3-nitrotyrosine and Spinal-Cord-Injuries

The extent that age-dependent mitochondrial dysfunction drives neurodegeneration is not well understood. This study tested the hypothesis that mitochondria contribute to spinal cord injury (SCI)-induced neurodegeneration in an age-dependent manner by using 2,4-dinitrophenol (DNP) to uncouple electron transport, thereby increasing cellular respiration and reducing reactive oxygen species (ROS) production. We directly compared the effects of graded DNP doses in 4- and 14-month-old (MO) SCI-mice and found DNP to have increased efficacy in mitochondria isolated from 14-MO animals. In vivo, all DNP doses significantly exacerbated 4-MO SCI neurodegeneration coincident with worsened recovery. In contrast, low DNP doses (1.0-mg/kg/day) improved tissue sparing, reduced ROS-associated 3-nitrotyrosine (3-NT) accumulation, and improved anatomical and functional recovery in 14-MO SCI-mice. By directly comparing the effects of DNP between ages we demonstrate that mitochondrial contributions to neurodegeneration diverge with age after SCI. Collectively, our data indicate an essential role of mitochondria in age-associated neurodegeneration.

Topics: 2,4-Dinitrophenol; Aging; Animals; Cell Survival; Disease Progression; Female; Mice; Mice, Inbred C57BL; Mitochondria; Neurodegenerative Diseases; Neurons; Oxidative Stress; Oxygen Consumption; Reactive Oxygen Species; Recovery of Function; Spinal Cord Injuries; Tyrosine; Uncoupling Agents

Red/near-infrared light therapy (R/NIR-LT) has been developed as a treatment for a range of conditions, including injury to the central nervous system (CNS). However, clinical trials have reported variable or sub-optimal outcomes, possibly because there are few optimized treatment protocols for the different target tissues. Moreover, the low absolute, and wavelength dependent, transmission of light by tissues overlying the target site make accurate dosing problematic.. In order to optimize light therapy treatment parameters, we adapted a mouse spinal cord organotypic culture model to the rat, and characterized myelination and oxidative stress following a partial transection injury. The ex vivo model allows a more accurate assessment of the relative effect of different illumination wavelengths (adjusted for equal quantal intensity) on the target tissue. Using this model, we assessed oxidative stress following treatment with four different wavelengths of light: 450 nm (blue); 510 nm (green); 660 nm (red) or 860 nm (infrared) at three different intensities: 1.93 × 10(16) (low); 3.85 × 10(16) (intermediate) and 7.70 × 10(16) (high) photons/cm(2)/s. We demonstrate that the most effective of the tested wavelengths to reduce immunoreactivity of the oxidative stress indicator 3-nitrotyrosine (3NT) was 660 nm. 860 nm also provided beneficial effects at all tested intensities, significantly reducing oxidative stress levels relative to control (p ≤ 0.05).. Our results indicate that R/NIR-LT is an effective antioxidant therapy, and indicate that effective wavelengths and ranges of intensities of treatment can be adapted for a variety of CNS injuries and conditions, depending upon the transmission properties of the tissue to be treated.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Immunohistochemistry; Infrared Rays; Mice; Microscopy, Confocal; Neurodegenerative Diseases; Oxidative Stress; Phototherapy; Random Allocation; Rats; Spinal Cord; Spinal Cord Injuries; Tissue Culture Techniques; Tyrosine

Substantial experimental evidence supports that reactive species mediate secondary damage after traumatic spinal cord injury (SCI) by inducing oxidative stress. Removal of reactive species may reduce secondary damage following SCI. This study explored the effectiveness of a catalytic antioxidant - Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) - in removing reactive oxygen species (ROS), reducing oxidative stress, and improving functional recovery in vivo in a rat impact SCI model. The efficiency of MnTBAP was also compared with that of methylprednisolone - the only drug used clinically in treating acute SCI.. In vivo measurements of time courses of ROS production by microdialysis and microcannula sampling in MnTBAP, methylprednisolone, and saline (as vehicle control)-treated SCI rats showed that both agents significantly reduced the production of hydrogen peroxide, but only MnTBAP significantly reduced superoxide elevation after SCI. In vitro experiments further demonstrated that MnTBAP scavenged both of the preceding ROS, whereas methylprednisolone had no effect on either. By counting the immuno-positive neurons in the spinal cord sections immunohistochemically stained with anti-nitrotyrosine and anti-4-hydroxy-nonenal antibodies as the markers of protein nitration and membrane lipid peroxidation, we demonstrated that MnTBAP significantly reduced the numbers of 4-hydroxy-nonenal-positive and nitrotyrosine-positive neurons in the sections at 1.55 to 2.55 mm and 1.1 to 3.1 mm, respectively, rostral to the injury epicenter compared to the vehicle-treated animals. By behavioral tests (open field and inclined plane tests), we demonstrated that at 4 hours post-SCI treatment with MnTBAP and the standard methylprednisolone regimen both significantly increased test scores compared to those produced by vehicle treatment. However, the outcomes for MnTBAP-treated rats were significantly better than those for methylprednisolone-treated animals.. This study demonstrated for the first time in vivo and in vitro that MnTBAP significantly reduced the levels of SCI-elevated ROS and that MnTBAP is superior to methylprednisolone in removing ROS. Removal of ROS by MnTBAP significantly reduced protein nitration and membrane lipid peroxidation in neurons. MnTBAP more effectively reduced neurological deficits than did methylprednisolone after SCI - the first most important criterion for assessing SCI treatments. These results support the therapeutic potential of MnTBAP in treating SCI.

Topics: Aldehydes; Analysis of Variance; Animals; Blood-Brain Barrier; Cell Count; Disease Models, Animal; Hydrogen Peroxide; Locomotion; Male; Metalloporphyrins; Methylprednisolone; Microdialysis; Nervous System Diseases; Neuroprotective Agents; Oxidative Stress; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Recovery of Function; Spinal Cord Injuries; Time Factors; Tyrosine

Functional deficits following spinal cord injury (SCI) arise from both mechanical injury and from secondary tissue reactions involving inflammation. Natural almond skins (NS) were tested to evaluate anti-inflammatory effects on an animal model of SCI.. SCI was induced by the application of vascular clips to the dura via a four-level T5-T8 laminectomy. In the present study, to elucidate whether the protective effects of NS are related to the total phenolic content, we also investigated the effect of a blanched (BS) almond skins (industrially obtained by removing bran from the nut) in SCI. NS and BS (30 mg/kg respectively) were administered per os, 1 h and 6 h, after SCI.. SCI in mice resulted in severe injury characterized by edema, tissue damage, production of inflammatory mediators and apoptosis (measured by Bax, Bcl-2 and Tunel assay). NS treatment, 1 and 6 h after SCI, reduced all parameters of inflammation as neutrophil infiltration, NF-κB activation, PAR formation, iNOS expression and apoptosis. However, treatment with BS did not exert any protective effect.. Our results suggest that NS treatment, reducing the development of inflammation and tissue injury, may be useful in the treatment of SCI.

Topics: Analysis of Variance; Animals; Anti-Inflammatory Agents; Apoptosis; Disease Models, Animal; Inflammation; Lipid Peroxidation; Male; Mice; Neuroprotective Agents; Neutrophil Infiltration; NF-kappa B; Plant Extracts; Plant Structures; Prunus; Spinal Cord Injuries; Tyrosine

NADPH-oxidase is an enzyme responsible for reactive oxygen species production, and inhibition of this enzyme represents an attractive therapeutic target for the treatment of many diseases. The aim of this study was to investigate the effects of Apocynin, NADPH-oxidase inhibitor, in the modulation of secondary injury in the spinal cord. The injury was induced by application of vascular clips to the dura via a four-level T5-T8 laminectomy in mice. Treatment with Apocynin 1 and 6 h after the trauma significantly decreased (1) the degree of spinal cord inflammation and tissue injury, (2) neutrophil infiltration, (3) adhesion molecule expression, (4) nuclear transcription factor-κB expression, (5) nitrotyrosine and poly-ADP-ribose formation, (6) pro-inflammatory cytokines production, (7) apoptosis and (8) mitogen-activated protein kinase activation. Moreover, Apocynin significantly ameliorated the loss of limb function (evaluated by motor recovery score). Thus, it is proposed that Apocynin may be useful in the treatment of inflammation associated with spinal cord trauma.

Topics: Acetophenones; Animals; Apoptosis; Cell Adhesion Molecules; Cytokines; Gene Expression; Immunohistochemistry; Laminectomy; Male; Mice; Mitogen-Activated Protein Kinases; Models, Animal; Motor Activity; Myelitis; NADPH Oxidases; Neutrophil Infiltration; NF-kappa B; Poly Adenosine Diphosphate Ribose; Reactive Oxygen Species; Recovery of Function; Signal Transduction; Spinal Cord; Spinal Cord Injuries; Tyrosine

Spinal cord injury (SCI) stimulates an inflammatory reaction that causes substantial secondary damage inside the injured spinal tissue. The purpose of this study was to determine the anti-inflammatory effects of epigallocatechin gallate (EGCG) on traumatized spinal cord.. Rats were randomly divided into four groups of 12 rats each as follow: sham-operated group, trauma group, and EGCG-treatment groups (50 mg/kg, i.p., immediately and 1 hour after SCI). Spinal cord samples were taken 24 hours after injury and studied for determination of myeloperoxidase (MPO) activity, histopathological assessment and immunohistochemistry of tumor necrosis factor-Alpha (TNF-Alpha), interleukin-1Beta (IL-1Beta), Nitrotyrosine, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and poly(ADP-ribose) polymerase (PARP).. The results showed that MPO activity was significantly decreased in EGCG-treatment groups. Attenuated TNF-Alphaα, IL-1Beta, Nitrotyrosine, iNOS, COX-2, and PARP expression could be detected in the EGCG treated rats. Also, EGCG attenuated myelin degradation.. On the basis of these findings, we propose that EGCG may be effective in protecting rat spinal cord from secondary damage by modulating the inflammatory reactions.

Topics: Animals; Anti-Inflammatory Agents; Catechin; Cyclooxygenase 2; Densitometry; Immunohistochemistry; Interleukin-1beta; Male; Myelin Sheath; Neutrophil Infiltration; Nitric Oxide Synthase Type II; Peroxidase; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Tumor Necrosis Factor-alpha; Tyrosine

L-Carnosine is an endogenously synthesized dipeptide composed of beta-alanine and L-histidine. It acts as a free radical scavenger and possesses antioxidant properties. L-Carnosine reduces proinflammatory and profibrotic cytokines such as transforming growth factor-beta (TGF-beta), interleukin (IL)-1, and tumor necrosis factor (TNF)-alpha in different experimental settings. In the present study, we investigated the efficacy of L and D-carnosine on the animal model of spinal cord injury (SCI). The spinal cord was exposed via a four-level T5-T8 laminectomy and SCI was produced by extradural compression of the spinal cord at level T6-T7 using an aneurysm clip with a closing force of 24 g. Treatment with D-carnosine (150 mg/kg administered i.p., 1 h and 6h, after SCI), but not L-carnosine significantly decreased (a) the degree of spinal cord inflammation and tissue injury (histological score), (b) neutrophil infiltration (myeloperoxidase activity), (c) nitrotyrosine formation, inducible NO synthase (iNOS) and Hsp70 expression, (d) proinflammatory cytokines, and (e) apoptosis (TUNEL staining, Fas ligand, Bax, and Bcl-2 expression). Furthermore, D-carnosine (150 mg/kg administered i.p., 1 h and 6 h, after SCI) significantly ameliorated the loss of limb function (evaluated by motor recovery score). Taken together, our results demonstrate the strong difference between L-carnosine and D-carnosine. The result strongly suggests that D-carnosine treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Topics: Animals; bcl-2-Associated X Protein; Carnosine; Dose-Response Relationship, Drug; Fas Ligand Protein; Gene Expression Regulation; Male; Mice; Proto-Oncogene Proteins c-bcl-2; Spinal Cord Injuries; Tyrosine

Liver X receptor alpha (LXRalpha) and LXRbeta are members of the nuclear receptor superfamily of ligand-activated transcription factors. The aim of this study was to investigate the effects of T0901317, a potent LXR receptor ligand, in a mouse model of spinal cord injury (SCI). SCI was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy in mice. Treatment with T0901317, 1 and 6 h after the SCI, significantly decreased (i) the degree of spinal cord inflammation and tissue injury (histological score); (ii) neutrophil infiltration (myeloperoxidase activity); (iii) inducible nitric oxide synthase expression; (iv) nitrotyrosine, lipid peroxidation, and poly-ADP-ribose formation; (v) pro-inflammatory cytokines expression; (vi) nuclear factor-kappa B activation; and (vii) apoptosis (terminal deoxynucleotidyltransferase-mediated UTP end labeling staining, FAS ligand, Bax, and Bcl-2 expression). Moreover, T0901317 significantly ameliorated the loss of limb function (evaluated by motor recovery score). These data suggest that LXR ligand may be useful in the treatment of inflammation associated with SCI.

Topics: Animals; Anti-Inflammatory Agents; bcl-2-Associated X Protein; Cytokines; Disease Models, Animal; Fas Ligand Protein; Hydrocarbons, Fluorinated; In Situ Nick-End Labeling; Laminectomy; Lipid Peroxidation; Liver X Receptors; Male; Mice; Movement Disorders; Myelitis; Neutrophil Infiltration; NF-kappa B; Nitric Oxide Synthase Type II; Orphan Nuclear Receptors; Peroxidase; Poly Adenosine Diphosphate Ribose; Proteins; Proto-Oncogene Proteins c-bcl-2; Spinal Cord Injuries; Statistics, Nonparametric; Sulfonamides; Time Factors; Tyrosine

Several lines of evidence suggest a biological role for peroxisome proliferator-activated receptor (PPAR)-beta/delta in the pathogenesis many diseases. The aim of the present study was to evaluate the contribution of PPAR-beta/delta in the secondary damage in experimental spinal cord injury (SCI) in mice. To this purpose, we used 4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy]acetic acid (GW0742), a high-affinity PPAR-beta/delta agonist. Spinal cord trauma was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5 to T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of inflammatory mediators, tissue damage, and apoptosis. GW0742 treatment (0.3 mg kg(-1) i.p.) 1 and 6 h after the SCI significantly reduced 1) the degree of spinal cord inflammation and tissue injury (histological score), 2) neutrophil infiltration (myeloperoxidase activity), 3) nitrotyrosine formation, 4) proinflammatory cytokines expression, 5) nuclear factor-kappaB activation, 6) inducible nitric-oxide synthase expression, and 6) apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, FasL, Bax, and Bcl-2 expression). Moreover, GW0742 significantly ameliorated the recovery of limb function (evaluated by motor recovery score). To elucidate whether the protective effects of GW0742 are related to activation of the PPAR-beta/delta receptor, we also investigated the effect of PPAR-beta/delta antagonist methyl 3-({[2-(methoxy)-4 phenyl]amino}sulfonyl)-2-thiophenecarboxylate (GSK0660) on the protective effects of GW0742. GSK0660 (1 mg/kg i.p. 30 min before treatment with GW0742) significantly blocked the effect of the PPAR-beta/delta agonist and thus abolished the protective effect. Our results clearly demonstrate that GW0742 treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Topics: Animals; Apoptosis; Cytokines; Enzyme Induction; Fas Ligand Protein; Male; Mice; Neutrophil Infiltration; Nitric Oxide Synthase; Peroxidase; PPAR delta; PPAR-beta; Spinal Cord Injuries; Sulfones; Thiazoles; Thiophenes; Tumor Necrosis Factor-alpha; Tyrosine

In this study we evaluated the effect of glycosylated phenylpropanoid verbascoside (VB), isolated from cultured cells of the medicinal plant Syringa vulgaris (Oleaceae) in experimental animal model of spinal cord injury (SCI). SCI was induced by the application of vascular clips to the dura via a four-level T5-T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, tissue damage, and apoptosis. At 1 and 6 h after injury, the mice were treated with VB extract, administered at the dose of 2 mg/kg with intraperitoneal administration. Immunohistochemical examination demonstrated a marked increase on expression for nitrotyrosine, inducible nitric oxide synthase, poly(ADP-ribose), and apoptosis events (increase of Bax and Bcl-2 expression) in the spinal cord tissue. Additionally, we demonstrate that these inflammatory events were associated with the cytokines expression (TNF-α and IL-1β), neutrophil infiltration (myeloperoxidase), and activation of NF-κB. In contrast, all of these parameters of inflammation were attenuated by treatment with VB. In a separate set of experiment, we have clearly demonstrated that VB treatment significantly ameliorated the recovery of function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with VB extract reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Culture Techniques; Chromatography, High Pressure Liquid; Disease Models, Animal; Glucosides; Immunohistochemistry; In Situ Nick-End Labeling; Injections, Intraperitoneal; Interleukin-1beta; Male; Mice; Mice, Inbred Strains; Motor Activity; Nitric Oxide Synthase Type II; Phenols; Plant Extracts; Poly Adenosine Diphosphate Ribose; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Spinal Cord; Spinal Cord Injuries; Syringa; Tumor Necrosis Factor-alpha; Tyrosine

Glycyrrhizin, a major active constituent of liquorice root (Glycyrrhiza glabra), has a free radical scavenging property, and its effects were evaluated on an animal model of spinal cord injury (SCI) induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, tissue damage, and apoptosis (measured by terminal deoxynucleotidyltransferase-mediated dUTP-biotin end labeling staining, Bax, and Bcl-2 expression). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine, iNOS, and poly(adenosine diphosphate-ribose) in the spinal cord tissue. Additionally, we demonstrate that these inflammatory events were associated with the activation of nuclear factor-kappaB. In contrast, the degree of (1) spinal cord inflammation and tissue injury (histological score), (2) nitrotyrosine and poly(adenosine diphosphate [ADP] ribose) formation, (3) iNOS expression, (4) nuclear factor-kappaB activation, and (5) apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP-biotin end labeling, Bax, and Bcl-2) was markedly reduced in spinal cord tissue obtained from mice treated with glycyrrhizin extract (10 mg/kg, i.p., 30 min before and 1 and 6 h after SCI). In a separate set of experiments, we have clearly demonstrated that glycyrrhizin extract treatment significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with glycyrrhizin extract reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

Topics: Animals; Anti-Inflammatory Agents; bcl-2-Associated X Protein; Glycyrrhizic Acid; Immunohistochemistry; In Situ Nick-End Labeling; Inflammation; Male; Mice; Mice, Inbred Strains; Motor Activity; Nitric Oxide Synthase Type II; Proto-Oncogene Proteins c-bcl-2; Spinal Cord Injuries; Tyrosine

Primary impact to the spinal cord results in stimulation of secondary processes that potentiate the initial trauma. In the present study, we hypothesized that the altered expression of nitric oxide synthase (NOS) may contribute to these effects. Recent evidence indicates that nicotine can exert potent antioxidant and neuroprotective effects in spinal cord injury (SCI). Therefore, the aim of the present study was to evaluate whether the administration of nicotine can influence expression of inducible NOS (iNOS) and/or neuronal NOS (nNOS) in injured spinal cords. Adult male Long-Evans rats were subjected to a moderate contusion model of SCI and received a single intraperitoneal injection of either saline or nicotine (0.35, 3.5, or 7 mg/kg) 2 hr after trauma. SCI dramatically increased iNOS (but not nNOS) mRNA and protein levels in microglial cells in the thoracic and lumbar regions of spinal cords. iNOS overexpression resulted in increased nitrotyrosine formation, decreased number of NeuN (neuronal nuclei)-immunoreactive cells, and up-regulation of inflammatory genes. Most importantly, these effects were markedly attenuated by nicotine acting via a receptor-mediated mechanism. These data may have significant therapeutic implications for the targeting of nicotine receptors in the treatment of compressive spinal cord trauma.

Topics: Animals; Disease Models, Animal; Enzyme Inhibitors; Guanidines; Injections, Intraperitoneal; Male; Microglia; Neuroprotective Agents; Nicotine; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Rats; Rats, Long-Evans; RNA, Messenger; Spinal Cord; Spinal Cord Injuries; Tyrosine

The aim of the present study was to evaluate in a mouse model of spinal cord injury (SCI) the effect of the treatment with ethyl pyruvate (EP). Spinal cord injury was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy in mice. Treatment with EP (75, 25, or 8.5 mg/kg) 1 and 6 h after the SCI significantly decreased (a) the degree of spinal cord inflammation and tissue injury (histological score), (b) neutrophil infiltration (myeloperoxidase activity), (c) nitrotyrosine formation and iNOS expression, (d) proinflammatory cytokines expression, (e) nuclear factor kappaB activation, (f) extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase phosphorylation, and (g) apoptosis (TUNEL staining, Fas ligand, Bax, and Bcl-2 expression). Moreover, EP (75, 25, or 8.5 mg/kg) significantly ameliorated in a dose-dependent manner the loss of limb function (evaluated by motor recovery score). Taken together, our results demonstrate that EP treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Disease Models, Animal; Dose-Response Relationship, Drug; Fas Ligand Protein; Gene Expression Regulation; Humans; Inflammation; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neutrophil Infiltration; Nitric Oxide Synthase Type II; Peroxidase; Phosphorylation; Pyruvates; Spinal Cord Injuries; Time Factors; Tyrosine

In this study, we evaluated the effect of green tea extract (that was administered 25 mg/kg intraperitoneal at 1 and 6 h after injury) in experimental animal model of spinal cord injury. The spinal cord trauma was induced by the application of vascular clips to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterised by oedema, neutrophilic infiltration and apoptosis. Also, immunohistochemical examination demonstrated a marked increase in immune reactivity for nitrotyrosine. All parameters of inflammation were attenuated by green tea extract. The degree of spinal cord inflammation, nitrotyrosine, poli (ADP-ribosio) synthetase (PARS) and neutrophilic infiltration was markedly reduced. Green tea extract significantly ameliorated the recovery of limb function. Values shown are mean +/- SE mean of ten mice for each group. *p < 0.01 versus sham, degrees p < 0.01 versus spinal cord injury. Taken together, our results clearly demonstrate that green tea extract treatment ameliorates spinal cord injury oxidative stress.

Topics: Animals; Apoptosis; Disease Models, Animal; Edema; Immunohistochemistry; Inflammation; Male; Mice; Neutrophil Infiltration; Oxidative Stress; Plant Extracts; Poly(ADP-ribose) Polymerases; Spinal Cord Injuries; Tea; Time Factors; Tyrosine

Glucocorticoids (GCs) are effective anti-inflammatory agents widely used in therapeutic approach to treatment of spinal cord trauma. Previous results suggest that peroxisome proliferator activated receptor alpha (PPAR-alpha), an intracellular transcription factor activated by fatty acids, plays a role in control of secondary inflammatory process associated with spinal cord injury (SCI). With the aim to characterize the role of PPAR-alpha in GC-mediated anti-inflammatory activity, we tested the efficacy of dexamethasone (DEX), a synthetic GC specific for glucocorticoid receptor (GR), in an experimental model of spinal cord trauma induced in mice by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy, and comparing mice lacking PPAR-alpha (PPAR-alphaKO) with wild type (WT) mice. Results indicate that DEX-mediated anti-inflammatory activity is weakened in PPAR-alphaKO mice, as compared to WT controls. In particular, DEX was less effective in PPAR-alphaKO, compared to WT mice, as evaluated by inhibition of the degree of spinal cord inflammation and tissue injury, neutrophil infiltration, nitrotyrosine formation, pro-inflammatory cytokine expression, NF-kappaB activation, inducible nitric-oxide synthase (iNOS) expression; and apoptosis. This study indicates that PPAR-alpha can contribute to the anti-inflammatory activity of GCs in SCI.

Topics: Animals; Anti-Inflammatory Agents; Apoptosis; bcl-2-Associated X Protein; Dexamethasone; Disease Models, Animal; Inflammation Mediators; Interleukin-1beta; Mice; Mice, Knockout; NF-kappa B; Nitric Oxide Synthase Type II; PPAR alpha; Proto-Oncogene Proteins c-bcl-2; Spinal Cord Injuries; Tumor Necrosis Factor-alpha; Tyrosine

The aim of the present study was to assess the effect of a metalloporphyrinic peroxynitrite decomposition catalyst, ww-85, in the pathophysiology of spinal cord injury (SCI) in mice. Spinal cord trauma was induced by the application of vascular clips to the dura via a four-level T5-T8 laminectomy. SCI in mice resulted in severe trauma characterized by oedema, neutrophil infiltration, production of inflammatory mediators, tissue damage and apoptosis. ww-85 treatment (30-300 microg/kg, i.p. 1 h after the SCI) significantly reduced in a dose-dependent manner: (1) the degree of spinal cord inflammation and tissue injury, (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation and PARP activation, (4) pro-inflammatory cytokines expression, (5) NF-kappaB activation and (6) apoptosis. Moreover, ww-85 significantly ameliorated the recovery of limb function (evaluated by motor recovery score) in a dose-dependent manner. The results demonstrate that ww-85 treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cytokines; Disease Models, Animal; Immunoenzyme Techniques; In Situ Nick-End Labeling; Lipid Peroxidation; Male; Mice; Motor Activity; Neutrophil Infiltration; NF-kappa B; Peroxidase; Peroxynitrous Acid; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Spinal Cord Injuries; Thiobarbituric Acid Reactive Substances; Tyrosine

Therapeutic impact of neural stem cells (NSCs) for acute spinal cord injury (SCI) has been limited by the rapid loss of donor cells. Neuroinflammation is likely the cause. As there are close temporal-spatial correlations between the inducible nitric oxide (NO) synthase expression and the donor NSC death after neurotrauma, we reasoned that NO-associated radical species might be the inflammatory effectors which eliminate NSC grafts and kill host neurons. To test this hypothesis, human NSCs (hNSCs: 5 x 10(4) to 2 x 10(6) per milliliter) were treated in vitro with "plain" medium, 20 microM glutamate, or donors of NO and peroxynitrite (ONOO(-); 100 and 400 microM of spermine or DETA NONOate, and SIN-1, respectively). hNSC apoptosis primarily resulted from SIN-1 treatment, showing ONOO(-)-triggered protein nitration and the activation of p38 MAPK, cytochrome c release, and caspases. Therefore, cell death following post-SCI (p.i.) NO surge may be mediated through conversion of NO into ONOO(-). We subsequently examined such causal relationship in a rat model of dual penetrating SCI using a retrievable design of poly-lactic-co-glycolic acid (PLGA) scaffold seeded with hNSCs that was shielded by drug-releasing polymer. Besides confirming the ONOO(-)-induced cell death signaling, we demonstrated that cotransplantation of PLGA film embedded with ONOO(-) scavenger, manganese (III) tetrakis (4-benzoic acid) porphyrin, or uric acid (1 micromol per film), markedly protected hNSCs 24 hours p.i. (total: n = 10). Our findings may provide a bioengineering approach for investigating mechanisms underlying the host microenvironment and donor NSC interaction and help formulate strategies for enhancing graft and host cell survival after SCI.

Topics: Acute Disease; Animals; Annexin A5; Blotting, Western; Caspases; Cell Death; Cell Line; Cytoprotection; Drug Delivery Systems; Enzyme Activation; Fluoresceins; Free Radical Scavengers; Glycolates; Humans; Immunohistochemistry; In Situ Nick-End Labeling; Lactic Acid; Neurons; Nitric Oxide; Peroxynitrous Acid; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Signal Transduction; Spinal Cord Injuries; Stem Cells; Tyrosine

The reactive nitrogen species peroxynitrite (PN) has been suggested to be an important mediator of the secondary oxidative damage that occurs following acute spinal cord injury (SCI). The PN decomposition products nitrogen dioxide (*NO(2)), hydroxyl radical (*OH), and carbonate radical (*CO(3)) are highly reactive with cellular lipids and proteins. In this immunohistochemical study, we examined the temporal (3, 24, and 72 h, and 1 and 2 weeks) and spatial relationships of PN-mediated oxidative damage in the contusion-injured rat thoracic spinal cord (IH device, 200 kdyn, T10) using 3-nitrotyrosine (3-NT), a marker for protein nitration by PN-derived *NO(2) and 4-hydroxynonenal (4-HNE), an indicator of lipid peroxidation (LP) initiated by any of the PN radicals. Minimal 3-NT or 4-HNE immunostaining was seen in sham, non-injured spinal cords. In contrast, both markers showed a substantial increase at 3 h post-injury at the epicenter, that extended throughout the gray matter and into the surrounding white matter. At 24 and 72 h, the oxidative damage expanded circumferentially to involve all but a small rim of white matter tissue at the injury site, and longitudinally as much as 6-9 mm in the rostral and caudal directions. The staining was observed in neuronal soma, axons, and microvessels. At all time points except 3 h, there was no significant difference in the mean rostral or caudal extent of 3-NT and 4-HNE staining. By 1, and more so at 2 weeks, the longitudinal extent of the oxidative damage staining was greatly decreased. The spatial and temporal overlap of 3-NT and 4-HNE staining supports the concept that PN is involved in both damage produced by lipid peroxidation and protein nitration, and that antioxidant agents that target PN or PN-derived radicals should be effective neuroprotectants for acute SCI if administered during the first post-injury hours.

Topics: Aldehydes; Animals; Female; Immunohistochemistry; Lipid Peroxidation; Neurons; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Thoracic Vertebrae; Time Factors; Tyrosine

Elevated concentrations of nitric oxide (NO) and peroxynitrite (ONOO(-)) are present within the CNS following neurotrauma and are implicated in the pathogenesis of the accompanying neurologic deficits. We tested the hypothesis that elevated extracellular concentrations of ONOO(-), introduced by the donor 3-morpholinosydnonimine (SIN-1), induce reversible axonal conduction deficits in neurons of the guinea-pig spinal cord. The compound action potential (CAP) and compound membrane potential (CMP) of excised ventral cord white matter were recorded before, during, and after, bathing the tissue (30 min) in varying concentrations (0.125-2.0 mM) of SIN-1 (3.75-60 microM ONOO(-)). The principal results were rapid onset, concentration-dependent, reductions in amplitude of the CAP (P<0.05). At a concentration of 0.25 mM of SIN-1 the reduction in CAP amplitude was fully reversible and was not accompanied by any changes in CMP. At higher concentrations of SIN-1 (> or =0.5 mM) the reversibility was incomplete and there was concurrent depolarization of the CMP. These electrophysiological changes were not evident when the donor had been a priori depleted of ONOO(-) by uric acid or was co-administered with the ONOO(-) scavenger ebselen (3 mM). Immuno-fluorescence staining for nitrotyrosine (Ntyr) revealed extensive nitration of tyrosine residues in neurons exposed to higher concentrations of SIN-1. These results are the first to demonstrate that ONOO(-) induces reversible conduction deficits within axons of the spinal cord. The dissociation of CAP and CMP changes at low concentrations of SIN-1, when the CAP changes were reversible and there was no evidence of nitration of tyrosine residues, is consistent with ONOO(-)-induced alteration in Na+ channel conductance in the axolemma. The results support the view that ONOO(-) contributes to both reversible and non-reversible neurologic deficits following neurotrauma. The reversal of immune-mediated conduction deficits may contribute to spontaneous neurologic deficits following neurotrauma.

Topics: Action Potentials; Animals; Axons; Cell Membrane; Dose-Response Relationship, Drug; Extracellular Fluid; Female; Guinea Pigs; Molsidomine; Nerve Degeneration; Neural Conduction; Neurons; Nitric Oxide; Nitric Oxide Donors; Nitro Compounds; Peroxynitrous Acid; Sodium Channels; Spinal Cord; Spinal Cord Injuries; Tyrosine

Because studies have shown that 17beta-estradiol (E2) produces anti-inflammatory effects after various adverse circulatory conditions, we examined whether administration of E2 before spinal cord injury (SCI) has any salutary effects in reducing SCI. Spinal cord injury was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. To gain a better insight into the mechanism of action of the anti-inflammatory effects of E2, the following end points of the inflammatory process were evaluated: (1) spinal cord inflammation and tissue injury (histological score); (2) neutrophil infiltration (myeloperoxidase activity); (3) expression of iNOS, nitrotyrosine, and COX-2; (4) apoptosis (terminal deoxynucleotidyltransferase-mediated UTP end labeling staining and Bax and Bcl-2 expression); and (5) tissue TNF-alpha, IL-6, IL-1beta, and monocyte chemoattractant protein 1 levels. In another set of experiments, the pretreatment or posttreatment with E2 significantly ameliorates the recovery of limb function (evaluated by motor recovery score). To elucidate whether the protective effects of E2 were mediated via the estrogen receptors, we investigated the effect of an estrogen receptor antagonist, ICI 182,780, on the protective effects of E2. ICI 182,780 (500 microg/kg, s.c., 1 h before treatment with E2) significantly antagonized the effect of the E2 and abolished the protective effect against SCI. Taken together, our results clearly demonstrate that administration of E2 before SCI reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Topics: Animals; Apoptosis; Base Sequence; bcl-2-Associated X Protein; Chemokines; Cyclooxygenase 2; Cytokines; DNA Primers; Estradiol; Male; Mice; Motor Activity; Neutrophils; Nitric Oxide Synthase Type II; Peroxidase; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Spinal Cord Injuries; Tyrosine

Peroxynitrite (PON, ONOO(-)), formed by nitric oxide synthase-generated nitric oxide radical ( NO) and superoxide radical (O(2) (-)), is a crucial player in post-traumatic oxidative damage. In the present study, we determined the spatial and temporal characteristics of PON-derived oxidative damage after a moderate contusion injury in rats. Our results showed that 3-nitrotyrosine (3-NT), a specific marker for PON, rapidly accumulated at early time points (1 and 3 h) and a significant increase compared with sham rats was sustained to 1 week after injury. Additionally, there was a coincident and maintained increase in the levels of protein oxidation-related protein carbonyl and lipid peroxidation-derived 4-hydroxynonenal (4-HNE). The peak increases of 3-NT and 4-HNE were observed at 24 h post-injury. In our immunohistochemical results, the co-localization of 3-NT and 4-HNE results indicates that PON is involved in lipid peroxidative as well as protein nitrative damage. One of the consequences of oxidative damage is an exacerbation of intracellular calcium overload, which activates the cysteine protease calpain leading to the degradation of several cellular targets including cytoskeletal protein (alpha-spectrin). Western blot analysis of alpha-spectrin breakdown products showed that the 145-kDa fragments of alpha-spectrin, which are specifically generated by calpain, were significantly increased as soon as 1 h following injury although the peak increase did not occur until 72 h post-injury. The later activation of calpain is most likely linked to PON-mediated secondary oxidative impairment of calcium homeostasis. Scavengers of PON, or its derived free radical species, may provide an improved antioxidant neuroprotective approach for the treatment of post-traumatic oxidative damage in the injured spinal cord.

Topics: Aldehydes; Animals; Biomarkers; Calcium Signaling; Calpain; Disease Progression; Female; Free Radical Scavengers; Free Radicals; Lipid Peroxidation; Nerve Degeneration; Nitric Oxide; Oxidative Stress; Peptide Fragments; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Spectrin; Spinal Cord Injuries; Time Factors; Tyrosine; Up-Regulation

Mitochondrial dysfunction following spinal cord injury (SCI) may be critical for the development of secondary pathophysiology and neuronal cell death. Previous studies have demonstrated a loss of mitochondrial bioenergetics at 24 h following SCI. To begin to understand the evolution and study the contribution of mitochondrial dysfunction in pathophysiology of SCI, we investigated mitochondrial bioenergetics in the mid-thoracic region at 6, 12, and 24 h following contusion SCI. It is widely accepted that increased free radical generation plays a critical role in neuronal damage after SCI. Hence, to ascertain the role of free radicals in SCI-induced mitochondrial dysfunction, markers for oxidative damage, including nitrotyrosine (3-NT), lipid peroxidation byproduct (4-hydroxynonenal [HNE]), and protein oxidation (protein carbonyls) were quantified in the same samples of isolated mitochondria during the 24-h time course. The results demonstrate that a significant decline in mitochondrial function begins to occur 12 h post-injury and persists for a least 24 h following SCI. Furthermore, there was a progressive increase in mitochondrial oxidative damage that preceded the loss of mitochondrial bioenergetics, suggesting that free radical damage may be a major mitochondrial secondary injury process. Based on the present results, the temporal profile of mitochondrial dysfunction indicates that interventions targeting mitochondrial oxidative damage and dysfunction may serve as a beneficial pharmacological treatment for acute SCI.

Topics: Aldehydes; Animals; Disease Models, Animal; Disease Progression; Energy Metabolism; Female; Free Radicals; Lipid Peroxidation; Mitochondria; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Spinal Cord; Spinal Cord Injuries; Time Factors; Tyrosine

The aim of the present study was to assess the contribution of peroxynitrite formation in the pathophysiology of spinal cord injury (SCI) in mice. To this purpose, we used a peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride (FeTSPP). Spinal cord trauma was induced by the application of vascular clips (force of 24g) to the dura via a four-level T5-T8 laminectomy. SCI in mice resulted in severe trauma characterized by edema, neutrophil infiltration, production of inflammatory mediators, tissue damage, and apoptosis. FeTSPP treatment (10-100 mg/kg, i.p.) significantly reduced in dose-dependent manner 1 and 4 h after the SCI (1) the degree of spinal cord inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) nitrotyrosine formation and poly-(ADP-ribose) polymerase activation, (4) proinflammmaory cytokines expression, (5) NF-kappaB activation, and (6) apoptosis (TUNEL staining, Bax and Bcl-2 expression). Moreover, FeTSPP significantly ameliorated the recovery of limb function (evaluated by motor recovery score) in a dose-dependent manner. Taken together, our results clearly demonstrate that FeTSPP treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma similarly to dexamethasone, a well-known antiinflammatory agent which we have used as positive control.

Topics: Animals; Catalysis; Disease Models, Animal; DNA; I-kappa B Proteins; Interleukin-1beta; Interleukin-2; Lipid Peroxidation; Male; Metalloporphyrins; Mice; Mitochondria; Neutrophil Infiltration; NF-kappa B; Peroxidase; Peroxynitrous Acid; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins c-bcl-2; RNA; Spinal Cord Injuries; T-Lymphocytes; Tumor Necrosis Factor-alpha; Tyrosine

GSH plays multiple roles in the nervous system including free radical scavenger, redox modulator of ionotropic receptor activity, and possible neurotransmitter. A lot of evidence suggests that GSH is involved in the pathogenesis of neurodegenerative disorders, like spinal cord injury (SCI). This study was undertaken to determine if the inhibition of endogenous glutathione, by L-buthionine-(S,R)-sulfoximine (BSO), affords protection against peroxynitrite-mediated toxicity in response to the spinal cord injury in vivo. The spinal cord of damaged animals showed a significant elevation of biochemical, immunohistochemical and functional parameters, increasing, respectively, neutrophils infiltration, lipid peroxidation, nitrotyrosine formation, PAR expression, apoptosis (measured by TUNEL staining) and loss of hind legs movement in SCI-operated mice. In contrast, the administration of BSO led to worsening of this already compromised setting, increasing the degree of (1) neutrophil infiltration, (2) lipid peroxidation, (3) histological damage, (4) apoptosis, (5) nitrotyrosine formation, (6) PAR expression, (7) apoptosis (measured by TUNEL staining) and (7) loss of hind legs movement. Thus, endogenous glutathione plays an important protective role against secondary damage after SCI.

Topics: Animals; Antimetabolites; Apoptosis; bcl-2-Associated X Protein; Buthionine Sulfoximine; DNA; Glutathione; Immunohistochemistry; In Situ Nick-End Labeling; Kinetics; Lipid Peroxidation; Male; Malondialdehyde; Mice; Neutrophil Infiltration; Peroxidase; Poly Adenosine Diphosphate Ribose; RNA; Spinal Cord Compression; Spinal Cord Injuries; Tyrosine

The aim of our study was to evaluate the therapeutic efficacy of combination therapy with etanercept and dexamethasone (DEX) in vivo in experimental murine model of spinal cord trauma, which was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and cytokine production followed by recruitment of other inflammatory cells, production of inflammation mediators, tissue damage, apoptosis and disease. Treatment of the mice with etanercept (1.25 mg/kg) and DEX (0.025 mg/kg) when administered as a combination therapy but not as a single treatment significantly reduced the degree of (1) spinal cord inflammation and tissue injury (histological score), (2) infiltration of neutrophils (MPO evaluation), (3) inducible nitric oxide synthase, nitrotyrosine, and cytokines expression (tumor necrosis factor-alpha and interleukin-1 beta), (4) and apoptosis (Terminal deoxynucleotidyltransferase-mediated UTP end labeling staining, Fas-ligand expression and Bax and Bcl-2 expression). In a separate set of experiments we have also clearly demonstrated that the combination therapy significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate for the first time that strategies targeting multiple proinflammatory pathways may be more effective than a single effector molecule for the treatment of spinal cord trauma.

Topics: Animals; Anti-Inflammatory Agents; bcl-2-Associated X Protein; Dexamethasone; Disease Models, Animal; Drug Therapy, Combination; Etanercept; Immunoglobulin G; In Situ Nick-End Labeling; Interleukin-1beta; Laminectomy; Male; Mice; Neutrophils; Nitric Oxide Synthase Type II; Peroxidase; Proto-Oncogene Proteins c-bcl-2; Receptors, Tumor Necrosis Factor; Spinal Cord Injuries; Tumor Necrosis Factor-alpha; Tyrosine

In the present study, we evaluated the effect of age, in a model of spinal cord injury that was induced by the application of vascular clips to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in old rats resulted in severe trauma characterized by edema and neutrophil infiltration. Immunohistochemical examination demonstrated an increase in immunoreactivity for nitrotyrosine. In contrast, the degree of: (a) spinal cord inflammation and tissue injury (histological score), (b) nitrotyrosine, (c) PARS, and (d) neutrophils infiltration was markedly reduced in spinal cord tissue obtained from young rats. We have also demonstrated that ageing significantly worsened the recovery of limb function and caused an increase in mortality rate when compared with young rats.

Topics: Age Factors; Analysis of Variance; Animals; Disease Models, Animal; Immunohistochemistry; Laminectomy; Male; Oxidation-Reduction; Peroxidase; Poly Adenosine Diphosphate Ribose; Rats; Spinal Cord Injuries; Time Factors; Tyrosine

Etanercept is a tumor necrosis factor antagonist with anti-inflammatory effects. The aim of our study was to evaluate for the first time the therapeutic efficacy of in vivo inhibition of tumor necrosis factor-alpha (TNF-alpha) in experimental model of spinal cord trauma, which was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration, and cytokine production that it is followed by recruitment of other inflammatory cells, such as production of a range of inflammation mediators, tissue damage, apoptosis, and disease. Treatment of the mice with etanercept significantly reduced the degree of 1) spinal cord inflammation and tissue injury (histological score); 2) neutrophil infiltration (myeloperoxidase evaluation); 3) inducible nitric-oxide synthase, nitrotyrosine, cyclooxygenase-2, and cytokines expression (TNF-alpha and interleukin-1beta); and 4) apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and Bax and Bcl-2 expression). In a separate set of experiment, we have also clearly demonstrated that TNF-alpha inhibitor significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results clearly demonstrate that treatment with etanercept reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cyclooxygenase 2; Etanidazole; Immunologic Factors; Interleukin-1; Male; Mice; Neutrophil Infiltration; Nitric Oxide Synthase Type II; Proto-Oncogene Proteins c-bcl-2; Spinal Cord Injuries; Tumor Necrosis Factor-alpha; Tyrosine

Oxidative stress results from an oxidant/antioxidant imbalance, an excess of oxidants, and/or a depletion of antioxidants. A considerable body of recent evidence suggests that oxidative stress and exaggerated production of reactive oxygen species play a major role in several aspects of inflammation. Hypericum perforatum is a medicinal plant species containing many polyphenolic compounds, namely, flavonoids and phenolic acids. Because polyphenolic compounds have high antioxidant potential, in this study, we evaluated the effect of H. perforatum (given at 30 mg . kg (-1)) in an experimental animal model of spinal cord injury, which was induced by the application of vascular clips to the dura via a four-level T5 through T8 laminectomy. The degree of (a) spinal cord inflammation and tissue injury (histological score), (b) nitrotyrosine, (c) poly(adenosine diphosphate-ribose), (d) neutrophils infiltration, and (e) the activation of signal transducer and activator transcription 3 was markedly reduced in spinal cord tissue obtained from H. perforatum extract-treated mice. We have also demonstrated that H. perforatum extract significantly ameliorated the recovery of limb function.

Topics: Animals; Hypericum; Inflammation; Male; Mice; Neutrophil Infiltration; Oxidative Stress; Phytotherapy; Plant Extracts; Poly Adenosine Diphosphate Ribose; Recovery of Function; Signal Transduction; Spinal Cord Injuries; Tyrosine

Melatonin is the principal secretory product of the pineal gland and its role as an immuno-modulator is well established. Recent evidence shows that melatonin is a scavenger of oxyradicals and peroxynitrite and exerts protective effects in septic shock, hemorrhagic shock and inflammation. In the present study, we evaluated the effect of melatonin treatment, in a model of spinal cord injury (SCI). SCI was induced by the application of vascular clips (force of 50 g) to the dura via a four-level T5-T8 laminectomy. SCI in rats resulted in severe trauma characterized by edema, neutrophil infiltration and apoptosis (measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling staining). Infiltration of spinal cord tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced lipid peroxidation (increased tissue levels of malondialdehyde). Immunohistochemical examination demonstrated a marked increase in immunoreactivity for nitrotyrosine and Poly(ADP-ribose) (PAR) in the spinal cord tissue. In contrast, the degree of (a) spinal cord inflammation and tissue injury (histological score), (b) nitrotyrosine and PAR formation, (c) neutrophils infiltration and (d) apoptosis was markedly reduced in spinal cord tissue obtained from rats treated with melatonin (50 mg/kg i.p., 30 min before SCI, 30 min, 6 hr, 12 hr and 24 hr after SCI). In a separate set of experiment we have clearly demonstrated that melatonin treatment significantly ameliorated the recovery of limb function (evaluated by motor recovery score). Taken together, our results demonstrate that treatment with melatonin reduces the development of inflammation and tissue injury events associated with spinal cord trauma.

Topics: Animals; Apoptosis; DNA; Enzyme Activation; Free Radical Scavengers; I-kappa B Proteins; Lipid Peroxidation; Male; Melatonin; Neutrophils; NF-kappa B; NF-KappaB Inhibitor alpha; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Tyrosine

We investigated mechanisms by which a monoclonal antibody (mAb) against the CD11d subunit of the leukocyte integrin CD11d/CD18 improves neurological recovery after spinal cord injury (SCI) in the rat. The effects of an anti-CD11d mAb treatment were assessed on ED-1 expression (estimating macrophage infiltration), myeloperoxidase activity (MPO, approximating neutrophil infiltration), lipid peroxidation, inducible nitric oxide synthase (iNOS) and nitrotyrosine (indicating protein nitration) expression in the spinal cord lesion after severe clip-compression injury. Protein expression was evaluated by western blotting and immunocytochemistry. Lipid peroxidation was assessed by thiobarbituric acid reactive substances (TBARS) production. After anti-CD11d mAb treatment, decreased ED-1 expression at 6-72 h after SCI indicated reduced macrophage infiltration. MPO activity (units/g tissue) was reduced significantly from 114 +/- 11 to 75 +/- 8 (- 34%) at 6 h and from 38 +/- 2 to 22 +/- 4 (- 42%) at 72 h. After SCI, anti-CD11d mAb treatment significantly reduced TBARS from 501 +/- 61 to 296 +/- 17 nm (- 41%) at 6 h and to approximately uninjured values (87 nm) at 72 h. The mAb treatment also attenuated the expression of iNOS and formation of nitrotyrosine at 6-72 h after SCI. These data indicate that anti-CD11d mAb treatment blocks intraspinal neutrophil and macrophage infiltration, reducing the intraspinal concentrations of reactive oxygen and nitrogen species. These effects likely underlie improved tissue preservation and neurological function resulting from the mAb treatment.

Topics: Animals; Anti-Inflammatory Agents; Antibodies, Monoclonal; CD11 Antigens; Cell Movement; Disease Models, Animal; Disease Progression; Female; Integrin alpha Chains; Lipid Peroxidation; Macrophages; Neutrophil Infiltration; Neutrophils; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peroxidase; Proteins; Rats; Rats, Wistar; Spinal Cord Injuries; Thiobarbituric Acid Reactive Substances; Tyrosine

To determine whether peroxynitrite at the concentration and duration present after spinal cord injury induces protein oxidation and nitration in vivo, the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) was administered into the gray matter of the rat spinal cord for 5 hr. The cords were removed at 6, 12, 24, and 48 hr after SIN-1 exposure, immunohistochemically stained with antibodies to dinitrophenyl (DNP) and nitrotyrosine (Ntyr), markers of protein oxidation and nitration, respectively, and the immunostained neurons were counted. The percentages of DNP-positive (P = 0.023-0.002) and Ntyr-positive (P < 0.001 for all) neurons were significantly higher in the SIN-1-exposed groups than in the ACSF controls at each time, suggesting that peroxynitrite induced intracellular oxidation and nitration of proteins. The percentages of DNP- and Ntyr-positive neurons were not significantly different over time in either SIN-1- or ACSF-exposed groups (P = 0.20-1.00). The percentage of DNP-positive neurons was 7.6 +/- 3% to 12 +/- 4.2% at 6-24 hr, and it was 14 +/- 2% to 19 +/- 2% at 6-24 hr for Ntyr-positive neurons after SIN-1-exposure, whereas both ranged over 2-3% in ACSF controls. Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP, a broad-spectrum scavenger of reactive species) significantly reduced the percentages of DNP- and Ntyr-positive neurons (P = 0.04 and 0.002, respectively) compared to a SIN-1-exposed, untreated group at 24 hr after SIN-1 exposure. There were no significant differences between MnTBAP-treated and ACSF controls (P = 0.7 for DNP and 0.2 for Ntyr). These results further demonstrate peroxynitrite-induced protein oxidation and nitration and the efficiency of MnTBAP in scavenging peroxynitrite.

Topics: 2,4-Dinitrophenol; Animals; Drug Interactions; Enzyme Inhibitors; Free Radical Scavengers; Immunohistochemistry; Male; Metalloporphyrins; Microdialysis; Molsidomine; Neurons; Oxidation-Reduction; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Time Factors; Tyrosine

Nitric oxide (NO) has been shown to play an important role in the pathophysiology of traumatic brain injury (TBI) and cerebral ischemia. However, its contribution to the pathogenesis of traumatic spinal cord injury (SCI) remains to be clarified. This study determined the time course of constitutive and inducible nitric oxide synthases (cNOS and iNOS, respectively) after SCI. Rats underwent moderate SCI at T10 using the NYU impactor device and were allowed to survive for 3, 6, or 24 h and 3 days after SCI (n = 5 in each group). For the determination of enzymatic activities, spinal cords were dissected into five segments, including levels rostral and caudal (remote) to the injury site. Other rats were perfusion fixed for the immunohistochemical localization of iNOS protein levels. cNOS activity was significantly decreased at 3 and 6 h within the traumatized T10 segment and at 3, 6, and 24 h at the rostral (T9) level (p < 0.05). Rostral (T8) and caudal (T11, T12) to the injury site cNOS activity was also decreased at 3 h after injury (p < 0.05). However, cNOS activity returned to control levels within 6 h at T8, T11 and T12 and at one day at T10 and T9 segments. iNOS enzymatic activity was elevated at all time points tested (p < 0.05), with the most robust increase observed at 24 h. Immunostaining for iNOS at 24 h revealed that a significant cellular source of iNOS protein appeared to be invading polymorphonuclear leukocytes (PMNLs). To assess the functional consequences of iNOS inhibition, aminoguanidine treatment was initiated 5 min after SCI and rats tested using the BBB open field locomotor score. Treated rats demonstrated significantly improved hindlimb function up to 7 weeks after SCI. Histopathological analysis of contusion volume showed that aminoguanidine treatment decreased lesion volume by 37% (p < 0.05). In conclusion, these results indicate that (1) cNOS and iNOS activities are regionally and temporally affected after moderate SCI, (2) the early accumulation of PMNLs are a potentially significant source of NO-induced cytotoxic products, and (3) acute aminoguanidine treatment significantly improves functional and histopathological outcome after SCI.

Topics: Animals; Enzyme Inhibitors; Female; Guanidines; Immunohistochemistry; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Tyrosine

Secondary tissue damage after spinal cord injury (SCI) may be due to inflammatory mediators. After SCI, the nuclear factor-kappaB (NF-kappaB) transcription factor can activate many pro-inflammatory genes, one of which is inducible nitric oxide synthase (iNOS). iNOS catalyzes the synthesis of nitric oxide (NO), a key inflammatory mediator, which in turn reacts with superoxide to generate peroxynitrite. Peroxynitrite is a strong oxidant that can damage cellular enzymes, membranes, and subcellular organelles through the nitration of tyrosine residues on proteins. The presence of nitrotyrosine (NT) is an indirect chemical indicator of toxic NO and peroxynitrite-induced cellular damage. Using a New York University (NYU) impactor to induce SCI in adult rats, we examined the temporal and cellular expression of iNOS and NT. We observed a progressive increase in iNOS expression in the injured cord starting at day 1 with maximal expression occurring at day 7, as determined by Western blot analysis. iNOS expression corresponded temporally to an increase in iNOS enzyme activity after SCI. In parallel with the progressive increase in iNOS activity, NT expression also increased with time after SCI. The iNOS and NT immunoreactivity was localized in neurons, astrocytes, endothelial cells and ependymal cells at the epicenter and adjacent to the region of spinal cord impact and injury. Results from the present study suggest that increased iNOS and peroxynitrite anion, as reflected by the progressive accumulation of NT in the injured impacted spinal cord, may contribute to the secondary injury process after SCI.

Topics: Animals; Blotting, Western; Female; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Long-Evans; Spinal Cord; Spinal Cord Injuries; Thoracic Vertebrae; Tyrosine

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.

Topics: Amino Acids; Animals; Immunohistochemistry; Male; Models, Biological; Nitrates; Nitric Oxide; Nitroarginine; Proteins; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Superoxide Dismutase; Tyrosine; Wounds, Nonpenetrating

In the present study, immunohistochemical stainings for OX-6, OX-42, nitric oxide synthase I and II as well as nitrotyrosine were used to investigate possible correlation among microglial reactivity, nitric oxide synthase upregulation, peroxynitrite involvement and neuronal death in the nucleus dorsalis and red nucleus following lower thoracic spinal cord hemisection. Significant neuronal loss was found in the ipsilateral nucleus dorsalis and contralateral red nucleus after cord hemisection. A distinctive microglial reaction for OX-42 could be observed from one to four weeks post axotomy in the ipsilateral nucleus dorsalis; by contrast, it was observed on both sides of the red nucleus from one to three weeks following cord hemisection. The activated microglial cells showed some degree of hypertrophy. From the microglial immunoreactivity as well as their appearance, it was speculated that microglial activation might be beneficial or protective to the axotomized neurons. In normal and sham-operated rats, neurons of the nucleus dorsalis were not nitric oxide synthase I reactive. Three weeks after cord hemisection, neurons in the ipsilateral nucleus dorsalis below the lesion showed strong immunoreactivity. Neurons in the red nucleus that normally displayed weak nitric oxide synthase I immunoreactivity showed an increase on both sides of the nucleus. These results suggested that nitric oxide synthase I expression in the nucleus dorsalis following axotomy was synthesized de novo and might act as a neurotoxic agent. However, the bilateral increase in expression of nitric oxide synthase I in the red nucleus after lower thoracic cord hemisection was due to up-regulation of the constitutive enzyme and might have some neuroprotective function. Our results also suggested that peroxynitrite played no or little role in the neurodegeneration in the nucleus dorsalis and red nucleus following axotomy.

Topics: Animals; Antibodies, Monoclonal; Antigens, CD; Antigens, Neoplasm; Antigens, Surface; Avian Proteins; Basigin; Blood Proteins; Functional Laterality; Gene Expression Regulation, Enzymologic; Immunohistochemistry; Male; Membrane Glycoproteins; Microglia; Nerve Degeneration; Neurons; Nitric Oxide Synthase; Rats; Rats, Wistar; Red Nucleus; Spinal Cord; Spinal Cord Injuries; Tegmentum Mesencephali; Tyrosine