Ethanol exposure of neonatal rats does not increase biomarkers of oxidative stress in isolated cerebellar granule neurons

Oxidative stress is a candidate mechanism for ethanol neuropathology in fetal alcohol spectrum disorders. Oxidative stress often involves production of reactive oxygen species (ROS), deterioration of the mitochondrial membrane potential (MMP), and cell death. Previous studies have produced conflicting results regarding the role of oxidative stress and the benefit of antioxidants in ethanol neuropathology in the developing brain. This study investigated the hypothesis that ethanol neurotoxicity involves production of ROS with negative downstream consequences for MMP and neuron survival. This was modeled in neonatal rats at postnatal day 4 (P4) and P14. It is well established that granule neurons in the rat cerebellar cortex are more vulnerable to ethanol neurotoxicity on P4 than at later ages. Thus, it was hypothesized that ethanol produces more oxidative stress and its negative consequences on P4 than on P14. A novel experimental approach was used in which ethanol was administered to animals in vivo (gavage 6 g/kg), granule neurons were isolated 2-24 h post-treatment, and ROS production and relative MMP were immediately assessed in the viable cells. Cells were also placed in culture and survival was measured 24 h later. The results revealed that ethanol did not induce granule cells to produce ROS, cause deterioration of neuronal MMP, or cause neuron death when compared to vehicle controls. Further, granule neurons from neither P4 nor P14 animals mounted an oxidative response to ethanol. These findings do not support the hypothesis that oxidative stress is obligate to granule neuron death after ethanol exposure in the neonatal rat brain. Other investigators have reached a similar conclusion using either brain homogenates or cell cultures. In this context, it is likely that oxidative stress is not the sole and perhaps not the principal mechanism of ethanol neurotoxicity for cerebellar granule neurons during this stage of brain development.