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

Abstract

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 employed in which ethanol was administered to animals in vivo (gavage 6g/kg), granule neurons were isolated 2-24 hr 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 hr 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 following 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.

Mechanisms of Ethanol-Induced Death of Cerebellar Granule Cells

“…Kane et al (2008) evaluate the effect of in vivo ethanol exposure on CGC in vitro . In this model, ethanol was administered to rats in vivo via gavage on PD4 (vulnerable period) and PD14 (resistant period) (92). CGC were isolated 2-24 hours post-treatment, and ROS production and relative mitochondrial membrane potential (MMP) were immediately assessed in viable cells.…”

Alcohol and thiamine deficiency trigger differential mitochondrial transition pore opening mediating cellular death

“…These results support the hypothesis that oxidative stress is involved in ethanol-induced neuroapoptosis. However, Kane et al [69] reported that in cerebellar granule cells, isolated from 4-day-old rats exposed to ethanol in vivo, neither ROS production nor neuron death are showed. In addition, 17β-estradiol protects more efficiently against the ethanol-dependent oxidative stress-induced MPT pore opening than PYC an efficient antioxidant [66].…”

Alcohol and Developing Neuronal Circuits

“…Exposure during the first-and second-trimester equivalent has been shown to reduce numbers of not only Purkinje cells but also granule cells (Maier & West, 2001;Servais et al, 2007). Binge-like ethanol exposure during the third-trimester equivalent has been consistently demonstrated to be detrimental to Purkinje and granule cells, causing a significant reduction in their numbers via multiple mechanisms, including oxidative stress, reduction in the activity of neurotrophic factors, alterations in cell migration and axonal outgrowth, and neuroimmune deficits (Chen & Charness, 2008;Goodlett, Horn, & Zhou, 2005;Hamre & West, 1993;Heaton, Mitchell, & Paiva, 1999;Heaton, Mitchell, & Paiva, 2000;Heaton, Paiva, Mayer, & Miller, 2002;Kane, Chang, Roberson, Garg, & Han, 2008;Kane et al, 2011;Kumada et al, 2010;Light, Brown, Newton, Belcher, & Kane, 2002). In addition, exposure during the third-trimester equivalent reduces the frequency of events generated by climbing fiber activation (i.e., complex spikes), an effect that may be a consequence of a reduction in cell numbers in the inferior olive or functional alterations at climbing fiber-to-Purkinje neuron synapses (Backman, West, Mahoney, & Palmer, 1998;Pierce, Hayar, Williams, & Light, 2011).…”