3-nitrotyrosine and Infarction--Middle-Cerebral-Artery

Preconditioning exercise can exert neuroprotective effects after stroke. However, the mechanism underlying these neuroprotective effects by preconditioning exercise remains unclear. We investigated the neuroprotective effects of preconditioning exercise on brain damage and the expression levels of the midkine (MK) and brain-derived neurotrophic factor (BDNF) after brain ischemia. Animals were assigned to one of 4 groups: exercise and ischemia (Ex), no exercise and ischemia (No-Ex), exercise and no ischemia (Ex-only), and no exercise and intact (Control). Rats ran on a treadmill for 30 min once a day at a speed of 25 m/min for 5 days a week for 3 weeks. After the exercise program, stroke was induced by a 60 min left middle cerebral artery occlusion using an intraluminal filament. The infarct volume, motor function, neurological deficits, and the cellular expressions levels of MK, BDNF, GFAP, PECAM-1, caspase 3, and nitrotyrosine (NT) were evaluated 48 h after the induction of ischemia. The infarct volume, neurological deficits and motor function in the Ex group were significantly improved compared to that of the No-Ex group. The expression levels of MK, BDNF, GFAP, and PECAM-1 were enhanced in the Ex group compared to the expression levels in the No-Ex group after brain ischemia, while the expression levels of activated caspase 3 and NT were reduced in the area surrounding the necrotic lesion. Our findings suggest that preconditioning exercise reduced the infract volume and ameliorated motor function, enhanced expression levels of MK and BDNF, increased astrocyte proliferation, increased angiogenesis, and reduced neuronal apoptosis and oxidative stress.

Topics: Animals; Brain Ischemia; Brain-Derived Neurotrophic Factor; Caspase 3; Cytokines; Glial Fibrillary Acidic Protein; Infarction, Middle Cerebral Artery; Male; Midkine; Nerve Growth Factors; Physical Conditioning, Animal; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Sprague-Dawley; Tyrosine

Stroke is a major health issue in women. Our previous studies in male rats showed decreased myogenic tone in middle cerebral arteries (MCAs) after ischemia and reperfusion (I/R), while tone in parenchymal arterioles (PAs) was increased. This vascular response may aggravate stroke damage in males by limiting reperfusion; however, the effect in females is not known. The current study investigated the effect of I/R and tissue plasminogen activator (tPA) on myogenic tone and reactivity of MCAs and PAs in female rats. Nitrosative stress by peroxynitrite and recruitment of inflammatory neutrophils to the microvasculature were also studied. Female rats were subjected to 2-h MCA filament occlusion (n = 16) or sham surgery (n = 17) and given tPA (1 mg/kg, i.v) or vehicle followed by 30-min reperfusion. Myogenic tone and reactivity were measured in isolated and pressurized MCAs and PAs from the same animals. Cerebrovascular F-actin, 3-nitrotyrosine (3-NT, peroxynitrite marker), and intravascular neutrophils were quantified. Myogenic tone and constriction to the nitric oxide synthase inhibitor Nω-nitro-L-arginine were decreased in MCAs but unchanged in PAs after I/R with no effect of tPA. F-actin and 3-NT expression were unaffected by I/R or tPA. Our study showed that MCAs from females, similar to what has been seen in males, are dilated after I/R and have decreased myogenic tone while tone in PAs was unchanged. Increased small vessel resistance may contribute to decreased reperfusion and worse outcome after stroke.

Topics: Actins; Amides; Animals; Cerebrovascular Circulation; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Fibrinolytic Agents; Infarction, Middle Cerebral Artery; Middle Cerebral Artery; Muscle, Smooth; Neutrophils; Ovariectomy; Pyridines; Rats; Rats, Wistar; Reperfusion; Reperfusion Injury; Tissue Plasminogen Activator; Tyrosine

The present study was designed to investigate the potential role of miR-23a-3p in experimental brain ischemia-reperfusion injury. Cerebral ischemia reperfusion was induced by transient middle cerebral artery occlusion (MCAO) for 1h in C57/BL6 mice. And miR-23a-3p angomir was transfected to upregulate the miR-23a-3p level. Our results showed that miR-23a-3p levels were transiently increased at 4h after reperfusion in the peri-infarction area, while markedly increased in the infarction core at reperfusion 4h and 24h. Importantly, in vivo study demonstrated that miR-23a-3p angomir treatment through intracerebroventricular injection markedly decreased cerebral infarction volume after MCAO. Simultaneously, miR-23a-3p reduced peroxidative production nitric oxide (NO) and 3-nitrotyrosine (3-NT), and increased the expression of manganese superoxide dismutase (MnSOD). In vitro study demonstrated that miR-23a-3p decreased hydrogen peroxide (H2O2)-induced lactate dehydrogenase (LDH) leakage dose-dependently, and reduced protein levels of activated caspase-3 in neuro-2a cells. In addition, miR-23a-3p reduced H2O2-induced production of NO and 3-NT dose-dependently, and reversed the decreased activity of total SOD and MnSOD in neuro-2a cells. Our study indicated that miR-23a-3p suppressed oxidative stress and lessened cerebral ischemia-reperfusion injury.

Topics: Animals; Brain; Brain Ischemia; Caspase 3; Cell Line; Disease Models, Animal; Hydrogen Peroxide; Infarction, Middle Cerebral Artery; L-Lactate Dehydrogenase; Male; Mice, Inbred C57BL; MicroRNAs; Nitric Oxide; Oxidative Stress; Reperfusion Injury; Superoxide Dismutase; Transfection; Tyrosine; Up-Regulation

Cerebral ischemia exacerbates neuronal death and neurological dysfunction. Evidence supports the involvement of oxidative/nitrative stress in the pathophysiology of cerebral ischemia. Heme oxygenase-1 (HO-1) is a rate-limiting enzyme in heme catabolism, possessing potent anti-oxidant and anti-apoptosis effects. In transgenic mice, HO-1 overproduction is neuroprotective against cerebral ischemia injury, but by unclear mechanisms. The present study determined whether treatment with adenoviral vector overexpressing HO-1 (Ad-HO-1) attenuates post-ischemic brain damage via reduction of oxidative/nitrative stress. After focal cerebral ischemia, Ad-HO-1 reduced lipid peroxidation and protein nitration, decreased infarct volume, and attenuated neurologic deficits. Zinc protoporphyrin IX (ZnPP IX, a specific HO-1 inhibitor) blocked Ad-HO-1 mediated effects against ischemic brain damage. Although Ad-HO-1 slightly reduced ischemic brain NO concentrations, Ad-HO-1 treatment significantly inhibited cerebral expression of iNOS protein expression, without significant effect upon nNOS or eNOS expression compared to vehicle after focal cerebral ischemia. Ad-HO-1 preserved NO bioavailability by increasing eNOS phosphorylation during ischemia compared to vehicle. Together, our results suggest that Ad-HO-1 attenuates post-ischemic brain damage via simultaneous reduction of oxidative/nitrative stress and preservation of NO bioavailability.

Topics: Animals; Blotting, Western; Brain Ischemia; Dependovirus; Genetic Therapy; Genetic Vectors; Heme Oxygenase-1; Infarction, Middle Cerebral Artery; Male; Nervous System Diseases; Nitric Oxide; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Phosphorylation; Rats; Rats, Sprague-Dawley; Reactive Nitrogen Species; Reactive Oxygen Species; Superoxides; Tyrosine; Up-Regulation

The hallmark of stroke injury is endothelial dysfunction leading to blood-brain barrier (BBB) leakage and edema. Among the causative factors of BBB disruption are accelerating peroxynitrite formation and the resultant decreased bioavailability of nitric oxide (NO). S-nitrosoglutathione (GSNO), an S-nitrosylating agent, was found not only to reduce the levels of peroxynitrite but also to protect the integrity of BBB in a rat model of cerebral ischemia and reperfusion (IR). A treatment with GSNO (3 μmol/kg) after IR reduced 3-nitrotyrosine levels in and around vessels and maintained NO levels in brain. This mechanism protected endothelial function by reducing BBB leakage, increasing the expression of Zonula occludens-1 (ZO-1), decreasing edema, and reducing the expression of matrix metalloproteinase-9 and E-selectin in the neurovascular unit. An administration of the peroxynitrite-forming agent 3-morpholino sydnonimine (3 μmol/kg) at reperfusion increased BBB leakage and decreased the expression of ZO-1, supporting the involvement of peroxynitrite in BBB disruption and edema. Mechanistically, the endothelium-protecting action of GSNO was invoked by reducing the activity of nuclear factor kappa B and increasing the expression of S-nitrosylated proteins. Taken together, the results support the ability of GSNO to improve endothelial function by reducing nitroxidative stress in stroke.

Topics: Animals; Blood-Brain Barrier; Brain; Brain Edema; Carrier Proteins; Disease Models, Animal; E-Selectin; Endothelial Cells; Evans Blue; Functional Laterality; Infarction, Middle Cerebral Artery; Male; Matrix Metalloproteinase 9; Neoplasm Proteins; Neurons; Neuroprotective Agents; NF-kappaB-Inducing Kinase; Nitric Oxide; Nitrites; Nucleocytoplasmic Transport Proteins; Peroxynitrous Acid; Phosphopyruvate Hydratase; Protein Serine-Threonine Kinases; Protein Transport; Rats; Rats, Sprague-Dawley; S-Nitrosoglutathione; Statistics, Nonparametric; Time Factors; Tyrosine; Zonula Occludens-1 Protein

NADPH oxidase is a major complex that produces reactive oxygen species (ROSs) during the ischemic period and aggravates brain damage and cell death after ischemic injury. Although many approaches have been tested for preventing production of ROSs by NADPH oxidase in ischemic brain injury, the regulatory mechanisms of NADPH oxidase activity after cerebral ischemia are still unclear. The aim of this study is identifying apocynin as a critical modulator of NADPH oxidase and elucidating its role as a neuroprotectant in an experimental model of brain ischemia in rat. Treatment of apocynin 5min before of reperfusion attenuated cerebral ischemia in rats. Administration of apocynin showed marked reduction in infarct size compared with that of control rats. Medial carotid artery occlusion (MCAo)-induced cerebral ischemia was also associated with an increase in, nitrotyrosine formation, as well as IL-1β expression, IκB degradation and ICAM expression in ischemic regions. These expressions were markedly inhibited by the treatment of apocynin. We also demonstrated that apocynin reduces levels of apoptosis (TUNEL, Bax and Bcl-2 expression) resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. This new understanding of apocynin induced adaptation to ischemic stress and inflammation could suggest novel avenues for clinical intervention during ischemic and inflammatory diseases.

Topics: Acetophenones; Animals; Apoptosis; bcl-2-Associated X Protein; Brain; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; I-kappa B Proteins; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Intercellular Adhesion Molecule-1; Interleukin-1beta; Male; NADPH Oxidases; Neurologic Examination; Peptide Fragments; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

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

Hyperglycemia is one of the major factors for hemorrhagic transformation after ischemic stroke. In this study, we tested the effect of hydrogen gas on hemorrhagic transformation in a rat focal cerebral ischemia model. Sprague-Dawley rats (n=72) were divided into the following groups: sham; sham treated with hydrogen gas (H(2)); Middle Cerebral Artery Occlusion (MCAO); and MCAO treated with H(2) (MCAO+H(2)). All rats received an injection of 50% dextrose (6 ml/kg i.p.) and underwent MCAO 15 min later. Following a 90 min ischemic period, hydrogen was inhaled for 2 h during reperfusion. We measured the level of blood glucose at 0 h, 0.5 h, 4 h, and 6 h after dextrose injection. Infarct and hemorrhagic volumes, neurologic score, oxidative stress (evaluated by measuring the level of 8 Hydroxyguanosine (8OHG), 4-Hydroxy-2-Nonenal (HNE) and nitrotyrosine), and matrix metalloproteinase (MMP)-2/MMP-9 activity were measured at 24 h after ischemia. We found that hydrogen inhalation for 2 h reduced infarct and hemorrhagic volumes and improved neurological functions. This effect of hydrogen was accompanied by a reduction of the expression of 8OHG, HNE, and nitrotyrosine and the activity of MMP-9. Furthermore, a reduction of the blood glucose level from 500+/-32.51 to 366+/-68.22 mg/dl at 4 h after dextrose injection was observed in hydrogen treated animals. However, the treatment had no significant effect on the expression of ZO-1, occludin, collagen IV or aquaporin4 (AQP4). In conclusion, hydrogen gas reduced brain infarction, hemorrhagic transformation, and improved neurological function in rats. The potential mechanisms of decreased oxidative stress and glucose levels after hydrogen treatment warrant further investigation.

Topics: Administration, Inhalation; Aldehydes; Animals; Antioxidants; Aquaporin 4; Brain Damage, Chronic; Cerebral Hemorrhage; Disease Progression; Drug Evaluation, Preclinical; Extracellular Matrix Proteins; Glucose; Hydrogen; Hyperglycemia; Infarction, Middle Cerebral Artery; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neuroprotective Agents; Random Allocation; Rats; Rats, Sprague-Dawley; Tyrosine

Endothelin-1 (ET-1) is up-regulated in the endothelial cells and astrocytes under ischemia. Transgenic mice with astrocytic ET-1 over-expression (GET-1) showed more severe neurological deficit and larger infarct after transient middle cerebral artery occlusion (MCAO). Here, the significance of endothelial ET-1 in ischemic brain injury was investigated using transgenic mice with the endothelial ET-1 over-expression (TET-1). Increased ET-1 level was observed in the TET-1 brain infarct core after transient MCAO. ET(A) receptor expression was induced in the penumbra and ET(A) antagonist (A-147627) partially normalized the infarct volume and neurological deficit. In the infarct core of TET-1 brain, superoxide, nitrotyrosine, and gp91(phox) levels were increased. TET-1 brain displayed increased matrix metalloproteinase-2 expression, water content, immunoglobulin leakage and decreased occludin level in the ipsilateral hemisphere indicative of BBB breakdown and hemispheric edema. Interestingly, AQP-4 expression was increased in the penumbra of TET-1 brain following transient MCAO leading to the water accumulation. Taken together, endothelial ET-1 over-expression and ETA receptor activation contributes to the increased oxidative stress, water accumulation and BBB breakdown after transient MCAO leading to more severe neurological deficit and increased infarct.

Topics: Animals; Aquaporin 4; Atrasentan; Blood-Brain Barrier; Brain; Capillary Permeability; Endothelin A Receptor Antagonists; Endothelin-1; Endothelium, Vascular; Immunoglobulins; Infarction, Middle Cerebral Artery; Matrix Metalloproteinase 2; Membrane Glycoproteins; Membrane Proteins; Mice; Mice, Transgenic; NADPH Oxidase 2; NADPH Oxidases; Occludin; Oxidative Stress; Promoter Regions, Genetic; Pyrrolidines; Receptor, Endothelin A; Receptor, TIE-1; Superoxides; Tyrosine; Water

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

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

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

This study investigated the effect of peroxynitrite (ONOO-) on pressure-induced myogenic activity and vascular smooth muscle (VSM) actin of isolated posterior cerebral arteries (PCAs).. Histochemical staining of nitrotyrosine (NT) was used to demonstrate the presence of ONOO- in the cerebrovasculature after 1 hour of middle cerebral artery occlusion with 30 minutes of reperfusion. To determine the effect of ONOO- on pressure-induced myogenic activity, third-order PCAs from nonischemic animals were isolated and mounted in an arteriograph chamber. Diameter in response to changes in pressure was determined in the absence and presence of ONOO- (10(-8) to 10(-4) mol/L). Filamentous actin (F-actin) and globular actin (G-actin) were quantified using confocal microscopy in PCAs with and without exposure to ONOO-.. NT staining of vascular cells was greater in ischemic brain versus sham animals (56+/-3% versus 35+/-3%; P<0.01). Addition of low concentrations of ONOO- (< or =10(-6) mol/L) to isolated PCAs caused constriction from 129+/-16 microm to 115+/-15 microm (P<0.01), whereas concentrations >10(-6) mol/L caused dilation of spontaneous tone and loss of myogenic activity in the physiological range of 50 to 125 mm Hg, increasing diameter from 130+/-6 to 201+/-5 microm at 75 mm Hg (P<0.01). In addition, the diminished myogenic activity was associated with a 4.5-fold decrease in F-actin content of VSM and a 27% increase in G-actin content (P<0.01).. This study demonstrates that ONOO- affects the myogenic activity of cerebral arteries and causes F-actin depolymerization in VSM, a consequence that could promote vascular damage during reperfusion injury and further brain injury.

Topics: Actins; Animals; Blood Pressure; Brain; Cerebral Arteries; Humans; Infarction, Middle Cerebral Artery; Ischemia; Male; Microscopy, Confocal; Muscle, Smooth, Vascular; Peroxynitrous Acid; Posterior Cerebral Artery; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Time Factors; Tyrosine

Tetramethylpyrazine (TMPZ) is an active ingredient isolated from a commonly used Chinese herb, Ligusticum wallichii Franchat, which has long been used in China for the treatment of vascular diseases. In the present study, TMPZ significantly attenuated middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Administration of TMPZ at 10 and 20 mg/kg produced concentration-dependent reductions in infarct size compared to that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in both nitrotyrosine and inducible nitric oxide synthase (iNOS) expression in ischemic regions. The expressions of nitrotyrosine and iNOS were markedly inhibited by TMPZ (20 mg/kg) treatment. Furthermore, TMPZ (100-250 microM) concentration-dependently inhibited respiratory bursts in human neutrophils stimulated by fMLP (800 nM) and PMA (320 nM). TMPZ (100-250 microM) also significantly inhibited neutrophil migration stimulated by fMLP (800 nM) and LTB4 (160 nM). An electron spin resonance (ESR) method was used to further study the scavenging activity of TMPZ on free radicals formed in human neutrophils. TMPZ (100 and 200 microM) greatly reduced the ESR signal intensity of hydroxyl radical formation. In conclusion, we demonstrate a neuroprotective effect of TMPZ in MCAO-induced focal cerebral ischemia in vivo. TMPZ mediates at least part of the free radical-scavenging activity and inhibits neutrophil activation, resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, TMPZ treatment may represent an ideal approach to lowering the risk of or improving function in ischemia-reperfusion brain injury-related disorders.

Topics: Animals; Brain Ischemia; Dose-Response Relationship, Drug; Free Radical Scavengers; Infarction, Middle Cerebral Artery; Ligusticum; Male; Neuroprotective Agents; Neutrophils; Nitric Oxide Synthase Type II; Phytotherapy; Plant Extracts; Pyrazines; Rats; Rats, Wistar; Respiratory Burst; Telencephalon; Tyrosine

In cerebral ischemic insults, activated inflammatory cells such as microglia and macrophages may be implicated in the pattern and degree of ischemic injury by producing various bioactive mediators. In the present study, we provide the evidence that activated microglia/macrophages accelerate cerebral ischemic injury by overexpression of inducible nitric oxide synthase (iNOS). To activate microglia/macrophages, a potent inflammation inducer lipopolysaccharide (LPS, 5 microg/5 microl) was microinjected into rat corpus callosum. Isolectin B4-positive microglia/macrophages were abundantly observed in ipsilateral hemisphere at 1 day after LPS injection. RT-PCR showed that LPS injection induced iNOS mRNA expression mostly in microglia/macrophages, peaking in intensity at 15 h after LPS injection. While ischemic injury was little evoked in control rats by 2-h middle cerebral artery occlusion (MCAO) followed by 3-h reperfusion, it was markedly increased in rats pre-injected with LPS 1 day before MCAO. However, no significant difference between control and LPS-pretreated groups was observed after 24-h reperfusion. The increased ischemic injury in LPS-treated rats was well correlated with iNOS level expressed over 3 orders of magnitude than in LPS-untreated rats. Immunohistochemical studies showed that iNOS- and nitrotyrosine (a peroxynitrite marker)-positive cells were prominent throughout the infarct area. NOS inhibitors aminoguanidine or N(G)-nitro-L-arginine, simultaneously injected with LPS, reduced the iNOS immunoreactivity and infarct volume, especially in penumbra regions. Total glutathione levels in ischemic regions were decreased more in LPS pre-injected rats than in control ones. Further defining the role of NO in cerebral ischemic insults would provide the rationale for new therapeutic strategies based on modulation of microglial and macrophageal NO production in the brain.

Topics: Animals; Brain Ischemia; Corpus Callosum; Enzyme Inhibitors; Fluoresceins; Fluorescent Dyes; Glutathione; Immunohistochemistry; Infarction, Middle Cerebral Artery; Lipopolysaccharides; Macrophage Activation; Macrophages; Male; Microglia; Microinjections; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Organic Chemicals; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tyrosine

Perfluorochemicals (PFCs) may exert a neuroprotective function in the early phase of ischemia by improving the oxygen supply to the endangered tissue. We have, therefore, investigated the effect of Oxycyte, a second-generation perfluorocarbon solution, on the extent of early ischemic brain damage in a model of permanent focal cerebral ischemia.. Eight hours of permanent focal cerebral ischemia was induced in isoflurane anesthetized male Sprague-Dawley rats by unilateral middle cerebral artery (MCA) thread occlusion under the control of laser Doppler flowmetry (LDF). Animals were assigned to one of the following treatment groups: nO2-NaCl and hO2-NaCl-NaCl (0.9%, 1 ml/100 g i.v.) and nO2-Oxycyte and hO2-Oxycyte-Oxycyte (1 ml/100 g i.v.). The injection of NaCl or Oxycyte was performed immediately after MCA occlusion. After injection, breathing was changed to pure oxygen in groups hO2-NaCl and hO2-Oxycyte while animals in groups nO2-NaCl and nO2-Oxycyte were allowed to breathe air. The necrotic volume was calculated from serial coronal sections stained with silver-nitrate. In addition, nitrotyrosine production was studied by immunohistochemistry.. Upon MCA occlusion, animals showed a reduction of cerebral blood flow of approximately 80% of the LDF signal in all groups. Hemodynamic and metabolic parameters were not affected by the infusion of Oxycyte. The total infarct volume was reduced in hO2-Oxycyte animals [group nO2-NaCl: 341+/-31 mm3 (mean+/-SD), group hO2-NaCl: 351+/-33 mm3, group nO2-Oxycyte: 354+/-24 mm3, and group hO2-Oxycyte: 300+/-29 mm3, p < 0.05 versus all other groups]. Moreover, hO2-Oxycyte animals showed lesser intensity of nitrotyrosine staining when compared with hO2-NaCl animals.. These results suggest that Oxycyte administered immediately after the onset of vascular occlusion may exert neuroprotective effects in the early phase of brain ischemia.

Topics: Analysis of Variance; Animals; Blood Gas Analysis; Brain Ischemia; Cerebrovascular Circulation; Disease Models, Animal; Drug Therapy, Combination; Fluorocarbons; Immunohistochemistry; Infarction, Middle Cerebral Artery; Laser-Doppler Flowmetry; Male; Necrosis; Neuroprotective Agents; Oxygen; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Silver Staining; Time Factors; Tyrosine

Green tea catechins possess potent antioxidative properties, and the preventive effects against various oxidative diseases have been reported. The purpose of this study is to examine preventive and protective effects of green tea catechins on various deteriorative processes following stroke.. Male Wistar rats were given ad libitum water with or without 0.25 and 0.5% tea catechin extract for 5 days prior to the operation and during the experiment. Right middle cerebral artery was occluded for 2 h, then reperfused for 22 h. Brain slices were stained with triphenyltetrazolim chloride to assess infarct area. Concentrations of plasma EGCg, and serum NOx were analyzed by HPLC. Detection of iNOS expression, neutrophil infiltration and peroxynitrite formation in the penumbra was performed by immunostain. Neurologic deficit was scored by posture reflex.. Tea catechins dose-dependently reduced the brain infarct area and volume. Infarct volume was inversely correlated with plasma EGCg concentration. Dark staining for iNOS, neutrophils and peroxynitrite were observed in vessel wall of small arteries in control ischemic hemisphere, while in catechins (0.5%)-treated rats iNOS was detected slightly, and staining for neutrophils and peroxynitrite was not seen. Catechin ingestion blocked a 3-fold increase in serum NOx concentration in the jugular vein, and also reduced by 35% a 2-fold increase of plasma lipid peroxide level seen in control rats after reperfusion. Neurologic deficits were significantly alleviated by 0.5% catechin ingestion.. Daily intake of green tea catechins efficiently protects the penumbra from irreversible damage due to cerebral ischemia, and consequent neurologic deficits.

Topics: Animals; Brain Ischemia; Catechin; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Granulocyte Colony-Stimulating Factor; Hematopoietic Cell Growth Factors; Infarction, Middle Cerebral Artery; Interleukin-3; Male; Malondialdehyde; Nitrates; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Plant Extracts; Rats; Rats, Wistar; Recombinant Fusion Proteins; Recombinant Proteins; Tea; 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

Free radicals appear to participate in the final common pathway of neuronal death in ischemia and may therefore be an adequate target for therapy. Tempol is a nitroxide antioxidant with proven protective efficacy in several animal models, including myocardial ischemia, that has not been previously tested in models of permanent cerebral ischemia. Spontaneously hypertensive rats underwent permanent middle cerebral artery occlusion (PMCAO). Following dose-response and time-window-finding experiments rats were given vehicle or tempol (50 mg/kg) subcutaneously 1 h after PMCAO (n = 10/group). Five animals in each group were evaluated with a motor scale 24 h after the infarct and were then sacrificed and the injury volume was measured. The remaining animals were examined daily with the motor scale and also with a Morris water maze test on days 26-30 after PMCAO and sacrificed on day 30. Motor scores at all time points examined were significantly better in the tempol-treated animals (P < 0.05 for all). Significantly better performance in the water maze test for performance on days 26-30 was noted in the tempol group compared with the vehicle-treated group (P < 0.05). Injury volumes at days 1 and 30 were significantly reduced in the tempol group (9.83 +/- 1.05 vs 19.94 +/- 1.43% hemispheric volume, P = 0.0009, and 13.2 +/- 2.97 vs 24.4 +/- 2.38% hemispheric volume, P = 0.02, respectively). In conclusion, treatment with tempol led to significant motor and behavioral improvement and reduced injured tissue volumes both in the short and in the long term after stroke.

Topics: Animals; Antioxidants; Behavior, Animal; Body Weight; Brain; Brain Chemistry; Brain Ischemia; Cyclic N-Oxides; Disease Models, Animal; Disease Progression; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Infarction, Middle Cerebral Artery; Male; Maze Learning; Motor Activity; Neuroprotective Agents; Rats; Rats, Inbred SHR; Spin Labels; Treatment Outcome; Tyrosine

Mild hypothermia is neuroprotective, but the reasons are not well known. Inflammation contributes to ischemic damage; therefore, we examined whether the protection by hypothermia may be attributable to alterations in the inflammation. We examined whether hypothermia might alter the inflammatory cell-associated inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) and peroxynitrite generation in experimental stroke and inflammation. Rats underwent 2 hr of middle cerebral artery occlusion (MCAO). Brain inflammation was modeled by intravenous lipopolysaccharide (LPS) (2 mg/kg) injection. Temperature was maintained at 33 degrees C for 2 hr immediately after MCAO and LPS injection, delayed 2 hr after MCAO or maintained at 38 degrees C. Cultured microglia were activated with LPS and then incubated at 33 or 37 degrees C. Both intraischemic and delayed mild hypothermia attenuated infarct size by 40% (p < 0.05). Immunohistochemistry was performed to identify cell type, iNOS, and peroxynitrite. The majority of iNOS- and peroxynitrite-positive cells were activated microglia-macrophages, and mild hypothermia significantly decreased the numbers of immunoreactive cells at 72 hr by >50% (p < 0.05). After ischemia, mild hypothermia decreased NO production by 40%. Similarly, hypothermia attenuated NO and iNOS in LPS-injected rats, as well as in cultured microglia. Aminoguanidine, an iNOS inhibitor, also attenuated infarct size and NO in ischemic and inflammation models. We conclude that mild hypothermia significantly inhibits the inflammatory response by affecting microglial iNOS-NO generation. Therapies directed against microglia or their activation may be useful in treating stroke.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Encephalitis; Hypothermia, Induced; Infarction, Middle Cerebral Artery; Lipopolysaccharides; Male; Microglia; Monocytes; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Reactive Nitrogen Species; Stroke; Tyrosine

We have characterized the temporal changes in iNOS, MnSOD and nitrotyrosine immune reactivity in a rat model of permanent middle cerebral artery occlusion under acute hyperglycemic or normoglycemic conditions followed by either 3- or 24-h recovery. We found that the macroscopic labeling pattern for all three antibodies colocalized with the ischemic core and penumbra which was determined by cresyl violet histological evaluation in adjacent sections. Hyperglycemia induced prior to ischemia resulted in earlier infarction which correlated with increased immunoreactivity for iNOS, MnSOD and nitrotyrosine. In the penumbral region of the frontal cortex, labeling of specific cell structures was largely limited to cortical neurons near the corpus callosum and was apparent earlier in the hyperglycemic rats. Increased polymorphonuclear leukocyte adhesion in blood vessels was observed at 24 h in the hyperglycemic group. At both of the recovery times studied, we observed only minor vascular staining for nitrotyrosine and none for iNOS. Our results are consistent with hyperglycemia resulting in an early and concomitant increase in both superoxide and nitric oxide production which can lead to peroxynitrite formation that then nitrates tyrosine residues. It would appear that hyperglycemic ischemia contributes to the early induction of key enzymes involved in nitric oxide bioavailability.

Topics: Animals; Blood Vessels; Brain Ischemia; Cerebral Cortex; Cerebrovascular Circulation; Disease Models, Animal; Hyperglycemia; Immunohistochemistry; Infarction, Middle Cerebral Artery; Male; Nerve Degeneration; Neuroglia; Neurons; Nitric Oxide; Nitric Oxide Synthase; Oxygen; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Tyrosine

Nitrotyrosine produced by NO-mediated reaction is a possible marker for cytotoxicity in brain ischemia. In this study, we aimed to determine whether iNOS is responsible for the nitrotyrosine formation and which type of cell is predominantly nitrated. Fifty-eight wild-type and 28 iNOS knockout male mice were used. Under halothane anesthesia the left middle cerebral artery was occluded for 2 h and reperfused for 0.5 or 15 h. The ratio of nitrotyrosine to total tyrosine (%NO2-Tyr) was measured by means of a hydrolysis/HPLC. After 0.5-h reperfusion, %NO2-Tyr in the ischemic cortex of wild-type and knockout mice amounted to 0.037 +/- 0.040% (n = 8) and 0.064 +/- 0.035% (n = 6), respectively, being significantly higher than that in the sham operation group (n = 7) (P < 0.05). After 15-h reperfusion, nitrotyrosine was detected only in wild-type mice (0.039 +/- 0.025%, n = 7), not in knockout or sham-operated mice (P < 0.05). Immunohistochemical reaction for nitrotyrosine was seen predominantly in the vascular wall in the peri-infarct region of the cerebral cortex in wild-type mice after 15-h reperfusion, but not in corresponding knockout mice. Our data suggest that iNOS is responsible for nitrotyrosine formation in the later phase of reperfusion, and that vascular endothelium is the major site of this reaction, at least in the case of 15-h reperfusion.

Topics: Acidosis; Animals; Blood Glucose; Blood Pressure; Carbon Dioxide; Cerebral Cortex; Cerebrovascular Circulation; Chromatography, High Pressure Liquid; Endothelium, Vascular; Infarction, Middle Cerebral Artery; Ischemic Attack, Transient; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxygen; Reperfusion Injury; Tyrosine

Reperfusion injury is one of the factors that unfavorably affects stroke outcome and shortens the window of opportunity for thrombolysis. Surges of nitric oxide (NO) and superoxide generation on reperfusion have been demonstrated. Concomitant generation of these radicals can lead to formation of the strong oxidant peroxynitrite during reperfusion.. We have examined the role of NO generation and peroxynitrite formation on reperfusion injury in a mouse model of middle cerebral artery occlusion (2 hours) and reperfusion (22 hours). The infarct volume was assessed by 2,3,5-triphenyl tetrazolium chloride staining; blood-brain barrier permeability was evaluated by Evans blue extravasation. Nitrotyrosine formation and matrix metalloproteinase-9 expression were detected by immunohistochemistry.. Infarct volume was significantly decreased (47%) in animals treated with the nonselective nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (L-NA) at reperfusion. The specific inhibitor of neuronal NOS, 7-nitroindazole (7-NI), given at reperfusion, showed no protection, although preischemic treatment with 7-NI decreased infarct volume by 40%. Interestingly, prereperfusion administration of both NOS inhibitors decreased tyrosine nitration (a marker of peroxynitrite toxicity) in the ischemic area. L-NA treatment also significantly reduced vascular damage, as indicated by decreased Evans blue extravasation and matrix metalloproteinase-9 expression.. These data support the hypothesis that in addition to the detrimental action of NO formed by neuronal NOS during ischemia, NO generation at reperfusion plays a significant role in reperfusion injury, possibly through peroxynitrite formation. Contrary to L-NA, failure of 7-NI to protect against reperfusion injury suggests that the source of NO is the cerebrovascular compartment.

Topics: Animals; Biomarkers; Blood-Brain Barrier; Coloring Agents; Endothelium, Vascular; Enzyme Inhibitors; Evans Blue; Indazoles; Infarction, Middle Cerebral Artery; Matrix Metalloproteinase 9; Mice; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxidants; Permeability; Reperfusion Injury; Tyrosine