In premature infants, oxygen free radicals generated following neonatal resuscitation are associated with subsequent diseases such as retinopathy of prematurity and bronchopulmonary dysplasia. Recent studies in brain tissue samples have shown that nonphysiologic oxygen levels play a key role in induction of apoptosis in the developing brain. Estrogen is a well-established agent in neuroprotection and, therefore, is thought to be neuroprotective even in the premature brain. Astrocytes appear to have a critical role in protection and survival of neurons in the brain. As one of the glial cell types, they have a great potential for possible involvement in the mediation of estrogen neuroprotective effects. The aim of our study was to analyze whether astrocytes in cell cultures are damaged by hyperoxia and whether 17beta-estradiol (E2) can protect them against apoptosis. Additionally, we investigated the mechanism of the protection by E2, hypothesizing that it is mediated through extracellular signal-regulated kinase (ERK1/2). Cells underwent eightfold more apoptosis when cultivated in hyperoxia compared with normoxia. Addition of E2 reduced apoptosis in hyperoxia by more than 50%. Levels of ERK1/2 and phosphorylated ERK1/2 were increased after hyperoxia compared with normoxia. Preincubation with E2 prior to exposure to hyperoxia resulted in decreased levels of ERK1/2 and pERK1/2. Hyperoxia induces apoptosis in C8-D1A cells, and E2 seems to be a protecting factor for astrocytes in hyperoxia. This effect is not mediated through up-regulation of pERK1/2.
Premature infants are at risk for bilirubin-associated brain damage. In cell cultures bilirubin causes neuronal apoptosis and necrosis. Ibuprofen is used to close the ductus arteriosus, and is often given when hyperbilirubinemia is at its maximum. Ibuprofen is known to interfere with bilirubin-albumin binding. We hypothesized that bilirubin toxicity to cultured rat embryonic cortical neurons is augmented by coincubation with ibuprofen. Incubation with ibuprofen above a concentration of 125 g/mL reduced cell viability, measured by methylthiazole tetrazolium reduction, to 68% of controls (p Ͻ 0.05). Lactate dehydrogenase (LDH) release increased from 29 to 38% (p Ͻ 0.01). The vehicle solution did not affect cell viability. Coincubation with 10 M unconjugated bilirubin (UCB)/human serum albumin in a molar ratio of 3:1 and 250 g/mL ibuprofen caused additional loss of cell viability and increased LDH release (p Ͻ 0.01), DNA fragmentation, and activated caspase-3. Preincubation with the pan-caspase inhibitor z-val-alaasp-fluoromethyl ketone abolished ibuprofen-and UCB-induced DNA fragmentation. The study demonstrates that bilirubin in low concentration of 10 M reduces neuron viability and ibuprofen increases this effect. Apoptosis is the underlying cell death mechanism. (Pediatr Res 65: 392-396, 2009)
Recent evidence suggests oxygen as a powerful trigger for cell death in the immature white matter, leading to periventricular leukomalacia (PVL) as a cause of adverse neurological outcome in survivors of preterm birth. This oligodendrocyte (OL) death is associated with oxidative stress, upregulation of apoptotic signaling factors (i.e., Fas, caspase-3) and decreased amounts of neurotrophins. In search of neuroprotective strategies we investigated whether the polysulfonated urea derivative suramin, recently identified as a potent inhibitor of Fas signaling, affords neuroprotection in an in vitro model of hyperoxia-induced injury to immature oligodendrocytes. Immature OLs (OLN-93) were subjected to 80% hyperoxia (48 h) in the presence or absence of suramin (0, 30, 60, 120 microM). Cell death was assessed by flow cytometry (Annexin V, caspase-3 activity assay) and immunohistochemistry for activated caspase-3. Immunoblotting for the death receptor Fas, cleaved caspase-8 and the phosphorylated isoform of the serine-threonin kinase Akt (pAkt) was performed. Suramin lead to OL apoptosis and potentiated hyperoxia-induced injury in a dose-dependent manner. Immunoblotting revealed increased Fas and caspase-8 expression by suramin treatment. This effect was significantly enhanced when suramin was combined with hyperoxia. Furthermore, pAkt levels decreased following suramin exposure, indicating interference with neurotrophin-dependent growth factor signaling. These data indicate that suramin causes apoptotic cell death and aggravates hyperoxia-induced cell death in immature OLs. Its mechanism of action includes an increase of previously described hyperoxia-induced expression of pro-apoptotic factors and deprivation of growth factor dependent signaling components.
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