title: 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.
abstract: 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.
authors.affiliation: Department of Neurology, University of Texas Medical Branch, 301 University Boulevard, Route 0881, Galveston, TX 77555-0881, USA; Department of Biochemistry & Molecular Biology, University of Texas Medical Branch, 301 University Boulevard, Route 0881, Galveston, TX 77555-0881, USA. Electronic address: dliu@utmb.edu.
authors.forename: D
authors.initials: D
authors.lastname: Liu
authors.affiliation: Department of Neurology, University of Texas Medical Branch, 301 University Boulevard, Route 0881, Galveston, TX 77555-0881, USA. Electronic address: baofeng03@yahoo.ca.
authors.forename: F
authors.initials: F
authors.lastname: Bao
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000502:physiology*; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004195:Disease Models, Animal; D005110:Extracellular Space / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000008:administration & dosage / Q000378:metabolism*; D008297:Male; D008665:Metalloporphyrins / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord / Q000187:drug effects / Q000473:pathology / Q000503:physiopathology*; D013119:Spinal Cord Injuries / Q000188:drug therapy / Q000473:pathology / Q000503:physiopathology*
chemical_list: D000975:Antioxidants; D008665:Metalloporphyrins; D010743:Phospholipids; D011506:Proteins; C097284:manganese(III)-tetrakis(4-benzoic acid)porphyrin; D004247:DNA; D006861:Hydrogen Peroxide; D020169:Caspases
keywords: Mn (III) tetrakis (4-benzoic acid) porphyrin; apoptotic cell death; hydrogen peroxide; membrane lipid peroxidation; proteins and DNA oxidation; spinal cord injury