (VBBB) was obtained as the difference of the ref-

erence electrodes in the brain and the body (Held et al., 1964; Woody

et al., 1970). Values of pH or VBBB in the two experimental paradigms

were compared using the Mann-Whitney U-test (two tailed). Data are

shown as mean � SD.

Assessment of blood–brain barrierpermeability using sodium fluoresceinFor the asphyxia group, 30min after termination of asphyxia, 4ml/kg

body weight of 10% sodium fluorescein (Sigma-Aldrich) was injected

intraperitoneally (Ferrari et al., 2010). Thirty minutes after sodium

fluorescein administration (Farkas et al., 1998), pups were anaesthe-

tized with isoflurane and then perfused with ice-cold 0.9% NaCl

through the left ventricle, with a cut in the right auricle for 25min,

a period of time that allows for the outflowing fluid to run clear.

Whole brains were dissected out, weighed and frozen in liquid nitro-

gen, and then kept at �80�C till further processing. For the negative

control group (with intact blood–brain barrier), rat pups were removed

from the nest and kept at 35�C for 90min, injected with sodium

fluorescein and, 30min later, processed as described earlier. For the

positive control group (with mannitol-induced damage of the blood–

brain barrier), rat pups were injected with sodium fluorescein; 30min

later, under isoflurane anaesthesia and on a heating pad set to 35�C, asmall thoracotomy was made to expose the tip of the left ventricle.

Over a period of 2min, 30 ml of 24% D-mannitol (w/w) in 0.9% NaCl

(Hooper et al., 1995) was infused into the left ventricle. The pups

were left under anaesthesia for a further 20min before being pro-

cessed, as described earlier.

Brain content of sodium fluorescein was measured as described

before (Lenzser et al., 2005) with some modifications as follows.

Brains were homogenized in 2.5ml of 50% trichloroacetic acid and

centrifuged for 15min at 10 000g. The supernatant was diluted with

5M NaOH, 1:0.8. Fluorometry was performed with a Victor2

(Perkin-Elmer) in plates with black frames and clear wells (IsoPlate

96, Perkin-Elmer).

Brain content of sodium fluorescein was compared between the

experimental paradigms using Mann-Whitney U-test (two-tailed).

Data are shown as mean � SD.

Behavioural assessment ofseizure burdenSeizure burden was quantified during the 2 h post-asphyxia period

from video recordings, based on the number of loss of righting

reflex in freely moving animals as described before (Helmy et al.,

2011). MIA was administered intraperitoneally 30min before the

onset of experimental asphyxia (Kendall et al., 2006). In some experi-

ments, amiloride (amiloride hydrochloride hydrate; Sigma-Aldrich), the

blood–brain barrier-impermeable parent drug of MIA (Sipos and Brem,

2000; Fisher, 2002; Liu et al., 2010), was administered at an identical

concentration. Each incidence of loss of righting reflex was given a

score of one. The scores for every pup were added to give the total

individual seizure burden. Seizure quantifications based on loss of

righting reflex were done in uninstrumented animals. Total seizure

burden was compared between the experimental paradigms using

the Mann-Whitney U-test (two-tailed). Data are shown as

mean � SD. Control experiments with vehicle alone (1.3mM acetic

acid in 0.9% NaCl, 100ml) did not have any effect.

Figure 1 An intraneuronal alkalosis is triggered after asphyxia and suppressed by graded restoration of normocapnia. The experimental

changes in inhaled CO2 and O2 are schematically shown above the recordings. (A) Two-photon in vivo measurements of intracellular pH

changes (mean �pH indicated by black line) in 40 layer 2/3 pyramidal neurons shown in colour from five Day 6–7 rat pups. Inset shows

BCECF-loaded neurons. Scale bar = 20 mm. (B) Intracellular �pH in 41 neurons from five rat pups in the graded restoration of normo-

capnia paradigm.

Neonatal asphyxia and blood–brain barrier function Brain 2012: 135; 3311–3319 | 3313

Effect of N-methyl-isobutyl-amilorideon acid extrusion in cortical pyramidalneuronsTo examine the effects of MIA on acid extrusion in neocortical

pyramidal neurons, measurements of intracellular pH based on

BCECF were done on acute slices in CO2/HCO�3 -buffered and CO2/

HCO�3 -free HEPES (20mM) buffered solutions. For further details on

methods and data, see Supplementary material and Ruusuvuori et al.

(2010).

ResultsThe intracellular pH of parietal cortex layer 2/3 neurons was

measured in Day 6 rat pups using in vivo two-photon microscopy

and the pH-sensitive dye BCECF (inset in Fig. 1A). When pups

were exposed to asphyxia (20% CO2 and 9% O2), a conspicuous

fall in fluorescence took place that levelled off by the end of the

1-h exposure period. This decrease in fluorescence corresponded

to an acidification of 0.30 � 0.08 pH units below the baseline

(n = 40 neurons from five pups; Fig. 1A). During the 2-h

post-asphyxia period, intracellular pH not only recovered from

acidosis but also showed a slower alkalosis with a plateau level

of 0.27 � 0.12 pH units above baseline pH. Importantly, this is a

time point when blood pH has already recovered from the

post-asphyxia acidosis and, moreover, blood pH does not

become alkaline at any point of time under the present experi-

mental conditions (Helmy et al., 2011). Hence, the above data

and our previous observations on brain extracellular pH (see also

Fig. 2A) clearly indicate that the post-asphyxia alkalosis is confined

to brain tissue.

We have previously shown that graded restoration of normo-

capnia brought about by gradually reducing CO2 in inhaled air

during recovery from asphyxia (Helmy et al., 2011) suppresses

the post-asphyxia alkalosis of brain extracellular pH, but it is not

known how graded restoration of normocapnia affects intracellular

pH. Therefore, we exposed rats to asphyxia, which again resulted

in a decrease in intracellular pH similar to that described above

(0.31 � 0.08 pH units, n = 41 neurons from five pups; Fig. 1B).

Importantly, during graded restoration of normocapnia, intracellu-

lar pH gradually recovered but with a marked suppression of the

overshoot of intracellular pH (cf. Fig. 1A), which was only

�0.03 � 0.10 pH units above baseline at 2-h (P50.0001, imme-

diate versus graded restoration of normocapnia) closely resembling

the suppressing action of graded restoration of normocapnia on

the overshoot of extracellular pH (Helmy et al., 2011).

As concluded earlier, the fact that brain extracellular and intra-

cellular pH undergo a post-asphyxia alkalosis indicates that there is

a net loss of acid equivalents from both compartments. Thus, the

most likely explanation that could account for the parallel increase

in extracellular and intracellular pH is an increase in net extrusion

of acid equivalents across the blood–brain barrier. We directly

tested this idea by simultaneous measurements of brain extracel-

lular pH and body pH (Fig. 2) using one pH-sensitive electrode

plus its reference in the brain parenchyma and a similar pair of

electrodes in body subcutaneous tissue (Voipio and Kaila, 1993).

To manipulate acid transport across the blood–brain barrier

(Pedersen et al., 2006), we used the Na/H exchange blocker,

MIA.

The brain extracellular pH changes during and after asphyxia

were similar to previous measurements (Helmy et al., 2011),

with a baseline level of 7.20 � 0.01, peak acidosis at

6.80 � 0.05 and plateau alkalosis at 7.57 � 0.04 (n = 6 pups).

Body pH during asphyxia decreased from 7.42 � 0.03 to

7.19 � 0.03 and recovered with no alkaline overshoot, at any in-

stant of time, to a level that was identical to the pre-exposure

baseline (7.42 � 0.01). The recordings in Fig. 2A show that the

time course of pH changes in response to asphyxia is strikingly

different in the two compartments, with the brain extracellular pH

changes being larger and faster than those in the body. Unlike

brain extracellular pH, body pH does not show a post-asphyxia

alkaline overshoot. This observation is in full agreement with the

blood acid–base data obtained in our previous study (Helmy et al.,

2011). A finding of crucial importance here is that the application

of MIA completely suppressed the post-asphyxia increase in brain

extracellular pH (P = 0.004, n = 5; Fig. 2B). Clearly, brain extracel-

lular pH and body pH behave as distinct compartments in re-

sponse to asphyxia and during the post-asphyxia period.

The only structure that could be responsible for the above pH

compartmentalization is the blood–brain barrier. The blood–brain

barrier in the Day 6 rats is likely tight enough with regard to

ionic movements to maintain such a compartmentalization (Butt

et al., 1990), which we found was the case. A trans-blood–brain

barrier potential difference (Woody et al., 1970; Voipio et al.,

2003), which was sensitive to the extracellular pH changes in a

manner similar to the mature human brain (Voipio et al., 2003),

was clearly seen in the electrophysiological recordings (Fig. 2A).

The average peak shift during the asphyxia-induced acidosis was

similar in the absence ( + 4.08 � 0.24mV, n = 6) and presence

( + 4.05 � 0.22mV, n = 5) of MIA (P = 0.9). At the end of the

2-h post-asphyxia period, the blood–brain barrier potential shift

was �3.08 � 0.37mV and 0 � 0.1mV in the asphyxia and as-

phyxia plus MIA-treated groups, respectively (P = 0.004; Fig.

2B), indicating that the steady negative shift in trans-blood–brain

barrier potential was abolished in parallel with the extracellular pH

overshoot in the presence of MIA.

To gain further information on blood–brain barrier permeability,

we used sodium fluorescein as an indicator (Fig. 2C). No signifi-

cant difference was found in brain fluorescein extravasation after

asphyxia when compared with control (128.1 � 38.7 and

129.9 � 33.1 ng sodium fluorescein/mg brain tissue, respectively,

n = 4, P = 0.9). In contrast, when the blood–brain barrier was dis-

rupted by injecting 30ml of 24% mannitol in saline into the left

ventricle, a large increase in sodium fluorescein extravasation was

observed (549.8 � 99.1 ng sodium fluorescein/mg brain tissue,

n = 5, P = 0.02). These data provide further evidence that

blood–brain barrier permeability is not altered under the present

experimental conditions.

If the increase in brain pH is caused by an enhanced efflux of

acid equivalents from the brain across the blood–brain barrier, one

might assume that it is large enough to cause a detectable tran-

sient in the body pH measurements. Indeed, we found that,

during the time window of maximum increase in brain


extracellular pH, a small deflection to the acid direction in body pH

could be observed in four out of six recordings, including the one

shown in Fig. 2A.

Consistent with a key role of the blood–brain barrier in the

functional compartmentalization of the brain pH during and

after asphyxia was that, when plotting brain extracellular pH

against body pH, a profound hysteresis was observed (Fig. 3A).

Blocking the blood–brain barrier Na/H exchange using the inhibi-

tor MIA completely inhibited the post-asphyxia overshoot alkalosis

of brain extracellular pH (Fig. 2B), which abolished the hysteresis

and, under these conditions, brain extracellular pH was related to

body pH in an almost linear manner. Thus, the trajectories of body

pH and brain extracellular pH in the presence and absence of MIA

in Fig. 3A directly illustrate the role of blood–brain barrier Na/H

exchange in dissociating brain extracellular pH regulation from

body pH during the post-asphyxia period. MIA had no effect on

either body or brain extracellular pH under control conditions

during 3.5 h post-injection, which covers the duration of the pre-

sent experiments (n = 3).

In line with the above and previous results (Kendall et al., 2006;

Helmy et al., 2011), we found that MIA reduced seizure burden

by 88% (P50.0002, n = 8; Fig. 3B). We repeated the behavioural

tests with the Na/H exchange inhibitor amiloride, because

amiloride penetration across the blood–brain barrier is known to

be poor (Sipos and Brem, 2000; Fisher, 2002; Liu et al., 2010).

Notably, the effects of amiloride and MIA were similar (Fig. 3B),

providing further support for the conclusion that activation of Na/

H exchange in the blood–brain barrier leads to brain alkalosis and

consequent seizures.

All the above evidence points to an action of the blockers of

Na/H exchange that is not mediated by changes in neuronal regu-

lation of intracellular pH. Moreover, intracellular pH regulation in

hippocampal pyramidal neurons is insensitive to amiloride and its

derivatives (Raley-Susman et al., 1991) but whether this is true for

neocortical neurons is not known (Mellergard et al., 1993;

Ou-yang et al., 1993; Chesler, 2003). Hence, we examined the

effects of MIA on layer 2/3 pyramidal neurons in neocortical slices

(Supplementary material). Consistent with previous results on hip-

pocampal pyramidal neurons (Schwiening and Boron, 1994; Baxter

and Church, 1996; Bevensee et al., 1996), acid extrusion following

an ammonium prepulse (Boron and De Weer, 1976) was much

more efficient (by a factor of �10) in the presence of HCO�3 than

in the HEPES-buffered solution, indicating a minor role for any

HCO�3 -independent acid extruder, such as Na/H exchange

(Supplementary Fig. 1). Moreover, application of 10 mM MIA

(Vinnikova et al., 2004) did not have any detectable effect on

Figure 2 Enhanced net acid extrusion across the blood–brain barrier takes place after asphyxia and is suppressed by the Na/H exchange

blocker, MIA. (A) From top to bottom: the experimental paradigm; sample recording of extracellular pH changes in the brain (red) and in

the body (black); sample recording of trans-blood–brain barrier potential difference (VBBB, calculated as Vbrain–Vbody); mean values � SD in

extracellular brain �pH (red triangles) and body �pH (black squares) in the asphyxia paradigm from six rats; mean values � SD of VBBB.

(B) Data from experiments on five rats where MIA was injected 30min before the experimental asphyxia. Note strict dependence of

steady-state values of VBBB on the pH changes across the blood–brain barrier. (C) Blood–brain barrier permeability is not disrupted by

experimental birth asphyxia, as indicated with sodium fluorescein (NaF) extravasation into the brain (n = 4 rat pups in each paradigm). For

further details see text.


steady-state intracellular pH or acid extrusion either in the pres-

ence (n = 8 cells) or absence (n = 10 cells) of CO2/HCO�3

(Supplementary material and Supplementary Fig. 1). Taken to-

gether, these data do not support the view that the actions of

MIA and amiloride on brain extracellular pH changes caused by

asphyxia are mediated by a block in neuronal Na/H exchange.

Here, it is also worth emphasizing that a cell-autonomous increase

in neuronal pH caused by acid extrusion during the post-asphyxia

period would impose an acid shift in extracellular pH (Siesjo, 1985;

Chesler and Kaila, 1992; Chesler, 2003), exactly the opposite of

what was seen in our present and previous (Helmy et al., 2011)

study.

DiscussionThe present work shows that post-asphyxia alkalosis in the

brain and the consequent seizures are attributable to an enhanced

efflux of acid equivalents from the brain that is suppressed by

blocking Na/H exchange located in the blood–brain barrier

(Pedersen et al., 2006; Lam et al., 2009). This conclusion is

based on (i) the post-asphyxia increase and alkaline overshoot

of both brain intracellular pH and extracellular pH, which indicates

a net loss of acid equivalents from brain tissue; (ii) direct meas-

urements of the extracellular pH levels and dynamics in the brain

and the rest of the body, which indicated a strict compartmental-

ization of brain extracellular pH by the blood–brain barrier under

normal conditions and in response to asphyxia in the Day 6 rats;

(iii) the specific block of the post-asphyxia brain alkalosis by MIA,

an inhibitor of Na/H exchange (Kendall et al., 2006; Pedersen

et al., 2006) and (iv) the equally strong inhibitory action on seiz-

ures by MIA and amiloride, whereof the latter is an Na/H

exchange inhibitor that has a very low permeability across the

blood–brain barrier (Sipos and Brem, 2000; Fisher, 2002; Liu

et al., 2010).

An increase in seizure burden after birth has an incremental,

deleterious effect on neurological outcome and therefore it is of

utmost importance to identify the mechanisms underlying the

pathophysiological post-asphyxia brain alkalosis. It is a well-

established fact that a rise in both intracellular and extracellular

pH enhances neuronal excitability (Chesler, 1990; Kaku et al.,

1993; Kaila, 1994; Casey et al., 2010; Enyedi and Czirjak, 2010;

Ruusuvuori et al., 2010). The immature brain appears to be par-

ticularly sensitive to changes in pH. Recent experiments on neo-

natal hippocampal slices have shown that a change of 0.05 pH

units has a profound effect on endogenous network activity

(Ruusuvuori et al., 2010). A robust cause–effect between brain

alkalosis and seizure induction in the immature rat brain was

shown in our work on experimental febrile seizures, where

tonic–clonic seizures were triggered following an increase in extra-

cellular pH of �0.2 pH units induced by hyperthermia-evoked

hyperventilation (Schuchmann et al., 2006, 2011). Moreover, an

intraperitoneal injection of sodium bicarbonate that produced an

increase in brain extracellular pH of �0.2 pH units triggered seiz-

ures in rat pups (Schuchmann et al., 2006).

Consistent with the present results, magnetic resonance spec-

troscopy following severe human birth asphyxia has shown an

abnormally high brain intracellular pH (Robertson et al., 2002).

A high magnitude and prolonged duration of alkalosis are predict-

ive of poor neurodevelopmental outcome including cerebral atro-

phy (Robertson et al., 1999). The elevated intracellular and

extracellular pH in our animal model (Helmy et al., 2011) as

well as the elevated intracellular pH in the newborn human

brain after birth asphyxia (Robertson et al., 2002) persist beyond

Figure 3 Brain extracellular pH and body pH are functionally compartmentalized in an Na/H exchanger-dependent manner as indicated

by the hysteresis in response to experimental asphyxia and by the MIA sensitive seizures. (A) The change in brain extracellular pH plotted

against the change in body pH. Pups exposed to asphyxia only are shown as blue circles; pups to which MIA was administered are shown

as green diamonds. Data taken from Fig. 2. (B) Total seizure burden [quantified as the number of loss of righting reflex (LRR) per pup]

after asphyxia and asphyxia with administration of MIA or amiloride, at equal doses, from eight rat pups in each paradigm.


the time of normalization of the post-asphyxia acid–base param-

eters in the blood (Brouillette and Waxman, 1997).

We hypothesized that among the acid–base transporters in the

blood–brain barrier (Pedersen et al., 2006), the Na/H exchanger is

the most relevant one under our experimental conditions. In full

agreement with this idea, we found that MIA abolished the

brain-specific post-asphyxia alkalosis and the associated seizures,

while having no effects on the changes in body pH or its basal

level under control conditions.

It is unlikely that neuronal Na/H exchange contributes to the

effects of asphyxia or to the actions of MIA and amiloride seen in

the present study. It has been shown that hippocampal neurons

are resistant to amiloride and its derivatives (Raley-Susman et al.,

1991; Chesler, 2003), and our present findings with MIA indicate

that this is also true with regard to neocortical neurons.

Furthermore, if the post-asphyxia increase in intracellular pH

would be caused by enhanced neuronal Na/H exchange (or by

any acid extruder), this would lead to a simultaneous decrease in

extracellular pH (Siesjo, 1985; Chesler and Kaila, 1992; Chesler,

2003) while the opposite was observed. Indeed, that an extracel-

lular alkalosis leads to an intracellular alkalosis has been demon-

strated in various kinds of mammalian neurons (Roos and Boron,

1981; Ou-yang et al., 1993; Kaila and Ransom, 1998; Ritucci

et al., 1998; Bouyer et al., 2004). The most parsimonious explan-

ation for the near-parallel increase in intracellular and extracellular

pH is that the blood–brain barrier Na/H exchanger-dependent rise

in extracellular pH causes a rise in neuronal pH.

Na/H exchange in the blood–brain barrier is prominently sensi-

tive to amiloride and MIA (Murphy and Johanson, 1990; Sipos

et al., 2005; Pedersen et al., 2006) and a high binding capacity

of MIA has been demonstrated in cerebral microvessels (Kalaria

et al., 1998). The endothelial isoforms of Na/H exchangers are

located on the luminal side of endothelial cells (Crone, 1986;

Goldstein et al., 1986; Redzic, 2011; Benarroch, 2012), and there-

fore, they are easily accessible to systemically applied Na/H ex-

change inhibitors, regardless of the drug permeability across the

blood–brain barrier. Amiloride is poorly permeable in the blood–

brain barrier (Sipos and Brem, 2000; Fisher, 2002; Liu et al.,

2010), and notably, our present study shows that amiloride is as

effective as MIA in abolishing post-asphyxia seizures.

The persistent activation of Na/H exchange in the blood–brain

barrier most likely involves a change in the kinetic set point (Roos

and Boron, 1981). This set point renders Na/H exchangers func-

tionally silent at normal intracellular pH and active when intracel-

lular pH becomes acidic (Casey et al., 2010). Notably, an alkaline

shift in the set point of intracellular pH occurs when Na/H ex-

changers are persistently stimulated by hormones and other neu-

roactive factors (Sardet et al., 1991; Orlowski and Grinstein, 2004;

Pedersen et al., 2006). It is well known that various signalling

molecules, including hormones, cytokines and growth factors,

are released following birth asphyxia and related states

(Lagercrantz and Slotkin, 1986; Yoon et al., 1996; Nordstrom

and Arulkumaran, 1998; Savman et al., 1998; Silveira and

Procianoy, 2003; Fliegel and Karmazyn, 2004; Gazzolo et al.,

2009), and an important question for future studies is to identify

the factor(s) that act on the Na/H exchanger in the blood–brain

barrier.

Interestingly, block of Na/H exchange by MIA administered

systemically before hypoxia/ischaemia insult has been shown to

have a neuroprotective effect (Kendall et al., 2006) that may be

at least partly attributable to the drug-induced block of seizures.

Another, mutually non-exclusive possibility is that the post-

asphyxia recovery of acidosis and the subsequent alkalosis trigger

cell damage by activating enzymes, such as proteases and

phospholipases (Currin et al., 1991). Analysing the contributions

of these mechanisms to neuronal damage following birth asphyxia

is an important topic for future studies.

An interesting hypothesis based on the enhanced efflux of acid

across the blood–brain barrier is that in human neonates suffering

from severe birth asphyxia, the net acid efflux from the brain

during the initial alkalosis might cause a slowing down of the

early recovery of the systemic acidosis as monitored using blood

samples. The total-body volume fraction of the newborn human

brain is higher than that of a Day 6 rat, and even in the rat pups,

we found evidence that the maximum rate of recovery of brain

extracellular pH after asphyxia was paralleled by a small acid tran-

sient in body pH (Fig. 2A). These findings predict that an increase

in the severity of birth asphyxia in human newborns is associated

with a progressively slower recovery of blood pH that might even

include a transient acid shift. This hypothesis can be readily tested

in future clinical work.

In conclusion, following neonatal asphyxia, extrusion of acid

equivalents across the blood–brain barrier generates a brain alkal-

osis that promotes seizures. Our results raise the important and

worrying question whether resuscitation paradigms where normo-

capnic conditions established in a fast manner will, in fact, lead to

the promotion of birth-asphyxia seizures. Graded restoration of

normocapnia and/or drugs targeting the Na/H exchange in the

blood–brain barrier offer an effective and straightforward means

to functionally suppress seizures and ameliorate other neurological

sequela after birth asphyxia (Rakhade and Jensen, 2009; Bonifacio

et al., 2011), and can be readily used in conjunction with

other treatment modalities, such as hypothermia (Johnston

et al., 2011) and optimization of blood oxygenation levels

(Saugstad, 2010).

FundingThis work was supported by grants from the Letten Foundation,

the Academy of Finland, the Sigrid Juselius Foundation, and

the Jane and Aatos Erkko Foundation. K.K. is a member of the

Finnish Center of Excellence in Molecular and Integrative

Neuroscience Research. P.O.K. is supported by National

Institutes of Health [R01DC009607], and the Alfred P. Sloan

Foundation. P.V.W is supported by National Institutes of Health

[NIH T32DC000046].

Supplementary materialSupplementary material is available at Brain online.


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Helmy et al. Supplementary Data

Material and Methods

Intracellular pH recordings from cortical pyramidal neurons in slice preparation

Rat pups (Day 5 - 7) were decapitated, and the brains were dissected in ice cold (0 - 4 °C)

oxygenated (95 % O2 and 5 % CO2) solution containing (in mM) 87 NaCl, 50 sucrose, 2.5

KCl, 0.5 CaCl2, 25 NaHCO3, 1.25 NaH2PO4, 7 MgCl2, and 25 D-glucose, and coronal brain

slices (400 µm) were cut with a vibrating microtome (Campden 7000smz, Campden

Instruments). Slices were let to recover in standard solution containing (in mM) 124 NaCl, 3

KCl, 2 CaCl2, 25 NaHCO3, 1.1 NaH2PO4, 2 MgSO4 and 10 D-glucose (equilibrated with 95

% O2, 5 % CO2, pH 7.4) at 34 oC for 30 minutes and then stored at room temperature. In the

bicarbonate free, HEPES-buffered solution 25 mM NaHCO3 was replaced with 20 mM

HEPES, 10 mM NaOH, and 10 mM NaCl (pH 7.4 at 32 oC), and oxygenated with 100 % O2.

Intracellular pH (pHi) recordings using the H+-sensitive indicator BCECF (Invitrogen) were

done as described in Ruusuvuori et al. (2010). In order to examine the properties of acid

extrusion, we used the ammonium prepulse technique (Boron and DeWeer, 1976; 20 or 7.5

mM NH4Cl substituted for equimolar NaCl in the CO2/HCO3- and HEPES buffered solutions,

respectively, for 3 minutes) and compared the pH recovery in the absence and presence of 10

µM N-methyl-isobutyl-amiloride (MIA; Sigma-Aldrich, St Louis, MO, stock solution 10 mM

in DMSO). The maximum rate of pHi recovery (ΔpHi/Δt) was measured immediately after

the peak acidosis by fitting a linear regression line using SigmaPlot 8.0.

Net acid-extrusion (JH+) was calculated from

( )2ii COH

pHJt

β β+Δ

= × +Δ

where the intrinsic buffering power (βi) had a value taken from Bevensee et al. (1996; see

also Chesler, 2003) and that of CO2/HCO3- buffering (βCO2) was estimated as βCO2 =

2.3x[HCO3]i (Chesler, 2003).

REFERENCES

Bevensee MO, Cummins TR, Haddad GG, Boron WF, Boyarsky G. pH regulation in single

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mitochondrial energy metabolism. J Neurosci 2010; 30: 15638-42.

RESULTS

An ammonium prepulse resulted in a transient intracellular acidosis which peaked at 6.88 ±

0.03 in the presence of CO2/HCO3- (n = 8) and at 6.93 ± 0.07 in the HEPES buffer (n = 10),

while the steady state pHi levels were 7.17 ± 0.05 and 7.12 ± 0.1, respectively. At the pHi

level of acid extrusion quantification, intracellular HCO3- concentration is about 10 mM,

yielding βCO2 of 23 mM (pH unit)-1, and the βi of 17 mM (pH unit)-1 was adopted from

Bevensee et al. (1996). Using these values of buffering power, the mean of maximum rate of

net acid extrusion under control conditions was approximately 10 times faster in the presence

of CO2/HCO3- than in the HEPES buffer (0.049 ± 0.01 mM s-1 vs. 6.8·10-3 ± 2.8·10-3 mM s-1,

respectively), indicating acid extrusion that is largely dependent on HCO3-. Application of 10

µM MIA had little effect on the baseline pHi (in CO2/HCO3- 7.18 ± 0.05 and in HEPES 7.08

± 0.1). The small reduction in the pHi recovery rate in the presence of MIA (~ 17 %) was

fully accounted for by rundown, since a similar change was seen when the ammonium

prepulse was repeated twice under control conditions. Taken together, these results indicate

that Na/H exchange in neonatal cortical layer 2/3 pyramidal neurons is insensitive to

amiloride derivatives.

Supplementary Figure 1: Acid extrusion in cortical pyramidal neurons is

insensitive to the NHE-inhibitor N-methyl-isobutyl-amiloride.

Bath application of N-methyl-isobutyl-amiloride (MIA; 10 µM) had little effect on the steady

state pHi and on pHi recovery from induced acidosis in the presence (A) or absence (B) of

CO2/HCO3-. Recordings in A and B are from single cortical layer 2/3 pyramidal neurons in

Day 7 slice preparations. Intracellular acidification was induced with 20 mM (A) and 7.5 mM

(B) NH4+ in order to generate acidifications of comparable amplitude.

mechanisms of birth asphyxia and a novel resuscitation

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