Spinal cord injury (SCI) is a major health care issue, causing immense hardships to its victims and their families. The initial injury is worsened by secondary """"""""autodestructive"""""""" processes caused by over production of harmful substances by injured cells. The PI's long-term focus in studying SCI is to identify endogenous toxic substances that cause secondary damage and to explore the pathways by which they cause cell death in order to develop therapeutic strategies to prevent such death. Methylprednisolone (MP) is the only drug approved for SCI treatment;however side effects limit its utility. Therefore it is urgent to discover more effective, less toxic therapeutic agents to reduce secondary injury. Reactive species (RS) identified as mediators of secondary injury after SCI induce cell death by two hypothesized pathways: 1) SCI-induced RS initiate oxidation of major cellular components, thereby destroying cells;2) Other SCI-induced toxic agents (e.g. glutamate) induce RS formation, which in turn potentiate toxicity of non-RS toxins. Due to this feedback amplification mechanism, RS are produced longer after SCI than are some non-RS toxins;this provides a longer therapeutic window for scavenging RS. Mn (III) tetrakis (benzoic acid) porphyrin (MnTBAP), a novel superoxide dismutase mimetic and a broad spectrum RS scavenger, is cell permeable, active, stable, nontoxic, and scavenges superoxide anion, hydrogen peroxide, and peroxynitrite. It appears significantly superior to MP. The goal of this project is thus to explore the therapeutic potential of MnTBAP for SCI treatment and its mechanism of action in vivo.
Specific Aim 1 is to explore the therapeutic potential and action site of MnTBAP to reduce secondary injury using a standard mechanical SCI model. The effect of MnTBAP will be compared with that of MP. The PI's group has demonstrated RS as initiators to induce oxidative damage and cell death.
Specific Aims 2 and 3 will explore the role of RS as death signaling messengers to potentiate toxicity of non-RS toxins and the action site of MnTBAP in this hypothesized pathway. A novel overall secondary chemical injury model will be used. It separates secondary chemical events from initial mechanical injury by sampling extracellular death signals in the extracellular fluid of an injured rat spinal cord and administering them directly into the cord of an uninjured rat by a microcannula inserted laterally through the cords of both rats to induce secondary cell death. To explore the role of RS and effect of MnTBAP in delayed Glu toxicity, Glu will be administered at the SCI-induced concentration and duration into the rat spinal cord through a microdialysis fiber to induce cell death. The ability of MnTBAP to scavenge RS, prevent oxidation of proteins, DNA and membrane lipids, reduce activation of caspases and death of different types of cells, particularly by apoptosis, and ameliorate neurological dysfunction will be assessed by comparing the results of MnTBAP- and saline-treated experiments. Production of RS and biomarkers of oxidation will be measured by HPLC, visualized by fluorescent probes and specific antibodies. Apoptosis of each cell type will be characterized by double staining with TUNEL and cell specific antibodies, Hoechst 33342 staining and electron microscopy. Activation of caspases in each cell types will be characterized by double staining. The immunohistochemical stained cells will be counted to determine the effect of MnTBAP on cell death. The therapeutic potential of MnTBAP will be assessed by behavioral tests.
