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Hypoxia-Induced Caspase-3 Activation and DNA Fragmentation in Cortical Neurons of Newborn Piglets: Role of Nitric Oxide

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Abstract

Hypoxia results in generation of nitric oxide (NO) free radicals, activation of caspase-3, and genomic DNA fragmentation. The present study tests the hypothesis that hypoxia-induced caspase-3 activation and DNA fragmentation are nitric oxide mediated. Studies were conducted in newborn piglets, divided into normoxic (n = 5), hypoxic (n = 5), and hypoxic-7-NINA (n = 6). Hypoxic-7-NINA group received the neuronal nitric oxide synthase inhibitor, 7-Nitroindazole (7-NINA). Caspase-3 activity was determined spectrofluorometrically using enzyme-specific substrates. Sections from the neocortex were stained with an antiserum recognizing active caspase-3. Purified DNA was separated by gel electrophoresis. Administration of 7-NINA resulted in decreased immunoreactivity of caspase-3 (mean LI: 20.2%) as compared to the untreated hypoxia group (mean LI: 57.5%) (P < 0.05). 7-NINA attenuated caspase-3 enzymatic activity as well in comparison to the untreated hypoxia group (P < 0.05). Furthermore, multiple low molecular weight bands corresponding to DNA fragments were present in the hypoxic but not in the normoxic or hypoxic-7-NINA groups. Inhibition of nNOS abates the hypoxia-induced increase in active caspase-3 immunoreactivity, as well as enzymatic activity in cortical neurons, and DNA fragmentation in brain homogenates. We conclude that the coordinate increase of capase-3 activity and fragmentation of nuclear DNA in the hypoxic newborn piglet brain are NO mediated.

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Authors and Affiliations

  1. Department of Pediatrics, Jefferson Medical College, Thomas Jefferson University Hospital, Philadelphia, Pennsylvania

    N. A. Parikh

  2. Presently with the Department of Pediatrics, University of Texas Houston Medical School, Houston, Texas

    N. A. Parikh

  3. Department of Pediatrics, Drexel University College of Medicine and St. Christopher's Hospital for Children, Philadelphia, Pennsylvania

    C. D. Katsetos, Q. M. Ashraf, S. H. Haider, A. Legido, M. Delivoria-Papadopoulos & O. P. Mishra

  4. Department of Pathology and Laboratory Medicine, St. Christopher's Hospital for Children, P, hiladelphia, Pennsylvania

    C. D. Katsetos

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  1. N. A. Parikh
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Parikh, N.A., Katsetos, C.D., Ashraf, Q.M. et al. Hypoxia-Induced Caspase-3 Activation and DNA Fragmentation in Cortical Neurons of Newborn Piglets: Role of Nitric Oxide. Neurochem Res 28, 1351–1357 (2003). https://doi.org/10.1023/A:1024992214886

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