Relationship between evolving epileptiform activity and delayed loss of mitochondrial activity after asphyxia measured by near-infrared spectroscopy in preterm fetal sheep

Prematurely born infants continue to have a high rate of neurodevelopmental handicap, including overt cerebral palsy and cognitive and learning deficits (Marlow et al. 2005). Although the underlying pathogenesis of injury is complex and multifactorial (Volpe, 2001), exposure to hypoxia–ischaemia before, during or after birth is a major factor that contributes to adverse neurodevelopmental outcomes (Volpe, 2001). In the near-term brain there is now extensive clinical and experimental evidence that perinatal hypoxia–ischaemia can lead to a biphasic pattern of impaired oxidative energy metabolism, with initial recovery from the acute hypoxic depression, in a transient ‘latent’ phase, before secondarily falling as late as 6–15 h after reperfusion in the piglet (Lorek et al. 1994), the neonatal rat (Vannucci et al. 2004), and clinically (Azzopardi et al. 1989; Roth et al. 1997). There is little information available on the timing of these phases of injury in relevant preterm models. This is of considerable practical importance, because, as recently reviewed, the latent phase appears to correspond broadly with the practical window of opportunity for treatment (Gunn et al. 2005). We and others have recently reported that exposure to severe hypoxia in the preterm fetal sheep is associated with diffuse white matter loss and severe subcortical neural injury (George et al. 2004; Welin et al. 2005; Dean et al. 2006), consistent with clinical observations (Barkovich & Sargent, 1995). Following this insult, the early recovery phase is marked by evolving abnormal neural activity, including frequent epileptiform transients (George et al. 2004; Dean et al. 2006), followed by delayed onset of large amplitude, stereotypical seizures, from approximately 10 h (Quaedackers et al. 2004a; Dean et al. 2006). The early EEG transients were maximal in the first 4 h after reperfusion, and were only seen in fetuses that later developed subcortical neuronal loss (George et al. 2004). Similar EEG changes have been observed in the preterm newborn; overall epileptiform transient activity is strongly associated with adverse outcome (Rowe et al. 1985; Hughes & Guerra, 1994; Vecchierini-Blineau et al. 1996; Marret et al. 1997; Biagioni et al. 2000; Okumura et al. 2003). In contrast, although seizures are associated with marked hypermetabolism with depletion of local metabolites, studies in rats suggest that the immature brain is relatively resistant to subsequent injury (Fernandes et al. 1999; Haas et al. 2001; Pereira de Vasconcelos et al. 2002). There is some evidence from near-term fetal sheep that delayed onset seizures after transient cerebral ischaemia are a marker or reflection of the onset of secondary failure of oxidative metabolism (Marks et al. 1996, 1999). There is considerable evidence suggesting that mitochondrial dysfunction, with loss of cytochrome oxidase (CytOx) activity and depletion of high energy phosphates, is either causally related to, or at least tightly coupled with, the onset of delayed energy failure after hypoxic–ischaemic injury (Vannucci et al. 2004). There is a close correlation between histological loss of CytOx and neuronal loss (Dimlich et al. 1990; Wagner et al. 1990a; Nelson & Silverstein, 1994), and between the timing of loss of CytOx activity after severe anoxia in the cat and subsequent delayed neurological deterioration (Wagner et al. 1990b). These data are supported by in vitro evidence that the well described increase in intracellular calcium levels during hypoxia/reoxygenation triggers subsequent delayed functional impairment and morphological disintegration of mitochondria (Schild et al. 2003). The time course of changes in CytOx can be monitored using near-infrared spectroscopy (NIRS). Studies using continuous NIRS monitoring in the newborn piglet have firstly confirmed evolving loss of CytOx after severe hypoxia–ischaemia (Chang et al. 1999), and secondly demonstrated a close correlation between CytOx and depletion of high energy metabolites on magnetic resonance spectroscopy (Tsuji et al. 1995; Chang et al. 1999; Peeters-Scholte et al. 2004). These data all relate to the term or post-term brain; there is no information on the evolution of mitochondrial dysfunction in the preterm brain, or when posthypoxic epileptiform transient activity and seizures occur in relation to the onset of failure of oxidative metabolism. The goal of the present study was to examine the hypothesis that early EEG epileptiform transient activity after severe hypoxia in the preterm fetus occurs prior to the onset of secondary mitochondrial failure, whereas the onset of overt seizures would correspond with the onset of secondary energy failure as measured by changes in cerebral oxygenation and mitochondrial activity using NIRS. In terms of cerebral maturity the 0.7 gestation fetal sheep is comparable to the human brain at term at 28–32 weeks of gestation (McIntosh et al. 1979), prior to the onset of cortical myelination (Barlow, 1969).