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(Stroke. 1996;27:327-332.)
© 1996 American Heart Association, Inc.

Articles

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

Claude A. Piantadosi, MD Jing Zhang, MD, PhD

From the Department of Medicine, Duke University Medical Center, Durham, NC.

Correspondence to C.A. Piantadosi, MD, Department of Medicine, Box 3315, Duke University Medical Center, Durham, NC 27710.

	Abstract

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Background and Purpose Brain 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.

Methods Ischemia-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.

Results Transient 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.

Conclusions The 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.

Key Words: cerebral ischemia, transient • oxygen radical • reperfusion • rats

	Introduction

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Mitochondria provide ATP for cellular function by means of a remarkably efficient respiratory chain that transports electrons from reduced pyridine nucleotides to molecular O₂.¹ Normally, mitochondria reduce O₂ completely to H₂O in a four-electron reaction catalyzed by cytochrome c oxidase (complex IV). This reaction accounts for as much as 90% of the oxygen used by the normal brain. One or two percent of the O₂ reduced by mitochondria generates ROS, ie, O₂ can be reduced by a single electron to form ·O₂^-.^{2 3} Superoxide is produced in at least two locations within the respiratory chain. These sites are in the NADH dehydrogenase (complex I) and in the ubiquinone-cytochrome b-c₁ region (complex III).⁴

Superoxide anion produced by mitochondria rapidly dismutates to H₂O₂ either spontaneously or enzymatically via manganese superoxide dismutase.⁵ The rate of ·O₂^- and/or H₂O₂ production increases as mitochondrial Ca²⁺ increases or pH decreases. Also, the rate of H₂O₂ production by mitochondria is a function of the oxidation-reduction state of the electron carriers; as the carriers become reduced, the rate of H₂O₂ production increases. H₂O₂ is converted to O₂ and H₂O by glutathione peroxidase⁵ ; however, mitochondria may also release H₂O₂ to other sites within the cell, which in the presence of incompletely coordinated transition metals, such as iron, can be reduced and produce highly reactive or OH·-like activity.⁶

The hypothesis that ROS contribute to brain injury after IR has been popular for several years because the brain, in part because of its relatively modest antioxidant defenses, is sensitive to oxidative damage.⁷ The exact role of ROS in the pathogenesis of ischemic brain injury has been difficult to assess because the mechanisms of ROS production that contribute to neuronal injury are often linked to nonradical-mediated events that also contribute to injury.⁸ This principle may also pertain to brain mitochondria, for aerobic production of ATP must be restored for the survival of the cell, but ROS generation may cause oxidative damage to essential proteins, DNA, and other mitochondrial components. Direct experimental evidence for this concept, however, has been lacking.⁹ In this report we tested the hypothesis that brain mitochondria produce ROS after ischemia at a rate sufficient to escape endogenous antioxidant defenses in vivo.

	Materials and Methods

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To determine whether ROS produced by mitochondria can contribute to OH· generated after brain ischemia, experiments were conducted in healthy male Sprague-Dawley rats (weight, 300±20 g). The animal protocol was approved by the Duke University Institutional Animal Care Committee. The animals were anesthetized with sodium pentobarbital (50 mg/kg IP), intubated, and ventilated with air with the use of a rodent respirator. Catheters were placed in both femoral arteries to monitor arterial blood pressure and to remove aliquots of blood (200 µL) for blood gas determinations (model 1304, Instrumentation Laboratories). A third catheter was placed in the femoral vein to administer supplemental anesthetics and fluids. Rectal temperature was monitored continuously and kept near 37°C with the use of a heating pad. Brain temperature was estimated with a nasopharyngeal probe and kept between 36°C and 37°C. Reversible forebrain ischemia was produced by transient occlusion of the carotid arteries and hemorrhage to a mean blood pressure of 35 mm Hg for 15 minutes.¹⁰ This procedure causes selective, delayed neuronal damage to the hippocampus and other vulnerable brain regions in the rat.¹¹

A microdialysis probe (Carnegie-Medicin) was placed into the hippocampus through a craniotomy under stereotaxic guidance and perfused continuously at 2 µL/min with artificial CSF at pH 7.4.¹² The CSF contained salicylic acid (5 mmol/L) as an OH· trap. After the animals had stabilized for 2 hours, they completed either a control or an ischemia protocol. Samples of dialysate were collected every 15 minutes with a microfraction collector. The concentrations of hydroxylated salicylic acid in the dialysate were measured as an indicator of OH· production.^{13 14} OH· attacks the benzene ring of salicylic acid to produce two major products, 2,3- and 2,5-DHBA. The 2,3-DHBA, which can only be formed nonenzymatically, was separated by reverse phase high-performance liquid chromatography and its concentration measured electrochemically (model 5100A, ESA) to determine OH· production.^{15 16}

In separate experiments, the concentrations of salicylic acid in the hippocampus were measured after 2 hours of microdialysis by rapid microdissection of the brain, homogenization of the tissue at 4°C, and extraction of salicylate.¹⁵ Salicylate concentration was measured fluorometrically with the use of an excitation wavelength of 312 nm and an emission wavelength of 420 nm (model FD-300, SpectroVision). Partitioning of salicylic acid between mitochondrial and postmitochondrial fractions was determined in broken cell preparations of rat forebrain 60 minutes after injection of animals with 1000 mg/kg IP sodium salicylate (see Reference 15). Nonspecific lipid peroxidation was measured ex vivo as TBARs in the hippocampus and expressed per milligram of protein.¹⁷

Mitochondrial inhibitor studies were conducted to assess the contribution of brain mitochondria to the OH· signal obtained during and after ischemia. The first series of experiments consisted of infusion of rotenone (10 µmol/L in ethanol) into the brain through the microdialysis catheter. These experiments were conducted both with and without succinic acid (2 mmol/L) in the CSF. The second series of animals was pretreated with haloperidol (3 mg/kg IP) 30 minutes before IR with or without succinic acid (2 mmol/L) infused into the hippocampus through the microdialysis catheter. Both rotonene and haloperidol are inhibitors of the NADH coenzyme Q reductase (complex I), which provides electrons to coenzyme Q from NADH derived from the enzymes of the tricarboxylic acid cycle.¹⁸

Data from experiments in the same protocol group were expressed as mean±SEM for each time point. Comparisons were made with commercially available software (Statview 512+Brainpower) with one-way ANOVA with corrections for multiple comparisons.

	Results

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The physiological responses of the groups of animals in the study are summarized in the Table. Neither IR nor IR with any of the tested interventions produced significant alterations in arterial blood gases, pH, or blood pressure. In addition, rectal and nasopharyngeal temperatures were maintained within the target ranges throughout the IR protocols (data not shown).

View this table: [in this window] [in a new window]   Table 1. Physiological Measurements During Cerebral IR

Infusions of salicylic acid into the brain via microdialysis produced ex vivo concentrations of the compound in the hippocampus of 452±67 µmol/L (n=5) after 2 hours. In separate experiments to determine the distribution of the probe to mitochondria, the peak concentration of salicylate reached 293±38 µmol/L in forebrain homogenates after intraperitoneal injections of the compound into the rats. In these studies, the distribution of salicylic acid in cytosol and mitochondrial fractions of forebrain was found to favor the cytosol in a ratio of approximately 4:1 (n=12). Using these data, we estimated mitochondrial concentrations of salicylic acid in vivo to be approximately 100 µmol/L during the microdialysis studies. Hence, the amount of salicylate in the mitochondria would not be expected to have a significant effect on coupling of electron transport to ATP generation in vivo, although it could interfere with cytosolic enzymatic processes in the brain that contribute to ROS generation after ischemia (see "Discussion").

In these in vivo experiments, transient global ischemia progressively increased the concentration of 2,3-DHBA recovered from the hippocampus during reperfusion (Fig 1A). After an hour of reperfusion, the mean concentration of 2,3-DHBA in the dialysate was more than five times greater than that recovered from control animals. Infusion of the complex I inhibitor rotenone inhibited the increase in 2,3-DHBA during reperfusion after ischemia by more than 90% (Fig 1B). Intracerebral infusion of rotenone into control animals gave results similar to those in ischemic animals (data not shown). These results implicated the mitochondria as a major source of the ROS measured in vivo on reperfusion of the brain after ischemia. The ischemia experiments with rotenone were repeated with the inclusion of succinic acid in the dialysate (Fig 1B). Succinic acid supports mitochondrial O₂ uptake in brain tissue by providing electrons for the respiratory chain through complex II via FADH₂, thereby bypassing complex I.¹⁹ The presence of succinic acid and rotenone in the dialysate during ischemia nearly completely restored the recovery of 2,3-DHBA from the hippocampus by microdialysis after reperfusion. In studies with rotenone and succinic acid, the concentrations of 2,3-DHBA recovered after 1 hour of reperfusion were not different by ANOVA from untreated IR animals after 1 hour of reperfusion. This effect of succinic acid was dependent on ischemia, since infusion of succinate alone did not alter the rate of 2,3-DHBA production in control animals (data not shown).

View larger version (12K): [in this window] [in a new window]   Figure 1. Line graphs show recovery of hydroxylated salicylate from brain interstitium after transient cerebral ischemia in the rat. Hydroxylated salicylic acid (2,3-DHBA) in brain dialysate was quantified by high-performance liquid chromatography and electrochemical detection. In control animals (n=5), the recovery of 2,3-DHBA was stable or declined during the experiments (A, bottom curve). In IR (n=5), 2,3-DHBA concentration increased progressively (A, top curve). The complex I inhibitor rotenone (10 µmol/L) in the artificial CSF during the ischemia experiments prevented accumulation of 2,3-DHBA in the interstitium (B, bottom curve; n=5). Rotenone (10 µmol/L) and succinic acid (2 mmol/L) in the CSF during the experiments restored the 2,3-DHBA concentration in the dialysate (B, top curve; n=5).

To further assess the hypothesis that mitochondria contribute to ROS production on reperfusion after brain ischemia, the experiments were repeated after systemic injection of the neuroleptic drug haloperidol. Haloperidol inhibits complex I in brain mitochondria at micromolar concentrations²⁰ ; it shares structural homology with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, whose active metabolite, 1-methyl-4-phenylpyridinium (MPP⁺), produces an irreversible Parkinson-like syndrome by destroying nigrostriatal dopaminergic neurons by a mechanism involving complex I.^{21 22} In rats pretreated with haloperidol, the production of 2,3-DHBA after IR was inhibited almost completely (Fig 2). As with the rotenone experiments, infusion of succinic acid reversed the inhibition of salicylate hydroxylation by haloperidol during IR. These data indicated that the neuroleptic drug had effects on 2,3-DHBA production that were specific for inhibition of complex I (Fig 2).

View larger version (14K): [in this window] [in a new window]   Figure 2. Line graph shows recovery of hydroxylated salicylate from brain interstitium after cerebral ischemia and treatment with haloperidol. Rats were pretreated with haloperidol (3 mg/kg IV) 30 minutes before cerebral ischemia. This dose of haloperidol produces tissue concentrations of the drug of 25 to 50 µmol/L, which are sufficient to inhibit complex I of the respiratory chain.20 Haloperidol-treated animals showed markedly reduced recovery of 2,3-DHBA from the brain interstitium after ischemia (bottom curve; n=5). When succinic acid (2 mmol/L) was included in the artificial CSF, the recovery of 2,3-DHBA after brain ischemia in haloperidol-treated animals (top curve; n=5) was similar to untreated animals during ischemia/reperfusion (Fig 1A, top curve, P<.05 vs haloperidol alone).

In addition to blocking 2,3-DHBA production in the hippocampus in vivo, haloperidol pretreatment 30 minutes before ischemia decreased the concentration of TBARs in the hippocampus 1 hour after ischemia (Fig 3). Therefore, an antioxidant effect of haloperidol was demonstrated. The possibility that haloperidol altered 2,3-DHBA production after IR by its effects on blocking dopamine reuptake was not supported by the results of the TBAR measurements since auto-oxidation of excess dopamine would have been expected to increase nonspecific lipid peroxidation.²³ The density of dopaminergic terminals in the hippocampus of the rat is only 0.5% of that in dopamine-rich regions such as the striatum,²⁴ and this appears to account for the observation that low concentrations of MPP⁺ inhibit O₂ consumption in slices of striatum but not hippocampus, while high concentrations of MPP⁺ inhibit O₂ consumption in slices from both regions.²²

View larger version (34K): [in this window] [in a new window]   Figure 3. Bar graph shows measurements of nonspecific peroxidation in the hippocampus after transient cerebral ischemia. Anesthetized rats in control, IR, and IR plus haloperidol groups were killed 60 minutes after control or ischemia periods, the brains perfused free of blood, and the hippocampus removed by microdissection. Values for TBARs are mean±SEM for n=8 in each group. OD indicates optical density. *Significant difference from control by one-way ANOVA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The data in this report directly support the hypothesis that ROS generated by mitochondrial electron transport escape cellular defenses after transient cerebral ischemia. This hypothesis is supported by two lines of evidence. First, we were able to suppress salicylate hydroxylation in the brain dramatically after ischemia using two different inhibitors of mitochondrial complex I. Second, salicylate hydroxylation could be restored in the presence of the complex I inhibitors during ischemia by the use of simultaneous infusions of succinic acid directly into the hippocampus. Succinic acid serves as a specific substrate for mitochondrial respiration in the brain by directly supplying electrons to complex II via FADH₂.¹⁹

An ancillary finding that also supported the specificity of the data for mitochondrial ROS production was that both rotenone and haloperidol increased the concentrations of 2,3-DHBA detected in the hippocampus before ischemia. This observation is consistent with the known effects of inhibiting complex I with rotenone in isolated mitochondria wherein ·O₂^- production increases proximal to its complex I binding site.²⁵ When complex I is inhibited at the time of ischemia in vivo, however, further increments in 2,3-DHBA production are prevented when the brain is reperfused. The reversal of the effects of rotenone and haloperidol by succinic acid implicates the ubiquinone-cytochrome b-c₁ region of the chain (complex III) in postischemic ROS production,²⁵ although a role for complex IV has not been excluded by these experiments. Of note, our results were achieved despite barbiturate anesthesia, which may also have inhibitory effects on complex I activity.¹⁸

Although the findings of this study are readily explained on the basis of mitochondrial production of ROS, the results raise a number of important questions. The salicylate trapping method assumes that salicylic acid diffuses freely from the microdialysis catheter into the brain interstitium and into neuronal cells. The hydroxylated products also must diffuse from the cells and interstitium into the dialysate. Factors that affect the concentrations and diffusion characteristics of these compounds in the brain and across the dialysis membrane will alter the quantitative recovery of the hydroxylated by-products. These variables, however, should be accounted for by the control experiments and qualitative interpretation of the data. In addition, 2,3-DHBA is water soluble and freely diffusible across cell membranes. Also, the complex I inhibitors used in the study would not be expected to compete with the probe since they were used in low concentrations relative to salicylate and because of the high reactivity of OH· with virtually all biomolecules. The salicylate method has an efficiency of detection for OH · activity of approximately 10%,^{1 16} and the efficiency is decreased by the presence of the dialysis membrane. Previous estimates indicate that salicylate trapping detects approximately 3% of the total OH·-like activity generated after brain ischemia in vivo¹⁴ ; hence, the method substantially underestimates the true extent of OH· activity.

The precise cellular compartmentation of the hydroxylation events measured in this study is unknown. Mitochondrial OH· production was underestimated to the degree that the lower concentrations of salicylic acid in mitochondria relative to cytosol in the distribution studies reflect the conditions in the microdialysis studies. Since the generation of OH· activity is site specific and the salicylate probe is distributed throughout the aqueous phase of the tissue, we can conclude only that the mitochondria provide a source of oxidizing power, presumably via H₂O₂, which leads to the hydroxylation of salicylate. It seems unlikely that H₂O₂ produced by mitochondria would escape from the cell in significant amounts; however, determining the exact sites at which hydroxylation occurs and the role of free transitional metals such as iron must await further studies.

The use of salicylic acid in this study also contributes to our inability to determine the importance of mitochondrial ROS production relative to some of the other known sources of oxidative stress. For instance, salicylic acid inhibits cyclooxygenase, which can produce ·O₂^- from arachidonic acid mobilized during ischemia. This mechanism contributes to extracellular ·O₂^- after brain ischemia.⁸ As a result, the relative importance of prostanoid versus mitochondrial ROS production cannot be determined because of potential inhibition of cyclooxygenase when the present method is used. Release of arachidonic acid from cell membranes in the hippocampus after ischemia is known to occur, as in other brain regions.²⁶

In conclusion, this is the first study to demonstrate that the mitochondrial respiratory chain is an important source of ROS generated at the time of reperfusion after brain ischemia in vivo. This mechanism, because it leads to OH· production, also may depend on the availability of incompletely coordinated transition metal in one or more intracellular compartments or in the interstitial spaces of the brain. Although we cannot determine the exact sites responsible for generating OH· activity, the mitochondrion is clearly a source of oxidative stress after transient ischemia that is not contained by endogenous antioxidant defenses. Such an oxidative stress is capable of direct cellular damage^{7 9} and may also trigger mechanisms for programmed cell death.²⁷ In theory, persistent mitochondrial production of ROS could be a critical factor in delayed neuronal death after transient brain ischemia.

	Selected Abbreviations and Acronyms

 

CSF = cerebrospinal fluid DHBA = dihydroxybenzoic acid FADH2 = flavin adenine dinucleotide H2 IR = ischemia/reperfusion MPP+ = 1-methyl-4-phenylpyridinium ·O2- = superoxide anion OH· = hydroxyl radical ROS = reactive oxygen species TBARs = thiobarbituric acid reactive substances

	Footnotes

 
Review of this manuscript was directed by Michael S. Wolin, PhD.

Received August 10, 1995; revision received November 6, 1995; accepted November 8, 1995.

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