Publication Date

2012

Document Type

Thesis

Committee Members

Thomas Brown (Advisor), Thomas Brown (Committee Member), David Cool (Committee Member), Courtney Sulentic (Committee Member)

Degree Name

Master of Science (MS)

Abstract

Sarin is a neurotoxin that has been used in terrorist attacks in Japan and is a potential bioterrorist weapon. It induces seizures by affecting the regulation of neurotransmitters in the brain. Seizures are directly correlated to neuronal damage. Two types of neuronal damage that can occur are apoptosis and necrosis. One of the main regions of the brain where neuronal death occurs is the hippocampus, which is involved in memory. Victims showed chronic decline of memory loss 3 and 7 years after the Tokyo's terrorist attack incident. There are treatments available that can break down sarin or can block the continuous activation of acetylcholine by acting as a competitive antagonist at the acetylcholine receptor, but there are no current treatments that prevent neuronal death in the brain after sarin exposure. Our overall hypothesis is that the broad spectrum caspase inhibitor, Quinoline-Val-Asp-Difluorophenoxymethyl ketone (Q-VD-OPh), can reduce or prevent caspase-activated neuronal death in the brain thereby, preventing memory loss. Q-VD-OPh has been shown to prevent all apoptotic pathways and is not toxic to cells. It is the most effective known caspase inhibitor and has the ability to cross the blood brain barrier. In order to validate its effectiveness in preventing neuronal death in sarin-exposed mice, we designed and synthesized the appropriate negative control by replacing aspartic acid in Q-VD-OPh with glutamic acid, making Q-VE-OPh. Q-VE-OPh did not prevent DNA laddering or the activation of cleaved caspase 8 or 9. In addition, we found Q-VE-OPh is not toxic in vitro. Q-VE-OPh closely resembles Q-VD-OPh which provides it as an optimal negative control. Sarin-exposed mice brains treated with Q-VD-OPh or Q-VE-OPh will be analyzed by immunohistochemistry. Due to the use of sarin being restricted to two locations in the United States by the US Department of Defense, all of our experiments must be done at one time and at the same facility. Therefore, it was necessary to optimize brain tissue processing techniques, storage conditions, and the immunohistochemistry assays in mice not exposed to sarin. The goal of the current work will focus on eliminating background in immunohistochemistry assays in order to prevent false labeling. The optimizations of these labeling procedures will be used to determine if neuronal death in the hippocampus is decreased in sarin-exposed mice treated with Q-VD-OPh compared to sarin-exposed mice treated with Q-VE-OPh.

Page Count

114

Department or Program

Microbiology and Immunology

Year Degree Awarded

2012

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