NSTI BioNano 2010

Neuroprotective effects of cerium oxide nanoparticles in an in-vitro mouse hippocampal brain slice model of ischemia

A.Y. Estevez, P. Chatani, A. Lynch, J.C. Leiter, E.S. Andreescu, J.S. Erlichman
St. Lawrence University, US

Keywords: stroke, cerium oxide, oxidative stress


Cerium oxide nanoparticles (ceria NPs) are widely used in industrial applications such as oxygen sensors and automotive catalytic converters. The best-known mechanism underlying the action of these NPs is thought to originate from their dual oxidation state and their ability to reversibly bind oxygen during reduction/oxidation reactions. More recently, ceria NPs have been shown to reduce oxidative damage in cultured cells and are undergoing clinical trials for use in the treatment of glaucoma. Given the efficacy of ceria NPs in these biological systems, we explored their therapeutic use in an oxidative stress disease model. Oxidative stress plays a prominent role in the pathology of cerebral ischemia or stroke, for which there are currently few effective neuroprotective drug treatments. In the present study we tested the hypothesis that ceria NPs decrease cell death in an in-vitro model of stroke. Transverse brain sections of the hippocampus were prepared from adult CD1 mice littermates and ischemia was induced by placing the slices in a hypoxic, hypoglycemic and acidic aCSF for 30 min. Slices were subsequently placed in culture for 1, 2, 4 or 24 hr and cell death assessed at the given time-points using the fluorescent vital exclusion dye, Sytox Blue. Cerium oxide nanoparticles (0.2 - 2 g/ mL), administered during the ischemic insult and present throughout the post-ischemic period, decreased cell death by approximately 50% compared to anatomically paired, non-treated ischemic controls (Figure 1). The neuroprotective effects of CeO2 were observed as long as the nanoparticles were added within 4 hours of the ischemic insult. In brain slices not exposed to ischemia, ceria nanoparticles did not affect cell viability at the concentrations and over the duration of exposure that we tested. To explore the biological mechanisms of action of the ceria NPs, we assessed their localization using transmission electron microscopy (TEM) and measured their effects on ischemia-induced accumulation of reactive oxygen species (ROS) using fluorescence microscopy. TEM analysis demonstrated that the ceria NPs accumulated in high densities around cellular membranes, mitochondria and neurofilaments. In addition, treatment with ceria NPs decreased ROS production by 32% measured at 1 hr post-ischemia (Figure 2). To investigate actions on specific free radicals, we measured the effects of ceria on ischemia-induced accumulation of superoxide anion (O2-), nitric oxide (NO) and hydroxyl radical (HO) using various fluorescent indicator dyes. Ceria NPs reduced the ischemia-induced accumulation of O2- and NO by 10% and 15%, respectively, but had no significant effect on HO accumulation. Molecular analysis of the downstream effectors of free radical activation demonstrated that ceria NPs significantly decreased tissue nitrosylation, a post-translational modification mediated by the presence of NO derivatives such as the peroxynitrite radical (ONOO-). Ceria treatment (1 g/mL) also significantly reduced the ischemia-induced expression of the program cell death protein, apoptosis inducing factor, in both the nuclear and mitochondrial fractions at 24 hr post-ischemia. Taken together, these data suggest that ceria NPs mitigate ischemic brain injury by multiple mechanisms and may be a useful therapeutic intervention to reduce oxidative/nitrosative tissue damage following a stroke. Nano-Medicine General
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