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

Authors

Dominic P. D'Agostino
James E. Olson, Wright State University - Main CampusFollow
Javier Cuevas
Jay B. Dean

Document Type

Conference Proceeding

Publication Date

4-2009

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Abstract

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 (HBO₂). AFM was used to resolve changes in membrane ultrastructure in response to oxidative stimuli (e.g. hyperoxia, H₂O₂). Fluorescence microscopy was used to detect superoxide with Dihydroethidium (DHE). An amperometric biosensor (ISO-HPO-100; WPI) was used to measure nM levels of H₂O₂. Results from AFM scans of hyperoxia-treated glutaraldehyde-fixed (1%) U87 glioblastoma cells revealed nanoscopic membrane surface blebbing. Hyperoxia (95% O₂ and 3.5 ATA O₂) 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 H₂O₂ (0.2-2mM). Amperometric biosensors detected increased cellular H₂O₂ production during hyperoxia, which was elevated 156 ± 8% (95% O₂) and 276 ± 52% (4 ATA O₂) above baseline (20% O₂). 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).

Comments

Presented at the 2009 Federation of American Societies for Experimental Biology (FASEB) Science Research Conference.

Presentation Number 617.3.

Repository Citation

D'Agostino, D. P., Olson, J. E., Cuevas, J., & Dean, J. B. (2009). Analysis of Oxidative Stress in CNS Cells by Integration of Atomic Force Microscopy (AFM), Fluorescence Microscopy and Amperometry. The FASEB Journal, 23 (Meeting Abstract Supplement).
https://corescholar.libraries.wright.edu/ncbp/902