Interaction of inflammation and hyperoxia in a rat model of neonatal white matter damage.

Intrauterine infection and inflammation are major reasons for preterm birth. The switch from placenta-mediated to lung-mediated oxygen supply during birth is associated with a sudden rise of tissue oxygen tension that amounts to relative hyperoxia in preterm infants. Both infection/inflammation and hyperoxia have been shown to be involved in brain injury of preterm infants. Hypothesizing that they might be additive or synergistic, we investigated the influence of a systemic lipopolysaccharide (LPS) application on hyperoxia-induced white matter damage (WMD) in newborn rats. Three-day-old Wistar rat pups received 0.25 mg/kg LPS i.p. and were subjected to 80% oxygen on P6 for 24 h. The extent of WMD was assessed by immunohistochemistry, western blots, and diffusion tensor (DT) magnetic resonance imaging (MRI). In addition, the effects of LPS and hyperoxia were studied in an in vitro co-culture system of primary rat oligodendrocytes and microglia cells. Both noxious stimuli, hyperoxia, and LPS caused hypomyelination as revealed by western blot, immunohistochemistry, and altered WM microstructure on DT-MRI. Even so, cellular changes resulting in hypomyelination seem to be different. While hyperoxia induces cell death, LPS induces oligodendrocyte maturity arrest without cell death as revealed by TUNEL-staining and immunohistological maturation analysis. In the two-hit scenario cell death is reduced compared with hyperoxia treated animals, nevertheless white matter alterations persist. Concordantly with these in vivo findings we demonstrate that LPS pre-incubation reduced premyelinating-oligodendrocyte susceptibility towards hyperoxia in vitro. This protective effect might be caused by upregulation of interleukin-10 and superoxide dismutase expression after LPS stimulation. Reduced expression of transcription factors controlling oligodendrocyte development and maturation further indicates oligodendrocyte maturity arrest. The knowledge about mechanisms that triggered hypomyelination contributes to a better understanding of WMD in premature born infants.

Erythropoietin attenuates hyperoxia-induced oxidative stress in the developing rat brain.

Oxygen toxicity contributes to the pathogenesis of adverse neurological outcome in survivors of preterm birth in clinical studies. In infant rodent brains, hyperoxia triggers widespread apoptotic neurodegeneration, induces pro-inflammatory cytokines and inhibits growth factor signaling cascades. Since a tissue-protective effect has been observed for recombinant erythropoietin (rEpo), we hypothesized that rEpo would influence hyperoxia-induced oxidative stress in the developing rat brain. The aim of this study was to investigate the level of glutathione (reduced and oxidized), lipid peroxidation and the expression of heme oxygenase-1 (HO-1) and acetylcholinesterase (AChE) after hyperoxia and rEpo treatment. Six-day-old Wistar rats were exposed to 80% oxygen for 2-48 h and received 20,000 IU/kg rEpo intraperitoneally (i.p.). Sex-matched littermates kept under room air and injected with normal saline or rEpo served as controls. Treatment with rEpo significantly reduced hyperoxia-induced upregulation of oxidized glutathione (GSSG) and malondialdehyde, a product of lipid breakdown, whereas reduced glutathione (GSH) was upregulated by rEpo. In parallel, hyperoxia-treated immature rat brains revealed rEpo-suppressible upregulation of synaptic AChE-S as well as of the stress-inducible AChE-R variant, together predicting rEpo-protected cholinergic signaling and restrained inflammatory reactions. Furthermore, treatment with rEpo induced upregulation of HO-1 on mRNA, protein and activity level in the developing rat brain. Our results suggest that rEpo generates its protective effect against oxygen toxicity by a reduction of diverse oxidative stress parameters and by limiting the stressor-inducible changes in both HO-1 and cholinergic functions.

Erythropoietin attenuates hyperoxia-induced cell death by modulation of inflammatory mediators and matrix metalloproteinases.

Oxygen toxicity appears to contribute to the pathogenesis of adverse neurological outcome in survivors of preterm birth. In infant rodent brains, hyperoxia triggers widespread apoptotic neurodegeneration, induces proinflammatory cytokines and inhibits growth factor signaling cascades. Since a tissue-protective effect has been observed for recombinant erythropoietin (rEpo), we hypothesized that rEpo would influence the expression of proinflammatory cytokines and matrix metalloproteinase (MMP)-2 and MMP-9. Six-day-old Wistar rats were exposed to 80% oxygen for 2-48 h and received 20,000 IU rEpo i.p. Sex-matched littermates kept in room air and injected with normal saline or rEpo served as controls. Treatment with rEpo significantly reduced hyperoxia-induced upregulation of the proinflammatory cytokines IL-1beta and IL-18 in infant rodent brains on the mRNA and protein levels. In parallel, gelatin zymography in hyperoxia-treated immature rat brains revealed an upregulation of active MMP-2 which was reduced by concomitant rEpo treatment. Furthermore, hyperoxia induced upregulation of MMP-9 following 12 h of oxygen exposure and this was attenuated by rEpo treatment. Our results suggest that rEpo generates its protective effect against oxygen toxicity through a reduction of proinflammatory mediator levels.