Peroxynitrite generated in the rat spinal cord induces apoptotic cell death and activates caspase-3

We previously demonstrated that the peroxynitrite concentration increases after impact spinal cord injury. This study tests whether spinal cord injury-elevated peroxynitrite induces apoptotic cell death. Peroxynitrite was generated at the concentration and duration produced by spinal cord injury by administering S-morpholinosydnonimine through a microdialysis fiber into the gray matter of the rat spinal cord. Fragmented DNA was visualized by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling. Transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive neurons were quantitated by counting the transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and neuron-specific enolase double-stained neurons along the fiber track in the sections removed at 6, 12, 24 and 48 h post-peroxynitrite exposure. Peroxynitrite significantly increased transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive neurons at all time points examined (P≤0.001) compared with artificial cerebrospinal fluid controls (Two-way analysis of variance followed by Tukey test), peaking at 24 h post-exposure. Electron microscopic observation of characteristic features of apoptosis confirmed peroxynitrite-induced neuronal apoptosis. Total transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling-positive cells were counted in areas near and 0.2 mm away from the fiber track. The counts both peaked at 24 h with no significant difference between the two areas. However, at 6 and 12 h post-exposure the counts were significantly higher near than away from the fiber track (P=0.03 and P=0.007 respectively, paired t test). Immunohistochemical staining indicates caspase-3 was activated by peroxynitrite; this activation peaked at 6 h post-exposure, suggesting that activation of caspase-3 might be an early event in the apoptotic cell death cascade. We conclude that 1) peroxynitrite generated in the cord at the level produced by spinal cord injury induces neuronal apoptosis, indicating a role for peroxynitrite in secondary spinal cord injury; 2) caspase activation might be involved in peroxynitrite-induced neuronal apoptosis; 3) therefore removal of peroxynitrite should reduce secondary cell death after spinal cord injury.