Traumatic spinal cord injury often results in complete loss of voluntary motor and sensory function below the site of injury. The long-term neurological deficits after spinal cord trauma may be due in part to widespread apoptosis of neurons and oligodendroglia in regions distant from and relatively unaffected by the initial injury. The caspase family of cysteine proteases regulates the execution of the mammalian apoptotic cell death program. Caspase-3 cleaves several essential downstream substrates involved in the expression of the apoptotic phenotype in vitro, including gelsolin, PAK2, fodrin, nuclear lamins and the inhibitory subunit of DNA fragmentation factor. Caspase-3 activation in vitro can be triggered by upstream events, leading to the release of cytochrome c from the mitochondria and the subsequent transactivation of procaspase-9 by Apaf-1. We report here that these upstream and downstream components of the caspase-3 apoptotic pathway are activated after traumatic spinal cord injury in rats, and occur early in neurons in the injury site and hours to days later in oligodendroglia adjacent to and distant from the injury site. Given these findings, targeting the upstream events of the caspase-3 cascade has therapeutic potential in the treatment of acute traumatic injury to the spinal cord.

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We report here that activation of the caspase-3 apoptotic cascade in spinal cord injury is regulated, in part, by calcineurin-mediated BAD dephosphorylation. BAD, a proapoptotic member of the bcl-2 gene family, is rapidly dephosphorylated after injury, dissociates from 14-3-3 in the cytosol, and translocates to the mitochondria of neurons where it binds to Bcl-x(L). Pretreatment of animals with FK506, a potent inhibitor of calcineurin activity, or MK801, an NMDA glutamate receptor antagonist, blocked BAD dephosphorylation and abolished activation of the caspase-3 apoptotic cascade. These findings extend previous in vitro observations and are the first to implicate the involvement of glutamate-mediated calcineurin activation and BAD dephosphorylation as upstream, premitochondrial signaling events leading to caspase-3 activation in traumatic spinal cord injury.

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Traumatic injury to the spinal cord initiates a host of pathophysiological events that are secondary to the initial insult. One such event is the accumulation of free radicals that damage lipids, proteins, and nucleic acids. A major reactive product formed following lipid peroxidation is the aldehyde, 4‐hydroxynonenal (HNE), which cross‐links to side chain amino acids and inhibits the function of several key metabolic enzymes. In the present study, we used immunocytochemical and immunoblotting techniques to examine the accumulation of protein‐bound HNE, and synaptosomal preparations to study the effects of spinal cord injury and HNE formation on glutamate uptake. Protein‐bound HNE increased in content in the damaged spinal cord at early times following injury (1–24 h) and was found to accumulate in myelinated fibers distant to the site of injury. Immunoblots revealed that protein‐bound HNE levels increased dramatically over the same postinjury interval. Glutamate uptake in synaptosomal preparations from injured spinal cords was decreased by 65% at 24 h following injury. Treatment of control spinal cord synaptosomes with HNE was found to decrease significantly, in a dose‐dependent fashion, glutamate uptake, an effect that was mimicked by inducers of lipid peroxidation. Taken together, these findings demonstrate that the lipid peroxidation product HNE rapidly accumulates in the spinal cord following injury and that a major consequence of HNE accumulation is a decrease in glutamate uptake, which may potentiate neuronal cell dysfunction and death through excitotoxic mechanisms.

Abstract:Immunocytochemical and immunoblotting techniques were used to investigate calpain I activation and the stability of the calpain-sensitive cytoskeletal proteins microtubule-associated protein 2 (MAP2) and spectrin at 1, 4, and 24 h after contusion injury to the spinal cord. Spinal cord injury resulted in the activation of calpain I at all time points examined, with the highest level of activation occurring at 1 h. At the same early time point, there was a loss of dendritic MAP2 staining in spinal cord sections, accompanied by pronounced perikaryal accumulation. The loss in MAP2 staining in the injured spinal cord progressed over the 24-h survival period to affect regions 3 mm distant to the site of injury. The presence of calpain I-specific spectrin degradation was apparent in neuronal cell bodies and fibers as early as 1 h after injury, with the most intense staining occurring within and juxtaposed to the injury site. Spectrin breakdown products in neuronal cell bodies declined rapidly at 4 h and were nearly undetectable at 24 h after injury. Immunoblot studies confirmed the immunocytochemical results by demonstrating a significant increase in calpain I activation, a significant decrease in MAP2 levels, and a significant increase in spectrin breakdown. Finally, treatment of animals with riluzole, an inhibitor of glutamate release, before surgery reduced significantly the loss of MAP2 levels observed at 24 h after injury. These results demonstrate that Ca 2~-dependentprotease activation and degradation of critical cytoskeletal proteins are early events after spinal cord injury and that treatments that minimize the actions of glutamate may limit their breakdown. Key Words: Microtubule-associated protein 2-Spectrin -Cytoskeleton -Glutamate-Calcium -Calpain l-RiluzoIe-CNS injury.

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The potential use of riluzole (a glutamate release inhibitor) alone or in combination with methyl-prednisolone (MP) in treating acute spinal cored injury (SCI) was examined. Rats received a contusion injury to the spinal cord using the NYU impactor and were treated with vehicle, riluzole (8 mg/kg), MP(30 mg/kg), or riluzole + MP at 2 and 4 h following injury. Animals continued to receive riluzole treatment (8 mg/kg) for a period of 1 week. The animals were then tested weekly for functional recovery using the BBB open field locomotor score. At the end of testing (6 weeks after injury), each spinal cord was examined for the amount of remaining tissue at the injury site and a myelination index was used to quantify remaining axons in the ventromedial white matter. In this study, only the combination treatment was found to significantly improve behavioral recovery as assessed using the BBB open field locomotor scale. In addition, the combination treatment promoted tissue sparing at the lesion epicenter, but had no clear effect on the index of myelination. The results of this study clearly demonstrate the potential beneficial effects of a combination approach in the treatment of traumatic SCI.