Differential vulnerability of immature murine neurons to oxygen-glucose deprivation

Abstract

In vivo studies support selective neuronal vulnerability to hypoxia-ischemia (HI) in the developing brain. Since differences in intrinsic properties of neurons might be responsible, pure cultures containing immature neurons (6–8 days in vitro) isolated from mouse cortex and hippocampus, regions chosen for their marked vulnerability to oxidative stress, were studied under in vitro ischemic conditions—oxygen-glucose deprivation (OGD). Twenty-four hours of reoxygenation after 2.5 h of OGD induced significantly greater cell death in hippocampal than in cortical neurons (67.8% vs. 33.4%, P = 0.0068). The expression of neuronal nitric oxide synthase (nNOS) protein, production of nitric oxide (NO), and reactive oxygen species (ROS), as well as glutathione peroxidase (GPx) activity and intracellular levels of reduced glutathione (GSH), were measured as indicators of oxidative stress. Hippocampal neurons had markedly higher nNOS expression than cortical neurons by 24 h of reoxygenation, which coincided with an increase in NO production, and significantly greater ROS accumulation. GPx activity declined significantly in hippocampal but not in cortical neurons at 4 and 24 h after OGD. The decrease in GSH level in hippocampal neurons correlated with the decline of GPx activity. Our data suggest that developing hippocampal neurons are more sensitive to OGD than cortical neurons. This finding supports our in vivo studies showing that mouse hippocampus is more vulnerable than cortex after neonatal HI. An imbalance between excess prooxidant production (increased nNOS expression, and NO and ROS production) and insufficient antioxidant defenses created by reduced GPx activity and GSH levels may, in part, explain the higher susceptibility to OGD of immature hippocampal neurons.

Introduction

Hypoxia-ischemia (HI) from a variety of causes such as neonatal stroke or perinatal mechanical disruption of cerebral blood flow results in severe brain injury and permanent neurologic disability in children (Fullerton et al., 2002, Lynch et al., 2002). There is a growing awareness based on MR brain imaging and laboratory investigations that HI causes injury to specific brain structures or groups of neurons rather than uniform or global brain injury, the concept of selective vulnerability (Haddad and Jiang, 1993, Johnston, 1998, Pulsinelli, 1985, Schmidt-Kastner and Freund, 1991). In the neonatal brain, the basal ganglia are the most vulnerable region at term, whereas the periventricular white matter is the region susceptible in the preterm (Barkovich et al., 1995, Ferriero, 2001). In vivo animal experiments have shown that the neostriatum, hippocampal field CA1, and neocortical layers III, V, and VI appear to be particularly prone to develop neuronal damage after HI both in the mature and immature brain (Guzzetta et al., 2000, Johnston et al., 2001, Schmidt-Kastner and Freund, 1991).

The precise mechanisms responsible for such selectivity in the face of a global insult are not fully understood. It is assumed to relate to enhanced excitatory synaptic function as well as to enhanced energy metabolism in the neonatal basal ganglia. The regional distribution of immature glutamate receptors corresponds to regions of selective vulnerability to HI (Greenamyre et al., 1987, Mitani et al., 1998). Regions that express neuronal nitric oxide synthase (nNOS) correspond to the regions that overexpress NMDA receptors and correlate with regions of the developing rat brain that are selectively vulnerable to HI (Black et al., 1995). In addition, regional differences among hippocampus, cortex, and striatum in prooxidant and antioxidant defenses (Candelario-Jalil et al., 2001, Khan and Black, 2003), the calcium sensitivity of the mitochondrial permeability transition (Friberg et al., 1999), DNA damage and repair capacity (Cardozo-Pelaez et al., 2000), and levels of expression of apoptosis-regulation genes (Cao et al., 2003, Xu et al., 2001) may also account for the differential susceptibility. Among these possible mechanisms, we hypothesized that insufficient antioxidant defense during oxidative stress is a major contributor to regional specificity in the immature brain after ischemia.

Oxidative stress is an integral part of the cascade of events leading to cell death following HI (Chan, 2001, Coyle and Puttfarcken, 1993). It implies imbalance in the neutralization of reactive oxygen species (ROS) such as superoxide (·O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (·OH), which are normally scavenged by the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (Cat). SOD converts ·O₂⁻ to H₂O₂, which is further detoxified by GPx or Cat. GPx is far more prevalent in the brain than is Cat, and GPx, not Cat, is assumed to be the critical downstream enzyme rescuing neuronal cells from H₂O₂ toxicity. Among other small molecular antioxidants, reduced glutathione (GSH) has been reported to be present in relatively high concentrations in brain and is considered vital for cell survival. With the activity of GPx and glutathione reductase, it serves to detoxify H₂O₂ to H₂O and molecular oxygen. It helps maintain the reduced state of the cysteinyl-thiol groups of proteins and rescues cells from apoptosis by buffering an endogenously induced oxidative stress (Filomeni et al., 2002, Schulz et al., 2000). It has been postulated that decreased levels of reduced GSH would be a marker for increased susceptibility to oxidant injury. Brain damage associated with an oxidative stress has been reported following GSH depletion (Gupta et al., 2000, Mytilineou et al., 2002, Papadopoulos et al., 1997, Vexler et al., 2003). There is mounting evidence that different brain regions have different activities and responses of antioxidant enzymes to oxidative stress (Candelario-Jalil et al., 2001, Cardozo-Pelaez et al., 2000, de Haan et al., 1994).

In neonatal brain, from previous studies of our and other laboratories, the severity of brain HI injury varies between and within regions, with the hippocampus being the most affected region in CD1 mice and other inbred strains (C57 BL6 et al.) (Schmidt-Kastner and Freund, 1991, Sheldon et al., 1998, Sheldon et al., 2004). In an in vitro study, primary neurons cultured from hippocampus were five times more vulnerable than neurons from cortex after 50 μM H₂O₂ exposure (Koshy et al., 1998). These results suggest that developing mouse hippocampus may be more sensitive and vulnerable than cortex during oxidative stress. But whether this difference is due to the intrinsic properties of neurons, and the mechanisms underlying this difference are still unclear. In the current study, using oxygen-glucose deprivation (OGD) as an in vitro ischemic injury paradigm, we compared the responses of cortical versus hippocampal neurons. To investigate the contribution of prooxidant enzymes and antioxidant defense in oxidative stress-induced neuronal cell death after OGD, we studied the protein expression of nNOS, copper/zinc SOD (SOD1), production of nitric oxide (NO), and ROS, as well as GPx activity and intracellular GSH levels. Since we had previously shown that there was no change in Cat activity, but a decrease in GPx activity in vivo after HI in hippocampus (Fullerton et al., 1998) and cortex (Sheldon et al., 2004), we chose to study the temporal pattern of GPx activity after OGD.