Low doses of ethanol and hypoxia administered together act synergistically to promote the death of cortical neurons

The majority of clinically relevant injuries to the developing brain are increasingly being recognized as the result of a combination of least two insults. Evidence from epidemiologic and clinical studies suggests that many of these insults do not cause injury in and of themselves. Rather, they act as predisposing factors (pre-conditioning cytotoxic factors, such as chronic ethanol consumption), increasing the susceptibility to a second, injury-producing (precipitating), unfavorable event (such as hypoxia-ischemia). In this study we make use of primary neuronal cultures to test, in a reproducible system, this two-hit hypothesis. We show that, when administered in combination, low levels of two clinically important environmental insults, hypoxia and ethanol, despite having minimal effects on their own, can act synergistically produce a significant degree of neuronal injury. We further show that one indicator of apoptotic cell death, activated caspase 3, undetectable in neurons subjected to low levels of either insult alone, is detectable when they are administered together.