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(Stroke. 2003;34:1015.)
© 2003 American Heart Association, Inc.

Original Contributions

Opening of Mitochondrial ATP-Sensitive Potassium Channels Is a Trigger of 3-Nitropropionic Acid–Induced Tolerance to Transient Focal Cerebral Ischemia in Rats

Takashi Horiguchi, MD; Bela Kis, PhD; Nishadi Rajapakse, BS; Katsuyoshi Shimizu, MD David W. Busija, PhD

From the Department of Physiology and Pharmacology (T.H., B.K., N.R., D.W.B.) and Molecular Medicine Graduate Program (N.R.), Wake Forest University School of Medicine, Winston-Salem, NC, and Department of Neurosurgery, Tachikawa Hospital, Tokyo, Japan (K.S.).

Correspondence to Takashi Horiguchi, MD, Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1083. E-mail takaholy{at}aol.com

	Abstract

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Background and Purpose— The role of mitochondrial ATP-sensitive potassium channels (mitoK_ATP) in ischemic tolerance has been well documented in heart, but little work has been done in brain. To investigate the involvement of mitoK_ATP activation in chemical preconditioning in brain, we examined the effect of 5-hydroxydecanoate (5-HD), a selective mitoK_ATP blocker, on neurotoxin 3-nitropropionic acid (3-NPA)–induced ischemic tolerance to transient focal cerebral ischemia in rats.

Methods— Male Wistar rats were administrated 3-NPA (20 mg/kg IP; n=16) or vehicle (saline; n=16) 3 days before temporary occlusion (120 minutes) of the middle cerebral artery; 5-HD (40 mg/kg IP; n=16) was injected 20 minutes before 3-NPA administration. Infarct volumes were measured 4 days after reperfusion. To directly investigate whether chemical preconditioning activates mitoK_ATP, we tested the effect of prior incubation with 1 mmol/L 5-HD on 300 µmol/L 3-NPA–induced alterations of mitochondrial membrane potential (_m) in cultured neurons and astrocytes using the fluorescent dye tetramethylrhodamine ethyl ester.

Results— Treatment with 3-NPA exhibited a 16% reduction (P<0.05) and 23% reduction in infarct volume (P<0.01) for total brain and cortex, respectively. Pretreatment with 5-HD completely abolished the neuroprotective effect of chemical preconditioning. In cultured cells, 3-NPA resulted in mitochondrial depolarization. This change of _m was completely blocked by 5-HD pretreatment.

Conclusions— These results strongly suggest that opening of mitoK_ATP plays a key role as the trigger in the development of 3-NPA–induced ischemic tolerance in brain.

Key Words: brain ischemia • middle cerebral artery occlusion • mitochondria • potassium channels • rats

	Introduction

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Ischemic tolerance" is a phenomenon in which preconditioning with sublethal stresses or stimuli such as brief ischemia,¹ spreading depression,² or pharmacological agents³ can induce resistance to subsequent lethal ischemia. It is clear in the heart and brain that activation of ATP-sensitive potassium channels (K_ATP) is an essential initiator of the mechanisms resulting in the development of ischemic tolerance.^4,5 K_ATP are found in many places within cells, including the surface membrane (sK_ATP) and the inner mitochondrial membrane (mitoK_ATP), which have different pharmacological and functional properties.⁶ Recently, in heart, a pivotal role of mitoK_ATP in development of ischemic tolerance was established.⁷ In contrast, the correlation between mitoK_ATP and ischemic tolerance in the brain has been relatively unstudied. Recent studies have revealed that diazoxide, a selective agonist of mitoK_ATP, applied approximately 30 minutes before index ischemia, ameliorates neuronal dysfunction and damage.^8–10 However, the role of mitoK_ATP in delayed tolerance, which appears 24 to 72 hours after preconditioning, is unclear and apparently unstudied in brain.

Fungal toxin 3-nitropropionic acid (3-NPA) has direct effects on mitochondria as the inhibitor of succinate dehydrogenase, associated with the tricarboxylic acid cycle and complex II in the respiratory chain.¹¹ In the brain, the striatum is the most vulnerable region to systemic intoxication with high doses¹² or repeated injections of low-dose 3-NPA.¹³ On the other hand, several studies have shown that a single dose of 20 mg/kg 3-NPA given systemically confers no histopathological abnormalities but provides delayed cerebral ischemic tolerance.^3,12,14,15 Although a previous study reported that 3-NPA has the potential to activate sK_ATP in hippocampal cells,¹⁶ the role of mitoK_ATP in mediating the delayed ischemic tolerance with 3-NPA has not been documented in intact brain. The purpose of this study was to examine the involvement of mitoK_ATP opening in the generation of delayed ischemic tolerance with 3-NPA in brain. We tested whether the selective K_ATP blocker glibenclamide or selective mitoK_ATP blocker 5-hydroxydecanoate (5-HD) would prevent delayed protection by 3-NPA in rats after transient middle cerebral artery occlusion (MCAO). To directly investigate whether chemical preconditioning with 3-NPA induces opening of mitoK_ATP, we examined the effects of 5-HD on alterations of mitochondrial membrane potential (_m) produced by 3-NPA in cultured neurons.

	Materials and Methods

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Animals
We used 88 male Wistar rats (weight, 250 to 320 g) for the MCAO experiments and embryonic day-18 Wistar rats for culturing neurons. All animal protocols were approved by the institutional animal care and use committee.

Focal Cerebral Ischemia
Transient focal cerebral ischemia was induced by the MCAO filament model as previously described with some modifications.¹⁷ Briefly, rats were fasted overnight with free access to water before surgery. After anesthesia was induced with 5% halothane in oxygen, rats were ventilated spontaneously with 1.0% halothane in a 70:30 gas mixture of N₂O and O₂. The tail artery was cannulated to monitor mean blood pressure, blood sugar, and blood gases. During surgery, the rectal temperature was maintained in a range between 36.5°C and 37.5°C by a heating pad. The right external and internal carotid arteries (ECA and ICA, respectively) were dissected from surrounding connective tissue through a midline neck incision. The branches of the ECA were ligated and cut. Then the ECA was cut between the double ligations at the distal portion. Two microvascular clips were placed across the common carotid artery and the ICA. A 4-0 monofilament nylon suture (Ethicon), its tip coated with silicon (Rhodoia RTV 1556 A and B), was introduced into the ICA via the ECA stump gently and advanced in the ICA approximately 20 mm from the carotid bifurcation after the proximal microvascular clip was removed. The suture around the ECA stump was tightened. After the incision was closed, anesthesia was discontinued, and rats were kept in the cage during 120 minutes of MCAO. The animals were reanesthetized 2 hours after induction of ischemia to remove the suture and to ligate the stump of the ECA. After reperfusion, rats were awakened quickly, kept in the cage, and allowed free access to food and water. In several rats, local cerebral blood flow (CBF) of the assumed infarct core was measured with laser-Doppler flowmetry (Multichannel Laser Doppler System, PERIMED). The bone of the skull was drilled out at 0.5 mm posterior and 4.5 mm lateral to the bregma without injuring the dura under the surgical microscope. Local CBF was measured at 4 time points, ie, before ischemia (baseline), just after insertion of the occluder, just before reperfusion, and after reperfusion. Four days after recovery from MCAO, all animals were anesthetized with halothane 5% in O₂ and decapitated.

Measurement of Infarct Volume
The rat brain was quickly removed and sliced into coronal sections at 2-mm intervals. Each slice was immersed in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma) for 20 minutes and then fixed in 10% buffered formaldehyde solution. The infarction area in each brain slice was determined on the NIH Image program (version 1.62) according to the indirect method proposed by Swanson and others.¹⁸ Total infarct volumes in cortex and basal ganglia expressed in cubic millimeters were calculated by summation of the infarcted area in 6 brain slices (2 to 14 mm from frontal pole) and integrated by the thickness (2 mm).

Experimental Design
The rats were randomly assigned to 1 of the following 5 groups (Figure 1). A single dose of 20 mg/kg 3-NPA is documented to confer significant neuroprotection but not abnormalities in neurological and histopathological outcomes.^3,12,14,15 In group 1 (n=32), animals were chemically preconditioned with 3-NPA (Sigma; 20 mg/kg, diluted in sodium chloride 0.9% to a concentration of 1 mg/mL, buffered [pH 7.4] with NaOH, IP) 24 hours (n=8), 48 hours (n=8), or 72 hours (n=16) before MCAO. In the following groups, all drugs were given 72 hours before MCAO. Group 2 (n=16) consisted of rats injected with only saline (sodium chloride 0.9%, IP). In group 3 (n=16), animals received glibenclamide (Sigma; 0.1 mg/kg, dissolved in 20 µL dimethyl sulfoxide) into the left lateral ventricle under stereotaxic guidance as described by Nishimura et al¹⁹ 20 minutes before 3-NPA administration. In group 4 (n=16), animals were treated with 5-HD (Sigma; 40 mg/kg, in sodium chloride 0.9% to a concentration of 1 mg/mL, buffered [pH 7.4] with NaOH, IP) 20 minutes before administration of 3-NPA. Group 5 (n=16) represented rats treated with 5-HD alone.

View larger version (28K): [in this window] [in a new window]   Figure 1. Experimental protocol indicating time of drug application, induction of index ischemia, and TTC staining.

Cell Culture
Primary rat cortical neurons were cultured on the basis of the method of Deadwyler et al.^20,21 Pregnant Wistar rats were anesthetized with pentobarbital (50 mg/kg), and E18 fetuses were recovered by caesarian section. After brains were removed from the fetuses, the pial membranes were peeled off. The collected cortical pieces were placed in ice-cold Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL) and incubated with dispase I (2 U/mL; Roche) for 35 minutes at 37°C after they were washed twice in DMEM supplemented with penicillin (100 U/mL; Sigma) and streptomycin (100 µg/mL; Sigma). The cells were washed twice with DMEM to stop the enzymatic reaction and then were dissociated by 2 series of gentle trituration via 200- and 20-µL pipette tips and plated at a density of 10⁵ cells per square centimeter onto poly-D-lysine–coated glass coverslips (Becton-Dickinson); they were maintained in Neurobasal medium (Gibco BRL) supplemented with B27 (2%; Gibco BRL), L-glutamine (0.5 mmol/L; Sigma), ß-mercaptoethanol (55 µmol/L; Gibco BRL), and potassium chloride (25 mmol/L). Cultures were grown at 37°C in a humidified atmosphere containing 5% CO₂ in air, and medium was changed on every third day. Cultures consisted of >98% of neurons verified by positive immunostaining for microtubule-associated protein-2 (Becton-Dickinson) and negative immunostaining for glial fibrillary acidic protein (Chemicon). Cells were used for experiments at 7 to 9 days in vitro.

Analysis of Mitochondrial Membrane Potential
We monitored _m using the _m-sensitive dye tetramethylrhodamine ethyl ester (TMRE) (Molecular Probes). Neuronal cultures were loaded in the dark at 37°C in the 5% CO₂ incubator with 0.5 µmol/L TMRE in Neurobasal medium for 15 minutes. After they were loaded, the cells were washed 3 times with phosphate-buffered saline, and cell cultures were then placed on the stage of the confocal microscope in phosphate-buffered saline. Experiments were performed at 22°C. Confocal images of cellular TMRE fluorescence were acquired on a Zeiss LSM 510 laser scanning microscope with the use of a x63, numerical aperture 1.2, Zeiss C-Apochromat water immersion objective. Fields of cells were randomly selected under differential interference contrast optics. The cells were treated with 3-NPA (300 µmol/L) with or without prior incubation with 5-HD (1 mmol/L) for 1 minute, and fluorescent images were obtained with excitation =543 and emission >560 nm (560 nm long-pass filter). Images were recorded every minute for 5 minutes after treatment, and the average pixel intensity in individual cell bodies was determined with the use of a computerized image analysis system (NIH Image, version 1.62). The estimated brain concentration after systemic administration of 20 mg/kg 3-NPA is 300 µmol/L.³ Additionally, to confirm that 3-NPA–induced cellular consequences occurred in mitochondria, we also used the mitochondrion-selective dye chloromethyl-tetramethyl-rosamine (CMTMROS) (MitoTracker Orange, Molecular Probes) with the same confocal microscope. Cultured cells were loaded with 2 µmol/L CMTMROS in Neurobasal medium for 20 minutes. Then the cells were rinsed 3 times with phosphate-buffered saline. Fluorescent images at before and 5 minutes after 3-NPA application were obtained with or without 5-HD preincubation, at excitation =554 and emission >576 nm.

Statistical Analysis
All data are expressed as mean±SEM. A 1-way ANOVA followed by the Fisher least significant difference test was performed to assess statistical differences for infarct volumes, physiological parameters, and local CBF. To compare the change of _m, a 2-tailed unpaired t test was performed. A probability value <0.05 was regarded as statistically significant.

	Results

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Physiological Parameters
The physiological parameters during surgery among the 5 groups showed no statistically significant differences (Table 1).

View this table: [in this window] [in a new window]   TABLE 1. Physiological Parameters of Rats Before and After Surgery

Cerebral Blood Flow
Local CBF during MCAO showed no significant differences among the 5 groups (Table 2).

View this table: [in this window] [in a new window]   TABLE 2. Local Cerebral Blood Flow During MCAO

Infarct Volume
Pretreatment with 3-NPA at an interval of 24 hours (n=8) or 48 hours (n=8) before MCAO had no protective effect (infarct volume of 291.7±47.7 and 299.6±47.1 mm³ for 24 and 48 hours, respectively) (Figure 2). Only administration of 3-NPA 72 hours before MCAO consistently decreased infarct volume; the reduction in infarct volume for total brain was 16% and for the cortex was 23%. The subcortical infarct volumes were not significantly different. The administration of glibenclamide or 5-HD completely abolished the protective effect of 3-NPA. Treatment with 5-HD alone did not aggravate ischemic damage.

View larger version (36K): [in this window] [in a new window]   Figure 2. Glibenclamide (Glib) or 5-HD abolished 3-NPA–induced ischemic tolerance. A, Treatment of 3-NPA 72 hours before ischemia induced significant protective effect in cerebral cortex but not in subcortical area. Glibenclamide or 5-HD completely abolished 3-NPA–induced ischemic tolerance. Data are mean±SEM. *P<0.05 vs vehicle; **P<0.01 vs vehicle. B, Effect of 5-HD on 3-NPA–induced reduction of cortical infarct area. Data are mean±SEM. *P<0.05 vs 3-NPA; #P<0.05 vs vehicle.

Mitochondrial Membrane Potential
Administration of 3-NPA decreased _m (representing depolarization), as shown by the increment of TMRE fluorescence intensity in neurons (Figure 3). The measured fluorescence intensity of TMRE immediately increased after exposure to 3-NPA and reached a maximum by 1 minute in neurons before decreasing toward baseline values at 2 to 5 minutes (Figure 3A, gray bars). Pretreatment of 5-HD significantly prevented the mitochondrial depolarization in cultured neurons (Figure 3A, solid bars).

View larger version (66K): [in this window] [in a new window]   Figure 3. 3-NPA–induced mitochondrial membrane depolarization in neurons was completely inhibited by selective mitoKATP blocker 5-HD. A, Temporal profile of alterations of TMRE fluorescence intensity. Fluorescence intensity immediately increased after exposure to 3-NPA and reached a maximum by 1 minute. At 2 minutes after 3-NPA application, fluorescence intensity began to decrease toward baseline values (gray bars). Pretreatment with 5-HD significantly inhibited 3-NPA–induced alterations of TMRE fluorescence intensity (solid bars). *P<0.01. B, Representative confocal micrographs of 3-NPA–treated neurons with or without 5-HD pretreatment using TMRE. Top row, Before 3-NPA application, TMRE accumulated in mitochondria because of its negative charge (Before). One minute after 3-NPA exposure, fluorescence intensity increased in the cytoplasm because of depolarization-induced TMRE release from mitochondria. Three minutes after, TMRE fluorescence intensity began to decrease in the cytoplasm. At 5 minutes, fluorescence was detected in the same intracellular location as seen before 3-NPA treatment. Bottom row, 5-HD completely inhibited 3-NPA–induced m depolarization. C, Representative confocal micrographs of 3-NPA–treated neurons with or without 5-HD pretreatment using CMTMROS. Pretreatment with 5-HD completely inhibited 3-NPA–induced change of CMTMROS fluorescence intensity. Bar=10 µm.

The representative photographs in Figure 3B demonstrate the changes of intracellular distribution of TMRE fluorescence induced by 3-NPA in neurons. Before application of 3-NPA, TMRE fluorescence was detected in mitochondria regardless of 5-HD pretreatment. At 1 minute after exposure to 3-NPA, TMRE was released into the cytoplasm and increased the fluorescence intensity at the whole cell level. TMRE fluorescence was redistributed into the mitochondria at 5 minutes after 3-NPA treatment. Pretreatment with 5-HD inhibited 3-NPA–induced alterations of TMRE fluorescence intensity in both cell types.

Additionally, 3-NPA induced increases in CMTMROS fluorescence intensity, which was inhibited by the preincubation of 5-HD, as shown in Figure 3C.

	Discussion

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In this study we provided the first evidence that administration of 3-NPA protects the brain in situ via mechanisms that involve activation of mitoK_ATP. The effects of 3-NPA in limiting infarct volume were not correlated with the restoration of local CBF during ischemia and were blocked by administration of 5-HD or glibenclamide. Furthermore, in cultured cells 3-NPA resulted in mitochondrial depolarization, and these effects were also blocked by 5-HD. In numerous studies, 5-HD has been used as a selective antagonist of mitoK_ATP.^5,8–10 Recently, in heart, it was reported that 5-HD may also stimulate ß-oxidation.²² However, 3-NPA itself is reported to inhibit ß-oxidation in the central nervous system.²³ Therefore, our present results using 5-HD, together with those involving glibenclamide administration, suggest a key role of mitoK_ATP as a trigger in the development of ischemic tolerance induced by 3-NPA in brain.

Systemic injection of 3-NPA 20 mg/kg IP has been shown to decrease succinate dehydrogenase enzymatic activity by 70% for several days, resulting in a decrease in brain ATP levels initially of up to 50%.³ Previously, glibenclamide has been reported to eliminate the 3-NPA–induced hyperpolarization of the cultured neuronal cells¹⁶ or prolongation of hypoxic depolarization in hippocampal slices.²⁴ These findings agree with our results that glibenclamide abolishes the reduction of the infarct area after 3-NPA pretreatment. Therefore, like ischemic preconditioning, potassium channel activation is thought to play a key role in chemical preconditioning. Recent pharmacological studies revealed that glibenclamide inhibited not only sK_ATP but also mitoK_ATP.⁶ In the present study, to extend these important earlier findings, we report that selective blockade of mitoK_ATP prevents delayed chemical preconditioning with 3-NPA using an in vivo model. Similar results have recently been published for the heart.⁷ However, the precise details of this relationship among 3-NPA pretreatment, activation of mitoK_ATP, depolarization of mitochondria, and delayed protection against ischemic challenge are unclear.

In the present fluorescence experiments using TMRE, we showed that 300 µmol/L 3-NPA depolarized the mitochondria and that this effect was blocked with 5-HD. Similar responses were observed in astrocytes (data not shown). Since Riepe et al¹⁶ failed to show that single application of 0.1 to 10 mmol/L 3-NPA induced currents in the plasma membrane of cultured neurons, the increase of TMRE fluorescence intensity elicited by 300 µmol/L 3-NPA in our experiments suggested a direct effect on mitochondria. Our data with TMRE and CMTMROS indicate that the 3-NPA–induced changes in _m were caused by opening of mitoK_ATP.

The mechanisms involved in the present chemical preconditioning remain obscure. Wiegand et al³ showed that administration of a free radical scavenger or protein synthesis inhibitor completely abolished chemical preconditioning with 3-NPA in brain. Additionally, Sugino et al²⁵ demonstrated that preconditioning with 3-NPA activated c-jun N-terminal kinases (JNK) that induce transcriptional factors such as c-Jun or ATF2.²⁶ In heart, recent studies showed free radical formation after activation of mitoK_ATP.²⁷ Taken together, it is possible to speculate that mitoK_ATP opening leads to oxygen radical release, enhanced protein synthesis, and resultant ischemic tolerance. It is not clear at the present time which proteins are involved in development of preconditioning associated with 3-NPA, but such diverse proteins as the adenosine A3 receptor²⁸ or anti-apoptosis proteins like bcl-2¹⁵ have been suggested.

In our model, the chemical preconditioning conferred no protective effect on striatum despite its regional vulnerability to 3-NPA toxicity. This result is consistent with the previous data that ischemic preconditioning prevents neuronal injury in neocortex but not in subcortex after transient MCAO with the intraluminal thread model.²⁹ Memezawa et al³⁰ reported that selective neuronal necrosis in the caudoputamen was observed after only 15 minutes of MCAO, while neocortex was intact. Therefore, we believe that the limitation of 3-NPA–induced neuroprotection in striatum is due either to lack of collateral blood flow during MCAO or to increased vulnerability to ischemia in this region.

In conclusion, we have provided evidence to indicate that the activation of mitoK_ATP is involved in development of the delayed ischemic tolerance induced by a sublethal dose of the neurotoxin 3-NPA. We believe that mitoK_ATP is a novel target for study to clarify the mechanisms of endogenous protection from ischemia in brain.

	Acknowledgments

 
This work was supported by grants HL30260, HL46558, and HL50587 from the National Institutes of Health and American Heart Association Bugher Foundation Award 0270114N. The authors would like to thank Ken Grant for excellent technical assistance in confocal microscopic experiments.

Received July 22, 2002; revision received October 9, 2002; accepted November 4, 2002.

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