title: Hydroxyl radicals generated in the rat spinal cord at the level produced by impact injury induce cell death by necrosis and apoptosis: protection by a metalloporphyrin.
abstract: We previously measured the time courses of hydrogen peroxide (H2O2), hydroxyl radical (*OH), and catalytic iron increases following traumatic spinal cord injury (SCI). This study determines whether the SCI-elevated level of *OH causes cell death. OH was generated by administering H2O2 and Fe2+ at the concentrations attained following SCI, each through a separate microdialysis fiber inserted laterally into the gray matter of the cord. The duration of *OH generation mimics the duration of its elevation after SCI. The death of neurons and astrocytes was characterized at 24 h post-*OH exposure and quantitated by counting surviving cells along the fiber track in sections stained with Cresyl Violet, or immunohistochemically stained with anti-neuron-specific enolase (anti-NSE) and anti-glial fibrillary acidic protein (anti-GFAP). DNA fragmentation in neurons was characterized by double staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) and anti-NSE. Using a one way ANOVA followed by the Tukey test, we demonstrated that *OH generated in the cord induced significant losses of neurons in both Cresyl Violet (P<0.001) and anti-NSE-stained sections (P<0.001), and of astrocytes in GFAP-stained sections (P=0.001). *OH generated in the cord increased numbers of TUNEL-positive neurons compared with Ringer's solution administered as a control (P=0.001). Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic and a broad spectrum reactive species scavenger, significantly reduced *OH-induced death of neurons (P<0.001 in anti-NSE stained sections and P=0.002 in the Cresyl Violet-stained sections) and astrocytes (P=0.03). It also reduced the numbers of TUNEL-positive neurons (P=0.01). Electron microscopy confirmed that generated *OH induced neuronal and glial death with characteristic features of both necrosis and apoptosis. We conclude that 1) SCI-elevated *OH is sufficient to induce both necrosis and apoptosis, criteria for identifying an endogenous secondary damaging agent; 2) MnTBAP reduces *OH-induced cell death, perhaps by removing H2O2 administered in the tissue, thereby blocking formation of *OH, and also by scavenging downstream reactive species.
authors.affiliation: Department of Neurology, 301 University Boulevard, University of Texas Medical Branch, Galveston, TX 77555-0653, USA.
authors.forename: F
authors.initials: F
authors.lastname: Bao
authors.forename: D
authors.initials: D
authors.lastname: Liu
mesh_terms: D000818:Animals; D000870:Anterior Horn Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D017665:Hydroxyl Radical / Q000378:metabolism*; D008297:Male; D008665:Metalloporphyrins / Q000494:pharmacology* / Q000627:therapeutic use; D009336:Necrosis; D018696:Neuroprotective Agents / Q000494:pharmacology / Q000627:therapeutic use; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D013119:Spinal Cord Injuries / Q000378:metabolism* / Q000473:pathology / Q000517:prevention & control
chemical_list: D008665:Metalloporphyrins; D018696:Neuroprotective Agents; C097284:manganese(III)-tetrakis(4-benzoic acid)porphyrin; D017665:Hydroxyl Radical