Robert D. Azbill

Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury.

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 (refs. 13, 14, 15). 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.

Impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury

Glutamate-induced excitotoxicity involving the formation of reactive oxygen species (ROS) has been implicated in neuronal dysfunction and cell loss following ischemic and traumatic injury to the central nervous system (CNS). ROS are formed in mitochondria when energy metabolism is compromised, and are inactivated by the ROS scavengers superoxide dismutase (SOD), catalase, and glutathione (GSH). ROS can impair the function of several cellular components including proteins, nucleic acids, and lipids. In the present study, we measured indicators of mitochondrial metabolic activity, ROS formation, lipid peroxidation, and antioxidant enzyme activities in synaptosomes obtained from rat spinal cord at early times following traumatic injury. Mitochondrial metabolic activity was found to significantly decrease as early as 1 h following injury, and continued to be compromised over the remaining postinjury time points. ROS formation was found to be significantly increased at 4 and 24 h following injury, while lipid peroxidation levels were found to be significantly increased in the injured spinal cord at 1 and 24 h, but not 4 h following injury. SOD enzyme activity was unchanged at all postinjury time points, while catalase activity and GSH levels were significantly increased at 24 h following injury. These findings indicate that impaired mitochondrial function, ROS, and lipid peroxidation occur soon after traumatic spinal cord injury, while the compensatory activation of molecules important for neutralizing ROS occurs at later time points. Therapeutic strategies aimed at facilitating the actions of antioxidant enzymes or inhibiting ROS formation and lipid peroxidation in the CNS may prove beneficial in treating traumatic spinal cord injury, provided such treatments are initiated at early stages following injury.

4‐Hydroxynonenal, a Lipid Peroxidation Product, Rapidly Accumulates Following Traumatic Spinal Cord Injury and Inhibits Glutamate Uptake

Abstract: 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.

Riluzole increases high-affinity glutamate uptake in rat spinal cord synaptosomes

The purpose of this study was to examine the effect of the anti-convulsant agent, riluzole, on high-affinity glutamate uptake as measured in rat spinal cord synaptosomes. The rate of glutamate uptake was significantly increased in the presence of 0.1 microM and 1.0 microM riluzole, but not at the higher concentrations examined. Kinetics analysis demonstrated that riluzole (0.1 microM) decreased the apparent K(m) by 21% and increased the V(max) by 31%. Glutamate uptake also was significantly increased in spinal cord synaptosomes obtained from rats treated with 8 mg/kg (i.p.) of riluzole and sacrificed 4 h later. The increase in glutamate uptake in vitro was not affected by pretreatment either with H7, an inhibitor of PKA and PKC, or with the PKC activating phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Previous studies have shown that some of the actions of riluzole are mediated by G proteins sensitive to pertussis toxin. Surprisingly, treatment of synaptosomes with pertussis toxin alone increased the rate of glutamate uptake, while having no effect on uptake in the presence of riluzole. However, pretreatment with cholera toxin was found to completely block the effects of riluzole on glutamate uptake. These results reveal an additional mechanism by which riluzole can affect glutamatergic neurotransmission, and provides further support that riluzole may prove beneficial in the treatment of traumatic central nervous system injuries involving the excitotoxic actions of glutamate.

A rapid and sensitive assay for measuring mitochondrial metabolic activity in isolated neural tissue

In the present study, we used the oxidation-reduction sensitive dye Alamar Blue, a fluorometric/colorimetric indicator of metabolic activity, as a tool for examining mitochondrial function in rat spinal cord synaptosomes. At 15 min following incubation, Alamar Blue fluorescence levels were found to increase by 3-fold, and could be detected in samples containing as little as 25 microg of protein. Alamar Blue is non-toxic, making it possible to obtain measures of the metabolic rate and the maximal functional capacity of mitochondria in a single sample. The findings of this study demonstrate that Alamar Blue fluorescence levels increased in a near linear fashion when samples were measured every 15 min for a period of 1 h. To document that the changes in Alamar Blue fluorescence are directly related to mitochondrial function, synaptosomes were pre-incubated with antimycin A (10 microM) or malonate (50 mM), both of which are potent inhibitors of mitochondrial function. Pretreatment with either compound significantly reduced the Alamar Blue fluorometric signal at all time points examined. These results provide evidence that Alamar Blue is a valuable analytical tool for examining mitochondrial function in synaptosomal preparations from neural tissue. Moreover, the properties of Alamar Blue are such that it provides a more sensitive and simpler indicator compared to indicators used in existing assays.

Riluzole improves measures of oxidative stress following traumatic spinal cord injury.

Rats received a contusion injury to the spinal cord followed by treatment with riluzole (a glutamate release inhibitor, 8 mg/kg), methylprednisolone (MP 30 mg/kg) or both. At 4 h following injury, spinal cords were removed and synaptosomes prepared and examined using five measures of oxidative stress. Riluzole treatment was found to improve mitochondrial function, and enhance glutamate and glucose uptake. As expected, MP treatment was found to reduce lipid peroxidation, but also improved glutamate and glucose uptake. Interestingly, the combination treatment was found to be effective in improving all five measures of oxidative stress. The results of this study clearly demonstrate the potential beneficial effects of a combination approach in the treatment of oxidative stress events in traumatic spinal cord injury.

Caspase-3 apoptotic signaling following injury to the central nervous system.

Abstract Apoptotic cell death is a fundamental and highly regulated biological process in which a cell is instructed to participate actively in its own demise. This process of cellular suicide is activated by developmental and environmental cues and normally plays an essential role in eliminating superfluous, damaged, and senescent cells of many tissue types. In recent years, a number of experimental studies have provided evidence of widespread neuronal and glial apoptosis following injury to the central nervous system (CNS). These studies indicate that injury-induced apoptosis can be detected from hours to days following injury and may contribute to neurological dysfunction. Given these findings, understanding the biochemical signaling events controlling apoptosis is a first step towards developing therapeutic agents which would target this cell death process. This review will focus on the molecular cell death pathways responsible for generating the apoptotic phenotype, summarize what is currently known about apoptotic signals activated in the injured CNS, and what potential strategies might be pursued to reduce this cell death process as a means to promote functional recovery.

NBQX Treatment Improves Mitochondrial Function and Reduces Oxidative Events after Spinal Cord Injury

The purpose of this study was to examine the effects of inhibiting ionotropic glutamate receptor subtypes on measures of oxidative stress events at acute times following traumatic spinal cord injury (SCI). Rats received a moderate contusion injury and 15 min later were treated with one of two doses of 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzol[f]quinoxaline-7-sulfonamide disodium (NBQX), MK-801, or the appropriate vehicle. At 4 h following injury, spinal cords were removed and a crude synaptosomal preparation obtained to examine mitochondrial function using the MTT assay, as well as measures of reactive oxygen species (ROS), lipid peroxidation, and glutamate and glucose uptake. We report here that intraspinal treatment with either 15 or 30 nmol of NBQX improves mitochondrial function and reduces the levels of ROS and lipid peroxidation products. In contrast, MK-801, given intravenously at doses of 1.0 or 5.0 mg/kg, was without effect on these same measures. Neither drug treatment had an effect on glutamate or glucose uptake, both of which are reduced at acute times following SCI. Previous studies have documented that drugs acting on non-N-methyl-D-aspartate (NMDA) receptors exhibit greater efficacy compared to NMDA receptor antagonists on recovery of function and tissue sparing following traumatic spinal cord injury. The results of this study provide a potential mechanism by which blockade of the non-NMDA ionotropic receptors exhibit positive effects following traumatic SCI.