Jing Zhang

Mitochondrial Generation of Reactive Oxygen Species After Brain Ischemia in the Rat

BACKGROUND AND PURPOSEnBrain mitochondria have a substantial capacity to generate reactive oxygen species after ischemia when the components of the respiratory chain are reduced and molecular oxygen is present. We tested the hypothesis that brain mitochondria in vivo produce reactive oxygen species after ischemia/reperfusion (IR) in rats at a rate sufficient to escape endogenous antioxidant defenses.nnnMETHODSnIschemia-dependent production of hydroxyl radical in the hippocampus of the anesthetized rat was monitored with the use of intracerebral microdialysis. Transient global ischemia was produced by bilateral carotid artery occlusion and hemorrhagic hypotension to a mean arterial pressure of 35 mm Hg for 15 minutes followed by reperfusion for 60 minutes. Salicylic acid was infused into the hippocampus during the experiments, and changes in the recovery of its hydroxylated product, 2,3-dihydroxybenzoic acid (2,3-DHBA), were used to assess the effects of inhibitors of mitochondrial complex I on formation of hydroxyl radical during IR. Hydroxylation data from control groups of animals were compared with data from animals undergoing IR during treatment with either a mitochondrial complex I inhibitor alone or the inhibitor plus succinic acid.nnnRESULTSnTransient ischemia led to a fivefold increase in the recovery of 2,3-DHBA by microdialysis after 1 hour relative to control animals (P < .05). Inhibition of mitochondrial complex I prevented 2,3-DHBA formation after IR; this effect could be reversed by infusion of succinic acid by microdialysis during IR.nnnCONCLUSIONSnThe data indicate that reactive oxygen species generated by mitochondrial electron transport escape cellular antioxidant defenses and promote highly damaging hydroxyl radical activity after transient brain ischemia in the rat.

Brain Temperature Alters Hydroxyl Radical Production During Cerebral Ischemia/Reperfusion in Rats

Selective neuronal cell death in the CA, pyramidal cells of the hippocampus and neurons of the dorsolateral striatum as a consequence of brain ischemia/reperfusion (IR) can be ameliorated with brain hypothermia. Since postischemic injury is mediated partially by chemical production of reactive oxygen species (ROS), decreased ROS production may be one of the mechanisms responsible for cerebral protection by hypothermia. To determine if ischemic brain temperature alters ROS production, reversible IR was produced in rats by occlusion of both carotid arteries with hemorrhagic hypotension. After 15 min of ischemia, circulation was restored for 60 min. Brain temperature was maintained during ischemia at either 30, 36, or 39°C and kept at 36–37°C after reperfusion. Using cerebral microdialysis, we measured nonenzymatic hydroxylation of salicylate by HPLC with electrochemical detection in the hippocampus. CBF was also compared among the groups during IR. The results were that normothermic animals during reperfusion had persistently increased levels of the salicylate hydroxylation product, 2,3-dihydroxybenzoic acid (2,3-DHBA), reaching 251% of control at 60 min. This increase in 2,3-DHBA production was potentiated after 60 min of reperfusion (406% of control) with ischemic hyperthermia. In hypothermic ischemia, 2,3-DHBA production at 60 min was attenuated to 160% of control. CBF decreased to ∼5% of baseline value during ischemia, but increased three- to four-fold relative to control in all three groups. Therefore, the effects of ischemic brain temperature on 2,3-DHBA production did not correlate with changes in CBF during IR. We conclude that brain-temperature-related changes in OH · production are readily detected in the rat and decreased ROS generation may contribute to cerebral protection afforded by hypothermia during brain ischemia.

Mitochondrial oxidative stress after carbon monoxide hypoxia in the rat brain.

To better understand the mechanisms of tissue injury during and after carbon monoxide (CO) hypoxia, we studied the generation of partially reduced oxygen species (PROS) in the brains of rats subjected to 1% CO for 30 min, and then reoxygenated on air for 0-180 min. By determining H2O2-dependent inactivation of catalase in the presence of 3-amino-1,2,4-triazole (ATZ), we found increased H2O2 production in the forebrain after reoxygenation. The localization of catalase to brain microperoxisomes indicated an intracellular site of H2O2 production; subsequent studies of forebrain mitochondria isolated during and after CO hypoxia implicated nearby mitochondria as the source of H2O2. In the mitochondria, two periods of PROS production were indicated by decreases in the ratio of reduced to oxidized glutathione (GSH/GSSG). These periods of oxidative stress occurred immediately after CO exposure and 120 min after reoxygenation, as indicated by 50 and 43% decreases in GSH/GSSG, respectively. The glutathione depletion data were supported by studies of hydroxyl radical generation using a salicylate probe. The salicylate hydroxylation products, 2,3 and 2,5-dihydroxybenzoic acid (DHBA), were detected in mitochondria from CO exposed rats in significantly increased amounts during the same time intervals as decreases in GSH/GSSG. The DHBA products were increased 3.4-fold immediately after CO exposure, and threefold after 120 min reoxygenation. Because these indications of oxidative stress were not prominent in the postmitochondrial fraction, we propose that PROS generated in the brain after CO hypoxia originate primarily from mitochondria. These PROS may contribute to CO-mediated neuronal damage during reoxygenation after severe CO intoxication.

Apoptosis and Delayed Neuronal Damage after Carbon Monoxide Poisoning in the Rat

Delayed neurological damage after CO hypoxia was studied in rats to determine whether programmed cell death (PCD), in addition to necrosis, is involved in neuronal death. In rats exposed to either air or CO (2500 ppm), microdialysis in brain cortex and hippocampus was performed to determine the extent of glutamate release and hydroxyl radical generation during the exposures. Groups of control and CO-exposed rats also were tested in a radial maze to assess the effects of the CO exposures on learning and memory. At 3, 7, and 21 days after CO exposure brains were perfusion-fixed and hematoxylin-eosin (H&E) was used to assess injury and to select regions for further examination. DNA fragmentation was sought by examining cryosections with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) reaction. We found significant increases in glutamate release and .OH generation during and immediately after CO hypoxia. CO-exposed rats showed learning and memory deficits after exposure associated with heterogeneous cell loss in cortex, globus pallidus, and cerebellum. The frontal cortex was affected most seriously; the damage was slight at Day 3, increased at Day 7, and persistent at Day 21 after CO exposure. TUNEL staining was positive at all three time points, and TUNEL-labeled cells were distributed similarly to eosinophilic cells. The number of cells stained by TUNEL was less than by H&E and amounted to 2 to 5% of all cell nuclei in regions of injury. Ultrastructural features of both neuronal necrosis and apoptosis also were observed readily by electron microscopy. These findings indicate that both necrosis and apoptosis (PCD) contribute to CO poisoning-induced brain cell death.

Hydrogen Peroxide Production by Monoamine Oxidase during Ischemia-Reperfusion in the Rat Brain

Monoamine oxidase (MAO) as a source of hydrogen peroxide (H2O2) was evaluated during ischemiareperfusion in vivo in the rat brain. H2O2 production was assessed with and without inhibition of MAO during and after 15 min of ischemia. Metabolism of H2O2 by catalase during ischemia and reperfusion was measured in forebrain homogenates using aminotriazole (ATZ), an irreversible H2O2-dependent inhibitor of catalase. Catecholamine and glutathione concentrations in forebrain were measured with and without MAO inhibitors. During ischemia, forebrain blood flow was reduced to 8% of baseline and H2O2 production decreased as measured at the microperoxisome. During reperfusion, a rapid increase in H2O2 generation occurred within 5 min as measured by a threefold increase in oxidized glutathione (GSSG). The H2O2-dependent rates of ATZ inactivation of catalase between control and ischemia–reperfusion were similar, indicating that H2O2 was more available to glutathione peroxidase than to catalase in this model. MAO inhibitors eliminated the biochemical indications of increased H2O2 production and increased the catecholamine concentrations. Mortality was 67% at 48 h after ischemiareperfusion, and there was no improvement in survival after inhibition of MAO. We conclude that MAO is an important source of H2O2 generation early in brain reperfusion, but inhibition of the enzyme does not improve survival in this model despite ablating H2O2 production.

Cerebral amino acid, norepinephrine and nitric oxide metabolism in CNS oxygen toxicity.

CNS oxygen (O2) toxicity is complex, and the etiology of its most severe manifestation, O2 convulsions, is yet to be determined. A role for depletion of the brain GABA pool has been proposed, although recent data have implicated production of reactive O2 species, e.g. H2O2, in this process. We hypothesized that the production of H2O2 and NH3 produced by monoamine oxidase (MAO) would lead to depletion of GABA and production of nitric oxide (NO.) respectively, and thereby enhance CNS O2 toxicity. In this study, rats treated with an MAO inhibitor (pargyline) or a nitric oxide synthase inhibitor (LNNA) were protected against O2-induced convulsions. Selected cerebral amino acids including arginine were measured in control and O2 treated rats (6 ATA, 20 min) with or without drug pretreatment. After O2 exposure, the cerebral pools of glutamate, aspartate, and GABA decreased significantly while glutamine content increased relative to control (P < 0.05). After treatment with either enzyme inhibitor, glutamine, glutamate and aspartate concentrations were maintained near control levels. Remarkably, GABA depletion by O2 was not prevented despite protection from seizures by both pargyline and LNNA. The NO. precursor, arginine, was increased significantly in the brain by toxic O2 exposure, but both pargyline and LNNA inhibited this effect. Simultaneous norepinephrine measurements indicated that its storage substantially decreased during hyperoxia (P < 0.05), but this effect too was blocked by either pargyline or LNNA. These data indicate that protection against O2 by these inhibitors is not related to preservation of the GABA pool. More importantly, O2 dependent norepinephrine metabolism and NO. synthesis appear to be interactive during CNS O2 toxicity.

Hydroxyl radical production in the brain after CO hypoxia in rats

Reactive oxygen species (ROS) have been implicated in the pathogenesis of neuronal injury after carbon monoxide (CO) poisoning. Severe CO poisoning is treated with hyperbaric oxygen (HBO), which eliminates CO quickly from hemoglobin and body tissue stores, but has a potential to increase ROS generation. In this study, the effects of HBO on generation of highly reactive hydroxyl radical (HO.) in the brain after CO poisoning in rats was investigated using nonenzymatic hydroxylation of salicylic acid to 2,3 dihydroxybenzoic acid (2,3-DHBA) as a probe. In control studies, the concentrations of 2,3-DHBA after HBO in brain mitochondria and postmitochondrial supernatant (cytosol) were similar to air-exposed animals. After CO poisoning, 2,3-DHBA concentration increased in brain mitochondria but not in the cytosol. After CO exposure and HBO administration at 1.5 atmospheres absolute (ATA), a decrease in 2,3-DHBA production was detected in brain mitochondria. After CO and HBO at 2.5 ATA, 2,3-DHBA concentration increased in both mitochondria and cytosol. The oxidant scavenger dimethylthiourea (DMTU) and the monoamine oxidase (MAO) inhibitor pargyline, administered to CO poisoned rats after HBO at 2.5 ATA, diminished 2,3-DHBA production in both subcellular compartments. These findings indicate that brain HO. production can be either diminished or accelerated after severe CO poisoning depending on the oxygen partial pressure employed during therapy.

Production of Hydroxyl Radical in the Hippocampus After CO Hypoxia or Hypoxic Hypoxia in the Rat

Carbon monoxide poisoning produces both immediate and delayed neuronal injury in selective regions of the brain that is not readily explained on the basis of tissue hypoxia. One possibility is that cellular injury during and after CO poisoning is related to the production of reactive oxygen species (ROS) by the brain. In this study, we hypothesized that the extent of ROS generation in the brain would be greater after CO than after hypoxic hypoxia due to intracellular uptake of CO. We assessed hydroxyl radical (OH.) production by comparing the nonenzymatic hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid (2,3-DHBA) in the hippocampus of the rat by microdialysis during either CO hypoxia or an exposure to hypoxic hypoxia that produced similar PO2 and cerebral blood flow (CBF) values in the region of microdialysis. We found neither control animals nor animals exposed to 30 min of hypoxic hypoxia at a mean tissue PO2 of 15 mmHg demonstrated significant increases in 2,3-DHBA production in the hippocampus over the 2-h the exposure. In contrast, CO exposed rats which also developed brain PO2 values in the range of 15 mmHg showed highly significant increases in 2,3-DHBA production. We conclude that cerebral oxidative stress in the hippocampus of the rat during CO hypoxia in vivo is not a direct effect of decreased tissue oxygen concentration.

Nitric Oxide Synthase Inhibition and Extracellular Glutamate Concentration After Cerebral Ischemia/Reperfusion

BACKGROUND AND PURPOSEnTransient cerebral ischemia in rats results in selective loss of neuronal viability, eg, hippocampal CA1 neurons. The neurochemical variables responsible for this selective vulnerability to ischemia/reperfusion (IR) appear to involve excitatory amino acids. In brain IR, excitatory amino acid toxicity may be modulated by endogenous nitric oxide (NO.) gas. To investigate NO. in global brain IR, we measured the effects of NO. synthase (NOS) inhibition on interstitial excitatory amino acids in rats. Changes in postischemic cerebral blood flow and blood-brain barrier function also were evaluated.nnnMETHODSnForebrain ischemia was produced by systemic hypotension and occlusion of both carotid arteries for 15 minutes. Blood flow was restored for 60 minutes by unclamping the carotids and reinfusing with blood. A microdialysis probe was placed into the cortex and hippocampus using a stereotaxic device. Interstitial glutamate concentration was measured during IR with high-performance liquid chromatography. A competitive NOS inhibitor, N omega-nitro-L-arginine methyl ester (L-NAME), was given intraperitoneally 30 minutes before ischemia in doses of 1, 4, and 20 mg/kg. Changes in cerebral blood flow and blood-brain barrier during IR were determined using laser-Doppler flowmetry and microdialysis with sodium fluorescein.nnnRESULTSnGlutamate in the dialysate during IR increased transiently 10-fold and returned to baseline levels by 30 minutes of reperfusion. Animals treated with L-NAME 30 minutes before ischemia also showed increases in glutamate concentration during ischemia, but glutamate remained elevated during reperfusion. The increase in glutamate concentration during reperfusion caused by L-NAME was prevented by L-arginine. The administration of L-arginine and L-NAME together decreased extracellular glutamate concentration during ischemia. Cerebral blood flow decreased to about 5% of baseline values during ischemia but increased approximately fourfold relative to control values on reperfusion. The hyperemic responses after ischemia were not different between IR groups treated with or without L-NAME. Brain ischemia increased the permeability of the blood-brain barrier to fluorescein; however, this change was attenuated by L-NAME administration at 20 mg/kg.nnnCONCLUSIONSnNOS inhibition did not attenuate extracellular glutamate accumulation during ischemia and increased its concentration on reperfusion. The elevated glutamate concentration after IR in L-NAME-treated rats did not appear to be due to either a decrease in cerebral blood flow response after ischemia or increases in local blood-brain barrier permeability. For the most part, the blood-brain barrier was spared in the immediate postischemic period by L-NAME treatment. These data suggest that NO. production may oppose synaptic excitatory amino acid accumulation and presumably excitotoxicity during IR.

Prolonged production of hydroxyl radical in rat hippocampus after brain ischemia-reperfusion is decreased by 21-aminosteroids

Transient global ischemia may lead to persistent production of reactive oxygen species in selected brain regions thereby contributing to selective vulnerability to ischemia. Using cerebral microdialysis, we assessed the production of the highly reactive hydroxyl radical (OH.) in rat hippocampus during global ischemia and reperfusion (IR). During IR, perfusate containing salicylic acid was collected and analyzed for non-enzymatic hydroxylation of salicylate to 2,3-DHBA. Since 21-aminosteroids can attenuate excitatory amino acid-mediated OH. production in the brain, we repeated the experiments after administration of the 21-aminosteroid, U-74389G. The data indicate that 2,3-DHBA level increased progressively between 15 and 60 min after reperfusion, reaching values nearly three times the baseline value at 60 min. U-74389G, given 30 min before ischemia, greatly attenuated the increase in 2,3-DHBA during reperfusion. This is the first evidence for prolonged OH. production in the hippocampus after reperfusion in vivo which can be prevented by 21-aminosteroids.