3-nitrotyrosine and Hypoxia-Ischemia--Brain

Oxidative stress (OS) plays a key role in perinatal brain damage. The aim of this study is to evaluate the effectiveness of melatonin as a neuroprotective drug by investigating the influence of melatonin on OS and inflammation biomarkers in an animal model of cerebral hypoxia-ischemia.. Five minutes after hypoxic-ischemic (HI) injury melatonin was administered to 28 rats (HI-Mel group). At the same time, 28 hypoxic-ischemic rats were vehicle-treated (V-HI group). Five rats were used as sham operated controls (CTL). OS biomarkers: isoprostanes (IsoPs), neuroprostanes (NPs) and neurofurans (NFs), and microglial activation markers (glial fibrillary acidic protein [GFAP] and monoclonal antirat CD68 [ED1]) were measured in the cerebral cortex of the two lobes.. A significant increase of IsoPs on the left lobe was observed in V-HI after 1 hour (h) from HI injury (p < 0.001); a significant increase of NPs on both side (p < 0.05) and a significant increase of NFs on the left (p < 0.05) were also observed in V-HI after 24 h. A significant increase of IsoPs on the left (p < 0.05) and of NPs on both lobes (p < 0.05) were observed in HI-Mel after 48 h. The ED1 and GFAP expression was lower in the HI-Mel brain tissue.. Melatonin reduces OS and inflammatory cells recruitment and glial cells activation in cerebral cortex after neonatal HI damage. These results lay the groundwork for future clinical studies in infants.

Topics: Animals; Animals, Newborn; Antioxidants; Biomarkers; Drug Evaluation, Preclinical; Female; Hypoxia-Ischemia, Brain; Melatonin; Microglia; Monocytes; Oxidative Stress; Pregnancy; Rats; Rats, Sprague-Dawley; Tyrosine

Selective cerebral perfusion (SCP) is commonly applied during the correction of complex congenital cardiac defects. In this study, we assessed the impact of different flow levels of SCP on potential brain ischaemia.. Fifteen piglets (7-10 kg, age 3-4 weeks) received SCP via the right common carotid artery during cardiopulmonary bypass at 25°C for 90 min. Regular brain perfusion (1 ml/g brain weight/min), moderate hypoperfusion (0.5 ml/g/min) and extensive hypoperfusion (0.25 ml/g/min) were evaluated. Clinical parameters and tissue oxygenation index (TOI) were registered online until 3 h of reperfusion. Hematoxylin and eosin (HE) staining and immunohistological analyses for apoptosis inducing factor (AIF) and nitrotyrosine (NO-Tyr) were performed on sections of the hippocampus.. Intracerebral pressure remained stable throughout the study. Haemodynamic parameters, blood gas and lactate measurements were stable until the end of the study. Extensive hypoperfusion led to a moderate reduction of TOI. NO-Tyr immuno-positive cells were 15.7% at regular cerebral perfusion, 23.9% at moderate hypoperfusion (P = n.s.) and 46.1% at extensive hypoperfusion (P < 0.05). AIF immuno-positive nuclei were present in 8.3% of the hippocampus cells after regular perfusion, in 10.8% after moderate hypoperfusion (P = n.s.) and in 17.9% after extensive hypoperfusion (P < 0.05).. SCP using a moderate SCP flow regime demonstrates comparable results to normal brain perfusion while after extensive hypoperfusion significant morphological brain injury could be found. Thus moderate, but not extensive, hypoperfusion might have the potential to prevent perfusion-related cerebral oedema and an increasing risk of brain injury.

Topics: Animals; Apoptosis Inducing Factor; Biomarkers; Brain; Cardiopulmonary Bypass; Carotid Artery, Common; Hippocampus; Hypothermia, Induced; Hypoxia-Ischemia, Brain; Intracranial Pressure; Intraoperative Complications; Monitoring, Intraoperative; Perfusion; Random Allocation; Swine; Treatment Outcome; Tyrosine

This study aimed to explore the mechanisms by which andrographolide protects against hypoxia-induced oxidative/nitrosative brain injury provoked by cerebral ischemic/reperfusion (CI/R) injury in mice. Hypoxia IN VITRO was modeled using oxygen-glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone CI/R injury with andrographolide (10-100 µg/kg, i. v.) at 1 h after hypoxia ameliorated CI/R-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. CI/R induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (NOX2), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b cells due to activation of nuclear factor-kappa B (NF- κB) and hypoxia-inducible factor 1-alpha (HIF-1 α). All these changes were significantly diminished by andrographolide. In BV-2 cells, OGD induced ROS and nitric oxide production by upregulating NOX2 and iNOS via the phosphatidylinositol-3-kinase (PI3K)/AKT-dependent NF- κB and HIF-1 α pathways, and these changes were suppressed by andrographolide and LY294002. Our results indicate that andrographolide reduces NOX2 and iNOS expression possibly by impairing PI3K/AKT-dependent NF- κB and HIF-1 α activation. This compromises microglial activation, which then, in turn, mediates andrographolide's protective effect in the CI/R mice.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Cerebral Infarction; Disease Models, Animal; Diterpenes; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Ischemia, Brain; Male; Mice; Mice, Inbred ICR; Microglia; NADPH Oxidases; NF-kappa B; Nitric Oxide Synthase Type II; Nitrosation; Phosphatidylinositol 3-Kinase; Reactive Oxygen Species; Stroke; Superoxides; Tyrosine; Up-Regulation

The optimum antihypertensive treatment for prevention of hypertensive stroke has yet to be elucidated. This study was undertaken to examine the benefit of a combination of valsartan, an angiotensin II type 1 (AT1) receptor blocker, and amlodipine, a calcium channel blocker, in prevention of high-salt-induced brain injury in hypertensive rats. High-salt-loaded stroke-prone spontaneously hypertensive rats (SHRSPs) were given 1) vehicle, 2) valsartan (2 mg/kg/day), 3) amlodipine (2 mg/kg/day), or 4) a combination of valsartan and amlodipine for 4 weeks. The effects on brain injury were compared between all groups. High-salt loading in SHRSPs caused the reduction of cerebral blood flow (CBF), cerebral hypoxia, white matter lesions, glial activation, AT1 receptor up-regulation, endothelial nitric-oxide synthase (eNOS) uncoupling, inducible nitric-oxide synthase induction, and nitroxidative stress. Valsartan, independently of blood pressure, enhanced the protective effects of amlodipine against brain injury, white matter lesions, and glial activation in salt-loaded SHRSPs. These beneficial effects of valsartan added to amlodipine were associated with an additive improvement in CBF and brain hypoxia because of an additive improvement in cerebral arteriolar remodeling and vascular endothelial dysfunction. Furthermore, valsartan added to amlodipine enhanced the attenuation of cerebral nitroxidative stress through an additive suppression of eNOS uncoupling. Valsartan, independently of blood pressure, augmented the protective effects of amlodipine against brain injury in salt-loaded hypertensive rats through an improvement in brain circulation attributed to nitroxidative stress. Our results suggest that the combination of valsartan and amlodipine may be a promising strategy for the prevention of salt-related brain injury in hypertensive patients.

Topics: Amlodipine; Angiotensin II Type 1 Receptor Blockers; Animals; Blood Pressure; Brain; Calcium Channel Blockers; Calcium Channels; Carotid Arteries; Drug Therapy, Combination; Endothelial Cells; Hypertension; Hypoxia-Ischemia, Brain; Male; Nitric Oxide; Nitric Oxide Synthase Type III; Random Allocation; Rats; Rats, Inbred SHR; Receptor, Angiotensin, Type 1; Sodium Chloride, Dietary; Tetrazoles; Tyrosine; Valine; Valsartan

This study aimed to explore the mechanisms by which prodigiosin protects against hypoxia-induced oxidative/nitrosative brain injury induced by middle cerebral artery occlusion/reperfusion (MCAo/r) injury in mice. Hypoxia in vitro was modeled using oxygen-glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone MCAo/r injury with prodigiosin (10 and 100μg/kg, i.v.) at 1h after hypoxia ameliorated MCAo/r-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. MCAo/r induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (gp91(phox)), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b leukocytes due to breakdown of blood-brain barrier (BBB) by activation of nuclear factor-kappa B (NF-κB). All these changes were significantly diminished by prodigiosin. In BV-2 cells, OGD induced ROS and nitric oxide production by up-regulating gp91(phox) and iNOS via activation of the NF-κB pathway, and these changes were suppressed by prodigiosin. In conclusion, our results indicate that prodigiosin reduces gp91(phox) and iNOS expression possibly by impairing NF-κB activation. This compromises the activation of microglial and/or inflammatory cells, which then, in turn, mediates prodigiosin's protective effect in the MCAo/r mice.

Topics: Animals; Blotting, Western; Dose-Response Relationship, Drug; Hypoxia-Ischemia, Brain; Male; Membrane Glycoproteins; Mice; Mice, Inbred ICR; NADPH Oxidase 2; NADPH Oxidases; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Synthase Type II; Nitrosation; Oxidative Stress; Prodigiosin; Reactive Oxygen Species; Tyrosine

Inflammation induced by hypoxia-ischemia (HI) contributes to the development of injury in the newborn brain. In this study, we investigated the role of galectin-3, a novel inflammatory mediator, in the inflammatory response and development of brain injury in a mouse model for neonatal HI. Galectin-3 gene and protein expression was increased after injury and galectin-3 was located in activated microglia/macrophages. Galectin-3-deficient mice (gal3-/-) were protected from injury particularly in hippocampus and striatum. Microglia accumulation was increased in the gal3-/- mice but accompanied by decreased levels of total matrix metalloproteinase (MMP)-9 and nitrotyrosine. The protection and increase in microglial infiltration was more pronounced in male gal3-/- mice. Trophic factors and apoptotic markers did not significantly differ between groups. In conclusion, galectin-3 contributes to neonatal HI injury particularly in male mice. Our results indicate that galectin-3 exerts its effect by modulating the inflammatory response.

Topics: Animals; Animals, Newborn; Brain; Disease Models, Animal; Encephalitis; Female; Galectin 3; Gliosis; Hypoxia-Ischemia, Brain; Inflammation Mediators; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Nerve Degeneration; Tyrosine

To compare the features of brain injury in neonatal rats with different severities of hypoxia-ischemia (HI), and explore the role of microglial activation and cytokines.. One hundred and twenty 7-day-old rats were randomized to three groups: sham control, mild HI, and severe HI. The rats in the HI groups were subjected to right carotid artery occlusion and 8% oxygen hypoxia exposure (40 minutes, 34.5 Celsius degree in the mild HI group; 65 minutes, 35.5 Celsius degree in the severe HI group). MRI, microtubule associated protein (MAP2) and TUNEL staining were used to confirm the severity of brain injury. Changes in expression of activated microglia (ED1) and signs of cytokine involvement or oxidative stress (TNF-alpha, nitrotyrosine) were assessed immunohistochemically.. In the mild HI group, MRI scans demonstrated increased T2 values in the ipsilateral subcortical white matter and a slight loss of T2 values in the cortex, corresponding to a medium loss of MAP2 in the ipsilateral cortex. There was an increase in the number of TUNEL positive cells compared to the control group within the subcortical white matter. In the severe HI group, the T2 value increased in the majority of the hemisphere, corresponding to a severe loss of staining for MAP2 in the ispilateral hemisphere. The number of TUNEL positive cells significantly increased in the ipsilateral cortex and white matter. In the mild HI group, ED1, TNF-alpha and nitrotyrosine expression increased only in the acute stage and was only observed in subcortical white matter. In contrast, after severe HI, the increase in ED1, TNF-alpha and nitrotyrosine expression was observed in the whole ipsilateral hemisphere and prolonged for weeks.. Following a mild HI a relatively selective white matter injury compares to the pannecrosis in the cortex and white matter following a severe HI. Microglial activation and over-expression of cytokines might contribute to the development of hypoxic-ischemic brain damage.

Topics: Animals; Animals, Newborn; Apoptosis; Hypoxia-Ischemia, Brain; Magnetic Resonance Imaging; Microglia; Microtubule-Associated Proteins; Rats; Tumor Necrosis Factor-alpha; Tyrosine

Cerebral ischemia initiates various injurious processes including neuroinflammatory responses such as activation of microglia and increases in cytokine and nitric oxide release. Evidence primarily from in vitro studies, indicates that neuroinflammatory effects can be either beneficial or harmful, possibly related to stimulus strength. We investigated using in vivo models, the effect of a mild or substantial cerebral hypoxia-ischemia on: cerebral microglial/macrophage activation (ED1), pro-inflammatory cytokines (tumor necrosis factor-alpha), nitrosative stress (nitrotyrosine) and permanent brain damage. A mild insult produced a transient (1-2 days post) increase in activated microglia/macrophages within subcortical white and not gray matter but transiently increased cytokine or nitrotyrosine expression in cortex and not white matter. There was also prolonged scattered cell death in cortex and white matter over weeks along with loss of myelin/axons and cortical atrophy at 4 weeks post-insult. In contrast, a substantial insult produced white and gray matter necrosis, cyst formation and atrophy, along with increases in tumor necrosis factor and nitrotyrosine staining within both white and gray matter starting at 1-2 days post-insult. Microglial/macrophage staining was increased starting at 1-week post a substantial insult and remained elevated for weeks thereafter. Thus, a transient neuroinflammatory response occurs following a mild insult whereas prolonged scattered cell death occurs for weeks, particularly in white matter. Insult severity also affects the progression of the neuroinflammatory response, which is prolonged after a substantial insult. Effective therapy will need to be customized for insult severity and timing; and, monitoring the injury processes with imaging or biomarkers may help guide treatment.

Topics: Animals; Brain; Cell Death; Glial Fibrillary Acidic Protein; Hypoxia-Ischemia, Brain; Inflammation; Myelin Basic Protein; Random Allocation; Rats; Tumor Necrosis Factor-alpha; Tyrosine

Spinal cord injury has been reported after perinatal asphyxia in full-term neonates.. To examine the role of excessive nitric oxide production in perinatal spinal cord injury.. Tissue samples of 18 full-term neonates who died of hypoxic-ischemic encephalopathy were analyzed for the presence of nitrotyrosine (NT).. NT was demonstrated in 5 of these 18 neonates. In addition, activated caspase 3, a marker of apoptosis, and CD68, as a marker of inflammation, could be demonstrated in some infants.. excessive nitric oxide production and subsequent NT formation is seen in spinal cord tissue after severe perinatal asphyxia. This finding may be relevant for the development of neuroprotective strategies.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Apoptosis; Asphyxia Neonatorum; Autopsy; Caspase 3; Female; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Inflammation; Male; Nitric Oxide; Spinal Cord; Tyrosine

Hydrogen peroxide (H2O2) and nitric oxide (NO) contribute to the pathogenesis of cerebral hypoxic-ischemic injury. We evaluated the neuroprotective effect of N-acetyl-l-cysteine (NAC, a free radical scavenger) against oxidative stress and perfusion in a model of neonatal hypoxia-reoxygenation (H-R). Piglets (1-3 d, 1.6-2.3 kg) were randomized into a sham-operated group (without H-R) (n = 5) and two H-R experimental groups (2 h normocapnic alveolar hypoxia followed by 4 h reoxygenation) (n = 7/group). Five minutes after reoxygenation, piglets were given either i.v. saline (H-R controls) or NAC (30 mg/kg bolus then 20 mg/kg/h infusion) in a blinded-randomized fashion. Heart rate, mean arterial pressure, carotid arterial blood flow (transit-time ultrasonic probe), cerebral cortical H2O2 and NO production (electrochemical sensor), cerebral tissue glutathione and nitrotyrosine levels (enzyme-linked immunosorbent assay) were examined. Hypoxic piglets were acidotic (pH 6.88-6.90), which recovered similarly in the H-R groups (p > 0.05 versus shams). Postresuscitation NAC treatment significantly attenuated the increase in cortical H2O2, but not NO, concentration during reoxygenation, with lower cerebral oxidized glutathione levels. NAC-treated piglets had significantly higher carotid oxygen delivery and lower cerebral lactate levels than that of H-R controls with corresponding changes in carotid arterial flow and vascular resistance. In newborn piglets with H-R, postresuscitation administration of NAC reduced cerebral oxidative stress and improved cerebral perfusion.

Topics: Acetylcysteine; Animals; Animals, Newborn; Carotid Arteries; Cerebral Cortex; Disease Models, Animal; Free Radical Scavengers; Free Radicals; Glutathione; Hydrogen Peroxide; Hypoxia-Ischemia, Brain; Lactates; Nitric Oxide; Oxidative Stress; Oxygen; Random Allocation; Regional Blood Flow; Swine; Tyrosine

We have shown earlier that 2-iminobiotin (2-IB) reduces hypoxia-ischemia (HI)-induced brain damage in neonatal rats, and presumed that inhibition of nitric oxide synthases (NOS) was the underlying mechanism. We now investigated the effect of 2-IB treatment in P7 rat pups to determine the role of gender and the neuroprotective mechanism. Pups were subjected to HI (occlusion of right carotid artery and 120 mins FiO(2) 0.08) and received subcutaneous (s.c.) 10 mg/kg 2-IB at 0, 12 and 24 h after hypoxia. After 6 weeks, neuronal damage was assessed histologically. We determined cerebral nitrite and nitrate (NO(x)) and nitrotyrosine, heat-shock protein 70, cytosolic cytochrome c, cleaved caspase 3, nuclear translocation of apoptosis-inducing factor (AIF) and the effect of 2-IB on NOS activity in cultured cells. 2-Iminobiotin treatment reduced long-term brain damage in female but not male rats. Unexpectedly, 2-IB treatment did not reduce cerebral NO(x) or nitrotyrosine levels, and did not inhibit NOS activity in vitro. The gender-dependent neuroprotective effect of 2-IB was reflected in inhibition of the HI-induced increase in cytosolic cytochrome c and cleaved caspase 3 in females only. Hypoxia-ischemia-induced activation of AIF was observed in males only and was not affected by 2-IB. Post-HI treatment with 2-IB provides gender-specific long- and short-term neuroprotection in female P7 rats via inhibition of the cytochrome c-caspase 3 neuronal death pathway. 2-Iminobiotin did not alter cerebral NO(x) nor inhibited NOS in intact cells. Therefore, we conclude that it is highly unlikely that the neuroprotective effect of 2-IB involves NOS inhibition.

Topics: Animals; Animals, Newborn; Biotin; Blotting, Western; Caspases; Enzyme Inhibitors; Female; Guanidines; HSP70 Heat-Shock Proteins; Hypoxia-Ischemia, Brain; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; omega-N-Methylarginine; Pregnancy; Rats; Rats, Wistar; Sex Characteristics; Signal Transduction; Tyrosine

2,2,5,7,8-Pentamethyl-6-hydroxychromane (PMC) is the most potent analogue of alpha-tocopherol for anti-oxidation. It is more hydrophilic than other alpha-tocopherol derivatives and has potent free radical-scavenging activity. In the present study, PMC significantly attenuated middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Administration of PMC at 20mg/kg, showed marked reductions in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in HIF-1alpha, active caspase-3, iNOS, and nitrotyrosine expressions in ischemic regions. These expressions were markedly inhibited by treatment with PMC (20mg/kg). In addition, PMC (4-12 microM) inhibited respiratory bursts in human neutrophils stimulated by fMLP (800 nM) and PMA (320 nM). Furthermore, PMC (6, 12, and 60 microM) also significantly inhibited neutrophil migration stimulated by leukotriene B(4) (160 nM). An electron spin resonance (ESR) method was conducted on the scavenging activity of PMC on the free radicals formed. PMC (12 microM) greatly reduced the ESR signal intensities of superoxide anion, hydroxyl radical, and methyl radical formation. In conclusion, we demonstrate a potent neuroprotective effect of PMC on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by inhibition of free radical formation, followed by inhibition of HIF-1alpha activation, apoptosis formation (active caspase-3), neutrophil activation, and inflammatory responses (i.e., iNOS and nitrotyrosine expressions), resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, PMC treatment may represent a novel approach to lowering the risk or improving function in ischemia-reperfusion brain injury-related disorders.

Topics: alpha-Tocopherol; Animals; Antioxidants; Caspase 3; Chromans; Gene Expression Regulation; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Ischemia, Brain; Male; Molecular Structure; Neuroprotective Agents; Neutrophil Activation; Neutrophils; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

Dopamine receptors regulate glutamatergic neurotransmission and Na(+),K(+)-ATPase via protein kinase A (PKA) and dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32)-dependent signaling. Consequently, dopamine receptor activation may modulate neonatal hypoxic-ischemic (H-I) neuronal damage in the selectively vulnerable putamen enriched with dopaminergic receptors. Piglets subjected to two durations of hypoxia followed by asphyxic cardiac arrest were treated with a D1-like (SCH23390) or D2-like (sulpiride) receptor antagonist. At 4 days of recovery from less severe H-I, the remaining viable neurons in putamen were 60% of control, but nearly completely salvaged by pretreatment with SCH23390 or sulpiride. After more severe H-I in which only 18% of neurons were viable, partial neuroprotection was seen with SCH23390 pretreatment (50%) and posttreatment (39%) and with sulpiride pretreatment (35%), but not with sulpiride posttreatment (24%). Dopamine was significantly elevated in microdialysis samples from putamen during asphyxia and the first 15 mins of reoxygenation. Pretreatment with SCH23390 or sulpiride largely attenuated the increased nitrotyrosine and the decreased Na(+),K(+)-ATPase activity that occurred at 3 h after severe H-I. Pretreatment with SCH23390, but not sulpiride, also attenuated H-I-induced increases in PKA-dependent phosphorylation of Thr34 on DARPP-32, Ser943 on the alpha subunit of Na(+),K(+)-ATPase, and Ser897 of the N-methyl-D-aspartate (NMDA) receptor NR1 subunit. These findings indicate that D1 and D2 dopamine receptor activation contribute to neuronal death in newborn putamen after H-I in association with increased protein nitration and decreased Na(+),K(+)-ATPase activity. Furthermore, mechanisms of D1 receptor toxicity may involve DARPP-32-dependent phosphorylation of NMDA receptor NR1 and Na(+),K(+)-ATPase.

Topics: Adenosine Triphosphatases; Animals; Animals, Newborn; Blotting, Western; Corpus Striatum; Dopamine; Dopamine and cAMP-Regulated Phosphoprotein 32; Dopamine Antagonists; Hypoxia-Ischemia, Brain; Immunohistochemistry; Microdialysis; Neurons; Neuroprotective Agents; Phosphorylation; Receptors, Dopamine; Receptors, N-Methyl-D-Aspartate; Swine; Tyrosine

Maternal inflammation/infection alone or in combination with birth asphyxia increases the risk for perinatal brain injury. Free radicals are implicated as major mediators of inflammation and hypoxia-ischemia (HI)-induced perinatal brain injury. This study evaluated the neuroprotective efficacy of a scavenging agent, N-acetylcysteine (NAC), in a clinically relevant model.. Lipopolysaccharide (LPS)-sensitized HI brain injury was induced in 8-day-old neonatal rats. NAC was administered in multiple doses, and brain injury was evaluated at 7 days after HI.. NAC (200mg/kg) provided marked neuroprotection with up to 78% reduction of brain injury in the pre+post-HI treatment group and 41% in the early (0 hour) post-HI treatment group, which was much more pronounced protection than another free radical scavenger, melatonin. Protection by NAC was associated with the following factors: (1) reduced isoprostane activation and nitrotyrosine formation; (2) increased levels of the antioxidants glutathione, thioredoxin-2, and (3) inhibition of caspase-3, calpain, and caspase-1 activation.. NAC provides substantial neuroprotection against brain injury in a model that combines infection/inflammation and HI. Protection by NAC was associated with improvement of the redox state and inhibition of apoptosis, suggesting that these events play critical roles in the development of lipopolysaccharide-sensitized HI brain injury.

Topics: Animals; Animals, Newborn; Apoptosis; Calpain; Caspases; Cystine; Enzyme Activation; Glutathione; Hypoxia-Ischemia, Brain; Isoprostanes; Lipopolysaccharides; Membrane Proteins; Neuroprotective Agents; Oxidation-Reduction; Rats; Rats, Wistar; Thioredoxins; 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

The short- and long-term neuroprotective effects of 2-iminobiotin, a selective inhibitor of neuronal and inducible nitric oxide synthase, were studied in 12-day-old rats following hypoxia-ischemia. Hypoxia-ischemia was induced by occlusion of the right carotid artery followed by 90 minutes of hypoxia (FiO2 0.08). Immediately on reoxygenation, 12 and 24 hours later the rats were treated with vehicle or 2-iminobiotin at a dose of 5.5, 10, 30, or 60 mg/kg per day. Histologic analysis of brain damage was performed at 6 weeks after hypoxia-ischemia. To assess early changes of cerebral tissue, levels of HSP70, nitrotyrosine, and cytochrome c were determined 24 hours after reoxygenation. Significant neuroprotection was obtained using a dose of 30 mg/kg per day of 2-iminobiotin. Levels of HSP70 were increased in the ipsilateral hemisphere in both groups (P<0.05), but the increase was significantly (P<0.05) less in the rats receiving the optimal dose of 2-iminobiotin (30 mg/kg per day). Hypoxia-ischemia did not lead to increased levels of nitrotyrosine, nor did 2-iminobiotin influence levels of nitrotyrosine. In contrast, hypoxia-ischemia induced an increase in cytochrome c level that was prevented by 2-iminobiotin. In conclusion, 2-iminobiotin administered after hypoxia-ischemia provides long-term neuroprotection. This neuroprotection is obtained by mechanisms other than a reduction of nitrotyrosine formation in proteins.

Topics: Animals; Animals, Newborn; Biotin; Brain Chemistry; Cytochromes c; Female; HSP70 Heat-Shock Proteins; Hypoxia-Ischemia, Brain; Male; Neuroprotective Agents; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidation-Reduction; Rats; Rats, Wistar; Tyrosine

Production of nitric oxide is thought to play an important role in neuroinflammation. Previously, we have shown that combined inhibition of neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) can reduce hypoxia-ischemia-induced brain injury in 12-day-old rats. The aim of this study was to analyze changes in expression of nNOS, iNOS and endothelial NOS (eNOS), and nitrotyrosine (NT) formation in proteins in neonatal rats up to 48 h after cerebral hypoxia-ischemia.. Twelve-day-old rats were subjected to unilateral carotid artery occlusion and hypoxia, resulting in unilateral cerebral damage. NOS and nitrotyrosine expression were determined by immunohistochemistry and Western blot analysis at 30 min-48 h after hypoxia-ischemia.. nNOS was increased in both hemispheres from 30 min to 3 h after hypoxia-ischemia. In the contralateral hemisphere, eNOS was decreased 1-3 h after hypoxia-ischemia. In the ipsilateral hemisphere, eNOS was decreased at 0.5 h after hypoxia-ischemia, normalized at 1-3 h and was increased 6-12 h after hypoxia-ischemia. At 24 and 48 h after hypoxia-ischemia, eNOS levels normalized. Surprisingly, iNOS expression did not change from 30 min up to 48 h after hypoxia-ischemia in the ipsi- or contralateral hemisphere. In addition, the regional expression of iNOS in the brain as determined by immunohistochemistry did not change after hypoxia-ischemia. Expression of nitrotyrosine was slightly increased in both hemispheres only at 30 min after hypoxia-ischemia.. In 12-day-old rat pups, cerebral hypoxia-ischemia induced a transient increase in nNOS, eNOS, and nitrotyrosine in proteins, but no change in iNOS expression up to 48 h after the insult.

Topics: Animals; Animals, Newborn; Blotting, Western; Disease Models, Animal; Female; Functional Laterality; Gene Expression Regulation; Hypoxia-Ischemia, Brain; Imino Pyranoses; Immunohistochemistry; Male; Nitric Oxide Synthase; Piperidines; Rats; Statistics, Nonparametric; Time Factors; Tyrosine

Excessive nitric oxide (NO) production after cerebral hypoxia-ischaemia (HI) may induce cellular injury in various ways, including reaction with superoxide to form the highly reactive peroxynitrite. We characterized the spatial and temporal formation of peroxynitrite through immunohistochemical detection of nitrosylated proteins. Nitrotyrosine immunoreactivity peaked around 3 h post-HI and was detected in areas of injury, as judged by the loss of microtubule-associated protein-2 (MAP-2) staining, in neurones, glia and endothelial cells. Nitrotyrosine staining co-localized with three other cellular markers of injury, active caspase-3, nuclear translocation of apoptosis-inducing factor (AIF) and an oligonucleotide hairpin probe detecting specific DNA strand breaks. The number of nitrotyrosine-positive cells at early time points outnumbered the cells positive for the other three markers of injury, indicating that nitrosylation preceded caspase-3 activation. Pharmacological inhibition of neuronal and inducible nitric oxide synthase (nNOS and iNOS) using 2-iminobiotin, which has been demonstrated earlier to be neuroprotective, significantly reduced nitrotyrosine formation and caspase-3 activation, but not nuclear translocation of AIF, in cortex and striatum of the ipsilatral hemisphere. In summary, nitrotyrosine is an early marker of cellular injury and inhibition of nNOS and iNOS is a promising strategy for neuroprotection after perinatal HI.

Topics: Animals; Animals, Newborn; Apoptosis Inducing Factor; Biomarkers; Biotin; Caspase 3; Caspases; Cell Count; Disease Models, Animal; DNA Damage; Enzyme Activation; Enzyme Inhibitors; Female; Flavoproteins; Hypoxia-Ischemia, Brain; Male; Membrane Proteins; Neurons; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Protein Transport; Rats; Rats, Wistar; Tyrosine

Acute inflammatory reactions cause neuronal damage in cerebral ischemia-reperfusion. Urinary trypsin inhibitor (UTI), a serine protease inhibitor, is cytoprotective against ischemia-reperfusion injury in the liver, intestine, kidney, heart, and lung through its antiinflammatory activity. Neuroprotective action of UTI on transient global cerebral ischemia has been documented. This is the first study to determine whether UTI is neuroprotective against transient focal cerebral ischemia.. Adult male Wistar rats were randomly assigned to the following treatment groups: 0.9% saline (control, n = 9); 100,000 U/kg UTI (n = 9); and 300,000 U/kg UTI (n = 9). Treatments were performed intravenously 10 min before right middle cerebral artery occlusion for 2 h and subsequent reperfusion. Ninety-six hours after the onset of reperfusion, the motor neurologic deficit and the cerebral infarct size were evaluated. Furthermore, immunohistochemical staining for myeloperoxidase and nitrotyrosine to count infiltrating neutrophils and nitrated cells, respectively, was performed on the brain sections.. Infarct volume in the 300,000 U/kg UTI group was smaller than in the 100,000 U/kg UTI and saline control groups (P < 0.05). Treatment with 300,000 U/kg UTI showed a trend to improve neurologic outcome but did not reach statistical significance (P = 0.0693). The significant decrease in neutrophil infiltration was observed in the ischemic hemisphere treated with 300,000 U/kg UTI compared with saline control (P < 0.05). Nitrotyrosine deposition in the ischemic hemisphere was significantly reduced in the 300,000 U/kg UTI group compared with saline control and 100,000 U/kg UTI groups (P < 0.05).. Intravenous pretreatment with 300,000 U/kg UTI reduces focal ischemia-reperfusion injury in the rat brain, potentially opening a novel therapeutic avenue for the treatment of cerebral ischemia.

Topics: Animals; Behavior, Animal; Brain; Cell Count; Cerebrovascular Circulation; Glycoproteins; Hypoxia-Ischemia, Brain; Immunohistochemistry; Infarction, Middle Cerebral Artery; Laser-Doppler Flowmetry; Male; Nervous System Diseases; Neuroprotective Agents; Neutrophils; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Trypsin Inhibitors; Tyrosine

Peroxynitrite is assumed to play a crucial role in brain damage associated with the overproduction of nitric oxide (NO). The purpose of this study is to examine time-dependent changes of nitrite and nitrate (NOx) concentration in the circulation, and peroxynitrite formation as well as the expression of inducible nitric oxide synthase (iNOS) in the penumbra of rat brains during transient middle cerebral artery occlusion (MCAO) of Wistar rat for 2 h and reperfusion for 4-70 h. NOx concentration in the circulation was continuously monitored at the right jugular vein by microdialysis. The expression of iNOS was detected at 22-70 h after reperfusion in vascular walls and the cortex. Nitrotyrosine, a marker of peroxynitrite, appeared 4 h after reperfusion in the cortex, increasing substantially at 22-46 h in vascular walls. NOx level in dialysate increased immediately after MCAO. After a gradual decrease, the level increased again 4 h after reperfusion, reaching a maximum at 46 h. Brain myeloperoxidase activity, a marker of neutrophil infiltration, was not detected 4 h after reperfusion, but greatly increased at 22 h and then decreased. These results suggest that a marked increase of NOx level in the circulation might reflect the expression of iNOS, while neuronal NOS may contribute to peroxynitrite formation in the cortex observed at an earlier phase of reperfusion. This study indicates that monitoring NOx level in the circulation serves to assess the progress of stroke, and to determine appropriate therapeutic measures.

Topics: Animals; Cerebral Cortex; Cerebral Infarction; Cerebrovascular Circulation; Chemotaxis, Leukocyte; Disease Models, Animal; Hypoxia-Ischemia, Brain; Infarction, Middle Cerebral Artery; Male; Neutrophils; Nitric Oxide; Nitric Oxide Synthase; Peroxidase; Peroxynitrous Acid; Rats; Rats, Wistar; Reaction Time; Reperfusion Injury; Tyrosine

The objective of this study was to determine the role of cerebral nitric oxide and its powerful oxidant peroxynitrite following mild birth asphyxia. The cerebrospinal fluid levels of nitric oxide and 3-nitrotyrosine as a marker for peroxynitrite are measured in neonates with mild hypoxic-ischemic encephalopathy. Based on the classification of Sarnat and Sarnat, term neonates with mild hypoxic-ischemic encephalopathy and neurologically normal neonates suspected of sepsis were taken as the control group. Nitric oxide measurements were done by chemiluminescence, and nitrotyrosine measurements were made by high-performance liquid chromatography. The Mann Whitney U-test was used, and a Pvalue < .05 was considered significant. Eleven patients with grade 1 hypoxic-ischemic encephalopathy and nine controls were included. The gestational age and birthweights were similar in both groups. Neither of the cerebrospinal fluid levels of nitric oxide (8.60 +/- 0.49 micromol/L) and nitrotyrosine (0.45 +/- 0.33 micromol/L) of the neonates with hypoxic-ischemic encephalopathy showed significant differences from that of the means of nitric oxide (8.66 +/- 1.07 micromol/L) and nitrotyrosine levels (0.25 +/- 0.13 micromol/L) of the controls. These data suggest that the oxidative stress is not overexpressed to lead nitric oxide and peroxynitrite to play a pathologic role in the early phase of mild hypoxic-ischemic encephalopathy of the newborn.

Topics: Chromatography, High Pressure Liquid; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Luminescent Measurements; Nitric Oxide; Tyrosine

A perfusion model of global cerebral ischemia was used for the immunohistochemical study of changes in the glutamate-nitric oxide (NO) system in the rat cerebellum and cerebellar nuclei during a 0-14 h reperfusion period after 30 min of oxygen and glucose deprivation, with and without administration of 1.5 mM N(omega)-nitro-L-arginine methyl ester (L-NAME). While immunostaining for N-methyl-D-aspartate receptor subunit 1 (NMDAR1) showed no marked changes during the reperfusion period, neuronal NO synthase (nNOS) immunostaining increased in stellate and basket cells, granule cells and neurons of the cerebellar nuclei. However, global cerebellar nNOS concentrations determined by Western blotting remained largely unchanged in comparison with actin expression. Inducible NOS (iNOS) immunostaining appeared in Purkinje cells and neurons of the cerebellar nuclei after 2-4 h of reperfusion and intensified during the 6-14 h period. This was reflected by an increase in global cerebellar iNOS expression determined by Western blotting. Immunostaining for protein nitrotyrosine was seen in Purkinje cells, stellate and basket cells, neurons of the cerebellar nuclei and glial cells in controls, and showed a progressive translocation in Purkinje cells and neurons of the cerebellar nuclei from an initial perinuclear or nuclear location towards the periphery. At the end of the reperfusion period the Purkinje cell apical dendrites were notably retracted and tortuous. Prior and concurrent L-NAME administration eliminated nitrotyrosine immunostaining in controls and blocked or reduced most of the postischemic changes observed. The results suggest that while nNOS expression may be modified in certain cells, iNOS is induced after a 2-4 h period, and that changes in protein nitration may be associated with changes in cell morphology.

Topics: Animals; Blotting, Western; Cerebellum; Disease Models, Animal; Enzyme Inhibitors; Glucose; Hypoxia-Ischemia, Brain; Immunohistochemistry; Male; Neurons; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Purkinje Cells; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Tyrosine

To determine whether, and if so how, iNOS expresses and affects brain injury induced by hypoxia-ischemia in an immature brain.. Seven-day-old Wistar rat pups were exposed to right common carotid artery ligation followed by 1.5 hours of hypoxia. The time course of iNOS mRNA expression, enzymatic activity, and protein production in the cerebral cortex were determined. The extent of the infarct area in the cerebral cortex and the production of 3-nitrotyrosine (a biomarker of peroxynitrite) were compared between the control pups and pups treated with S-methyl-isothiourea (a selective iNOS inhibitor).. In the cortex ipsilateral to carotid ligation, iNOS mRNA appeared from 6 hours to 24 hours after hypoxia-ischemia and disappeared at 48 hours. The iNOS protein and its activity also increased at 12 hours and reached a maximum level at 48 hours after the insult. The percentage of damage in the cerebral cortex was significantly higher in the control pups than in treated pups (31.9 vs 10.6%). Tri-nitrotyrosine following iNOS expression-positive cells were located predominantly at the infarct and peri-infarct regions.. iNOS expression might be an important determinant of ischemic immature brain injury.

Topics: Animals; Animals, Newborn; Brain Diseases; Carotid Arteries; Hypoxia-Ischemia, Brain; Kinetics; Ligation; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Wistar; RNA, Messenger; Tyrosine

The peroxynitrite contributions to hypoxic damage in brain slices that arise from N-methyl-D-aspartate (NMDA) receptor activation were studied by following the temporal-spatial course of nitrotyrosine (NT) formation during six conditions: hypoxia (pO(2)<5 mmHg) with or without 10 microM MK-801 treatment; with exposure to 10, 100 and 1000 microM NMDA; and no treatment (control). In each experiment, twenty 350-micrometer thick cerebrocortical slices, obtained from the parietal lobes of ten 7-day-old Sprague-Dawley rats, were metabolically recovered and allowed to respire in a well-oxygenated perfusion system. Thirty minutes exposures to hypoxia or NMDA were followed by 2 h of oxygenated reperfusion. MK-801 administration began 15 min prior to hypoxia and was discontinued during reperfusion. Anti-NT serum immunohistochemistry stains in 20-micrometer frozen sections of slices taken during oxygenated reperfusion, after hypoxia or NMDA exposure, were positive in both neurons and endothelial cells. NT-positive neurons were detected sooner after hypoxia than after NMDA exposure, suggesting that mechanisms of superoxide generation were different in both groups. After hypoxia and even more so after NMDA exposure, more intense NT-positive staining was observed in endothelial cells than in neurons. Cell damage after hypoxia was attenuated by MK-801. MK-801 decreased post-hypoxia counts of NT-stained endothelial cells by 78.5% (p<0. 001) and NT-stained neurons by 54.1% (p<0.05). Our findings suggest that NMDA receptor activation in hypoxic brain slices is associated with increased post-hypoxic peroxynitrite production that contributes to acute neuronal death and endothelial cell injury. Peroxynitrite injury to endothelial cells, caused either by increased peroxynitrite from within or from increased vulnerability to peroxynitrite from without, might play an important role in hypoxic-ischemic brain injury and NMDA-induced brain injury.

Topics: Animals; Brain; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hypoxia-Ischemia, Brain; N-Methylaspartate; Neurons; Nitrates; Nitric Oxide; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Superoxides; Tyrosine