Date of Award

2012

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Beulig, Alfred

Keywords

Neuroinflammation, Reactive Oxygen Species, Microglia

Area of Concentration

Chemistry

Abstract

The pro-inflammatory microglial response is widely believed to exacerbate brain injury following ischemia-reperfusion, whereas the anti-inflammatory microglial response is reported to improve neurological outcome. Because brain levels of reactive oxygen species (ROS) and the transcription factor p53 are known to increase following ischemia-reperfusion, and these molecules have been shown to bias microglia towards the pro-inflammatory response following other insults, ROS and p53 are prime candidates in the search for molecules that promote transcription of pro-inflammatory factors such as interleukin-1Beta (IL-1Beta) in response to ischemia-reperfusion. Importantly, ROS are known to activate p53 and are known to result from p53 activation in various cell types, but the existence of this positive feedback loop and its influence on microglial transcription have not been examined. By utilizing the redox-sensitive dye 2',7'-dichlorodihydrofluorescein, this thesis demonstrates that murine and BV-2 cell line microglia subjected to oxygen-glucose deprivation (OGD) exhibit ~30% greater ROS production upon reperfusion as compared to microglia maintained in normoxic-normoglycemic media. The same OGD-reperfusion paradigm elicited an increase in p53 activity in earlier studies, meaning that increased ROS production possibly contributed to the previously-observed increase in p53 activity. �No significant differences in ROS production were observed in microglia harvested from p53 knockout mice, suggesting that ROS produced upon reperfusion are not a result of p53 activity. Pre-treating cells with the radical scavenger N-acetylcysteine (NAC) abrogated ROS production in all conditions. The amount of IL-1Beta transcript measured by quantitative PCR was approximately 1500-fold higher in microglia subjected to OGD-reperfusion when compared to controls, an effect that was greatly inhibited by NAC, indicating that ROS are largely responsible for the observed increase in IL-1Beta transcription after OGD-reperfusion. Transcription of the pro-inflammatory marker macrophage receptor with collagenous structure (MARCO) showed similar trends, but with much less robust differences between groups. These findings extend the role of ROS in activating the pro-inflammatory microglial phenotype to the context of ischemia-reperfusion. Future studies are needed to confirm the role of ROS in activating p53, and to examine if p53 activity mediates the ROS-induced increases in pro-inflammatory transcription

Rights

This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.

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