Hypothermic Inhibition of Apoptotic Pathways for Combined Neurotoxicity of Iron and Ascorbic Acid in Differentiated PC12 Cells: Reduction of Oxidative Stress and Maintenance of the Glutathione Redox State

Recent clinical trials have demonstrated the efficacy and safety of therapeutic hypothermia for neonatal hypoxic ischemic encephalopathy (HIE). We previously reported that the levels of non-protein-bound iron and ascorbic acid (AA) are increased in the CSF of infants with HIE. In this study, we investigated the effect of hypothermia on the combined cytotoxicity of Fe and AA for differentiated PC12 cells. The optimal settings for hypothermic treatment were a temperature of 30-32 degrees C, rescue time window of less than 6 h, and minimum duration of at least 24 h. Hypothermia effectively prevented the loss of the mitochondrial transmembrane potential from 6 h to 72 h (end of the study period) and attenuated the release of apoptotic proteins (cytochrome c and apoptosis-inducing factor) at 6 h of exposure to Fe-AA. Activation of caspase-3 was also delayed until 24 h. Akt was transiently activated, although no influence of temperature was observed. Elevation of oxidative stress markers, including ortho-, meta-, and di-tyrosine (markers of protein oxidation) and 4-hydroxynonenal (lipid peroxidation) was significantly attenuated when the temperature was reduced by 5 degrees C. The half-cell reduction potential (Ehc) of GSSG/2GSH redox couple ranged from -220 to -180 mV in unstressed differentiated PC12 cells, and apoptosis was triggered when Ehc exceeded -180 mV. Hypothermia prevented Ehc from rising above -180 mV within 24 h of exposure to Fe-AA. In conclusion, hypothermia prevented cell death due to Fe-AA toxicity by inhibiting apoptotic pathways through maintenance of a reduced cellular environment, as well as by alleviating oxidative stress.