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

We previously measured the time courses of hydrogen peroxide (H ₂O₂), 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.