Danxia Liu

Excitatory amino acids rise to toxic levels upon impact injury to the rat spinal cord

The release of glutamate, aspartate, glutamine and asparagine upon impact injury to the rat spinal cord was characterized by sample collection from the site of injury by microdialysis. Injury caused dramatic and long-lasting increases in the concentrations of the excitatory amino acids. Determination of the relationship between unperturbed extracellular levels and the levels of amino acids in the collected fluids indicates that the concentrations of these amino acids were probably high enough to kill neurons for longer than one hour following impact injury to the spinal cord. Increases in the concentrations of the metabolically related non-neurotransmitters asparagine and glutamine were considerably smaller. The latter observations suggest that much of the increase in levels of the excitatory amino acids resulted from neuronal activity rather than from simple damage.

The roles of free radicals in amyotrophic lateral sclerosis: reactive oxygen species and elevated oxidation of protein, DNA, and membrane phospholipids

To explore whether reactive oxygen species (ROS) play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS), a unique microdialysis or microcannula sampling technique was used in mice transfected with a mutant Cu,Zn‐super‐oxide dismutase (SOD1) gene from humans with familial ALS, mice transfected with the normal human SOD1 gene, and normal mice. We demonstrate for the first time that the levels of hydrogen peroxide (H2O2) and the hydroxyl radical (OH) are significantly higher, and the level of the superoxide anion (O2•‐) is significantly lower in ALS mutant mice than in controls, supporting by in vivo evidence the hypothesis that the mutant enzyme catalyzes OH formation by the sequence: O2‐ → H2O2 → OH. This removes doubts regarding the relevance of elevated ROS in FALS raised by in vitro experiments. The levels of oxidation products are also significantly higher in the mutant mice than in controls, consistent with some previous reports. Only the superoxide concentration differs between two controls among all the measurements. Our findings correlate in vivo a gene mutation to both elevated H2O2 and OH and increased oxidation of cellular constituents. The elevated H2O2 in mutant mice indicates impairment of its detoxification pathways, perhaps by changed interactions between SOD1 and H2O2 detoxification enzymes.—Liu, D., Wen, J., Liu, J., Li, L. The roles of free radicals in amyotrophic lateral sclerosis: reactive oxygen species and elevated oxidation of protein, DNA, and membrane phospholipids. FASEB J. 13, 2318–2328 (1999)

The role of reactive nitrogen species in secondary spinal cord injury: Formation of nitric oxide, peroxynitrite, and nitrated protein

Abstract: To determine whether reactive nitrogen species contribute to secondary damage in CNS injury, the time courses of nitric oxide, peroxynitrite, and nitrotyrosine production were measured following impact injury to the rat spinal cord. The concentration of nitric oxide measured by a nitric oxide‐selective electrode dramatically increased immediately following injury and then quickly declined. Nitro‐L‐arginine reduced nitric oxide production. The extracellular concentration of peroxynitrite, measured by perfusing tyrosine through a microdialysis fiber into the cord and quantifying nitrotyrosine in the microdialysates, significantly increased after injury to 3.5 times the basal level, and superoxide dismutase and nitro‐L‐arginine completely blocked peroxynitrite production. Tyrosine nitration examined immunohistochemically significantly increased at 12 and 24 h postinjury, but not in sham‐control sections. Mn(III) tetrakis(4‐benzoic acid)‐porphyrin (a novel cell‐permeable superoxide dismutase mimetic) and nitro‐L‐arginine significantly reduced the numbers of nitrotyrosine‐positive cells. Protein‐bound nitrotyrosine was significantly higher in the injured tissue than in the sham‐operated controls. These results demonstrate that traumatic injury increases nitric oxide and peroxynitrite production, thereby nitrating tyrosine, including protein‐bound tyrosine. Together with our previous report that trauma increases superoxide, our results suggest that reactive nitrogen species cause secondary damage by nitrating protein through the pathway superoxide + nitric oxide peroxynitrite protein nitration.

ELEVATION OF HYDROGEN PEROXIDE AFTER SPINAL CORD INJURY DETECTED BY USING THE FENTON REACTION

To reveal whether reactive oxygen species (ROS) play a role after spinal cord injury, we developed a unique method for assaying hydrogen peroxide (H2O2) and determined the time course of its concentration changes following impact injury to the rat spinal cord. Microdialysis was used to sample H2O2 in the extracellular space and the dialysates were collected into a vial containing salicylate and ferrous chloride (FeCl2). H2O2 collected in the vial was converted to hydroxyl radicals (*OH) by FeCl2 catalysis. 2,3- and 2,5-dihydroxybenzoic acid produced by reaction of *OH with salicylate in the collecting vial were measured by HPLC and calibrated to H2O2 concentrations. The postinjury levels of H2O2 were significantly increased (p = 0.02) for over 11 h. FeCl2 administered through the dialysis fiber catalyzes H2O2 conversion in the cord to *OH. This *OH does not reach the collecting vial due to its extremely short lifetime (nanoseconds). The reduced H2O2 levels in the vials validate the measurement of H2O2. The relatively long-lasting formation of H2O2 and superoxide reported herein and previously suggests that ROS may be important in secondary spinal cord damage and that removal of ROS may be a realistic treatment strategy for reducing injury caused by free radicals.

Superoxide production after spinal injury detected by microperfusion of cytochrome c

Highly reactive oxygen-containing species may form upon CNS injury and cause oxidative damage to important cellular components, thereby destroying cells. To test this hypothesis, free radical formation following such insults should be characterized first. In this study, we measured the time course of superoxide production following impact injury to the rat spinal cord using a novel microcannula perfusion technique developed by us. Cytochrome c (50 microM in artificial cerebrospinal fluid) was perfused into the rat spinal cord through the cannula inserted laterally into the gray matter of the cord, and reduced cytochrome c was measured from perfusates spectrophotometrically. We found that the levels of superoxide in the extracellular space increased to approximately twice the basal level and remained elevated for over 10 h. Superoxide dismutase (60 U/ml) significantly reduced the elevation of superoxide levels (p = .016) and ferric chloride (0.1 mM)/EDTA (0.25 mM) infused together with cytochrome c completely removed the superoxide measured, validating the measurement of superoxide. The relatively long-lasting formation of superoxide reported herein suggests that removal of superoxide may be a realistic treatment strategy for reducing injury caused by free radicals.

Methylprednisolone reduces excitatory amino acid release following experimental spinal cord injury

Administration of methylprednisolone within several hours after injury to the spinal cord has been shown to reduce subsequent impairment in humans and experimental animals. Secondary damage following initial trauma is probably caused in part by the toxicity of released excitatory amino acids. We demonstrate here that methylprednisolone reduces the release of excitatory amino acids following experimental spinal cord injury in rats.

Protein and DNA oxidation in spinal injury: Neurofilaments - An oxidation target

This study measured the time courses of protein and DNA oxidation following spinal cord injury (SCI) in rats and characterized oxidative degradation of proteins. Protein carbonyl content-a marker of protein oxidation-significantly increased at 3-9 h postinjury and the ratio 8-hydroxy-2-deoxyguanosine/deoxyguanosine-an indicator of DNA oxidation-was significantly higher at 3-6 h postinjury in the injured cords than in the sham controls. This suggests that oxidative modification of proteins and DNA contributes to secondary damage in SCI. Densities of selected bands on coomassie-stained gels indicated that most proteins were degraded. Neurofilament protein (NFP) was particularly evaluated immunohistochemically; its light chain (NFP-68) was gradually degraded in nerve fibers, neuron bodies, and large dendrites following SCI. A mixture of Mn (III) tetrakis (4-benzoic acid) porphyrin (10 mg/kg)-a novel SOD mimetic-and nitro-L-arginine (1 mg/kg)-an inhibitor of nitric oxide synthase-injected intraperitoneally, increased NFP-68 immunoreactivity and the numbers of NFP-positive nerve fibers post-SCI, correlating NFP degradation in SCI to free radical-triggered oxidative damage for the first time. Therefore, blockage of protein and DNA oxidation in the secondary injury stage may improve long-term recovery-important information for development of the SCI therapies.

The Time Course of Hydroxyl Radical Formation following Spinal Cord Injury: The Possible Role of the Iron-Catalyzed Haber-Weiss Reaction

This study explores whether the hydroxyl radical (*OH)-one of the most destructive reactive oxygen species-plays a role in secondary spinal cord injury (SCI). First, we measured the time course of *OH formation in rat spinal tissue after impact SCI by administering salicylate as a trapping agent into the intrathecal space of the cord and measuring the hydroxylation products of salicylate, 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA) by HPLC. The 2,3-DHBA concentration was significantly higher in injured spinal tissue than in sham controls at 5 min, 1 and 3 h, but not at 5 h post-injury. Second, we generated *OH by administering H(2)O(2) and FeCl(2)/EDTA (Fentons reagents) at the concentrations produced by SCI into the gray matter of the cord for 4 h and found that it induced significant cell loss at 24 h post-*OH exposure. Mn (III) tetrakis (4-benzoic acid) porphyrin(MnTBAP)-a broad spectrum reactive species scavenger-significantly reduced *OH-induced cell death. Finally, we generated superoxide and administered FeCl(3)/EDTA in the intrathecal space of the cord at the concentration produced by SCI and measured extracellular *OH formation in the gray matter of the cord by microdialysis sampling. We found that the levels of *OH significantly increased compared to the pre-administration level, indicating that *OH can be produced in vivo by the iron-catalyzed Haber-Weiss reaction. All together, we demonstrated that *OH is an endogenous secondary damaging agent following SCI and the metal-catalyzed Haber-Weiss reaction may contribute to early *OH formation after SCI.

Mutation of superoxide dismutase elevates reactive species: comparison of nitration and oxidation of proteins in different brain regions of transgenic mice with amyotrophic lateral sclerosis.

As part of our effort to study the role of reactive species in amyotrophic lateral sclerosis (ALS), the goal of this work is to explore the correlation between nitration and oxidation of proteins and mutation of Cu, Zn-superoxide dismutase (SOD1) in ALS. Transgenic mice overexpressing the mutant Cu, Zn-superoxide dismutase (mSOD1) gene from humans with familial ALS, wild-type mice overexpressing the normal human SOD1 gene and normal mice without gene overexpression were used. Brain sections from different regions of three groups of mice were double immunohistochemically stained with anti-neurofilament plus anti-nitrotyrosine or treated with 2,4-dinitrophenylhydrazine to label protein carbonyls, then double stained with anti-neurofilament plus anti-2,4-dinitrophenyl (anti-DNP). Neurons containing nitrated and oxidized proteins were visualized only in mSOD1 mice in the motor cortex, the cerebellar cortex and nucleus of hypoglossal nerves (regions related with movement). This correlates mutation of SOD1 to nitration and oxidation of neurons in the movement regions. By counting double-stained neurons, we demonstrated that the number of nitrotyrosine- and DNP-positive neurons was significantly higher in the brain sections of both motor and sensory cortex in mSOD1 mice than in the corresponding regions of control mice (P=0.005 to <0.001), further correlating nitration and oxidation of proteins to SOD1 mutation. Neurons underwent significantly more nitration and oxidation in the motor cortex than in the sensory cortex in mSOD1 mice (P=0.002 and 0.02 respectively), indicating enhanced susceptibility of the motor cortex to nitration and oxidation of proteins and thereby targeting oxidation and nitration of proteins in neurons of the motor cortex in ALS. Significantly elevated protein nitration and nitric oxide synthesis were also demonstrated biochemically in the brain tissues and in cerebrospinal fluid of mutant SOD1 mice. Our in vivo evidence correlates mutation of the SOD1 gene to increased nitric oxide, nitration and oxidation of proteins in ALS.

Paraquat — A superoxide generator — Kills neurons in the rat spinal cord

Microdialysis was used to administer paraquat into the spinal cord of the anesthetized rat to determine the effects of the in vivo generation of the superoxide anion (O2.-) on neurons. Exposure to paraquat caused blockage of axonal conduction, destruction of the cell bodies of neurons, and a general release of amino acids. Thus, paraquat is quite harmful to neuronal tissue. Similarities between paraquat-induced damage and that previously observed from the hydroxyl radical are consistent with paraquat, causing damage by generation of reactive oxygen species and the widespread belief that generation of O2.- initiates the formation of destructive reactive oxygen species in a wide variety of traumas and other disorders.