Atomic Force Microscopy (AFM) Analysis of Oxidative Stress in Human U87 Glioblastoma Cells Treated with Hyperoxia

Abstract

Atomic Force Microscopy (AFM) has been used to image membrane surface topography at nanometer resolution. In this study we used the AFM to characterize the physical changes resulting from hyperoxia on cultured human glioblastoma cells (U87). U87 cells were exposed to air (21% O2), hyperoxia (95% O2) or hyperbaric oxygen (HBO2, 3.5ata O2) for 30 min. H2O2 (200µM) was used as a positive control. Following treatment, the cells were fixed with 2% glutaraldehyde for 20 min prior to AFM imaging. Three dimensional surface plots of U87 cells revealed a significant distortion of the plasma membrane (membrane blebbing) caused by HBO2 and H2O2, which is thought to be indicative of lipid peroxidation. Individual cells from each group were analyzed to assess Mean Roughness (Ra) and Maximum Roughness (Rmax). Ra was 40 ± 4 nm in 21% O2, 52 ± 5 nm in 95%O2, 64 ± 7 nm in HBO2, and 91 ± 9 nm in H2O2 treated cells. Rmaxwas 253 ± 27 nm in 21% O2, 302 ± 30 nm in 95% O2, 366 ± 35 nm in HBO2, and 608 ± 39 nm in H2O2 treated cells. In conclusion, these data show that oxidative damage in the plasma membrane is proportional to oxygen concentration. Moreover, the AFM is capable of characterizing subtle changes in membrane topography from oxidative damage