Production of reactive oxygen species after reperfusion in vitro and in vivo: protective effect of nitric oxide

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Object. Thrombolytic treatments for ischemic stroke can restore circulation, but reperfusion injury, mediated by oxygen free radicals, can limit their utility. The authors hypothesized that, during reperfusion, nitric oxide (NO) provides cytoprotection against oxygen free radical species.

Methods. Levels of NO and oxygen free radicals were determined in both reoxygenation in vitro and reperfusion in vivo models using an NO electrochemical probe and high-performance liquid chromatography with the 2,3- and 2,5-dihydroxybenzoic acid trapping method, before and after addition of the NO donor diethanolamine nitric oxide (DEA/NO).

Reoxygenation after anoxia produced a twofold increase in NO release by human fetal astrocytes and cerebral endothelial cells (p < 0.005). In both cell lines, there was also a two- to threefold increase in oxygen free radical production (p < 0.005). In human fetal astrocytes and cerebral endothelial cells given a single dose of DEA/NO, free radical production dropped fivefold compared with peak ischemic levels (p < 0.001). In a study in which a rat global cerebral ischemia model was used, NO production in a vehicle-treated group increased 48 ± 16% above baseline levels after reperfusion. After intravenous DEA/NO infusion, NO reached 1.6 times the concentration of the postischemic peak in vehicle-treated animals. In vehicle-treated animals during reperfusion, free radical production increased 4.5-fold over basal levels (p < 0.01). After intravenous DEA/NO infusion, free radical production dropped nearly 10-fold compared with peak levels in vehicle-treated animals (p < 0.006). The infarct volume in the vehicle-treated animals was 111 ± 16.9 mm3; after DEA/NO infusion it was 64.8 ± 23.4 mm3 (p < 0.01).

Conclusions. The beneficial effect of early restoration of cerebral circulation after cerebral ischemia is limited by reperfusion injury. These results indicate that NO release and oxygen free radical production increase during reperfusion, and suggest a possible early treatment of reperfusion injury using NO donors.

Article Information

Address reprint requests to: Edward H. Oldfield, M.D., Surgical Neurology Branch, Building 10, Room 5D37, 10 Center Drive, Bethesda, Maryland 20892–1414. email: oldfield@box-o.nih.gov.

© AANS, except where prohibited by US copyright law.

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Figures

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    Graphs depicting concentrations of NO in human fetal astrocytes (left) and human cerebral endothelial cells (right) in control tissue cultures during 1- and 2-hour periods of anoxia and a 24-hour period of reoxygenation. In human fetal astrocytes (left) in response to anoxia the production of NO decreased by 32% (p < 0.005). It increased rapidly in response to reoxygenation, reaching levels 32% above control levels after 1 hour of anoxia (p < 0.005) and 95% higher than control levels after 2 hours of anoxia (p < 0.005). In human cerebral endothelial cells (right) in response to anoxia the concentration of NO decreased by 23% (p < 0.005). It increased rapidly in response to reoxygenation, reaching levels 15% above control levels after 1 hour of anoxia (p < 0.005) and 75% higher than control levels after 2 hours of anoxia (p < 0.005).

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    Graphs demonstrating production of ROS by human fetal astrocytes (left) and human cerebral endothelial cells (right) during 2 hours of reoxygenation after either 1 or 2 hours of anoxia, as measured using HPLC and the 2,3′- and 2,5′-DHBA trapping methods. Left: In human fetal astrocytes in response to reoxygenation the production of ROS increased twofold after 1 hour (p < 0.001) and 2 hours (p < 0.001) of anoxia compared with the level before anoxia. In response to administration of DEA/NO (10−6 M) to the medium simultaneous with beginning reoxygenation, ROS decreased by 90% after 1 hour (p < 0.001) and 2 hours (p < 0.001) of anoxia. Right: In human cerebral endothelial cells in response to reoxygenation the production of ROS increased 1.7-fold after 1 hour (p < 0.001) and almost threefold after 2 hours (p < 0.001) of anoxia compared with the level before anoxia. In response to administration of DEA/NO (10−6 M) to the medium simultaneous with beginning reoxygenation, ROS decreased by 97% after 1 hour (p < 0.001) and 2 hours (p < 0.001) of anoxia.

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    Graph depicting the picomolar concentrations of ROS in human fetal astrocytes after addition of hydrogen peroxide (10−6 M) for 2 hours in the presence of increasing concentrations of DEA/NO (range 10−12–10−5 M). The ROS levels were measured using HPLC and the 2,3′- and 2,5′-DHBA trapping methods. The ROS concentrations diminished as the NO concentrations increased (correlation coefficient = −0.7, Z = −1.9; p < 0.05). The curve represents a fifth-order polynomial regression fit.

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    Bar graph showing the percentages of cell death in human astrocytes after addition of hydrogen peroxide (10−6 M) for 2 hours and in response to additions of DEA/NO in concentrations ranging from 10−12 to 10−5 M. The cells were washed in PBS and trypsinized; viable cells were counted at the end of the 2-hour exposure. The number of cells was compared with the number of cells observed after exposure to hydrogen peroxide alone (control). Cell death decreased after addition of increasing concentrations of DEA/NO (range 10−12–10−5 M). * Significant increase in cell death (p < 0.05) compared with cells not exposed to DEA/NO.

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    Graph depicting levels of NO measured directly in the rat brain during global brain ischemia lasting 5 minutes and later during reperfusion, with and without intravenous administration of DEA/NO (10−6 M) at the time of reperfusion. The baseline cerebral NO level in vivo was 213 ± 34 nM. During ischemia, production of NO decreased fourfold (p < 0.001). It started to increase immediately at reperfusion, peaking at 25 minutes of reperfusion (46% above preischemic levels; p < 0.05). The levels then declined to approximately preischemic levels within 1.5 hours. In response to a single intravenous bolus of DEA/NO, brain NO levels increased twofold (p < 0.001) and then slowly decreased throughout the experiment.

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    Graph demonstrating increased ROS production in the rat brain after 5 minutes of global ischemia, as measured in microdialysis samples obtained using the HPLC and the 2,3′- and 2,5′-DHBA trapping methods. Baseline cerebral ROS levels in vivo were 153.5 ± 33.8 pM in control (untreated) and 134 ± 26.7 pM in treated animals. In the control animals, ROS production increased 4.5-fold above the baseline at 70 minutes after reperfusion (p < 0.001) and then declined for several hours. In response to a single intravenous bolus of DEA/NO (10−6 M) at reperfusion, ROS levels decreased to approximately 50% of preischemic levels (p < 0.006) and remained depressed throughout the experiment.

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