A major problem in the study of animal models of neurodegenerative disease is the lack of an unambiguous marker to identify dying neurons. A new method, the terminal transferase-mediated dUTP-biotin nick end labeling (TUNEL) technique involves the end labeling of fragmented DNA associated with dying cells with biotinylated nucleotides, after which cells are visualized via avidin-conjugated peroxidase. The method has been used successfully to label cells undergoing programmed death in the digestive, lymphatic, and reproductive systems and skin. TUNEL has not been applied to studies of cell death in the nervous system, and in this Pilot, we propose a series of experiments designed to investigate the validity and usefulness of this technique as a tool to enhance our understanding of neuronal cell death. Initially, we will validate the selectivity and specificity of the technique on tissues from the spinal cord of rats at embryonic day 16, a time point at which naturally occurring motor neuron death has been reported. Subsequently, we will examine three animal models of retrograde neuronal degeneration that are well established in our laboratory, one involving neonatal and the other two involving adult neurons. The studies described in this Pilot will allow us to determine the usefulness of TUNEL in identifying degenerating neurons. This strategy will then be used to investigate these issues in other models, including transgenic mice in our Alzheimer's Disease Research Center (ADRC), and eventually, to focus this approach on processes that lead to death of neurons in the human brain.
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