4-hydroxy-2-nonenal and Brain-Injuries

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

This study aims to determine if erythromycin provides neuroprotective effects against ischemic injury following permanent focal cerebral ischemia.. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO). Each animal received a single subcutaneous injection of erythromycin lactobionate (EM, 50 mg/kg) or vehicle immediately after ischemia. The infarct volume, edema index and neurological performance were evaluated at 24 and 72 h after MCAO. The cerebral blood flow (CBF) was measured with an MRI system at 30 min after MCAO. TUNEL staining and immunohistochemical analyses for oxidative stress (4-HNE, 8-OHdG) and inflammation (Iba-1, TNF-α) in the cortex were conducted at 24 and 72 h after MCAO.. The CBF did not differ between the EM-treated and vehicle-treated groups. The EM treatment significantly reduced the infarct volume (p < 0.01) at 24 and 72 h after MCAO and significantly reduced the edema index (p < 0.01) at 24 h. The EM treatment significantly improved the neurological deficit scores (p < 0.05) at 24 and 72 h. EM also significantly suppressed the accumulation of 4-HNE (p < 0.01) and 8-OHdG (p < 0.01) and markedly reduced Iba-1 (p < 0.01) and TNF-α expression (p < 0.05) at both time points. The EM treatment significantly reduced TUNEL-positive cells (p < 0.01) at both time points.. These findings suggest that EM can protect against the neuronal damage caused by cerebral ischemia by alleviating inflammation and reducing oxidant stress.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Blood Pressure; Body Temperature; Brain Edema; Brain Infarction; Brain Injuries; Calcium-Binding Proteins; Cerebrovascular Circulation; Deoxyguanosine; Disease Models, Animal; Erythromycin; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Magnetic Resonance Imaging; Microfilament Proteins; Neuroprotective Agents; Rats; Statistics, Nonparametric; Time Factors; Tumor Necrosis Factor-alpha

The importance of free radical-induced oxidative damage after traumatic brain injury (TBI) has been well documented. Despite multiple clinical trials with radical-scavenging antioxidants that are neuroprotective in TBI models, none is approved for acute TBI patients. As an alternative antioxidant target, Nrf2 is a transcription factor that activates expression of antioxidant and cytoprotective genes by binding to antioxidant response elements (AREs) within DNA. Previous research has shown that neuronal mitochondria are susceptible to oxidative damage post-TBI, and thus the current study investigates whether Nrf2-ARE activation protects mitochondrial function when activated post-TBI. It was hypothesized that administration of carnosic acid (CA) would reduce oxidative damage biomarkers in the brain tissue and also preserve cortical mitochondrial respiratory function post-TBI. A mouse controlled cortical impact (CCI) model was employed with a 1.0mm cortical deformation injury. Administration of CA at 15 min post-TBI reduced cortical lipid peroxidation, protein nitration, and cytoskeletal breakdown markers in a dose-dependent manner at 48 h post-injury. Moreover, CA preserved mitochondrial respiratory function compared to vehicle animals. This was accompanied by decreased oxidative damage to mitochondrial proteins, suggesting the mechanistic connection of the two effects. Lastly, delaying the initial administration of CA up to 8h post-TBI was still capable of reducing cytoskeletal breakdown, thereby demonstrating a clinically relevant therapeutic window for this approach. This study demonstrates that pharmacological Nrf2-ARE induction is capable of neuroprotective efficacy when administered after TBI.

Topics: Abietanes; Adenosine Diphosphate; Aldehydes; Analysis of Variance; Animals; Antioxidants; Brain; Brain Injuries; Cytoskeleton; Disease Models, Animal; Dose-Response Relationship, Drug; Lipid Peroxidation; Male; Mice; Mitochondrial Diseases; Oxidative Stress; Plant Extracts; Succinic Acid

Transplantation of bone marrow mononuclear cells (BMMCs) exerts neuroprotection against cerebral ischemia. We examined the therapeutic timepoint of allogeneic BMMC transplantation in a rat model of focal cerebral ischemia, and determined the effects of repeated transplantation outside the therapeutic window.. Male Sprague-Dawley rats were subjected to 90 minute focal cerebral ischemia, followed by intravenous administration of 1 × 10(7) allogeneic BMMCs or vehicle at 0, 3 or 6 h after reperfusion or 2 × 10(7) BMMCs 6 h after reperfusion. Other rats administered 1 × 10(7) BMMCs at 6 h after reperfusion received additional BMMC transplantation or vehicle 9 h after reperfusion. Infarct volumes, neurological deficit scores and immunohistochemistry were evaluated 24 or 72 h after reperfusion.. Infarct volumes at 24 h were significantly decreased in transplantation rats at 0 and 3 h, but not at 6 h, after reperfusion, compared to vehicle-treatment. Even high dose BMMC transplantation at 6h after reperfusion was ineffective. Repeated BMMC transplantation at 6 and 9h after reperfusion reduced infarct volumes and significantly improved neurological deficit scores at 24 and 72 h. Immunohistochemistry showed repeated BMMC transplantation reduced ionized calcium-binding adapter molecule 1, 4-hydroxy-2-nonenal and 8-hydroxydeoxyguanosine expression at 24 and 72 h after reperfusion.. Intravenous allogeneic BMMCs were neuroprotective following transient focal cerebral ischemia, and the therapeutic time window of BMMC transplantation was >3 h and <6 h after reperfusion in this model. Repeated transplantation at 6 and 9 h after reperfusion suppressed inflammation and oxidative stress in ischemic brains, resulting in improved neuroprotection.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Bone Marrow Transplantation; Brain Injuries; Calcium-Binding Proteins; Deoxyguanosine; Disease Models, Animal; Immunohistochemistry; Inflammation; Ischemic Attack, Transient; Leukocytes, Mononuclear; Male; Microfilament Proteins; Oxidative Stress; Rats; Rats, Sprague-Dawley; Time Factors; Transplantation, Homologous

Our previous study showed that lipopolysaccharide (LPS)-induced brain injury in the neonatal rat is associated with nitrosative and oxidative stress. The present study was conducted to examine whether melatonin, an endogenous molecule with antioxidant properties, reduces systemic LPS-induced nitrosative and oxidative damage in the neonatal rat brain. Intraperitoneal (i.p.) injection of LPS (2mg/kg) was administered to Sprague-Dawley rat pups on postnatal day 5 (P5), and i.p. administration of melatonin (20mg/kg) or vehicle was performed 5min after LPS injection. Sensorimotor behavioral tests were performed 24h after LPS exposure, and brain injury was examined after these tests. The results show that systemic LPS exposure resulted in impaired sensorimotor behavioral performance, and acute brain injury, as indicated by the loss of oligodendrocyte immunoreactivity and a decrease in mitochondrial activity in the neonatal rat brain. Melatonin treatment significantly reduced LPS-induced neurobehavioral disturbances and brain damage in neonatal rats. The neuroprotective effect of melatonin was associated with attenuation of LPS-induced nitrosative and oxidative stress, as indicated by the decreased nitrotyrosine- and 4-hydroxynonenal-positive staining in the brain following melatonin and LPS exposure in neonatal rats. Further, melatonin significantly attenuated LPS-induced increases in the number of activated microglia in the neonatal rat brain. The protection provided by melatonin was also associated with a reduced number of inducible nitric oxide synthase (iNOS)+ cells, which were double-labeled with ED1 (microglia). Our results show that melatonin prevents the brain injury and neurobehavioral disturbances induced by systemic LPS exposure in neonatal rats, and its neuroprotective effects are associated with its impact on nitrosative and oxidative stress.

Topics: Aldehydes; Amyloid beta-Protein Precursor; Animals; Animals, Newborn; Antioxidants; Brain Injuries; Cyclooxygenase 2; Disease Models, Animal; Electron Transport Complex I; Enzyme-Linked Immunosorbent Assay; Female; Interleukin-1beta; Lipopolysaccharides; Male; Melatonin; Muscle Strength; Pregnancy; Rats; Reaction Time; Reflex

Rett syndrome (RTT) is a rare neurodevelopmental disorder affecting almost exclusively females, caused in the overwhelming majority of the cases by loss-of-function mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). High circulating levels of oxidative stress (OS) markers in patients suggest the involvement of OS in the RTT pathogenesis. To investigate the occurrence of oxidative brain damage in Mecp2 mutant mouse models, several OS markers were evaluated in whole brains of Mecp2-null (pre-symptomatic, symptomatic, and rescued) and Mecp2-308 mutated (pre-symptomatic and symptomatic) mice, and compared to those of wild type littermates. Selected OS markers included non-protein-bound iron, isoprostanes (F2-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes) and 4-hydroxy-2-nonenal protein adducts. Our findings indicate that oxidative brain damage 1) occurs in both Mecp2-null (both -/y and stop/y) and Mecp2-308 (both 308/y males and 308/+ females) mouse models of RTT; 2) precedes the onset of symptoms in both Mecp2-null and Mecp2-308 models; and 3) is rescued by Mecp2 brain specific gene reactivation. Our data provide direct evidence of the link between Mecp2 deficiency, oxidative stress and RTT pathology, as demonstrated by the rescue of the brain oxidative homeostasis following brain-specifically Mecp2-reactivated mice. The present study indicates that oxidative brain damage is a previously unrecognized hallmark feature of murine RTT, and suggests that Mecp2 is involved in the protection of the brain from oxidative stress.

Topics: Aldehydes; Analysis of Variance; Animals; Arachidonic Acid; Brain Injuries; Disease Models, Animal; Docosahexaenoic Acids; Female; Gas Chromatography-Mass Spectrometry; Isoprostanes; Male; Methyl-CpG-Binding Protein 2; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Nestin; Neuroprostanes; Oxidative Stress; Rett Syndrome

This study addresses the relationship between cochlear oxidative damage and auditory cortical injury in a rat model of repeated noise exposure. To test the effect of increased antioxidant defenses, a water-soluble coenzyme Q10 analog (Qter) was used. We analyzed auditory function, cochlear oxidative stress, morphological alterations in auditory cortices and cochlear structures, and levels of coenzymes Q9 and Q10 (CoQ9 and CoQ10, respectively) as indicators of endogenous antioxidant capability. We report three main results. First, hearing loss and damage in hair cells and spiral ganglion was determined by noise-induced oxidative stress. Second, the acoustic trauma altered dendritic morphology and decreased spine number of II-III and V-VI layer pyramidal neurons of auditory cortices. Third, the systemic administration of the water-soluble CoQ10 analog reduced oxidative-induced cochlear damage, hearing loss, and cortical dendritic injury. Furthermore, cochlear levels of CoQ9 and CoQ10 content increased. These findings indicate that antioxidant treatment restores auditory cortical neuronal morphology and hearing function by reducing the noise-induced redox imbalance in the cochlea and the deafferentation effects upstream the acoustic pathway.

Topics: Accessory Atrioventricular Bundle; Acoustic Stimulation; Aldehydes; Analysis of Variance; Animals; Antioxidants; Auditory Pathways; Brain Injuries; Cochlea; Disease Models, Animal; Ethidium; Evoked Potentials, Auditory, Brain Stem; Hair Cells, Auditory; Hearing Loss, Noise-Induced; Male; Oxidative Stress; Rats; Rats, Wistar; Silver Staining; Ubiquinone; Visual Cortex

To evaluate retina and optic nerve damage following experimental blast injury.. Healthy adult mice were exposed to an overpressure blast wave using a custom-built blast chamber. The effects of blast exposure on retina and optic nerve function and structure were evaluated using the pattern electroretinogram (pERG), spectral domain optical coherence tomography (OCT), and the chromatic pupil light reflex.. Assessment of the pupil response to light demonstrated decreased maximum pupil constriction diameter in blast-injured mice using red light or blue light stimuli 24 hours after injury compared with baseline in the eye exposed to direct blast injury. A decrease in the pupil light reflex was not observed chronically following blast exposure. We observed a biphasic pERG decrease with the acute injury recovering by 24 hours postblast and the chronic injury appearing at 4 months postblast injury. Furthermore, at 3 months following injury, a significant decrease in the retinal nerve fiber layer was observed using OCT compared with controls. Histologic analysis of the retina and optic nerve revealed punctate regions of reduced cellularity in the ganglion cell layer and damage to optic nerves. Additionally, a significant upregulation of proteins associated with oxidative stress was observed acutely following blast exposure compared with control mice.. Our study demonstrates that decrements in retinal ganglion cell responses can be detected after blast injury using noninvasive functional and structural tests. These objective responses may serve as surrogate tests for higher CNS functions following traumatic brain injury that are difficult to quantify.

Topics: Aldehydes; Amyloid beta-Peptides; Animals; Blast Injuries; Brain Injuries; Disease Models, Animal; Electroretinography; Immunohistochemistry; Light; Male; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; Optic Nerve Injuries; Reflex, Pupillary; Retina; Retinal Ganglion Cells; Tears; Tomography, Optical Coherence

Long-chain n-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have been shown to reduce ischemic neuronal injury. We investigated the effects of ethyl-EPA (EPA-E) on ischemic brain damage using a rat transient focal cerebral ischemia model. Male Sprague-Dawley rats (n=105) were subjected to 90 min of focal cerebral ischemia. EPA-E (100mg/kg/day) or vehicle was administered once a day for 3, 5 or 7 days prior to ischemia. Different withdrawal intervals of 3, 5, and 7 days prior to ischemia following 7-day pretreatment with EPA-E or vehicle were also examined. In addition, post-ischemic administration of EPA-E was investigated. Pretreatment with EPA-E for 7 and 5 days, but not 3 days, showed significant infarct volume reduction and neurological improvements when compared with vehicle pretreatment. In addition, withdrawal of EPA-E administration for 3 days, but not 5 and 7 days, also demonstrated significant infarct volume reduction and neurological improvements when compared with vehicle treatment. Post-ischemic treatment of EPA-E did not show any neuroprotection. Immunohistochemistry revealed that 7-day pretreatment with EPA-E significantly reduced cortical expression of 8-hydroxydeoxyguanosine (maker for oxidative DNA damage), 4-hydroxy-2-nonenal (maker for lipid peroxidation), phosphorylated adducin (marker for Rho-kinase activation) and von Willebrand factor (endothelial marker) when compared with vehicle pretreatment. In addition, phosphorylated adducin expression co-localized with von Willebrand factor immunoreactivity. The present study established the neuroprotective effect of EPA-E on ischemic brain damage following transient focal cerebral ischemia in rats, which may be involved in the suppression of oxidative stress and endothelial Rho-kinase activation.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Analysis of Variance; Animals; Brain Infarction; Brain Injuries; Deoxyguanosine; Disease Models, Animal; Dose-Response Relationship, Drug; Eicosapentaenoic Acid; Ischemic Attack, Transient; Magnetic Resonance Imaging; Male; Neurologic Examination; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Time Factors; von Willebrand Factor

Progressive age-associated increases in cerebral dysfunction have been shown to occur following traumatic brain injury (TBI). Moreover, levels of neuronal mitochondrial antioxidant enzymes in the aged brain are reduced, resulting in free radical-induced cell death. It was hypothesized that cognitive impairment after TBI in the aged progresses to a greater degree than in younger individuals, and that damage involves neuronal degeneration and death by free radicals. In this study, we investigated the effects of free radicals on neuronal degeneration, cell death, and cognitive impairment in 10-week-old (young group) and 24-month-old rats (aged group) subjected to TBI. Young and aged rats received TBI with a pneumatic controlled injury device. At 1, 3 and 7 days after TBI, immunohistochemistry, lipid peroxidation and behavioral studies were performed. At 1, 3 and 7 days post-TBI, the number of 8-hydroxy-2'-deoxyguanosine-, 4-hydroxy-2-nonenal- and single-stranded DNA (ssDNA)-positive cells, and the levels of malondialdehyde around the damaged area after TBI significantly increased in the aged group when compared with the young group (P < 0.05). In addition, the majority of ssDNA-positive cells in both groups co-localized with neuronal cells around the damaged area. There was a significant decrease in the number of surviving neurons and an increase in cognitive impairment after TBI in the aged group when compared with the young group (P < 0.05). These results indicate that following TBI, high levels of free radicals are produced in the aged rat brain, which induces neuronal degeneration and apoptotic cell death around the damaged area, resulting in cognitive impairment.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Age Factors; Aldehydes; Animals; Antigens, Nuclear; Apoptosis; Behavior, Animal; Brain; Brain Injuries; Cognition; Cognition Disorders; Deoxyguanosine; Disease Models, Animal; DNA Breaks, Single-Stranded; DNA, Single-Stranded; Immunohistochemistry; Lipid Peroxidation; Male; Malondialdehyde; Maze Learning; Motor Activity; Nerve Tissue Proteins; Neurons; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Time Factors

Genetic factors are important for outcome after traumatic brain injury (TBI), although exact knowledge of relevant genes/pathways is still lacking. We here used an unbiased approach to define differentially activated pathways between the inbred DA and PVG rat strains. The results prompted us to study further if a naturally occurring genetic variation in glutathione-S-transferase alpha 4 (Gsta4) affects the outcome after TBI.. Survival of neurons after experimental TBI is increased in PVG compared to the DA strain. Global expression profiling analysis shows the glutathione metabolism pathway to be the most regulated between the strains, with increased Gsta4 in PVG among top regulated transcripts. A congenic strain (R5) with a PVG genomic insert containing the Gsta4 gene on DA background displays a reversal of the strain pattern for Gsta4 expression and increased survival of neurons compared to DA. Gsta4 is known to effectively reduce 4-hydroxynonenal (4-HNE), a noxious by-product of lipid peroxidation. Immunostaining of 4-HNE was evident in both rat and human TBI. Intracerebral injection of 4-HNE resulted in neurodegeneration with increased levels of a marker for nerve injury in cerebrospinal fluid of DA compared to R5.. These findings provide strong support for the notion that the inherent capability of coping with increased 4-HNE after TBI affects outcome in terms of nerve cell loss.. A naturally occurring variation in Gsta4 expression in rats affects neurodegeneration after TBI. Further studies are needed to explore if genetic variability in Gsta4 can be associated to outcome also in human TBI.

Topics: Aldehydes; Animals; Brain Injuries; Glutathione; Glutathione Transferase; Humans; Isoenzymes; Lipid Peroxidation; Male; Mice; Mice, Transgenic; Neurons

We have previously reported free radical production after traumatic brain injury (TBI), which induces neural stem cell (NSC) degeneration and death. However, the effects of aging on NSC proliferation around the damaged area following TBI have not been investigated. Therefore, in this study, we used 10-week (young group) and 24-month-old (aged group) rat TBI models to investigate the effects of aging on NSC proliferation around damaged tissue using immunohistochemical and ex vivo techniques. Young and aged rats received TBI. At 1, 3 and 7 days after TBI, immunohistochemical and lipid peroxidation studies were performed. Immunohistochemistry revealed that the number of nestin-positive cells around the damaged area after TBI in the aged group decreased significantly when compared with those in the young group (P < 0.01). However, the number of 8-hydroxy-2'-deoxyguanosine-, 4-hydroxy-2-nonenal- and single-stranded DNA (ssDNA)-positive cells and the level of peroxidation around the damaged area after TBI significantly increased in the aged group, compared with those in the young group (P < 0.01). Furthermore, almost all ssDNA-positive cells in young and aged groups co-localized with NeuN and nestin staining. Ex vivo studies revealed that neurospheres, which differentiated into neurons and glia in culture, could only be isolated from injured brain tissue in young and aged groups at 3 days after TBI. These results indicate that, although there were fewer NSCs that have the potential to differentiate into neurons and glia, these NSCs escaped free radical-induced degeneration around the damaged area after TBI in the aged rat brain.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aging; Aldehydes; Animals; Brain Injuries; Cell Differentiation; Deoxyguanosine; Disease Models, Animal; DNA, Single-Stranded; Fluorescent Antibody Technique; Immunohistochemistry; Intermediate Filament Proteins; Lipid Peroxidation; Male; Nerve Tissue Proteins; Nestin; Neural Stem Cells; Rats; Rats, Wistar

Our previous study indicated that consuming (-)-epigallocatechin gallate (EGCG) before or after traumatic brain injury (TBI) eliminated free radical generation in rats, resulting in inhibition of neuronal degeneration and apoptotic death, and improvement of cognitive impairment. Here we investigated the effects of administering EGCG at various times pre- and post-TBI on cerebral function and morphology. Wistar rats were divided into five groups and were allowed access to (1) normal drinking water, (2) EGCG pre-TBI, (3) EGCG pre- and post-TBI, (4) EGCG post-TBI, and (5) sham-operated group with access to normal drinking water. TBI was induced with a pneumatic controlled injury device at 10 weeks of age. Immunohistochemistry and lipid peroxidation studies revealed that at 1, 3, and 7 days post-TBI, the number of 8-Hydroxy-2'-deoxyguanosine-, 4-Hydroxy-2-nonenal- and single-stranded DNA (ssDNA)-positive cells, and levels of malondialdehyde around the damaged area were significantly decreased in all EGCG treatment groups compared with the water group (P < 0.05). Although there was a significant increase in the number of surviving neurons after TBI in each EGCG treatment group compared with the water group (P < 0.05), significant improvement of cognitive impairment after TBI was only observed in the groups with continuous and post-TBI access to EGCG (P < 0.05). These results indicate that EGCG inhibits free radical-induced neuronal degeneration and apoptotic death around the area damaged by TBI. Importantly, continuous and post-TBI access to EGCG improved cerebral function following TBI. In summary, consumption of green tea may be an effective therapy for TBI patients.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Brain Edema; Brain Injuries; Catechin; Deoxyguanosine; Disease Models, Animal; DNA, Single-Stranded; Drug Administration Schedule; Glial Fibrillary Acidic Protein; Lipid Peroxidation; Male; Maze Learning; Neurons; Neuroprotective Agents; Phosphopyruvate Hydratase; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Time Factors

Phenelzine (PZ) is a scavenger of the lipid peroxidation (LP)-derived reactive aldehyde 4-hydroxynonenal (4-HNE) due to its hydrazine functional group, which can covalently react with 4-HNE. In this study, we first examined the ability of PZ to prevent the respiratory depressant effects of 4-HNE on normal isolated brain cortical mitochondria. Second, in rats subjected to controlled cortical impact traumatic brain injury (CCI-TBI), we evaluated PZ (10 mg/kg subcutaneously at 15 minutes after CCI-TBI) to attenuate 3-hour post-TBI mitochondrial respiratory dysfunction, and in separate animals, to improve cortical tissue sparing at 14 days. While 4-HNE exposure inhibited mitochondrial complex I and II respiration in a concentration-dependent manner, pretreatment with equimolar concentrations of PZ antagonized these effects. Western blot analysis demonstrated a PZ decrease in 4-HNE in mitochondrial proteins. Mitochondria isolated from peri-contusional brain tissue of CCI-TBI rats treated with vehicle at 15 minutes after injury showed a 37% decrease in the respiratory control ratio (RCR) relative to noninjured mitochondria. In PZ-treated rats, RCR suppression was prevented (P<0.05 versus vehicle). In another cohort, PZ administration increased spared cortical tissue from 86% to 97% (P<0.03). These results suggest that PZ's neuroprotective effect is due to mitochondrial protection by scavenging of LP-derived 4-HNE.

Topics: Aldehydes; Animals; Brain Injuries; Electron Transport Complex I; Electron Transport Complex II; Lipid Peroxidation; Male; Mitochondria; Mitochondrial Proteins; Monoamine Oxidase Inhibitors; Nerve Tissue Proteins; Neuroprotective Agents; Oxygen Consumption; Phenelzine; Rats; Rats, Sprague-Dawley

Edaravone is a novel free radical scavenger used clinically in patients with acute cerebral infarction; however, it has not been assessed in traumatic brain injury (TBI). We investigated the effects of edaravone on cerebral function and morphology following TBI. Rats received TBI with a pneumatic controlled injury device. Edaravone (3 mg/kg) or physiological saline was administered intravenously following TBI. Numbers of 8-OHdG-, 4-HNE-, and ssDNA-positive cells around the damaged area after TBI were significantly decreased in the edaravone group compared with the saline group (P < 0.01). There was a significant increase in neuronal cell number and improvement in cerebral dysfunction after TBI in the edaravone group compared with the saline group (P < 0.01). Edaravone administration following TBI inhibited free radical-induced neuronal degeneration and apoptotic cell death around the damaged area. In summary, edaravone treatment improved cerebral dysfunction following TBI, suggesting its potential as an effective clinical therapy.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Antipyrine; Apoptosis; Behavior, Animal; Brain Injuries; Deoxyguanosine; DNA, Single-Stranded; Edaravone; ELAV Proteins; Free Radical Scavengers; Glial Fibrillary Acidic Protein; Maze Learning; Rats; Rats, Wistar

We have assessed potential mechanisms associated with the deleterious effects of TBI on the integrity of plasma membranes in the hippocampus, together with consequences for behavioral function. In addition, we have investigated the efficacy of a dietary intervention based on a pyrazole curcumin derivative with demonstrated bioactivity and brain absorption, to re-establish membrane integrity. We report that moderate fluid percussion injury (FPI) increases levels of 4-Hydroxynonenal (HNE), an intermediary for the harmful effects of lipid peroxidation on neurons. A more direct action of FPI on membrane homeostasis was evidenced by a reduction in calcium-independent phospholipase A2 (iPLA₂) important for metabolism of membrane phospholipids such as DHA, and an increase in the fatty acid transport protein (FATP) involved in translocation of long-chain fatty acids across the membrane. A potential association between membrane disruption and neuronal function was suggested by reduced levels of the NR2B subunit of the transmembrane NMDA receptor, in association with changes in iPLA2 and syntaxin-3 (STX-3, involved in the action of membrane DHA on synaptic membrane expansion). In addition, changes in iPLA2, 4-HNE, and STX-3 were proportional to reduced performance in a spatial learning task. In turn, the dietary supplementation with the curcumin derivative counteracted all the effects of FPI, effectively restoring parameters of membrane homeostasis. Results show the potential of the curcumin derivative to promote membrane homeostasis following TBI, which may foster a new line of non-invasive therapeutic treatments for TBI patients by endogenous up-regulation of molecules important for neural repair and plasticity.

Topics: Aldehydes; Animals; Behavior, Animal; Blotting, Western; Brain Injuries; Cell Membrane; Cognition; Curcumin; Homeostasis; Immunohistochemistry; Lipid Peroxidation; Male; Maze Learning; Nerve Tissue Proteins; Neuronal Plasticity; Pyrazoles; Rats; Rats, Sprague-Dawley; Synapses

Recent studies have shown that sulforaphane, a naturally occurring compound that is found in cruciferous vegetables, offers cellular protection in several models of brain injury. When administered following traumatic brain injury (TBI), sulforaphane has been demonstrated to attenuate blood-brain barrier permeability and reduce cerebral edema. These beneficial effects of sulforaphane have been shown to involve induction of a group of cytoprotective, Nrf2-driven genes, whose protein products include free radical scavenging and detoxifying enzymes. However, the influence of sulforaphane on post-injury cognitive deficits has not been examined. In this study, we examined if sulforaphane, when administered following cortical impact injury, can improve the performance of rats tested in hippocampal- and prefrontal cortex-dependent tasks. Our results indicate that sulforaphane treatment improves performance in the Morris water maze task (as indicated by decreased latencies during learning and platform localization during a probe trial) and reduces working memory dysfunction (tested using the delayed match-to-place task). These behavioral improvements were only observed when the treatment was initiated 1h, but not 6h, post-injury. These studies support the use of sulforaphane in the treatment of TBI, and extend the previously observed protective effects to include enhanced cognition.

Topics: Aldehydes; Animals; Anticarcinogenic Agents; Brain Injuries; Cognition Disorders; Disease Models, Animal; Hippocampus; Isothiocyanates; Male; Maze Learning; Memory; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Space Perception; Sulfoxides; Thiocyanates; Time Factors

Edaravone is a novel free radical scavenger that is clinically employed in patients with acute cerebral infarction, but has not previously been used to treat traumatic brain injury (TBI). In this study, we investigated the effect of edaravone administration on rat TBI. In particular, we used immunohistochemistry to monitor neural stem cell (NSC) proliferation around the area damaged by TBI. Two separate groups of rats were administered saline or edaravone (3 mg/kg) after TBI and then killed chronologically. We also used ex vivo techniques to isolate NSCs from the damaged region and observed nestin-positive cells at 1, 3, and 7 days following TBI in both saline- and edaravone-treated groups. At 3 days following TBI in both groups, there were many large cells that morphologically resembled astrocytes. At 1 and 7 days following TBI in the saline group, there were a few small nestin-positive cells. However, in the edaravone group, there were many large nestin-positive cells at 7 days following TBI. At 3 and 7 days following TBI, the number of nestin-positive cells in the edaravone group increased significantly compared with the saline group. There were many single-stranded DNA-, 8-hydroxy-2'-deoxyguanosine-, and 4-hydroxy-2-nonenal-positive cells in the saline group following TBI, but only a few such cells in the edaravone group following TBI. Furthermore, almost all ssDNA-positive cells in the saline group co-localized with Hu, nestin, and glial fibrillary acidic protein (GFAP) staining, but not in the edaravone group. In the ex vivo study, spheres could only be isolated from injured brain tissue in the saline group at 3 days following TBI. However, in the edaravone group, spheres could be isolated from injured brain tissue at both 3 and 7 days following TBI. The number of spheres isolated from injured brain tissue in the edaravone group showed a significant increase compared with the saline group. The spheres isolated from both saline and edaravone groups were immunopositive for nestin, but not Tuj1 or vimentin. Moreover, the spheres differentiated into Tuj1-, GFAP-, and O4-positive cells after 4 days in culture without bFGF. This result indicated that the spheres were neurospheres composed of NSCs that could differentiate into neurons and glia. Edaravone administration inhibited production of free radicals known to induce neuronal degeneration and cell death after brain injury, and protected nestin-positive cells, including NSCs, with the potential to diff

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult Stem Cells; Aldehydes; Animals; Antipyrine; Behavior, Animal; Brain Injuries; Cell Count; Cell Differentiation; Cell Proliferation; Cells, Cultured; Deoxyguanosine; Disease Models, Animal; DNA, Single-Stranded; Edaravone; ELAV Proteins; Free Radical Scavengers; Linear Models; Lipid Peroxidation; Male; Nerve Tissue Proteins; Neurons; Rats; Rats, Wistar; Statistics, Nonparametric; Time Factors

We previously identified immunocytochemical evidence of nitrative and oxidative injury in premyelinating oligodendrocytes in periventricular leukomalacia (PVL). Here, we tested the hypothesis that free radical injury occurs in the overlying cerebral cortex and subplate neurons in PVL. We immunostained for nitrotyrosine, malondialdehyde, and hydroxynonenal adducts and scored neuron staining density in PVL (n = 11) and non-PVL (n = 15) cases (postconceptional ages from 34 to 109 weeks). Analysis of covariance controlled for age. Mean malondialdehyde scores in PVL cases were increased over controls (p = 0.005). Hydroxynonenal scores increased with age only in PVL cases (diagnosis vs age interaction; p = 0.024). Nitrotyrosine scores were not significantly increased. In 11 PVL and 23 control cases between 20 and 183 postconceptional weeks, cells morphologically consistent with subplate and Cajal-Retzius neurons showed qualitatively increased free radical modification in PVL over control cases with statistically significant odds ratios for hydroxynonenal and nitrotyrosine in both subplate neurons and Cajal-Retzius cells. Glial fibrillary acidic protein and CD68 scores for reactive astrocytes and microglia, respectively, were not significantly increased, suggesting a minimal inflammatory response. Thus, oxidative/nitrative damage to cortical and "pioneer" neurons, although mild overall, may contribute to cortical volume loss and cognitive/behavioral impairment in survivors of prematurity.

Topics: Aldehydes; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Astrocytes; Brain Injuries; Cell Count; Cerebral Cortex; Female; Glial Fibrillary Acidic Protein; Humans; Infant; Infant, Newborn; Leukomalacia, Periventricular; Logistic Models; Male; Neurons; Oxidation-Reduction

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

In the present study, we investigated effects of the dried, hot-water extract of Cordyceps militaris (CME) and its major metabolite (cordycepin) against ischemic damage. The repeated treatment with CME protected hippocampal CA1 pyramidal neurons from ischemic damage in gerbils. The treatment with CME or cordycepin in gerbils reduced 4-hydroxy-2-nonenal (a marker of lipid peroxidation) immunoreactivity and levels in the ischemic CA1 region. Glial fibrillary acidic protein immunoreactive astrocytes and ionized calcium-binding adapter molecule 1 immunoreactive microglia in the vehicle-treated ischemic group were activated in the CA1 region 4 days after ischemia/reperfusion, whereas in the CME- or cordycepin-treated ischemic group, their activation was significantly decreased. These results suggest that the repeated treatment with CME protects against neuronal damage from ischemia/reperfusion by reducing oxidative damage.

Topics: Adenine; Adenosine; Aldehydes; Animals; Antifungal Agents; Biological Products; Brain Injuries; Cordyceps; Deoxyadenosines; Gerbillinae; Hippocampus; Lipid Peroxidation; Male; Neurons

Nitric oxide (NO) released in response to hypoxia-ischemia (HI) in the newborn brain may mediate both protective and pathologic responses. We sought to determine whether pharmacologic increase of NO using an NO donor would reduce neurologic injury resulting from HI in the postnatal day 7 rat. We measured NO levels and CBF in the presence of either a NOS inhibitor, N-nitro-l-arginine methyl ester (L-NAME) or an NO donor (Z)-1-[N-(2-amino-ethyl)-N-(2-ammonio-ethyl)amino]diazen-1-ium-1,2-diolate (DETANONOate). Both inhibition of NOS and administration of an NO donor reduced neuropathologic injury after 7-day recovery. NO levels decreased in both ischemic and contralateral hemispheres during HI. This response was prevented by treatment with DETANONOate. Despite the decrease in NO, CBF increased during ischemia in the contralateral hemisphere but decreased when combined with brief hypoxia. Treatment with L-NAME abolished these increases, which were not altered by DETANONOate. Reduction of cellular metabolism by mild hypothermia also reduced both NO and CBF. Following prolonged HI, CBF remained decreased in the ischemic hemisphere up to 24-h recovery. This decrease was prevented by treatment with DETANONOate. These data show that administration of an NO donor reduces neurologic injury following HI in the newborn rat. This mechanism of this protection, in part, is due to an increase in the rate of recovery of CBF compared to vehicle-treated animals. Augmentation of NO-dependent increases in CBF may serve to improve neurologic outcome after perinatal asphyxia.

Topics: Aldehydes; Animals; Animals, Newborn; Brain Injuries; Brain Ischemia; Cerebrovascular Circulation; Enzyme Inhibitors; Functional Laterality; Laser-Doppler Flowmetry; Lipid Peroxidation; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Rats; Rats, Wistar; Recovery of Function

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

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

Hypoglycemia sometimes occurs in patients with diabetes mellitus who receive excessive doses of insulin. Severe hypoglycemia has been known to induce mitochondrial swelling followed by neuronal death in the brain. Since L-carnitine effectively preserves mitochondrial function in various cells both in vitro and in vivo, we investigated its effects on the neuronal damage induced by hypoglycemic insult in male Wistar rats. Animals were given L-carnitine-containing water (0.1%) for 1 week and then received insulin (20 U/kg, i.p.) to induce hypoglycemia. Although L-carnitine did not affect the mortality of animals that developed hypoglycemic shock, it improved the cognitive function of the survived animals as assessed by the Morris water-maze test. L-carnitine effectively inhibited the increase in oxidized glutathione and mitochondrial dysfunction in the hippocampus and prevented neuronal injury. L-carnitine also inhibited the decrease in mitochondrial membrane potential and the generation of reactive oxygen species in hippocampal neuronal cells cultured in glucose-deprived medium. These results suggest that L-carnitine prevents hypoglycemia-induced neuronal damage in the hippocampus, presumably by preserving mitochondrial functions. Thus, L-carnitine may have therapeutic potential in patients with hypoglycemia induced by insulin overdose.

Topics: Aldehydes; Analysis of Variance; Animals; Apoptosis; Benzimidazoles; Brain Injuries; Carbocyanines; Carnitine; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Embryo, Mammalian; Glucose; Glutathione; Hippocampus; Hypoglycemia; Immunohistochemistry; In Situ Nick-End Labeling; Insulin; Male; Maze Learning; Membrane Potentials; Mitochondria; Neurons; Rats; Rats, Wistar; Reaction Time; Reactive Oxygen Species; Respiration; Tetrazolium Salts; Thiazoles; Time Factors

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