8-hydroxy-2--deoxyguanosine and Brain-Ischemia

We evaluated changes in the levels of urinary 8-hydroxy-2-deoxyguanosine (8-OHdG) in patients undergoing mild hypothermia therapy and compared 8-OHdG expressions in those receiving whole-body cooling or selective head cooling.. The subjects were 15 patients undergoing mild hypothermia therapy following resuscitation after cardiac arrest in our intensive care unit. We divided the patients into 2 groups receiving either whole-body cooling or selective head cooling, according to their circulatory stability. We examined urinary 8-OHdG level for 1 week and neurological outcomes 28 days after admission.. We observed significant decreases in urinary 8-OHdG levels on days 6 and 7 compared with that on day 1 in the whole-body cooling group. Furthermore, we noted significantly lower urinary 8-OHdG levels after days 5, 6 and 7 in the whole-body cooling group than in the selective head-cooling group. Neurological outcomes were similar in both groups.. Mild hypothermia therapy with whole-body cooling had a greater effect on the suppression of free radical production than selective head cooling. However, selective head cooling might be an appropriate indication for patients with circulatory instability after resuscitation, because it provides neuroprotection similar to that of whole-body cooling.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adult; Brain Ischemia; Deoxyguanosine; Female; Head; Humans; Hypothermia, Induced; Male; Middle Aged; Rewarming; Time Factors; Treatment Outcome

Poststroke depression is independently associated with poor health outcomes, such as increased mortality, disability, anxiety, and lower quality of life. Identifying the potential biomarkers and detailed mechanisms of poststroke depression may improve the effectiveness of therapeutic intervention. In this cross-sectional study, the authors recruited patients with subacute ischemic stroke who were consecutively admitted for neurorehabilitation. Depression was assessed with the Patient Health Questionnaire-9 (PHQ-9), with a cutoff based on a summed-items score of 10. Polysomnography and laboratory tests for oxidative stress and inflammation were arranged. In total, 139 patients (97 men [69.8%] and 42 women [30.2%]; mean age: 63.2 years [±13.4]) with recent ischemic stroke were recruited and divided into two groups based on their depressive symptoms. Body mass index (BMI), the Barthel Index, percentage of antidepressant usage, and percentage of rapid eye movement (REM) sleep differed significantly between the two groups. The PHQ-9 score was significantly correlated with the levels of total antioxidant capacity, C-reactive protein, and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG). Urinary 8-OHdG, a marker of oxidative stress to DNA, remained significantly and positively correlated with PHQ-9 scores after adjusting for BMI, sleep-onset latency, Barthel Index, mean oxyhemoglobin saturation, age, antidepressant usage, and percentage of REM sleep by using multivariate linear regression. Depressive symptoms were related to increased oxidative DNA damage in patients with subacute ischemic stroke. Urinary 8-OHdG may serve as a potential biomarker for poststroke depression. Further longitudinal studies are needed to elucidate the causal relationship between poststroke depression and elevated oxidative stress level.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Biomarkers; Brain Ischemia; C-Reactive Protein; Cross-Sectional Studies; Deoxyguanosine; Depression; DNA Damage; Female; Humans; Interleukin-6; Male; Middle Aged; Oxidative Stress; Sleep Apnea, Obstructive; Stroke; Surveys and Questionnaires

The aim of this study was to investigate whether uric acid (UA) might exert neuroprotection via activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and regulating neurotrophic factors in the cerebral cortices after transient focal cerebral ischemia/reperfusion (FCI/R) in rats. UA was intravenously injected through the tail vein (16 mg/kg) 30 min after the onset of reperfusion in rats subjected to middle cerebral artery occlusion for 2 h. Neurological deficit score was performed to analyze neurological function at 24 h after reperfusion. Terminal deoxynucleotidyl transferase-mediated dNTP nick end labeling (TUNEL) staining and hematoxylin and eosin (HE) staining were used to detect histological injury of the cerebral cortex. Malondialdehyde (MDA), the carbonyl groups, and 8-hydroxyl-2'-deoxyguanosine (8-OHdG) levels were employed to evaluate oxidative stress. Nrf2 and its downstream antioxidant protein, heme oxygenase- (HO-) 1,were detected by western blot. Nrf2 DNA-binding activity was observed using an ELISA-based measurement. Expressions of BDNF and NGF were analyzed by immunohistochemistry. Our results showed that UA treatment significantly suppressed FCI/R-induced oxidative stress, accompanied by attenuating neuronal damage, which subsequently decreased the infarct volume and neurological deficit. Further, the treatment of UA activated Nrf2 signaling pathway and upregulated BDNF and NGF expression levels. Interestingly, the aforementioned effects of UA were markedly inhibited by administration of brusatol, an inhibitor of Nrf2. Taken together, the antioxidant and neuroprotective effects afforded by UA treatment involved the modulation of Nrf2-mediated oxidative stress and regulation of BDNF and NGF expression levels. Thus, UA treatment could be of interest to prevent FCI/R injury.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Blotting, Western; Brain Ischemia; Cerebral Infarction; Deoxyguanosine; Enzyme-Linked Immunosorbent Assay; Heme Oxygenase-1; Immunochemistry; In Situ Nick-End Labeling; Male; Malondialdehyde; NF-E2-Related Factor 2; Oxidative Stress; Rats; Rats, Sprague-Dawley; Uric Acid

Homocysteine (Hcy) has been shown to have a neurotoxic effect on ischemic brain cells; however, the underlying mechanisms remain incompletely understood. Here, we examined whether Hcy treatment influences mitochondria injury, oxidative stress, and mitochondrial STAT3 (mitoStat3) expression in rat ischemic brain. Our results demonstrated that Hcy treatment aggravated the damage of mitochondrial ultrastructure in the brain cortex and the dentate gyrus region of the hippocampus after focal cerebral ischemia. An elevated Hcy level was also accompanied by the significant inhibition of mitochondrial complex I-III enzymatic activities in addition to an increase in cytochrome c release. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) content and mitoStat3 protein phosphorylation level were increased in Hcy-treated animals, whereas AG490, a Jak2 inhibitor, inhibited mitoStat3 phosphorylation as well as 8-OHdG levels induced by Hcy. In vitro studies revealed that Hcy also markedly increased reactive oxygen species (ROS) and mitoStat3 levels. In addition, the inhibition of pSTAT3 reduced Hcy-mediated increase in ROS levels, whereas quenching ROS using the ROS inhibitor glutathione ethyl ester inhibited Hcy-mediated pSTAT3 overactivation in Neuro2a cells. These findings suggest that the development of therapies that interfere with the ROS/pSTAT3 pathway may be helpful for treating cerebral infarction-related diseases associated with Hcy.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Brain Ischemia; Cell Line; Cerebral Cortex; Dentate Gyrus; Deoxyguanosine; Gene Expression Regulation; Homocysteine; Male; Mitochondria; Mitochondrial Proteins; Oxidative Stress; Phosphorylation; Rats; Reactive Oxygen Species; STAT3 Transcription Factor; Tyrphostins

Deep hypothermic circulatory arrest (DHCA) is used to overcome the threat of cerebral ischemia during complex surgical operations of the heart and the aortic arch. Remote ischemic preconditioning (RIPC) has been shown to mitigate neurological damage.. We analyzed blood samples in a consecutive series of 52 piglets that underwent a 60-min period of DHCA with RIPC (the RIPC group) or without (the control group), to reveal whether the protective effect to oxidative stress could be seen by measuring serum 8-hydroxydeoxyguanosine (8-OHdG). The piglets were cannulated and cooled to 18°C using a heart-lung machine, for the DHCA. The piglets were then rewarmed to normothermic temperature. Blood sampling was taken at baseline, after 30 minutes of cooling, 2 hours postoperatively, and 8 hours postoperatively, and analyzed. 8-hydroxydeoxyguanosine (8-OHdG) from blood samples was analyzed by using Enzyme Linked Immunosorbent Assay (ELISA).. The serum 8-OHdG concentration was lower in the RIPC group after the cooling phase, 1.84 (1.44-2.17) ng/mL, and at 8 hours after HCA 1.48 (1.39-1.69) ng/mL, when compared with the control group, where the values were 2.14 (1.81-2.56) and 1.84 (1.62-2.44) ng/mL, respectively (P = .025) and (P = .004).. Remote ischemic preconditioning lowers oxidative stress during cardiopulmonary bypass.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Biomarkers; Brain Ischemia; Cardiopulmonary Bypass; Circulatory Arrest, Deep Hypothermia Induced; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Guanine; Ischemic Preconditioning; Oxidative Stress; Swine; Telemetry

The role of obstructive sleep apnea (OSA) in the mortality and further cardiovascular risk in subjects with ischemic stroke remains a contentious issue. Oxidative stress and inflammatory reaction due to OSA have seldom been studied in stable ischemic stroke patients.. This cross-sectional, prospective study involved 92 consecutive ischemic stroke patients who were admitted to the Rehabilitation ward. All subjects received polysomnography and laboratory tests for oxidative stress and inflammatory biomarkers, including: C-reactive protein (CRP), interleukin 6 (IL-6), total antioxidant capacity (TAC), and urinary 8-hydroxy-2-deoxyguanosine. Differences in study variables between patients with or without severe OSA were compared, and multivariate linear regression analyses were used to assess the relationship between OSA severity and target biomarkers.. Participants in the severe OSA group were significantly older (p = 0.002), had a significantly higher risk of hypertension (p = 0.021) and a lower level of CRP (p = 0.006). Among the subjects with ischemic stroke and severe OSA, the levels of CRP, IL-6, and TAC were positively correlated with the desaturation index (DI) and the TAC levels were negatively correlated with mean arterial oxygen saturation (SaO2). Regression analysis results indicated that the TAC levels remained significantly and negatively correlated with mean SaO2 levels. Moreover, the CRP levels remained significantly correlated with the apnea-hypopnea index and DI after controlling for covariates.. The present study demonstrated that a preferentially adaptive antioxidative response to hypoxia emerges, and the role of OSA with respect to inflammatory reaction is attenuated, in ischemic stroke patients with OSA.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aged; Biomarkers; Brain Ischemia; C-Reactive Protein; Cross-Sectional Studies; Deoxyguanosine; Female; Humans; Inflammation; Interleukin-6; Male; Middle Aged; Oxidative Stress; Polysomnography; Prospective Studies; Recurrence; Risk Factors; Severity of Illness Index; Sleep Apnea, Obstructive; Stroke

The phosphodiesterase (PDE) 3 inhibitor cilostazol, used as an anti-platelet drug, reportedly can also ameliorate ischemic brain injury. Here, we investigated the effects of cilostazol in a permanent focal ischemia mice model. Male Balb/c mice were subjected to permanent middle cerebral artery occlusion. Mice were then treated with either cilostazol (10 or 20mg/kg) or vehicle administered at 30min and 24h post-ischemia, and infarct volumes were assessed at 48h post-ischemia. Mice treated with 20mg/kg of cilostazol or vehicle were sacrificed at 6h or 24h post-ischemia and immunohistochemistry was used for brain sections. Treatment with 20mg/kg of cilostazol significantly reduced infarct volumes to 70.1% of those with vehicle treatment. Immunohistochemistry results for 8-hydroxydeoxyguanosine (OHdG) expression showed that some neurons underwent oxidative stress around the ischemic boundary zone at 6h post-ischemia. Cilostazol treatment significantly reduced the percentage of 8-OHdG-positive neurons (65.8±33.5% with vehicle and 21.3±9.9% with cilostazol). Moreover, NADPH oxidase (NOX) 2-positive neurons were significantly reduced with cilostazol treatment. In contrast, immunohistochemistry results for phosphorylated cyclic-AMP response element binding protein (pCREB) showed that there were significantly more pCREB-positive neurons around the ischemic boundary zone of cilostazol-treated mice than in those of vehicle-treated mice at 24h post-ischemia. These results suggested that cilostazol might have multiple mechanisms of action to ameliorate ischemic tissue damage, by attenuating oxidative stress mediated by suppressing NOX2 expression by ischemic neurons and an anti-apoptotic effect mediated through the pCREB pathway.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Brain; Brain Ischemia; Cilostazol; CREB-Binding Protein; Deoxyguanosine; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelial Cells; Immunohistochemistry; Infarction, Middle Cerebral Artery; Male; Membrane Glycoproteins; Mice, Inbred BALB C; NADPH Oxidase 2; NADPH Oxidases; Neurons; Neuroprotective Agents; Oxidative Stress; Phosphorylation; Tetrazoles; Time Factors

We evaluated the neuroprotective effects of an anti-oxidative nutrient rich enteral diet (AO diet) that contained rich polyphenols (catechins and proanthocyanidins) and many other anti-oxidative ingredients. Wistar rats were treated with either vehicle, normal AO diet (containing 100kcal/100mL, catechin 38.75mg/100mL and proanthocyanidin 19mg/100mL, 1mL/day), or high AO diet (containing 10 times the polyphenols of the normal AO diet) for 14 days, and were subjected to 90min of transient middle cerebral artery occlusion. The AO diet improved motor function, reduced cerebral infarction volume, and decreased both peroxidative markers such as 4-hydroxynonenal, advanced glycation end products, 8-hydroxy-2-deoxyguanosine and inflammatory markers such as monocyte chemotactic protein-1, ionized calcium-binding adapter molecule-1, and tumor necrosis factor-α. Our study has shown that an AO diet has neuroprotective effects through both anti-oxidative and anti-inflammatory mechanisms, indicating that nutritional control with polyphenols could be useful for patients with acute ischemic stroke.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Administration, Oral; Aldehydes; Animals; Antioxidants; Biomarkers; Brain Chemistry; Brain Damage, Chronic; Brain Ischemia; Cerebral Infarction; Deoxyguanosine; Diet; Glycation End Products, Advanced; Infarction, Middle Cerebral Artery; Inflammation; Male; Oxidative Stress; Proanthocyanidins; Rats; Rats, Wistar

Global cerebral ischemia and reperfusion (I/R) often result in high mortality. Free radicals have been reported to play an important role in global cerebral I/R, and therefore, reduction of these might improve the outcome. Here, we investigated the effect of hydrogen gas (H2) (a strong free radical scavenger) on the survival rate of mice following global cerebral I/R. We further examined the histopathological outcome and also the brain water content (as a possible determinant of mortality). Male C57BL/6J mice were subjected to global cerebral I/R by means of 45-min bilateral common carotid artery occlusion (BCCAO). A total of 160 mice were divided into three groups: sham surgery (sham group), BCCAO without H2 (BCCAO group), and BCCAO treated with 1.3% H2 (BCCAO + H2 group). We observed that H2 treatment significantly (P = 0.0232) improved the 7-day survival rate of mice, from 8.3% (BCCAO group, n = 12) to 50% (BCCAO + H2 group, n = 10). Histopathological analysis revealed that H2 treatment significantly attenuated neuronal injury and autophagy in the hippocampal cornu ammonis 1 sector and also brain edema, after 24 h of reperfusion. The beneficial effects of H2 treatment on brain injury were associated with significantly lower levels of oxidative stress markers (8-hydroxy-2'-deoxyguanosine and malondialdehyde) in the brain tissue. Thus, we believe that H2 may be an effective treatment for global cerebral I/R.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Administration, Inhalation; Animals; Body Water; Brain; Brain Ischemia; Capillary Permeability; Cerebrovascular Circulation; Deoxyguanosine; DNA Damage; Free Radical Scavengers; Hippocampus; Hydrogen; Lipid Peroxidation; Male; Malondialdehyde; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Reperfusion Injury; Serum Albumin; Survival Rate

The angiotensin II type 2 (AT(2)) receptor is expressed in bone marrow cells and may affect cell differentiation. We previously reported a beneficial role of the AT(2) receptor in ischemic brain damage. Here, we investigated the effect of AT(2) receptor stimulation in hematopoietic cells on ischemic brain injury using chimeric mice. Chimeric mice were generated by bone marrow transplantation into wild-type mice after irradiation. Bone marrow cells were prepared from wild-type (Agtr2(+)) or AT(2) receptor-deficient mice (Agtr2(-)). Six weeks after bone marrow transplantation, these chimeric mice were subjected to ischemia/reperfusion injury. Both Agtr2(+) and Agtr2(-) chimeric mice did not show a significant change in systolic and diastolic blood pressures, whereas body weight decreased in Agtr2(-) chimera. Twenty-four hours after ischemia/reperfusion injury, ischemic brain damage in Agtr2(-) chimera was exaggerated compared with that in Agtr2(+) chimera. Moreover, cerebral blood flow in the peripheral region before and after ischemia/reperfusion injury was decreased in Agtr2(-) chimera. The inflammatory response in the ipsilateral hemisphere was not significantly different, whereas tumor necrosis factor-α and monocyte chemoattractant protein 1 expressions tended to increase in the Agtr2(-) chimeric brain. Expression of methylmethane sulfonate 2, which has a neuroprotective effect, was lower in the brain of Agtr2(-) chimera. These results indicate that deletion of AT(2) receptor in blood cells has a harmful effect on ischemic brain injury.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Blood Pressure; Body Weight; Bone Marrow Cells; Bone Marrow Transplantation; Brain; Brain Ischemia; Cerebrovascular Circulation; Chemokine CCL2; Deoxyguanosine; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Receptor, Angiotensin, Type 2; Reperfusion Injury; Reverse Transcriptase Polymerase Chain Reaction; Transplantation Chimera; Tumor Necrosis Factor-alpha; Ubiquitin-Conjugating Enzymes

Different garlic products reduce the cerebral ischemic damage due to their antioxidant properties. In this work, we investigated the effect of aged garlic extract (AGE) on cyclooxygenase-2 (COX-2) protein levels and activity, and its role as a possible mechanism of neuroprotection in a cerebral ischemia model. Animals were subjected to 1 h of ischemia plus 24 h of reperfusion. AGE (1.2 ml/kg weight, i.p.) was administered at onset of reperfusion. To evaluate the damage induced by cerebral ischemia, the neurological deficit, the infarct area, and the histological alterations were measured. As an oxidative stress marker to deoxyribonucleic acid, 8-hydroxy-2-deoxyguanosine (8-OHdG) levels were determined. Finally, as inflammatory markers, TNFα levels and COX-2 protein levels and activity were measured. AGE treatment diminished the neurological alterations (61.6%), the infarct area (54.8%) and the histological damage (37.7%) induced by cerebral ischemia. AGE administration attenuated the increase in 8-OHdG levels (77.8%), in TNFα levels (76.6%), and in COX-2 protein levels (73.6%) and activity (30.7%) induced after 1 h of ischemia plus 24 h of reperfusion. These data suggest that the neuroprotective effect of AGE is associated not only to its antioxidant properties, but also with its capacity to diminish the increase in TNFα levels and COX-2 protein expression and activity. AGE may have the potential to attenuate the cerebral ischemia-induced inflammation.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Anti-Inflammatory Agents; Antioxidants; Brain Ischemia; Cerebral Infarction; Cyclooxygenase 2; Deoxyguanosine; Garlic; Male; Models, Animal; Oxidative Stress; Plant Extracts; Random Allocation; Rats; Rats, Wistar; Reperfusion; Time Factors; Tumor Necrosis Factor-alpha

We investigated the immunohistochemical changes of 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunoreactivity as a marker of DNA damage and single-strand DNA (ssDNA) immunoreactivity as a marker of apoptosis in the striatum from 1 up to 15 days after 90 min of focal cerebral ischemia caused by middle cerebral artery occlusion in rats. In the present study, marked loss of MAP2 immunostaining was observed in the ipsilateral striatum 3 days after focal cerebral ischemia. A significant increase in the number of ssDNA-immunoreactive apoptotic neurons was observed in the ipsilateral striatum 1 and 3 days after focal cerebral ischemia. In contrast, a significant increase in densities of 8-OHdG-immunopositive cells was observed in the ipsilateral striatum from 3 up to 15 days after focal cerebral ischemia. Our double-labeled immunochemical study showed that 8-OHdG immunoreactivity was observed in both isolectin B(4)-positive microglia and glial fibrillary acidic protein-immunopositive astrocytes in the ipsilateral striatum 7 days after focal cerebral ischemia. These results suggest that focal cerebral ischemia can cause a marked increase in the number of microglia and astrocytes with oxidative DNA damage in the ipsilateral striatum. Furthermore, our results show that most microglia and astrocytes in the ipsilateral striatum after focal cerebral ischemia may not die by apoptosis. Thus, our findings provide novel evidence that focal cerebral ischemia can cause oxidative DNA damage in most microglia and astrocytes.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Brain Ischemia; Corpus Striatum; Deoxyguanosine; DNA, Single-Stranded; Male; Neuroglia; Rats; Rats, Sprague-Dawley

Our previous studies have shown that ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) inhibits intercellular adhesion molecule-1 (ICAM-1) expression in the ischemic striatum after 2 h of reperfusion in a transient middle cerebral artery occlusion model in rats. The purpose of this study is to further investigate the neuroprotective effects of FA during reperfusion after cerebral ischemia. Rats were subjected to 90 min of ischemia; they were then sacrificed after 2, 10, 24 and 36 h of reperfusion. ICAM-1 and macrophage-1 antigen (Mac-1) mRNA were detected using semi-quantitative RT-PCR at 2 h of reperfusion. Mac-1, 4-hydroxy-2-nonenal (4-HNE), 8-hydroxy-2'-deoxyguanosine (8-OHdG), active caspase 3, neuronal nuclei (NeuN) and TUNEL positive cells were measured at 2, 10, 24 and 36 h of reperfusion. FA (100 mg/kg, i.v.) administered immediately after MCAo inhibited ICAM-1 and Mac-1 mRNA expression in the striatum at 2 h of reperfusion, and reduced the number of Mac-1, 4-HNE and 8-OHdG positive cells in the ischemic rim and core at 10, 24 and 36 h of reperfusion. FA decreased TUNEL positive cells in the penumbra at 10 h, and in the ischemic boundary and core at 24 and 36 h of reperfusion. FA curtailed active caspase 3 expression in the penumbra at 10 h and restored NeuN-labeled neurons in the penumbra and ischemic core at 36 h of reperfusion. FA decreased the level of ICAM-1 mRNA and the number of microglia/macrophages, and subsequently down-regulated inflammation-induced oxidative stress and oxidative stress-related apoptosis, suggesting that FA provides neuroprotection against oxidative stress-related apoptosis by inhibiting ICAM-1 mRNA expression after cerebral ischemia/reperfusion injury in rats.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Apoptosis; Brain Ischemia; Caspase 3; Coumaric Acids; Deoxyguanosine; Disease Models, Animal; DNA-Binding Proteins; Encephalitis; Free Radical Scavengers; Gene Expression; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Intercellular Adhesion Molecule-1; Macrophage-1 Antigen; Male; Nerve Tissue Proteins; Neuroprotective Agents; Nuclear Proteins; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reperfusion Injury; RNA, Messenger

Oxidative stress contributes to ischemia/reperfusion neuronal damage in a consecutive 2-phase pattern: an immediate direct cytotoxic effect and subsequent redox-mediated inflammatory insult. The present study was designed to assess the neuroprotective mechanisms of edaravone, a novel free radical scavenger, through antioxidative and anti-inflammatory pathways, from the early period to up to 7 days after ischemia/reperfusion in mice.. Mice were subjected to 60-minute ischemia followed by reperfusion. They were divided into the edaravone group (n=72; with different schedules for first administration) and the vehicle (control) group (n=36). Infarct volume and neurological deficit scores were evaluated at several time points after ischemia. Immunohistochemical analysis for 4-hydroxy-2-nonenal (HNE), 8-hydroxy-deoxyguanosine (8-OHdG), ionized calcium-binding adapter molecule 1 (Iba-1), inducible NO synthase (iNOS), and nitrotyrosine were performed at 24 hours, 72 hours, or 7 days after reperfusion.. Edaravone, even when administrated 6 hours after onset of ischemia/reperfusion, significantly reduced the infarct volume (68.10+/-6.24%; P<0.05) and improved the neurological deficit scores (P<0.05) at 24 hours after reperfusion. Edaravone markedly suppressed the accumulation of HNE-modified protein and 8-OHdG at the penumbra area during the early period after reperfusion (P<0.05) and reduced microglial activation, iNOS expression, and nitrotyrosine formation at the late period.. Our results indicated that edaravone exerts an early neuroprotective effect through the early free radicals scavenging pathway and a late anti-inflammatory effect and suggested that edaravone is important for expansion of the therapeutic time window in stroke patients.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Anti-Inflammatory Agents; Antioxidants; Antipyrine; Brain; Brain Ischemia; Deoxyguanosine; DNA Damage; Edaravone; Electrophoresis, Polyacrylamide Gel; Free Radical Scavengers; Immunoblotting; Immunohistochemistry; Inflammation; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; Oxidative Stress; Reperfusion Injury; Time Factors; Tyrosine

Grape seed extract (GSE) possess cardioprotective abilities by functioning as in vivo antioxidants and by virtue of their ability to directly scavenge ROS including hydroxyl and peroxyl radicals. In the present study, we investigated the neuroprotective effects of grape seed extract (GSE) in the gerbil hippocampus after 5 min transient forebrain ischemia. Neuronal cell density in GSE-treated ischemic animals was significantly increased as compared with vehicle-treated ischemic animals 4 days after ischemic insult. In the GSE-treated groups, about 60% of pyramidal cells of the sham-operated group were stained with cresyl violet 4 days after ischemic insult. In this study, we found that GSE had neuroprotective effects on neuronal injury by inhibiting DNA damage in the CA1 region after ischemia. In vehicle-treated groups, 8-hydroxy-2'-deoxyguanosine (8-OHdG) immunoreactivity was significantly changed time-dependently, whereas the immunoreactivity in the GSE-treated group was similar to the sham-operated group. In addition, we confirmed that astrocytes and microglia did not show significant activation in the CA1 region 4 days after ischemia-reperfusion, because many CA1 pyramidal cells were not damaged. Therefore, these results suggest that GSE can protect ischemic neuronal damage by inhibiting DNA damage after transient forebrain ischemia.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Astrocytes; Benzoxazines; Blotting, Western; Brain Ischemia; Cell Count; Deoxyguanosine; DNA Damage; Gerbillinae; Hippocampus; Immunohistochemistry; Male; Microglia; Neurons; Neuroprotective Agents; Oxazines; Plant Extracts; Pyramidal Cells; Seeds; Vitis

EPC-K1, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt, is a novel antioxidant. In this study, we investigated a reduction of oxidative neuronal cell damage with EPC-K1 by immunohistochemical analysis for 8-hydroxy-2'-deoxyguanosine (8-OHdG) in rat brain with 60 min transient middle cerebral artery occlusion, in association with terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL) and staining for total and active caspase-3. Treatment with EPC-K1 (20 mg kg(-1) i.v.) significantly reduced infarct size (p < 0.05) at 24 h of reperfusion. There were no positive cells for 8-OHdG and TUNEL in sham-operated brain, but numerous cells became positive for 8-OHdG, TUNEL and caspase-3 in the brains with ischemia. The number was markedly reduced in the EPC-K1 treated group. These reductions were particularly evident in the border zone of the infarct area, but the degree of reduction was less in caspase-3 staining than in 8-OHdG and TUNEL stainings. These results indicate EPC-K1 attenuates oxidative neuronal cell damage and prevents neuronal cell death.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Apoptosis; Ascorbic Acid; Brain; Brain Infarction; Brain Ischemia; Caspase 3; Caspases; Deoxyguanosine; DNA Damage; Free Radicals; Immunohistochemistry; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Male; Nerve Degeneration; Neurons; Neuroprotective Agents; Oxidative Stress; Rats; Rats, Wistar; Reperfusion Injury; Vitamin E

The repair enzyme 8-oxoguanine glycosylase/ apyrimidinic/apurinic lyase (OGG) removes 8-hydroxy-2'deoxyguanosine (oh8dG) in human cells. Our goal was to examine oh8dG-removing activity in the cell nuclei of male C57BL/6 mouse brains treated with either forebrain ischemia-reperfusion (FblR) or sham operations. We found that the OGG activity in nuclear extracts, under the condition in which other nucleases did not destroy the oligodeoxynucleotide duplex, excised oh8dG with the greatest efficiency on the oligodeoxynucleotide duplex containing oh8dG/dC and with less efficiency on the heteroduplex containing oh8dG/dT, oh8dG/dG, or oh8dG/dA. This specificity was the same as for the recombinant type 1 OGG (OGG1) of humans. We observed that the OGG1 peptide and its activity in the mouse brain were significantly increased after 90 min of ischemia and 20-30 min of reperfusion. The increase in the protein level and in the activity of brain OGG1 correlated positively with the elevation of FblR-induced DNA lesions in an indicator gene (the c-fos gene) of the brain. The data suggest a possibility that the OGG1 protein may excise oh8dG in the mouse brain and that the activity of OGG1 may have a functional role in reducing oxidative gene damage in the brain after FblR.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Base Sequence; Brain; Brain Ischemia; Cell Nucleus; Deoxyguanosine; DNA; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Male; Mice; Mice, Inbred C57BL; N-Glycosyl Hydrolases; Prosencephalon; Reperfusion Injury; RNA, Messenger; Up-Regulation

To address the role of oxidative DNA damage in focal cerebral ischemia lacking reperfusion, we investigated DNA base and strand damage in a rat model of permanent middle cerebral artery occlusion (MCAO). Contents of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and apurinic/apyrimidinic abasic sites (AP sites), hallmarks of oxidative DNA damage, were quantitatively measured in nuclear DNA extracts from brains obtained 4-72 h after MCAO. DNA single- and double-strand breaks were detected on coronal brain sections using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), respectively. Levels of 8-OHdG and AP sites were markedly elevated 16-72 h following MCAO in the frontal cortex, representing the peri-infarct region, but levels did not significantly change within the ischemic core regions of the caudateputamen and parietal cortex. PANT- and TUNEL-positive cells began to be detectable 4-8 h following MCAO in the caudate-putamen and parietal cortex and reached maximal levels at 72 h. PANT- and TUNEL-positive cells were also detected 16-72 h after MCAO in the lateral frontal cortex within the infarct border, where many cells also showed colocalization of DNA single-strand breaks and DNA fragmentation. In contrast, levels of PANT-positive cells alone were transiently increased (16 h after MCAO) in the medial frontal cortex, an area distant from the infarct zone. These data suggest that within peri-infarct brain regions, oxidative injury to nuclear DNA in the form of base and strand damage may be a significant and contributory cause of secondary expansion of brain damage following permanent focal ischemia.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Blood Flow Velocity; Brain; Brain Chemistry; Brain Infarction; Brain Ischemia; Cerebrovascular Circulation; Chromosome Breakage; Deoxyguanosine; Disease Models, Animal; DNA; DNA Damage; DNA Fragmentation; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Male; Oxidative Stress; Rats; Rats, Sprague-Dawley

Involvement of oxidative stress in ischemia/reperfusion-induced brain damage has been suggested. However, experimental support of this suggestion was limited partly because sensitive indices to assess oxidative consequences of ischemic brain damage were few. We have established biochemical assay systems to assess oxidative brain damage following ischemia. Mongolian gerbil brains were subjected to global ischemia/reperfusion, and the hippocampi were analyzed for oxidative damage by measuring temporal changes in glutathione and 8-ohdG following ischemia. Under oxidative stress, glutathione is known to be oxidized and subsequently depleted from cells. Therefore, glutathione content and its redox status can serve as sensitive indicators of oxidative damage. The accumulation of 8-ohdG has also been recognized as an excellent marker for oxidative DNA damage. The reduced and oxidized glutathione were measured by HPLC method following derivatization with 2,4-dinitrofluorobenzene. The 8-ohdG in DNA hydrolyzate was measured by HPLC with electrochemical detection. While total glutathione content decreased, glutathione oxidation ratio and 8-ohdG accumulation increased over a period of 30 min of reperfusion following ischemia. The results demonstrated that glutathione content, its oxidation ratio, and the accumulated 8-ohdG could be utilized as sensitive indices for the assessment of oxidative brain damage.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Biochemistry; Brain Ischemia; Chromatography, High Pressure Liquid; Deoxyguanosine; DNA; Electrochemistry; Gerbillinae; Glutathione; Glutathione Disulfide; Hippocampus; Male; Oxidation-Reduction

Eicosanoids accumulation and formation of oxygen free radicals have been implicated in the pathogenesis of ischemia/reperfusion brain injury. In the present study, we examined whether green tea extract protects against ischemia/reperfusion-induced brain injury by minimizing eicosanoid accumulation and oxygen radical-induced oxidative damage in the brain. Green tea extract (0.5%) was orally administered to Wistar rats for 3 weeks before induction of ischemia. Ischemia was induced by the occlusion of middle cerebral arteries for 60 min and reperfusion was achieved for 24 h. Infarction volume in the ipsilateral hemisphere of ischemia/reperfusion animals was 114 +/- 16 mm(3) in the 0.5% green tea pretreated animals compared to 180 +/- 54 mm(3) in left hemisphere of nontreated animals. Green tea extract (0.5%) also reduced ischemia/reperfusion-induced eicosanoid concentration: Leukotriene C(4) (from 245 +/- 51 to186 +/- 22), prostoglandin E(2) (from 306 +/- 71 to 212 +/- 43) and thromboxane A(2) (327 +/- 69 to 251 +/- 87 ng/mg protein). Ischemia/reperfusion-induced increases of hydrogen peroxide level (from 688 +/- 76 to 501 +/- 99 nmole/mg protein), lipid peroxidation products (from 1010 +/- 110 to 820 +/- 70 nmole/mg protein) and 8-oxodG formation (from 1.3 +/- 0.3 to 0.8 +/- 0.2 ng/microg DNA, x10(-2)) were also reduced. Moreover, 0.5% green tea extract also reduced the apoptotic cell number (from 44 +/- 11 to 29 +/- 1 in the striatum, and from 72 +/- 11 to 42 +/- 5 apoptotic cells/high power field in the cortex region). Green tea extract pretreatment also promoted recovery from the ischemia/reperfusion-induced inhibition of active avoidance. The present study shows that the minimizing effect of green tea extract on the eicosanoid accumulation and oxidative damage in addition to the reduction of neuronal cell death could eventually result in protective effect on the ischemia/reperfusion-induced brain injury and behavior deficit.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Apoptosis; Avoidance Learning; Brain; Brain Infarction; Brain Ischemia; Deoxyguanosine; Eicosanoids; Hydrogen Peroxide; Lipid Peroxidation; Neurons; Neuroprotective Agents; Plant Extracts; Rats; Rats, Wistar; Reperfusion Injury; Tea

To investigate whether melatonin reduces the susceptibility of the fetal rat brain to oxidative damage of lipids and DNA, we created a model of fetal ischemia/reperfusion using rats at day 19 of pregnancy. Fetal ischemia was induced by bilateral occlusion of the utero-ovarian artery for 20 min. Reperfusion was achieved by releasing the occlusion and restoring the circulation for 30 min. A sham operation was performed in control rats. Melatonin (10 mg/kg) or vehicle was injected intraperitoneally 60 min prior to the occlusion. We measured the concentration of thiobarbituric acid reactive substances (TBARS) in fetal brain homogenates, as well as levels of deoxyguanosine (dG) and 8-hydroxydeoxyguanosine (8-OHdG) in DNA extracted from those homogenates. Ischemia for 20 min did not significantly alter the levels of dG, 8-OHdG, and TBARS. Subsequent reperfusion, however, led to a significant reduction in the dG level (P < 0.05) and to significant increases in the levels of 8-OHdG (P < 0.05) and TBARS (P < 0.05), and in the 8-OHdG/dG ratio (P < 0.005). Melatonin administration prior to ischemia significantly reduced the ischemia/reperfusion-induced increases in the levels of 8-OHdG (14.33 +/- 6.52-5.15 +/- 3.28 pmol/mg of DNA, P < 0.001) and TBARS (11.61 +/- 3.85-4.73 +/- 3.80 nmol/mg of protein, P < 0.001) as well as in the 8-OHdG/dG ratio (7.19 +/- 2.49-1.61 +/- 0.98, P < 0.001). Furthermore, melatonin significantly increased the dG level (210.19 +/- 49.02-299.33 +/- 65.08 nmol/mg of DNA, P < 0.05). Results indicate that melatonin administration to the pregnant rat may prevent the ischemia/reperfusion-induced oxidative lipid and DNA damage in fetal rat brain.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Brain; Brain Ischemia; Deoxyguanosine; DNA; DNA Damage; Female; Fetus; Lipid Metabolism; Lipid Peroxidation; Melatonin; Oxidation-Reduction; Pregnancy; Rats; Rats, Wistar; Reperfusion Injury; Thiobarbituric Acid Reactive Substances