DNA Damage Profiling in Motor Neurons: A Single-Cell Analysis by Comet Assay |
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Authors: | Martin Lee J. Liu Zhiping |
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Affiliation: | (1) Department of Pathology, Division of Neuropathology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205;(2) Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, 21205 |
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Abstract: | We developed a method to measure DNA damage in single motor neurons (MN). A cell fraction enriched in viable -motor neurons was isolated from adult rat spinal cord. This cell preparation was used to measure the vulnerability of the MN genome to different reactive oxygen species (ROS). MN were exposed in vitro to hydrogen peroxide, nitric oxide and peroxynitrite. Specific types of DNA lesions (e.g., abasic sites, single-strand breaks, and double-strand breaks) were measured using single-cell gel electrophoresis (comet assay). The MN genome was very susceptible to attack by ROS. Different ROS induced different DNA damage profiles in MN. MN were also isolated from adult rats with sciatic nerve avulsions to show that DNA damage emerges early during their degeneration in vivo. This study demonstrates that the comet assay is a feasible method for profiling DNA lesions in the genome of single MN. Viable mature MN can be isolated and used for in vitro models of MN genotoxicity and can be isolated from in vivo models of MN degeneration for profiling DNA damage on a single-cell basis. |
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Keywords: | Amyotrophic lateral sclerosis apoptosis axotomy DNA single-strand breaks single-cell gel electrophoresis |
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