Hydrogen peroxide administered into the rat spinal cord at the level elevated by contusion spinal cord injury oxidizes proteins, DNA and membrane phospholipids, and induces cell death: Attenuation by a metalloporphyrin

We previously demonstrated that hydrogen peroxide concentration ([H₂O₂]) significantly increases after spinal cord injury (SCI). The present study explored (1) whether SCI-elevated [H₂O₂] is sufficient to induce oxidation and cell death, (2) if apoptosis is a pathway of H₂O₂-induced cell death, and (3) whether H₂O₂-induced oxidation and cell death could be reversed by treatment with the catalytic antioxidant Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP). H₂O₂ was perfused through a microcannula into the uninjured rat spinal cord to mimic the conditions induced by SCI. Protein and DNA oxidation, membrane phospholipids peroxidation (MLP), cell death and apoptosis were characterized by histochemical and immunohistochemical staining with antibodies against markers of oxidation and apoptosis. Stained cells were quantified in sections of H₂O₂-, or artificial cerebrospinal fluid (ACSF)-exposed with vehicle-, or MnTBAP-treated groups. Compared with ACSF-exposed animals, SCI-elevated [H₂O₂] significantly increased intracellular protein and DNA oxidation by threefold and MLP by eightfold in neurons, respectively. H₂O₂-elevated extracellular malondialdehyde was measured by microdialysis sampling. We demonstrated that SCI-elevated [H₂O₂] significantly increased extracellular malondialdehyde above pre-injury levels. H₂O₂ also significantly increased cell loss and the numbers of terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate-(dUTP)-biotin nick end labeling (TUNEL)-positive and active caspase-3-positive neurons by 2.3-, 2.8-, and 5.6-fold compared to ACSF controls, respectively. Our results directly and unequivocally demonstrate that SCI-elevated [H₂O₂] contributes to post-SCI MLP, protein, and DNA oxidation to induce cell death. Therefore, we conclude that (1) the role of H₂O₂ in secondary SCI is pro-oxidation and pro-cell death, (2) apoptosis is a pathway for SCI-elevated [H₂O₂] to induce cell death, (3) caspase activation is a mechanism of H₂O₂-induced apoptosis after SCI, and (4) MnTBAP treatment significantly decreased H₂O₂-induced oxidation, cell loss, and apoptosis to the levels of ACSF controls, further supporting MnTBAP's ability to scavenge H₂O₂ by in vivo evidence.