3-nitrotyrosine and Brain-Injuries

Traumatic brain injury (TBI) has become a growing epidemic but no approved pharmacological treatment has been identified. Our previous work indicates that mitochondrial oxidative stress/damage and loss of bioenergetics play a pivotal role in neuronal cell death and behavioral outcome following experimental TBI. One tactic that has had some experimental success is to target glutathione using its precursor N-acetylcysteine (NAC). However, this approach has been hindered by the low CNS bioavailability of NAC. The current study evaluated a novel, cell permeant amide form of N-acetylcysteine (NACA), which has high permeability through cellular and mitochondrial membranes resulting in increased CNS bioavailability. Cortical tissue sparing, cognitive function and oxidative stress markers were assessed in rats treated with NACA, NAC, or vehicle following a TBI. At 15days post-injury, animals treated with NACA demonstrated significant improvements in cognitive function and cortical tissue sparing compared to NAC or vehicle treated animals. NACA treatment also was shown to reduce oxidative damage (HNE levels) at 7days post-injury. Mechanistically, post-injury NACA administration was demonstrated to maintain levels of mitochondrial glutathione and mitochondrial bioenergetics comparable to sham animals. Collectively these data provide a basic platform to consider NACA as a novel therapeutic agent for treatment of TBI.

Topics: Acetylcysteine; Aldehydes; Animals; Brain Injuries; Cerebral Cortex; Disease Models, Animal; Double-Blind Method; Energy Metabolism; Glutathione; Male; Maze Learning; Mitochondria; Neuroprotective Agents; Oxidative Stress; Oxygen Consumption; Rats; Rats, Sprague-Dawley; Time Factors; Tyrosine

Stroke triggers a complex inflammatory process in which the balance between pro- and antiinflammatory mediators is critical for the development of the brain infarct. However, systemic changes may also occur in parallel with brain inflammation. Here we demonstrate that administration of recombinant IL-33, a recently described member of the IL-1 superfamily of cytokines, promotes Th2-type effects following focal ischemic stroke, resulting in increased plasma levels of Th2-type cytokines and fewer proinflammatory (3-nitrotyrosine+F4/80+) microglia/macrophages in the brain. These effects of IL-33 were associated with reduced infarct size, fewer activated microglia and infiltrating cytotoxic (natural killer-like) T cells, and more IL-10-expressing regulatory T cells. Despite these neuroprotective effects, mice treated with IL-33 displayed exacerbated post-stroke lung bacterial infection in association with greater functional deficits and mortality at 24 hours. Supplementary antibiotics (gentamicin and ampicillin) mitigated these systemic effects of IL-33 after stroke. Our findings highlight the complex nature of the inflammatory mechanisms differentially activated in the brain and periphery during the acute phase after ischemic stroke. The data indicate that a Th2-promoting agent can provide neuroprotection without adverse systemic effects when given in combination with antibiotics.

Topics: Animals; Brain Injuries; Brain Ischemia; Cytokines; Disease Models, Animal; Inflammation; Interleukin-10; Interleukin-33; Interleukin-4; Lung; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Stroke; Th1 Cells; Th2 Cells; Treatment Outcome; Tyrosine

A number of neurological disorders such as epidural hematoma can cause compression of cerebral cortex. We here tested the hypothesis that sustained compression of primary somatosensory cortex may affect stellate neurons and thalamocortical afferent (TCA) fibers. A rat model with barrel cortex subjected to bead epidural compression was used. Golgi-Cox staining analyses showed the shrinkage of dendritic arbors and the stripping of dendritic spines of stellate neurons for at least 3 months post-lesion. Anterograde tracing analyses exhibited a progressive decline of TCA fiber density in barrel field for 6 months post-lesion. Due to the abrupt decrease of TCA fiber density at 3 days after compression, we further used electron microscopy to investigate the ultrastructure of TCA fibers at this time. Some TCA fiber terminal profiles with dissolved or darkened mitochondria and fewer synaptic vesicles were distorted and broken. Furthermore, the disruption of mitochondria and myelin sheath was observed in some myelinated TCA fibers. In addition, expressions of oxidative markers 3-nitrotyrosine and 4-hydroxynonenal were elevated in barrel field post-lesion. Treatment of antioxidant ascorbic acid or apocynin was able to reverse the increase of oxidative stress and the decline of TCA fiber density, rather than the shrinkage of dendrites and the stripping of dendritic spines of stellate neurons post-lesion. Together, these results indicate that sustained epidural compression of primary somatosensory cortex affects the TCA fibers and the dendrites of stellate neurons for a prolonged period. In addition, oxidative stress is responsible for the reduction of TCA fiber density in barrels rather than the shrinkage of dendrites and the stripping of dendritic spines of stellate neurons.

Topics: Acetophenones; Afferent Pathways; Aldehydes; Animals; Antioxidants; Ascorbic Acid; Biotin; Brain Injuries; Dendrites; Dextrans; Disease Models, Animal; Electron Transport Complex IV; Epidural Space; Functional Laterality; Male; Neurons; Oxidative Stress; Rats; Somatosensory Cortex; Thalamus; Time Factors; Tyrosine

Traumatic brain injury (TBI) is the leading cause of mortality and morbidity in youth, but to date, effective therapies are still lacking. Previous studies revealed a marked response of apolipoprotein J (ApoJ) expression to the brain injury. The aim of this study was to determine the potential roles of ApoJ in functional recovery following TBI. After controlled cortex impact (CCI), a TBI model, in adult wild-type mice, ApoJ expression was up-regulated since 6 h post-injury and sustained for 5 days. Animals infused with recombinant human ApoJ intraventricularly at 30 min prior to CCI showed significantly reduced oxidative stress (3-nitrotyrosine, 4-hydroxynonenal) and complement activation (C5b-9). In addition, ApoJ treatment was shown to suppress the inflammatory response (glial activation, cytokine expression), blood-brain barrier disruption (Evans blue extravasation), and cerebral edema (water content) induced by CCI. Concomitantly, improved neuronal maintenance and neurological behavioral performance were observed in ApoJ-treated mice compared with the vehicle group. These findings support a neuroprotective role of ApoJ via multifunctional pathways, providing a novel and encouraging treatment strategy for TBI. Apolipoprotein J (ApoJ) was up-regulated after controlled cortical impact (CCI). Mice infused with human recombinant ApoJ prior to CCI showed reduced expression of complement and oxidative marker proteins as well as reduced inflammatory response and attenuated blood-brain barrier (BBB) disruption and cerebral edema. Neuronal maintenance and behavioral performance were improved by ApoJ infusion. These findings demonstrated the protective function of ApoJ for traumatic brain injury (TBI) therapy.

Topics: Aldehydes; Animals; Behavior, Animal; Blood-Brain Barrier; Brain Edema; Brain Injuries; Cerebral Cortex; Clusterin; Disease Models, Animal; Infusions, Intraventricular; Male; Mice, Inbred C57BL; Neuroprotective Agents; Tyrosine

Intracerebral hemorrhage (ICH)-induced brain injury leads to irreversible disruption of the blood-brain barrier (BBB) and fatality brain edema with massive cell death. Although secondary damage could, in principle, be preventable, no effective treatment approaches currently exist for patients with ICH. Tempol, a catalytic scavenger of peroxynitrite (ONOO)-derived free radicals, has been proven to ameliorate brain injury in several types of brain insults. This study aims to investigate the potential neuroprotective effect of tempol after ICH and to explore the underlying mechanisms. Collagenase-induced ICH was performed in rats. Tempol was administered immediately after ICH. The effects of tempol on ICH were evaluated by assessing neurological deficits, BBB permeability, brain edema, and apoptotic cell death. The mechanisms of action of tempol, with its clear ability on the derivative of ONOO [3-nitrotyrosine (3-NT), ONOO, and its derivative-mediated nitration marker] and expression of tight junction protein [zonula occludens-1 (ZO-1)], were also investigated. Perihematomal 3-NT increased significantly following ICH and expressed around vessels accompanied by reduced and discontinuous expression of ZO-1. Tempol treatment significantly suppressed 3-NT formation and preserved ZO-1 levels, and led to improvement in neurological outcomes and reduction of BBB leakiness, brain edema, and apoptosis. In conclusion, tempol has neuroprotective potential in experimental ICH and may help combat ICH-induced brain injury in patients.

Topics: Animals; Antioxidants; Apoptosis; Blood-Brain Barrier; Brain; Brain Edema; Brain Injuries; Cerebral Hemorrhage; Collagenases; Cyclic N-Oxides; Disease Models, Animal; Male; Neuroprotective Agents; Random Allocation; Rats, Sprague-Dawley; Spin Labels; Tyrosine; Zonula Occludens-1 Protein

It is known that mitochondria play an important role in the mechanisms of brain cells damage and death following traumatic brain injury (TBI). However, the relationship between the severity of brain damage following TBI and mitochondrial dysfunction are not well defined.. to study activities of NADN- and succinate dehydrogenases, a key enzyme of mitochondrial oxidative phosphorylation in children with TBI of varying severity and different outcomes; to detect ATP content in lymphocytes; the level of NOx and 3-nitrotyrosine in serum and plasma. Methods: all parameters were determined in the dynamics of one month following TBI, and in some cases up to the death ofpatients. The severity of TBI was scored by Glasgow Coma Scale (GCS), the outcome of TBI-Glasgow Outcome Scale (GOS). Based on the clinical examination children with TBI were divided into 3 groups: (1) mild TBI; (2) severe TBI and (3) severe TBI with fatal outcome.. we found that activity of dehydrogenases is significantly reduced only in patients with the poor neurologic outcome. The greatest decrease in these parameters was observed in patients with severe traumatic brain injury and fatal outcome. A direct correlation was found between the indices of dehydrogenases activity and A TP content in lymphocytes (r = 0.97, p = 0.005). The levels of NOx metabolites and 3-nitrotyrosine were significantly increased in children with severe TBI.. obtained results suggest that mitochondrial dysfunction, impaired cerebral energy metabolism and oxidative stress contribute to cell death in the brain and thus represent therapeutic targets for the treatment of TBI.

Topics: Brain Injuries; Child; Child, Preschool; Female; Glasgow Coma Scale; Humans; Infant; Lymphocytes; Male; Mitochondria; NADH Dehydrogenase; Nitric Oxide; Oxidative Phosphorylation; Statistics as Topic; Succinate Dehydrogenase; Tyrosine

Traumatic brain injury (TBI) initiates a neuroinflammatory cascade that contributes to neuronal damage and behavioral impairment. Toll-like receptors (TLRs) are signaling receptors in the innate immune system, although emerging evidence indicates their role in brain injury. We have therefore investigated the role played by TLR4 signaling pathway in the development of mechanisms of secondary inflammatory process in traumatic brain injury (TBI) differ in mice that lack a functional TLR4 signaling pathway.. Controlled cortical impact injury was performed on TLR4 knockout (KO) mice (C57BL/10ScNJ) and wild-type (WT) mice (C57BL/10ScNJ). TBI outcome was evaluated by determination of infarct volume and assessment of neurological scores. Brains were collected at 24 h after TBI. When compared to WT mice, TLR4 KO mice had lower infarct volumes and better outcomes in neurological and behavioral tests (evaluated by EBST and rotarod test). Mice that lacked TLR4 had minor expression of TBI-induced GFAP, Chymase, Tryptase, IL-1β, iNOS, PARP and Nitrotyrosine mediators implicated in brain damage. The translocation of expression of p-JNK, IκB-α and NF-κB pathway were also lower in brains from TLR4 KO mice. When compared to WT mice, resulted in significant augmentation of all the above described parameters. In addition, apoptosis levels in TLR4 KO mice had minor expression of Bax while on the contrary with Bcl-2.. Our results clearly demonstrated that absence of TLR4 reduces the development of neuroinflammation, tissues injury events associated with brain trauma and may play a neuroprotective role in TBI in mice.

Topics: Animals; Brain Infarction; Brain Injuries; Cerebral Cortex; Gene Expression Regulation; Glial Fibrillary Acidic Protein; I-kappa B Proteins; Inflammation; Interleukin-1beta; MAP Kinase Kinase 4; Mice; Mice, Knockout; Nerve Tissue Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase Type II; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Toll-Like Receptor 4; Tryptases; Tyrosine

Traumatic brain injury (TBI) is a major cause of death and disability in young people. No effective therapy is available to ameliorate its damaging effects. Our aim was to investigate the optimal therapeutic time window of edaravone, a free radical scavenger which is currently used in Japan. We also determined the temporal profile of reactive oxygen species (ROS) production, oxidative stress, and neuronal death. Male C57Bl/6 mice were subjected to a controlled cortical impact (CCI). Edaravone (3.0 mg/kg), or vehicle, was administered intravenously at 0, 3, or 6 hours following CCI. The production of superoxide radicals (O2 (∙-)) as a marker of ROS, of nitrotyrosine (NT) as an indicator of oxidative stress, and neuronal death were measured for 24 hours following CCI. Superoxide radical production was clearly evident 3 hours after CCI, with oxidative stress and neuronal cell death becoming apparent after 6 hours. Edaravone administration after CCI resulted in a significant reduction in the injury volume and oxidative stress, particularly at the 3-hour time point. Moreover, the greatest decrease in O2 (∙-) levels was observed when edaravone was administered 3 hours following CCI. These findings suggest that edaravone could prove clinically useful to ameliorate the devastating effects of TBI.

Topics: Animals; Antipyrine; Brain Injuries; Disease Models, Animal; Edaravone; Free Radical Scavengers; Japan; Male; Mice; Mice, Inbred C57BL; Neurons; Oxidative Stress; Reactive Oxygen Species; Tyrosine

Hydrogen gas has been demonstrated to neutralize free radicals and reduce oxidative stress recently. Our objective was to determine the therapeutic effect of H2 inhalation and its antioxidative activity on early brain injury after subarachnoid hemorrhage.. Controlled in vivo laboratory study.. Animal research laboratory.. One hundred thirty-seven adult male Sprague-Dawley rats weighing 280-350 g.. Subarachnoid hemorrhage was induced by endovascular perforation method in rats. Subarachnoid hemorrhage rats were treated with 2.9% hydrogen gas inhaled for 2 hrs after perforation. At 24 and 72 hrs, mortality, body weight, neurologic deficits, and brain water content were assessed. Blood-brain barrier permeability and apoptosis were also measured at 24 hrs. To investigate the antioxidative activity of hydrogen gas, the expression of malondialdehyde, nitrotyrosine, and 8-hydroxyguanosine, which are oxidative markers of lipid, protein, and DNA damage, respectively, were measured at 24 hrs.. Hydrogen gas significantly alleviated brain edema and blood-brain barrier disruption, reduced apoptosis, and improved neurologic function at 24 hrs but not 72 hrs after subarachnoid hemorrhage. These effects were associated with the amelioration of oxidative injury of lipid, protein, and DNA.. Hydrogen gas could exert its neuroprotective effect against early brain injury after subarachnoid hemorrhage by its antioxidative activity.

Topics: Administration, Inhalation; Animals; Antioxidants; Apoptosis; Blood-Brain Barrier; Brain; Brain Chemistry; Brain Injuries; Guanosine; Hydrogen; Lipid Peroxidation; Male; Malondialdehyde; Oxidative Stress; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Tyrosine; Water

Accumulating evidence has demonstrated that over-expression of Neuroglobin (Ngb) is neuroprotective against hypoxic/ischemic brain injuries. In this study we tested the neuroprotective effects of Ngb over-expression against traumatic brain injury (TBI) in mice.. Both Ngb over-expression transgenic (Ngb-Tg) and wild-type (WT) control mice were subjected to TBI induced by a controlled cortical impact (CCI) device. TBI significantly increased Ngb expression in the brains of both WT and Ngb-Tg mice, but Ngb-Tg mice had significantly higher Ngb protein levels at the pre-injury baseline and post-TBI. Production of oxidative tissue damage biomarker 3NT in the brain was significantly reduced in Ngb-Tg mice compared to WT controls at 6 hours after TBI. The traumatic brain lesion volume was significantly reduced in Ngb Tg mice compared to WT mice at 3 weeks after TBI; however, there were no significant differences in the recovery of sensorimotor and spatial memory functional deficits between Ngb-Tg and WT control mice for up to 3 weeks after TBI.. Ngb over-expression reduced traumatic lesion volume, which might partially be achieved by decreasing oxidative stress.

Topics: Analysis of Variance; Animals; Brain Injuries; Cerebral Cortex; Disease Models, Animal; Gait Disorders, Neurologic; Gene Expression Regulation; Globins; Maze Learning; Memory Disorders; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Tissue Proteins; Neuroglobin; Neuroprotective Agents; Seizures; Space Perception; Tyrosine

We have previously shown the pathophysiological importance of the reactive nitrogen species peroxynitrite (PN) formed from the reaction of nitric oxide (•NO) and superoxide (O(2)(•-)) radicals and its involvement in lipid peroxidation (LP) and protein nitration damage in brain tissue following traumatic brain injury (TBI). Nitric oxide is produced by at least three isoforms of the enzyme nitric oxide synthase (NOS) including: endothelial NOS (eNOS) in the CNS vasculature, neuronal NOS (nNOS), and inducible NOS (iNOS) in macrophages/microglia. In view of the requirement of •NO synthesis for PN formation, we sought to address the time course of NOS expression (mRNA by real time quantitative PCR and protein by western blot) after TBI in comparison with the time course of PN-mediated protein nitration (3-nitrotyrosine, 3-NT) in ipsilateral cortex (CTX) and hippocampus (HIPP) between 3 hours and 1 week post-injury using a controlled cortical impact (CCI) mouse model of TBI in young adult CF-1 mice. Protein nitration showed a progressive posttraumatic increase that became significant in CTX at 24 hours and then peaked at 72 hours in both CTX and HIPP. During the increase in PN-derived 3-NT, there was no increase in either CTX or HIPP eNOS mRNA levels, whereas eNOS protein levels were significantly (p<0.05) increased at 48 and 72 hours in both brain regions. There was a significant decrease in HIPP, but not CTX nNOS mRNA; however, nNOS protein did not change except for a significant increase in CTX at 1 week. There was significantly increased CTX and HIPP iNOS mRNA levels at 24, 48, and 72 hours (p<.05) post-injury. In contrast, no change was seen in CTX or HIPP iNOS protein at any timepoint. Taken together, eNOS protein expression and iNOS mRNA appear to bear a coincident temporal relationship to the time course of PN-mediated protein nitrative damage after CCI-TBI suggesting that both constitutive and inducible NOS isoforms contribute •NO for PN formation and 3-NT protein modification after TBI.

Topics: Analysis of Variance; Animals; Brain; Brain Injuries; Disease Models, Animal; Functional Laterality; Gene Expression Regulation; Mice; Nitric Oxide Synthase; Oxidative Stress; Peroxynitrous Acid; Protein Isoforms; RNA, Messenger; Time Factors; Tyrosine

The aim of the present study was to ascertain whether brain cooling causes attenuation of traumatic brain injury by reducing brain nitrostative and oxidative damage. Brain cooling was accomplished by infusion of 5 mL of 4°C saline over 5 minutes via the external jugular vein. Immediately after the onset of traumatic brain injury, rats were randomized into two groups and given 37°C or 4°C normal saline. Another group of rats were used as sham operated controls. Behavioral and biochemical assessments were conducted on 72 hours after brain injury or sham operation. As compared to those of the sham-operated controls, the 37°C saline-treated brain injured animals displayed motor deficits, higher cerebral contusion volume and incidence, higher oxidative damage (e.g., lower values of cerebral superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, but higher values of cerebral malondialdehyde), and higher nitrostative damage (e.g., higher values of neuronal nitric oxide synthase and 3-nitrotyrosine). All the motor deficits and brain nitrostative and oxidative damage were significantly reduced by retrograde perfusion of 4°C saline via the jugular vein. Our data suggest that brain cooling may improve the outcomes of traumatic brain injury in rats by reducing brain nitrostative and oxidative damage.

Topics: Animals; Body Temperature; Brain; Brain Infarction; Brain Injuries; Catalase; Glutathione Reductase; Hypothermia, Induced; Male; Malondialdehyde; Nitric Oxide Synthase Type II; Nitrosation; Oxidative Stress; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Tyrosine

Stachybotrys microspora triprenyl phenol-7 (SMTP-7) is a novel fibrinolytic agent with anti-inflammatory effect. Previous study demonstrated that SMTP-7 further ameliorated infarction volume in a mouse embolic stroke model compared with tissue type plasminogen activator (tPA), but the reason SMTP-7 has more beneficial effect than tPA has not yet been determined. In the present study, we investigated whether SMTP-7 has an intrinsic neuroprotective effect against transient focal cerebral ischemia (tFCI). Sprague-Dawley rats were subjected to tFCI by intraluminal middle cerebral artery occlusion for 2h. Following induction of tFCI, rats were randomized into two groups based on the agent administered: SMTP-7 group and vehicle group. We examined cerebral infarction volume 24h after reperfusion, and evaluated superoxide production, the expressions of nitrotyrosine and matrix metalloproteinase-9 (MMP-9), which play major roles in secondary brain injury and hemorrhagic transformation. The findings showed that SMTP-7 significantly suppressed superoxide production, the expression of nitrotyrosine and MMP-9 after tFCI, and consequently attenuated ischemic neuronal damage. These results suggest that SMTP-7 has an intrinsic neuroprotective effect on ischemia/reperfusion injury through the suppression of oxidative stress and MMP-9 activation.

Topics: Animals; Benzopyrans; Blotting, Western; Brain Injuries; Fibrinolytic Agents; Ischemic Attack, Transient; Male; Matrix Metalloproteinase 9; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reperfusion Injury; Stachybotrys; Superoxides; Tyrosine

Peroxynitrite is a powerful oxidant capable of nitrating phenolic moieties, such as tyrosine or tyrosine residues in proteins and increases after traumatic brain injury (TBI). First, we tested the hypothesis that TBI increases nitrotyrosine (NT) immunoreactivity in the brain by measuring the number of NT-immunoreactive neurons in the cerebral cortex and hippocampus of rats subjected to parasagittal fluid-percussion TBI. Second, we tested the hypothesis that treatment with L-arginine, a substrate for nitric oxide synthase, further increases NT immunoreactivity over TBI alone. Rats were anesthetized with isoflurane and subjected to TBI, sham TBI, or TBI followed by treatment with L-arginine (100 mg/kg). Twelve, 24, or 72 h after TBI, brains were harvested. Coronal sections (10 microm) were incubated overnight with rabbit polyclonal anti-NT antibody, rinsed, and incubated with a biotinylated secondary antibody. The antigen-antibody complex was visualized using a peroxidase-conjugated system with diaminobenzidine as the chromagen. The number of NT-positive cortical and hippocampal neurons increased significantly in both ipsilateral and contralateral hemispheres up to 72 h after TBI compared with the sham-injured group. Remarkably, treatment with L-arginine reduced the number of NT-positive neurons after TBI in both cortex and hippocampus. Our results indicate that L-arginine actually prevents TBI-induced increases in NT immunoreactivity.

Topics: Animals; Arginine; Brain Injuries; Cerebral Cortex; Hippocampus; Immunohistochemistry; Male; Neurons; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Tyrosine

Following traumatic brain injury (TBI), mitochondrial function becomes compromised. Mitochondrial dysfunction is characterized by intra-mitochondrial Ca(2+) accumulation, induction of oxidative damage, and mitochondrial permeability transition (mPT). Experimental studies show that cyclosporin A (CsA) inhibits mPT. However, CsA also inhibits calcineurin. In the present study, we conducted a dose-response analysis of NIM811, a non-calcineurin inhibitory CsA analog, on mitochondrial dysfunction following TBI in mice, and compared the effects of the optimal dose of NIM811 (10 mg/kg i.p.) against an optimized dose of CsA (20 mg/kg i.p.). Male CF-1 mice were subjected to severe TBI utilizing the controlled cortical impact model. Mitochondrial respiration was assessed from animals treated with either NIM811, CsA, or vehicle 15 min post-injury. The respiratory control ratio (RCR) of mitochondria from vehicle-treated animals was significantly (p<0.01) lower at 3 or 12 h post-TBI, relative to shams. Treatment of animals with either NIM811 or CsA significantly (p<0.03) attenuated this reduction. Consistent with this finding, both NIM811 and CsA significantly reduced lipid peroxidative and protein nitrative damage to mitochondria at 12 h post-TBI. These results showing the ability of NIM811 to fully duplicate the mitochondrial protective efficacy of CsA supports the conclusion that inhibition of the mPT may be sufficient to explain CsA's protective effects.

Topics: Acute Disease; Aldehydes; Animals; Biomarkers; Brain Injuries; Cyclosporine; Dose-Response Relationship, Drug; Immunoblotting; Lipid Peroxidation; Male; Mice; Mitochondrial Diseases; Oxidative Stress; Oxygen Consumption; Structure-Activity Relationship; Tyrosine

Ventilatory resuscitation with 100% O2 after severe traumatic brain injury (TBI) raises concerns about the increased production of reactive oxygen species (ROS). The product of peroxynitrite-meditated tyrosine residue nitration, 3-nitrotyrosine (3-NT), is a marker for oxidative damage to proteins. The authors hypothesized that posttraumatic resuscitation with hyperoxia (100% fraction of inspired oxygen [FiO2] concentration) results in increased ROS-induced damage to proteins compared with resuscitation using normoxia (21% FiO2 concentration).. Male Sprague-Dawley rats underwent controlled cortical impact (CCI) injury and resuscitation with either normoxic or hyperoxic ventilation for 1 hour (5 rats per group). Twenty-four hours after injury, rat hippocampi were evaluated using 3-NT immunostaining. In a second experiment, animals similarly underwent CCI injury and normoxic or hyperoxic ventilation for 1 hour (4 rats per group). One week after injury, neuronal counts were performed after neuronal nuclei immunostaining.. The 3-NT staining was significantly increased in the hippocampi of the hyperoxic group. The normoxic group showed a 51.0% reduction of staining in the CA1 region compared with the hyperoxic group and a 50.8% reduction in the CA3 region (p < 0.05, both regions). There was no significant difference in staining between the injured normoxic group and sham-operated control groups. In the delayed analysis of neuronal survival (neuronal counts), there was no significant difference between the hyperoxic and normoxic groups.. In this clinically relevant model of TBI, normoxic resuscitation significantly reduced oxidative damage to proteins compared with hyperoxic resuscitation. Neuronal counts showed no benefit from hyperoxic resuscitation. These findings indicate that hyperoxic ventilation in the early stages after severe TBI may exacerbate oxidative damage to proteins.

Topics: Animals; Brain Injuries; Disease Models, Animal; Hippocampus; Male; Oxidative Stress; Oxygen Inhalation Therapy; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Resuscitation; Tyrosine

Stroke affects a large number of people, especially in developed countries, but treatment options are limited. Over the years, it has become clear that nitric oxide (NO) plays a major role in this pathology and that treatments that either reduce or increase NO presence may provide an alternative route for reducing the sequelae of brain ischemia. The NO donor LA 419 previously has been shown to protect the brain tissue from ischemic damage in an experimental model of global brain ischemia. Here we study whether this holds true for focal ischemia, a condition closer to the more common form of human stroke. Ischemia was induced in rats by a stereotaxic injection of endothelin-1, a potent vasoconstrictor, in the striatum. Seven days after the injection, magnetic resonance imaging (MRI) found a significant elevation in apparent diffusion coefficient (ADC) in the injected striatum of untreated rats, due to ischemia-induced vascular edema. Animals that received LA 419 prior to injection with endothelin-1 showed an ADC undistinguishable from the contralateral striatum or from the striatum of rats not treated with LA 419. In addition, immunohistochemistry with antibodies against neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS), and nitrotyrosine showed a marked increase in the expression of these markers of NO production following ischemic treatment that was dampened by treatment with LA 419. In summary, our results clearly show that the NO donor LA 419 may be a useful compound for the prevention and/or treatment of focal brain ischemia.

Topics: Animals; Brain Injuries; Corpus Striatum; Disease Models, Animal; Ischemia; Isosorbide Dinitrate; Magnetic Resonance Imaging; Male; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Time Factors; Tyrosine

Previously, we reported that transgenic mice overexpressing endothelin-1 in astrocytes showed more severe neurological deficits and increased infarct after transient focal ischemia. In those studies, we also observed increased level of aldose reductase (AR), the first and rate-limiting enzyme of the polyol pathway, which has been implicated in osmotic and oxidative stress. To further understand the involvement of the polyol pathway, the mice with deletion of enzymes in the polyol pathway, AR, and sorbitol dehydrogenase (SD), which is the second enzyme in this pathway, were challenged with similar cerebral ischemic injury. Deletion of AR-protected animals from severe neurological deficits and large infarct, whereas similar protection was not observed in mice with SD deficiency. Most interestingly, AR(-/-) brains showed lowered expression of transferrin and transferrin receptor with less iron deposition and nitrotyrosine accumulation. The protection against oxidative stress in AR(-/-) brain was also associated with less poly(adenosine diphosphate-ribose) polymerase (PARP) and caspase-3 activation. Pharmacological inhibition of AR by Fidarestat also protected animals against cerebral ischemic injury. These findings are the first to show that AR contributes to iron- and transferrin-related oxidative stress associated with cerebral ischemic injury, suggesting that inhibition of AR but not SD may have therapeutic potential against cerebral ischemic injury.

Topics: Aldehyde Reductase; Animals; Apoptosis; Behavior, Animal; Brain; Brain Injuries; Brain Ischemia; Caspase 3; Gene Deletion; Imidazolidines; Infarction, Middle Cerebral Artery; Iron; L-Iditol 2-Dehydrogenase; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Mice, Transgenic; Oxidative Stress; Receptors, Transferrin; Transferrin; Tyrosine

Sublethal injurious stimuli induce tolerance to subsequent lethal insults, a phenomenon termed preconditioning. Inducible nitric oxide synthase (iNOS) is essential for the preconditioning induced by transient bilateral common carotid artery occlusion (BCCAO) or by systemic administration of the endotoxin lipopolysaccharide (LPS). We used a model of brain injury produced by neocortical injection of N-methyl-D-aspartate (NMDA) to investigate the mechanisms by which iNOS-derived nitric oxide (NO) contributes to tolerance induced by LPS or BCCAO. We found that the tolerance is blocked by the iNOS inhibitor aminoguanidine, is not observed in iNOS-null mice, and is rescued by the NO donor DTPA NONOate. Lipopolysaccharide failed to induce preconditioning in mice lacking the nox2 subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, suggesting that superoxide derived from NADPH oxidase is needed for the induction of the tolerance. Because superoxide reacts with NO to form peroxynitrite, we investigated the role of peroxynitrite. We found that LPS induces the peroxynitrite marker 3-nitrotyrosine in cortical neurons and that the peroxynitrite decomposition catalyst FeTPPS abolishes LPS-induced preconditioning. These results suggest that the protective effect of iNOS-derived NO is mediated by peroxynitrite formed by the reaction of NO with NADPH oxidase-derived superoxide. Thus, peroxynitrite, in addition to its well-established deleterious role in ischemic brain injury and neurodegeneration, can also be beneficial by inducing tolerance to excitotoxicity.

Topics: Animals; Body Temperature; Brain; Brain Injuries; Carotid Arteries; Guanidines; Ischemic Preconditioning; Ligation; Lipopolysaccharides; Male; Membrane Glycoproteins; Metalloporphyrins; Mice; Mice, Inbred C57BL; Mice, Knockout; N-Methylaspartate; NADPH Oxidase 2; NADPH Oxidases; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Superoxides; Tyrosine

We assessed the temporal and spatial characteristics of PN-induced oxidative damage and its relationship to calpain-mediated cytoskeletal degradation and neurodegeneration in a severe unilateral controlled cortical impact (CCI) traumatic brain injury (TBI) model. Quantitative temporal time course studies were performed to measure two oxidative damage markers: 3-nitrotyrosine (3NT) and 4-hydroxynonenal (4HNE) at 30 min, 1, 3, 6, 12, 24, 48, 72 h and 7 days after injury in ipsilateral cortex of young adult male CF-1 mice. Secondly, the time course of Ca(++)-activated, calpain-mediated proteolysis was also analyzed using quantitative western-blot measurement of breakdown products of the cytoskeletal protein alpha-spectrin. Finally, the time course of neurodegeneration was examined using de Olmos silver staining. Both oxidative damage markers increased in cortical tissue immediately after injury (30 min) and elevated for the first 3-6 h before returning to baseline. In the immunostaining study, the PN-selective marker, 3NT, and the lipid peroxidation marker, 4HNE, were intense and overlapping in the injured cortical tissue. alpha-Spectrin breakdown products, which were used as biomarker for calpain-mediated cytoskeletal degradation, were also increased after injury, but the time course lagged behind the peak of oxidative damage and did not reach its maximum until 24 h post-injury. In turn, cytoskeletal degradation preceded the peak of neurodegeneration which occurred at 48 h post-injury. These studies have led us to the hypothesis that PN-mediated oxidative damage is an early event that contributes to a compromise of Ca(++) homeostatic mechanisms which causes a massive Ca(++) overload and calpain activation which is a final common pathway that results in post-traumatic neurodegeneration.

Topics: Aldehydes; Animals; Brain; Brain Injuries; Calcium; Calpain; Cerebral Cortex; Cytoskeleton; Lipid Peroxidation; Male; Mice; Mice, Inbred Strains; Nerve Degeneration; Nerve Tissue Proteins; Nitrates; Oxidative Stress; Peroxynitrous Acid; Spectrin; Time Factors; Tissue Distribution; Tyrosine

Manganese superoxide dismutase (MnSOD) provides the first line of defense against superoxide generated in mitochondria. SOD competes with nitric oxide for reaction with superoxide and prevents generation of peroxynitrite, a potent oxidant that can modify proteins to form 3-nitrotyrosine. Thus, sufficient amounts of catalytically competent MnSOD are required to prevent mitochondrial damage. Increased nitrotyrosine immunoreactivity has been reported after traumatic brain injury (TBI); however, the specific protein targets containing modified tyrosine residues and functional consequence of this modification have not been identified. In this study, we show that MnSOD is a target of tyrosine nitration that is associated with a decrease in its enzymatic activity after TBI in mice. Similar findings were obtained in temporal lobe cortical samples obtained from TBI cases versus control patients who died of causes not related to CNS trauma. Increased nitrotyrosine immunoreactivity was detected at 2 h and 24 h versus 72 h after experimental TBI and co-localized with the neuronal marker NeuN. Inhibition and/or genetic deficiency of neuronal nitric oxide synthase (nNOS) but not endothelial nitric oxide synthase (eNOS) attenuated MnSOD nitration after TBI. At 24 h after TBI, there was predominantly polymorphonuclear leukocytes accumulation in mouse brain whereas macrophages were the predominant inflammatory cell type at 72 h after injury. However, a selective inhibitor or genetic deficiency of inducible nitric oxide synthase (iNOS) failed to affect MnSOD nitration. Nitration of MnSOD is a likely consequence of peroxynitrite within the intracellular milieu of neurons after TBI. Nitration and inactivation of MnSOD could lead to self-amplification of oxidative stress in the brain progressively enhancing peroxynitrite production and secondary damage.

Topics: Adolescent; Adult; Aged; Animals; Brain; Brain Injuries; Chemotaxis, Leukocyte; Encephalitis; Enzyme Activation; Enzyme Inhibitors; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Nerve Degeneration; Nitrates; Nitric Oxide Synthase Type I; Oxidative Stress; Peroxynitrous Acid; Superoxide Dismutase; Tyrosine

We previously demonstrated that fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARalpha) agonist, reduced the neurological deficit, the edema and the cerebral lesion induced by traumatic brain injury (TBI). In order to elucidate these beneficial effects, in the present study, we investigated, in the same TBI model, fenofibrate's effects on the inflammation and oxidative stress. Male Sprague Dawley rats were randomized in four groups: non-operated, sham-operated, TBI + vehicle, TBI + fenofibrate. TBI was induced by lateral fluid percussion of the temporoparietal cortex. Rats were given fenofibrate (50 mg/kg) or its vehicle (water containing 0.2% methylcellulose), p.o. 1 and 6 h after brain injury. A neurological assessment was done 24 h after TBI, then rats were killed and the brain COX2, MMP9 expression, GSx, GSSG levels were determined. The same schedule of treatment was used to evaluate the effect of fenofibrate on immunohistochemistry of 3NT, 4HNE and iNOS at 24 h post-injury. Our results showed that fenofibrate promotes neurological recovery by exerting anti-inflammatory effect evidenced by a decrease in iNOS, COX2 and MMP9 expression. In addition, fenofibrate showed anti-oxidant effect demonstrated by a reduction of markers of oxidative stress: loss of glutathione, glutathione oxidation ratio, 3NT and 4HNE staining. Our data suggest that PPARalpha activation could mediate pleiotropic effects and strengthen that it could be a promising therapeutic strategy for TBI.

Topics: Aldehydes; Animals; Anti-Inflammatory Agents; Antioxidants; Brain; Brain Injuries; Cyclooxygenase 2; Encephalitis; Fenofibrate; Glutathione; Male; Matrix Metalloproteinase 9; Nitric Oxide Synthase Type II; Oxidative Stress; PPAR alpha; Rats; Rats, Sprague-Dawley; Recovery of Function; Treatment Outcome; Tyrosine

Oxidative stress has been indicated as a mechanism of secondary neuronal injury in traumatic brain injury (TBI). Nitrotyrosine in the cerebrospinal fluid (CSF) may be an in vivo marker of oxidative nitric oxide damage. We tested the hypothesis that increased levels of nitrotyrosine correlate with poor neurologic outcomes in patients with TBI and attempted to identify the source of increased CSF nitrotyrosine.. This institutional-review-board-approved study included 10 adults with severe closed TBI (Glasgow Coma Scale score <8) and no documented hypoxic brain injury. These patients underwent routine evaluation and, when indicated, placement of an intraventricular catheter. CSF samples (n = 27) were collected 2 to 72 hours after TBI and were also obtained from four healthy individuals. Nitrotyrosine levels were measured, and immunohistochemistry was performed. Neurologic follow-up extended to 1 month after injury.. Nitrotyrosine was not detected in the control samples but was detected in 13 CSF samples from 7 TBI patients (range, 22.4-97.6 nM/mL). Seven patients had poor outcomes, and, in each, nitrotyrosine was detected. Nitrotyrosine immunoreactivity was detected in neurons and glia and confirmed in brain homogenate.. Oxidative stress contributes to secondary brain injury in patients with TBI. Poor neurologic outcome is associated with increased levels of nitrotyrosine in the CSF. Identifying patients or the stage at which oxidative stress is more active using CSF markers of oxidative injury may help in the development of more targeted treatments.

Topics: Adolescent; Adult; Biomarkers; Brain Injuries; Case-Control Studies; Cohort Studies; Craniotomy; Enzyme-Linked Immunosorbent Assay; Female; Glasgow Coma Scale; Humans; Immunohistochemistry; Injury Severity Score; Intracranial Pressure; Male; Middle Aged; Monitoring, Physiologic; Oxidative Stress; Prognosis; Reference Values; Sensitivity and Specificity; Tyrosine

The inflammatory response is thought to be important for secondary damage following traumatic brain injury (TBI). The inducible nitric oxide synthase (iNOS) isoform is a mediator in inflammatory reactions and may catalyze substantial synthesis of NO in the injured brain. This study was undertaken to analyze neuronal degeneration and survival, cellular apoptosis and formation of nitrotyrosine following treatment with the iNOS-inhibitor L-N-iminoethyl-lysine (L-NIL) in a model of brain contusion. A brain contusion was produced using a weight-drop device in 30 rats. The animals received treatment with L-NIL or NaCl at 15 min and 12 h after the injury and were sacrificed at 24 h or 6 days after trauma. iNOS activity was measured at 24 h post-trauma by the conversion of L-[U- ( 14 )C]arginine to L-[U-( 14 )C]citrulline and immunohistochemistry for iNOS. Peroxynitrite formation was indirectly assessed by nitrotyrosine (NT) immunohistochemistry. Neuronal degeneration and survival were assessed by Fluoro-Jade (FJ) and NeuN stainings, and cellular death by TUNEL staining. iNOS activity but not iNOS immunoreactivity was significantly reduced in animals that received L-NIL. Neuronal degeneration (FJ) and NT immunoreactivity were significantly reduced at 24 h. Neuronal survival was unchanged at 24 h but increased at 6 days in L-NIL-treated animals. Cellular apoptosis of ED-1 and NeuN positive cells was significantly reduced following L-NIL treatment at 6 days after trauma. We demonstrated neuroprotection by selective inhibition of iNOS after trauma. L-NIL appeared to protect the injured brain by limiting peroxynitrite formation. Our findings support a putative harmful role of iNOS induction early after TBI.

Topics: Animals; Apoptosis; Brain Injuries; Cell Survival; Disease Models, Animal; Female; Lysine; Nerve Degeneration; Neurons; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Time Factors; Tyrosine

Unilateral hypoxia-ischemia (HI) was induced in C57/BL6 male mice on postnatal day (P) 5, 9, 21 and 60, corresponding developmentally to premature, term, juvenile and adult human brains, respectively. HI duration was adjusted to obtain a similar extent of brain injury at all ages. Apoptotic mechanisms (nuclear translocation of apoptosis-inducing factor, cytochrome c release and caspase-3 activation) were several-fold more pronounced in immature than in juvenile and adult brains. Necrosis-related calpain activation was similar at all ages. The CA1 subfield shifted from apoptosis-related neuronal death at P5 and P9 to necrosis-related calpain activation at P21 and P60. Oxidative stress (nitrotyrosine formation) was also similar at all ages. Autophagy, as judged by the autophagosome-related marker LC-3 II, was more pronounced in adult brains. To our knowledge, this is the first report demonstrating developmental regulation of AIF-mediated cell death as well as involvement of autophagy in a model of brain injury.

Topics: Aging; Animals; Apoptosis; Apoptosis Inducing Factor; Autophagy; Brain Injuries; Calpain; Caspase 3; Caspases; Cell Death; Cytochromes c; Disease Models, Animal; Flavoproteins; Hypoxia-Ischemia, Brain; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Mitochondria; Necrosis; Neurons; Protein Transport; Tyrosine

Oxygen free radicals and nitric oxide (NO) participate in the pathogenesis of acute central nervous system (CNS) injury by forming peroxynitrite, which promotes oxidative damage and tyrosine nitration. Neuronal nitration is associated with cell death, but little is known of the characteristics and cell fate of nitrated astrocytes. In this study, we have used a postnatal excitotoxic lesion model (intracortical NMDA injection) and our aims were (i) to evaluate the temporal and spatial pattern of astroglial nitration in correlation with the neuropathological process and the sources of NO; and (ii) to establish, if any, the correlation among astrocyte nitration and other events such as expression of cytoskeletal proteins, antioxidant enzymes, and cell death markers to cope with nitration and/or undergo cell death. Our results show that after postnatal excitotoxic damage two distinct waves of nitration were observed in relation to astrocytes. At 24 h post-lesion, early-nitrated astrocytes were found within the neurodegenerating area, coinciding with the time of maximal cell death. These early-nitrated astrocytes are highly ramified protoplasmic cells, showing diffuse glial fibrillary acidic protein (GFAP) content and expressing inducible NOS. At later time-points, when astrogliosis is morphologically evident, nitrated hypertrophied reactive astrocytes are observed in the penumbra and the neurodegenerated area, displaying increased expression of GFAP and vimentin cytoskeletal proteins and of metallothionein I-II and Cu/Zn superoxide dismutase antioxidant proteins. Moreover, despite revealing activated caspase-3, they do not show TUNEL labeling. In summary, we show that nitrated astrocytes in vivo constitute a subpopulation of highly reactive astrocytes which display high resistance towards oxidative stress induced cell death.

Topics: Animals; Animals, Newborn; Astrocytes; Brain Injuries; Cytoskeletal Proteins; Disease Models, Animal; Female; Male; Metallothionein; Nerve Tissue Proteins; Neurotoxins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Rats; Rats, Long-Evans; Superoxide Dismutase; Tyrosine

Studies in experimental traumatic brain injury (TBI) suggest both deleterious and protective effects of inducible nitric oxide synthase (iNOS). Early after injury, iNOS may be detrimental via formation of peroxynitrite and iNOS inhibitors are protective. In contrast, we reported impaired long-term functional outcome after TBI in iNOS knockout (ko) versus wild-type (wt) mice. To elucidate potential neuroprotective and neurotoxic mechanisms for iNOS, we studied nitric oxide formation by electron paramagnetic resonance (EPR) spectroscopy using diethyldithiocarbamate-iron (DETC-Fe) as a spin trap and markers of nitrosative (S-nitrosothiol (RSNO, Fluorescent assay); nitrotyrosine (3NT, ELISA)) and oxidative stress (ascorbate, HPLC) at 72 h after controlled cortical impact (CCI) in iNOS ko and wt and in uninjured iNOS ko and wt mice. 3NT immunostaining with macrophage and myeloperoxidase (MPO) dual labeling was also assessed in brain sections. Brain DETC-Fe-NO low-temperature EPR signal intensity was approximately 2-fold greater in wt versus iNOS ko at 72 h after CCI. Ascorbate levels decreased in injured hemisphere in wt and iNOS ko versus uninjured -this decrease was more pronounced in iNOS ko. In wt mice, RSNO and 3NT levels were increased after CCI versus uninjured (50% and 400%, respectively, P < 0.05). RSNO levels were not increased in iNOS ko after CCI. Nitrotyrosine levels increased after CCI in wt and ko versus respective uninjured -this increase was more pronounced in wt (2.34 +/- 0.95 versus 1.27 +/- 0.49 pmol/mg protein, P < 0.05). Increased 3NT immunoreactivity was detected in wt versus iNOS ko at 72 h after CCI, and colocalized with macrophage marker and MPO. Our data support a role for iNOS-derived NO as an endogenous antioxidant after CCI. iNOS also contributes protein nitrosylation and nitration. Colocalization of 3NT with macrophages and MPO suggests generation of nitrating agents by macrophages and/or phagocytosis of nitrated proteins.

Topics: Animals; Brain; Brain Injuries; Electron Spin Resonance Spectroscopy; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidative Stress; Peroxidase; Tyrosine

Oxidative/nitrosative stress is involved in NMDA receptor-mediated excitotoxic brain damage produced by the glutamate analog quinolinic acid. The purpose of this work was to study a possible role of peroxynitrite, a reactive oxygen/nitrogen species, in the course of excitotoxic events evoked by quinolinic acid in the brain. The effects of Fe(TPPS) (5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinate iron (III)), an iron porphyrinate and putative peroxynitrite decomposition catalyst, were tested on lipid peroxidation and mitochondrial function in brain synaptic vesicles exposed to quinolinic acid, as well as on peroxynitrite formation, nitric oxide synthase and superoxide dismutase activities, lipid peroxidation, caspase-3-like activation, DNA fragmentation, and GABA levels in striatal tissue from rats lesioned by quinolinic acid. Circling behavior was also evaluated. Increasing concentrations of Fe(TPPS) reduced lipid peroxidation and mitochondrial dysfunction induced by quinolinic acid (100 microM) in synaptic vesicles in a concentration-dependent manner (10-800 microM). In addition, Fe(TPPS) (10 mg/kg, i.p.) administered 2 h before the striatal lesions, prevented the formation of peroxynitrite, the increased nitric oxide synthase activity, the decreased superoxide dismutase activity and the increased lipid peroxidation induced by quinolinic acid (240 nmol/microl) 120 min after the toxin infusion. Enhanced caspase-3-like activity and DNA fragmentation were also reduced by the porphyrinate 24 h after the injection of the excitotoxin. Circling behavior from quinolinic acid-treated rats was abolished by Fe(TPPS) six days after quinolinic acid injection, while the striatal levels of GABA, measured one day later, were partially recovered. The protective effects that Fe(TPPS) exerted on quinolinic acid-induced lipid peroxidation and mitochondrial dysfunction in synaptic vesicles suggest a primary action of the porphyrinate as an antioxidant molecule. In vivo findings suggest that the early production of peroxynitrite, altogether with the enhanced risk of superoxide anion (O2*-) and nitric oxide formation (its precursors) induced by quinolinic acid in the striatum, are attenuated by Fe(TPPS) through a recovery in the basal activities of nitric oxide synthase and superoxide dismutase. The porphyrinate-mediated reduction in DNA fragmentation simultaneous to the decrease in caspase-3-like activation from quinolinic acid-lesioned rats suggests a prevention in the r

Topics: Analysis of Variance; Animals; Behavior, Animal; Blotting, Southern; Brain Injuries; Caspase 3; Caspases; Chromatography, High Pressure Liquid; Corpus Striatum; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Electrophoresis, Agar Gel; gamma-Aminobutyric Acid; Huntington Disease; Lipid Peroxidation; Male; Mitochondria; Motor Activity; Nitric Oxide Synthase; Peroxynitrous Acid; Porphyrins; Quinolinic Acids; Radiation-Sensitizing Agents; Rats; Rats, Wistar; Rotarod Performance Test; Superoxide Dismutase; Synaptic Vesicles; Synaptosomes; Tyrosine

Nitric oxide (NO) and oxygen free radicals are implicated in the pathophysiology of traumatic brain injury (TBI). Peroxynitrite formation from NO and superoxide contributes to secondary neuronal injury but the neuroprotective effects of nitric oxide synthase (NOS)-inhibitors have been contradictory. This study was undertaken to examine whether PTtic administration of the (NOS)-inhibitor N-nitro-l-arginine methyl ester (L-NAME), and a combination of L-NAME and the nitrone radical scavenger 2-sulfo-phenyl-N-tert-butyl nitrone (S-PBN) favorable affects neuronal injury in a model of TBI.. A weight-drop model of TBI was used. The animals received L-NAME, S-PBN or a combination of the drugs 15 minutes prothrombin time (PT) and sacrificed after 24 hours or six days. NOS activity was measured by the conversion of L-[U-C]arginine to L-[U-C]citrulline. Peroxynitrite formation, cellular apoptosis, neuronal degeneration and survival were assessed by nitrotyrosine-, TUNEL-, Fluoro-Jade- and NeuN-stainings.. eNOS and nNOS activity was significantly reduced in animals that received L-NAME alone or the combination with S-PBN. iNOS activity or iNOS immunoreactivity was not affected. All treatments significantly reduced neuronal degeneration and nitrotyrosine immunoreactivity at 24 hours and increased neuronal survival at six days PT. No differences were detected between L-NAME and L-NAME + S-PBN groups.. NO from NOS contributes to secondary neuronal injury in this TBI-model. PTtic treatment does not inhibit early beneficial NO-related effects. L-NAME and S-PBN limit peroxynitrite formation, promoting neuronal survival. The combination of L-NAME and S-PBN was neuroprotective; surprisingly no additive effects were found on nitrotyrosine formation, apoptosis or neuronal survival.

Topics: Animals; Apoptosis; Benzenesulfonates; Brain; Brain Injuries; Cell Survival; Drug Combinations; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Immunohistochemistry; In Situ Nick-End Labeling; Male; Nerve Degeneration; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Organic Chemicals; Rats; Rats, Sprague-Dawley; Tyrosine

The role of reactive oxygen-induced oxidative damage to lipids (i.e., lipid peroxidation, LP) and proteins has been strongly supported in previous work. Most notably, a number of free radical scavengers and lipid antioxidants have been demonstrated to be neuroprotective in traumatic brain injury (TBI) models. However, the specific sources of reactive oxygen species (ROS), the time course of oxidative damage and its relationship to post-traumatic neurodegeneration in the injured brain have been incompletely defined. The present study was directed at an investigation of the role of the ROS, peroxynitrite (PON), in the acute pathophysiology of TBI and its temporal relationship to neurodegeneration in the context of the mouse model of diffuse head injury model. Male CF-1 mice were subjected to a moderately severe head injury and assessed at 1-, 3-, 6-, 12-, 24-, 48-, 72, 96- and 120-h post-injury for neurodegeneration using quantitative image analysis of silver staining and semi-quantitative analysis of PON-mediated oxidative damage to proteins (3-nitrotyrosine, 3-NT) and lipids (4-hydroxynonenal, 4-HNE). Significant evidence of silver staining was not apparent until 24-h post-injury, with peak staining seen between 72- and 120-h. This time-course of neurodegeneration was preceded by intense immunostaining for 3-NT and 4-HNE, which occurred within the first hour post-injury. The time course and staining pattern for 3-NT and 4-HNE were similar, with the highest staining intensity noted within the first 48-h in areas surrounding trauma-induced contusions. In the case of 3-NT, neuronal perikarya and processes and microvessels displayed staining. The temporal and spatial coincidence of protein nitration and LP damage suggests that PON is involved in both. However, lipid-peroxidative (4-HNE) immunoreactivity was broader and more diffuse than 3-NT, suggesting that other reactive oxygen mechanisms, such as iron-dependent LP, may also contribute to the more widespread 4-HNE immunoreactivity. This indicates that optimal pharmacological inhibition of post-traumatic oxidative damage in TBI may need to combine two functionalities: one to scavenge PON or PON-derived radicals, and the second to inhibit LP caused by multiple ROS species.

Topics: Aldehydes; Animals; Brain; Brain Injuries; Disease Models, Animal; Image Processing, Computer-Assisted; Immunohistochemistry; Lipid Peroxidation; Male; Mice; Nerve Degeneration; Peroxynitrous Acid; Proteins; Time Factors; Tyrosine

Reactive nitrogen species (RNS) cause nitration of protein-bound tyrosine that is used as biomarker for detection. We hypothesized that RNS are formed in fetal rabbit brain following acute placental insufficiency. Near-term pregnant rabbits were randomized to either repetitive uterine ischemia or no ischemia, and fetal brains obtained. Only one electrochemical HPLC method (of three tested) was successful in detecting brain nitrotyrosine. Protein nitrotyrosine was significantly increased following cumulative 40 min ischemia and 20 min reperfusion compared to controls. Repetitive hypoxia-ischemia results in the increased formation of RNS in near-term fetal brains.

Topics: Animals; Brain Injuries; Brain Ischemia; Female; Fetal Hypoxia; Fetus; Free Radicals; Hypoxia, Brain; Placental Insufficiency; Pregnancy; Rabbits; Reperfusion Injury; Tyrosine