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

Neuroscience. 2004;126(2):285-95. doi: 10.1016/j.neuroscience.2004.03.054.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Anterior Horn Cells / drug effects
  • Anterior Horn Cells / metabolism
  • Anterior Horn Cells / pathology
  • Apoptosis / drug effects
  • Apoptosis / physiology*
  • Cell Death / drug effects
  • Cell Death / physiology
  • Hydroxyl Radical / metabolism*
  • Male
  • Metalloporphyrins / pharmacology*
  • Metalloporphyrins / therapeutic use
  • Necrosis
  • Neuroprotective Agents / pharmacology
  • Neuroprotective Agents / therapeutic use
  • Rats
  • Rats, Sprague-Dawley
  • Spinal Cord / drug effects
  • Spinal Cord / metabolism*
  • Spinal Cord / pathology
  • Spinal Cord Injuries / metabolism*
  • Spinal Cord Injuries / pathology
  • Spinal Cord Injuries / prevention & control


  • Metalloporphyrins
  • Neuroprotective Agents
  • manganese(III)-tetrakis(4-benzoic acid)porphyrin
  • Hydroxyl Radical