3-nitrotyrosine and Cerebral-Infarction

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

Experimental evidence has shown that some garlic-derived products have a protective effect against ischemic brain injury. The present study was designed to investigate the effect of aged garlic extract (AGE), establish the therapeutic window, and determine its protective mechanism in a cerebral ischemia model. Animals were subjected to middle cerebral artery occlusion (MCAO) for 2h and treated with 1.2ml/kg body wt.(i.p.) of AGE 30min before, at the beginning of (0R), or 1h after reperfusion. The 0R treatment significantly reduced the size of the infarct area after 2h of reperfusion. Repeated doses subsequent to the 0R treatment (at 1, 2, or 3h after reperfusion) had no effect on the temporal window of protection. The protective 0R treatment with AGE prevented the increase in nitrotyrosine and the decrease in total superoxide dismutase, glutathione peroxidase, and extracellular superoxide dismutase activities induced by MCAO. These data indicate that AGE delays the effects of ischemia/reperfusion-induced neuronal injury. However, this treatment itself was not associated with a noticeable improvement in the neurological outcome, or with an effect on the inflammatory response. We conclude that the neuroprotective effect of AGE in the 0R treatment might be associated with control of the free-radical burst induced by reperfusion, preservation of antioxidant enzyme activity, and the delay of other pathophysiological processes.

Topics: Animals; Antioxidants; Brain; Brain Ischemia; Cerebral Infarction; Disease Models, Animal; Garlic; Glutathione Peroxidase; Infarction, Middle Cerebral Artery; Male; Neuroprotective Agents; Oxidative Stress; Phytotherapy; Plant Extracts; Rats; Rats, Wistar; Reperfusion Injury; Superoxide Dismutase; Tyrosine

Overproduction of neuronal nitric oxide synthase (nNOS)-derived NO is detrimental during cerebral ischemia. Normobaric hyperoxia (NBO) has been shown to be neuroprotective, extending the therapeutic time window for ischemic stroke, but the mechanism is not fully understood. In the present study, using a rat model of ischemic stroke, we investigated the effect of early NBO treatment on neuronal NO production. Male Sprague-Dawley rats were given normoxia (30% O(2)) or NBO (95% O(2)) during 10, 30, 60 or 90min filament occlusion of the middle cerebral artery. NO(x)(-) (nitrite plus nitrate) and 3-nitrotyrosine were measured in the ischemic cortex. Ischemia caused a rapid increase in the production of NO(x)(-), with a peak at 10min after ischemia onset, then gradually declining to the baseline level at 60min. NBO treatment delayed the NO(x)(-) production peak to 30min and attenuated the total amount of NO(x)(-). Ischemia also increased 3-nitrotyrosine formation, which was significantly reduced by NBO treatment. Inhibition of nNOS by pre-treatment with 7-nitroindazole had similar effect as NBO treatment on NO(x)(-) and 3-nitrotyrosine production, and when combined with NBO, no further reduction in NO production was observed. Furthermore, NBO treatment significantly decreased brain infarct volume. Taken together, our findings demonstrate that delaying and attenuating the early NO release from nNOS may be an important mechanism accounting for NBO's neuroprotection.

Topics: Animals; Brain Ischemia; Cerebral Infarction; Hyperoxia; Ischemic Attack, Transient; Male; Middle Cerebral Artery; Nitrates; Nitric Acid; Nitrites; Rats; Rats, Sprague-Dawley; Tyrosine

Reactive oxygen species (ROS) is massively produced in the brain after cerebral ischemia and reperfusion. It reacts strongly with cellular components, which has detrimental effects and leads to neuronal cell death. DJ-1, which was found to be the causative gene of familial Parkinson's disease PARK7, is a multifunction protein, which plays a key role in transcriptional regulation, and a molecular chaperone. In this study, we investigated the neuroprotective effect of DJ-1 against neurodegeneration caused by ischemia/reperfusion injury. Cerebral ischemia was induced in rats by 120 mins of middle cerebral artery occlusion (MCAO) using an intraluminal introduction method. The intrastriatal injection of recombinant glutathione S-transferase-tagged human DJ-1 (GST-DJ-1) markedly reduced infarct size in 2,3,5-triphenyltetrazolium chloride staining at 3 days after MCAO. In addition, we performed a noninvasive evaluation of ischemic size using magnetic resonance imaging and found a significant reduction of infarct size with the administration of GST-DJ-1. In GST-DJ-1-treated rats, behavioral dysfunction and nitrotyrosine formation were significantly inhibited. Furthermore, GST-DJ-1 markedly inhibited H(2)O(2)-mediated ROS production in SH-SY5Y cells. These results indicate that GST-DJ-1 exerts a neuroprotective effect by reducing ROS-mediated neuronal injury, suggesting that DJ-1 may be a useful therapeutic target for ischemic neurodegeneration.

Topics: Animals; Behavior, Animal; Cerebral Infarction; Humans; Intracellular Signaling Peptides and Proteins; Magnetic Resonance Imaging; Nerve Degeneration; Oncogene Proteins; Protein Deglycase DJ-1; Rats; Reactive Oxygen Species; Reperfusion Injury; Tyrosine

In the present study, we assessed the beneficial synergistic effects of concurrent treatment with low doses of cilostazol and probucol against focal cerebral ischemic infarct in rats. The ischemic infarct induced by 2-h occlusion of middle cerebral artery (MCA) and 22-h reperfusion was significantly reduced in rat brain that received cilostazol (20 mg/kg) and probucol (30 mg/kg) twice together with prominent improvement of neurological function compared to the effect of cilostazol or probucol monotherapy. Increased myeloperoxidase activity, a marker of neutrophil infiltration, observed in the penumbral zone of vehicle-treated brain was more significantly reduced by cilostazol plus probucol in combination. Increased superoxide-, nitrotyrosine (a marker of peroxynitrite)-, poly(ADP-ribose) [a marker for poly(ADP-ribose) polymerase activity]-, and cleaved caspase-3-positive cells (a proapoptotic marker) in the vehicle sample were significantly attenuated by the combination therapy, while individual treatment with low dose of cilostazol or probucol showed a marginal effect. Taken together, it is suggested that the neuroprotective potentials of combination therapy with low doses of cilostazol plus probucol may provide beneficial therapeutic intervention in reducing the focal cerebral ischemic infarct in rats.

Topics: Animals; Antioxidants; Apoptosis; Biomarkers; Brain Ischemia; Caspase 3; Cerebral Infarction; Chemotaxis, Leukocyte; Cilostazol; Dose-Response Relationship, Drug; Drug Combinations; Drug Synergism; Infarction, Middle Cerebral Artery; Male; Neuroprotective Agents; Peroxidase; Poly(ADP-ribose) Polymerases; Probucol; Rats; Rats, Sprague-Dawley; Superoxides; Telencephalon; Tetrazoles; Treatment Outcome; 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

Poly(ADP-ribose) synthase (PARS), an abundant nuclear protein, has been described as an important candidate for mediation of neurotoxicity by nitric oxide. However, in cerebral ischemia, excessive PARS activation may lead to energy depletion and exacerbation of neuronal damage. We examined the effect of inhibiting PARS on the (a) degree of cerebral injury, (b) process of inflammatory responses, and (c) functional outcomes in a neonatal rat model of focal ischemia. We demonstrate that administration of 3-aminobenzamide, a PARS inhibitor, leads to a significant reduction of infarct volume: 63 +/- 2 (untreated) versus 28 +/- 4 mm(3) (treated). The neuroprotective effects currently observed 48 h postischemia hold up at 7 and 17 days of survival time and attenuate neurological dysfunction. Inhibition of PARS activity, demonstrated by a reduction in poly(ADP-ribose) polymer formation, also reduces neutrophil recruitment and levels of nitrotyrosine, an indicator of peroxynitrite generation. Taken together, our results demonstrate that PARS inhibition reduces ischemic damage and local inflammation associated with reperfusion and may be of interest for the treatment of neonatal stroke.

Topics: Animals; Animals, Newborn; Benzamides; Brain Ischemia; Cell Death; Cerebral Infarction; Encephalitis; Female; Male; Motor Activity; Neurologic Examination; Neuroprotective Agents; Neutrophils; Nitrates; Poly(ADP-ribose) Polymerase Inhibitors; Polymers; Rats; Rats, Wistar; Reperfusion Injury; Signal Transduction; Stroke; Time Factors; Tyrosine

Nitric oxide (NO) is a new intercellular messenger that occurs naturally in the brain without causing overt toxicity. Yet, NO has been implicated as a mediator of cell death in cell death. One explanation is that ischemia causes overproduction of NO, allowing it to react with superoxide to form the potent oxidant peroxynitrite. To address this question, we used immunohistochemistry for citrulline, a marker for NO synthase activity, and 3-nitrotyrosine, a marker for peroxynitrite formation, in mice subjected to reversible middle cerebral artery occlusion. We show that ischemia triggers a marked augmentation in citrulline immunoreactivity but more so in the peri-infarct than the infarcted tissue. This increase is attributable to the activation of a large population (approximately 80%) of the neuronal isoform of NO synthase (nNOS) that is catalytically inactive during basal conditions, indicating a tight regulation of physiological NO production in the brain. In contrast, 3-nitrotyrosine immunoreactivity is restricted to the infarcted tissue and is not present in the peri-infarct tissue. In nNOS(Delta/Delta) mice, known to be protected against ischemia, no 3-nitrotyrosine immunoreactivity is detected. Our findings provide a cellular localization for nNOS activation in association with ischemic stroke and establish that NO is not likely a direct neurotoxin, whereas its conversion to peroxynitrite is associated with cell death.

Topics: Animals; Brain; Cerebral Infarction; Citrulline; Corpus Striatum; Enzyme Activation; Functional Laterality; Immunohistochemistry; Injections, Intraperitoneal; Ischemic Attack, Transient; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Microinjections; N-Methylaspartate; Neurons; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxidants; Tyrosine

Since ischemic insults lead to a deregulation of nitric oxide production which contributes to delayed neuronal death, we investigated changes in the distribution and amount of nitric oxide synthases I and II and in the appearance of nitrotyrosine caused by small, well-defined photothrombic lesions (2 mm in diameter) in the somatosensory cortex of rats. Four hours after lesioning, cell loss was evident in the core of the lesion and no nitric oxide synthase was present within this area, indicating that neurons expressing nitric oxide synthase I were lost or that nitric oxide synthase I was degraded. No increase in the number of neurons expressing nitric oxide synthase I was visible in the area surrounding the lesion, nor in other parts of the brain. One day after lesioning, NADPH-diaphorase- and nitric oxide synthase II-positive leucocytes had invaded the perilesional cortex and were accumulated in injured blood vessels. By two to three days post-lesion, layer V and VI pyramidal neurons, microglia, astrocytes and invading leucocytes had become strongly immunoreactive for nitric oxide synthase II within a perilesional rim. The number of cells expressing nitric oxide synthase I remained stable. Nitric oxide synthase II immunoreactivity and related NADPH-diaphorase had decreased by seven days post-lesion in most animals. However, the number of activated microglia or macrophages and astrocytes, as revealed by other markers, remained elevated. In addition, nitrotyrosine immunoreactivity was evident in the blood vessels close to the lesion, as well as in the ipsilateral hippocampus and thalamus. These findings indicate that no perilesional changes in the number of neurons expressing nitric oxide synthase I occur, but that a transient increase in nitric oxide synthase II does take place in the aftermath of small cortical lesions. The results suggest that increased nitric oxide production is limited to certain post-lesional intervals in this experimental model. It is also obvious that the vast majority of nitric oxide synthase-positive cells are nitric oxide synthase II-containing astrocytes three days after lesioning, suggesting that astrocyte-derived nitric oxide plays a significant role in delayed neuronal death. Such a condition points to an important aspect of post-lesional astrocytosis.

Topics: Animals; Cerebral Infarction; Immunohistochemistry; Ischemic Attack, Transient; Isoenzymes; Lectins; Male; NADPH Dehydrogenase; Nitric Oxide Synthase; Rats; Rats, Wistar; Somatosensory Cortex; Thrombosis; Tyrosine