Mild
therapeutic hypothermia is protective against several cellular stresses, but the mechanisms underlying this protection are not completely resolved. In the present study, we used an in vitro model to investigate whether
therapeutic hypothermia at 33°C applied following a
peroxide-induced oxidative stress would protect PC12 cells. A 1-hour exposure to
tert-butyl peroxide increased cell death measured 24 hours later. This cell death was dose-dependent in the range of 100-1000 μM
tert-butyl peroxide with ∼50% cell death observed at 24 hours from 500 μM
peroxide exposure. Cell survival/death was measured with an
alamarBlue viability assay, and
propidium iodide/Hoechst imaging for counts of living and dead cells.
Therapeutic hypothermia at 33°C applied for 2 hours postperoxide exposure significantly increased cell survival measured 24 hours postperoxide-induced stress. This protection was present even when delayed
hypothermia, 15 minutes after the
peroxide washout, was applied. Addition of any of the three FDA-approved
antioxidants (
Tempol,
EUK134,
Edaravone at 100 μM) in combination with
hypothermia improved cell survival. With the
therapeutic hypothermia treatment, a significant downregulation of caspases-3 and -8 and
tumor necrosis factor-α was observed at 3 and 24 hours poststress. Consistent with this, a cell-permeable pan-
caspase inhibitor
Z-VAD-FMK applied in combination with
hypothermia significantly increased cell survival. Overall, these results suggest that the
antioxidants quenching of
reactive oxygen species likely works with
hypothermia to reduce mitochondrial damage and/or apoptotic mechanisms. Further studies are required to confirm and extend these results to other cell types, including neuronal cells, and other forms of oxidative stress as well as to optimize the critical parameters of
hypothermia treatment such as target temperature and duration.