Analysis of Oxidative Stress in CNS Cells by Integration of Atomic Force Microscopy (AFM), Fluorescence Microscopy and Amperometry

Three sensitive methodologies were used to measure and characterize hyperoxia-induced oxidative stress in CNS cells (in vitro). A commercially available AFM (Bioscope SZ; Veeco) was mated with an inverted fluorescence microscope (Nikon TE2000-E) for simultaneous acquisition of AFM and fluorescence data. The system is housed in a hyperbaric chamber (Riemers Systems Inc.), allowing for cellular measurements at normobaric and hyperbaric pressures (0-85 psig), including hyperbaric oxygen (HBO ₂ ). AFM was used to resolve changes in membrane ultrastructure in response to oxidative stimuli ( e.g . hyperoxia, H ₂ O ₂ ). Fluorescence microscopy was used to detect superoxide with Dihydroethidium (DHE). An amperometric biosensor (ISO-HPO-100; WPI) was used to measure nM levels of H ₂ O ₂ . Results from AFM scans of hyperoxia-treated glutaraldehyde-fixed (1%) U87 glioblastoma cells revealed nanoscopic membrane surface blebbing. Hyperoxia (95% O ₂ and 3.5 ATA O ₂ ) induced a dose-dependent increase in average membrane roughness (Ra), which correlated with elevated malondialdehyde (MDA) production. Similar results in Ra levels and MDA production were observed with exogenous H ₂ O ₂ (0.2-2mM). Amperometric biosensors detected increased cellular H ₂ O ₂ production during hyperoxia, which was elevated 156 ± 8% (95% O ₂ ) and 276 ± 52% (4 ATA O ₂ ) above baseline (20% O ₂ ). In conclusion, these novel techniques (used alone or in combination) have provided a highly sensitive and complimentary means to detect reactive oxygen species (ROS) and assess oxidative stress from hyperoxia. ONR grant N000140610105 (DPD), ONR-DURIP equipment grant N000140210643 (JBD).