Application of in vivo ESR spectroscopy to measurement of cerebrovascular ROS generation in stroke

Free Radical Biology & Medicine, Vol. 35, No. 12, pp. 1619 –1631, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter

doi:10.1016/j.freeradbiomed.2003.09.013

Original Contribution APPLICATION OF IN VIVO ESR SPECTROSCOPY TO MEASUREMENT OF CEREBROVASCULAR ROS GENERATION IN STROKE MAYUMI YAMATO,* TORU EGASHIRA,†

and

HIDEO UTSUMI*

*Laboratory of Bio-function Sciences, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan; and † Department of Pharmacology, Oita Medical University, Oita, Japan (Received 15 April 2003; Revised 26 August 2003; Accepted 12 September 2003)

Abstract—This study used an in vivo ESR spectroscopy/spin probe technique to measure directly the generation of reactive oxygen species (ROS) in the brain after cerebral ischemia-reperfusion. Transient middle cerebral artery occlusion (MCAO) was induced in rats by inserting a nylon thread into the internal carotid artery for 1 h. The in vivo generation of ROS and its location in the brain were analyzed from the enhanced ESR signal decay data of three intra-arterially injected spin probes with different membrane permeabilities. The ESR signal decay of the probe with intermediate permeability was significantly enhanced 30 min after reperfusion following MCAO, whereas no enhancement was observed with the other probes or in the control group. The enhanced in vivo signal decay was significantly suppressed by superoxide dismutase (SOD). Brain damage was barely discernible until 3 h of reperfusion, and was clearly suppressed with the probe of intermediate permeability. The antioxidant MCI-186 completely suppressed the enhanced in vivo signal decay after transient MCAO. These results clearly demonstrate that ROS are generated at the interface of the cerebrovascular cell membrane when reperfusion follows MCAO in rats, and that the ROS generated during the initial stages of transient MCAO cause brain injury. © 2003 Elsevier Inc. Keywords—In vivo ESR, Focal ischemia reperfusion, Free radical, Reactive oxygen species, MCAO, MCI-186

INTRODUCTION

tive stress and other pathogenic mechanisms, such as excitotoxicity, calcium overload, mitochondrial cytochrome-c release, caspase activation, and apoptosis, in central nervous system ischemia [6,7]. Some studies have used microdialysis to demonstrate ROS generation during ischemia-reperfusion, by measuring salicylate hydroxylation [8 –10], chemiluminescence [11], and spin adduct signals [12,13] in the dialysate. However, a serious problem exists with microdialysis, in that insertion of the dialysis tube probably causes tissue damage, which may enhance the in vivo generation of free radicals [13]. Nitroxyl radicals react with O2⫺ in the presence of reducing agents [14,15] and with •OH [16,17], and hence suppress lipid peroxidation [18,19]. ROS and one- or twoelectron donating agents convert nitroxyl radicals to the corresponding hydroxylamines through one- or two-electron reduction [14,20]. These reactions were reported to reduce the electron spin resonance (ESR) signals for the nitroxyl radical [21,22], and we proposed the utilization of nitroxyl radicals as spin probes for in vivo ESR spectroscopy to determine ROS generation in vivo. When we administered nitroxyl radical probes to mice and rats in ex-

Stroke is a major cause of morbidity and mortality. Reactive oxygen species (ROS) and reactive nitrogen species are believed to be very important in ischemic nerve cell death. The involvement of ROS is suggested by the expression of superoxide dismutase (SOD) in nerve cells during cerebral ischemia [1], and by the relationship between the rise in H2O2 levels and edema formation in gerbils after ischemia-reperfusion [2]. In mice, SOD overexpression significantly reduced hippocampus failure after global cerebral ischemia-reperfusion [3], and glutathione peroxidase reduced the degree of infarction after transient middle cerebral artery occlusion (MCAO) [4]. Reduced SOD activity in mice exacerbated neuronal cell injury and edema formation after transient MCAO [5]. Using various mutant animals, Chan and colleagues implicated a combination of oxidaAddress correspondence to: Dr. Hideo Utsumi, Laboratory of Biofunction Sciences, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan; Tel: ⫹81 (92) 642-6621; Fax: ⫹81 (92) 642-6626; E-Mail: [email protected]. 1619

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M. YAMATO et al. Table 1. Structure, n-Octanol/Water Partition Coefficient, and Occurrence After Injection (% Dose of Injection) of Spin Probes Carboxy-PROXYL

Carbamoyl-PROXYL

Metoxycarbony-PROXYL

R Po/w Distribution

-COOH 0.02 Sham tMCAO

-CONH2 0.68

-COOCH3 8.7

Intact form (%) Total (%)

N.D. N.D.

N.D. N.D.

Basic structure R

Sham

tMCAO

Sham

tMCAO

0.019 ⫾ 0.010 0.049 ⫾ 0.006

0.008 ⫾ 0.009 0.054 ⫾ 0.026

0.015 ⫾ 0.005 0.10 ⫾ 0.024

0.036 ⫾ 0.013 0.31 ⫾ 0.11

N O

n-Octanol/water partition coefficient (Po/w) referred to the value of previous papers [31,36]. The occurrence of spin probe (% dose of injection) in ischemic hemisphere. At 15 min after the intra-arterially injection of spin probe, the animals were killed by transcardiac perfusion, and the brains were removed. The samples were evaluated with X-band ESR spectrometer at room temperature. N.D. ⫽ Not Determined. Each value represents the mean ⫾ SD of 5–7 rats.

perimental disease models, we observed enhanced ESR signal decay in vivo under conditions of hyperoxia [23], muscular ischemia-reperfusion [24], streptozotocin-induced diabetes [25,26], iron-overload [27], lung injury that was caused by diesel exhaust particles [21], and ammoniuminduced stomach injury [28]. In these cases, the administration of antioxidants, such as SOD, •OH scavengers, and iron chelators, suppressed the enhanced signal decay. The enhanced signal decay due to •OH has also been confirmed in in vitro experiments [22,29,30]. These results strongly indicate that the in vivo ESR/spin probe technique gives direct evidence of in vivo ROS generation as an index of the enhanced signal decay of nitroxyl probes. Recently, we succeeded in synthesizing 2,2,5,5,5-tetramethylpyrrolidine-l-oxyl (PROXYL) derivatives for use as spin probes with different permeabilities for the blood-brain barrier (BBB) and with different retention levels in the brain [31–33]. The ESR-CT images clarified that methoxycarbonyl-, carboxy-, and carbamoyl-PROXYL had maximal, zero-level, and intermediate membrane permeabilities for the BBB, respectively. These characteristics of the probes should be useful in localizing in vivo ROS generation in the brain. In this study, we applied for the first time the in vivo ESR/spin probe technique with three nitroxyl probes of different membrane permeabilities to transient MCAO rats, in order to provide direct evidence of in vivo ROS generation at the interface of the cerebrovascular cell membrane after reperfusion. The effect of MCI-186 (edaravone; 3-methyl-1-phenyl-2-pyrazolin-5-one), which is a newly developed antioxidant [34,35], on in vivo ROS generation, was also evaluated in the model rats. Furthermore, timedependent in vivo ROS generation was correlated with brain injury. MATERIALS AND METHODS

Chemicals Table 1 lists the three different nitroxyl radicals and their partition coefficients between phosphate-buffered saline

(PBS) and octanol, which were used as spin probes to detect ROS generation in the brain [31,36]. 3-Carbamoyl-2,2,5,5, 5-tetramethylpyrrolidine-l-oxyl (carbamoyl-PROXYL) and 3-carboxy-2,2,5,5,5-tetramethylpyrrolidine-l-oxyl(carboxyPROXYL) were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). 3-Methoxycarbonyl-2,2,5,5,5-tetramethylpyrrolidine-l-oxyl (methoxycarbonyl-PROXYL) was synthesized as described previously [32]. The isotonic solutions containing 100 mM of nitroxyl probes at a final concentration were prepared by mixing with saline, sterilized by filtration (0.2 ␮m), and stocked at ⫺20°C before use. The osmolarities of carbamoyl-, carboxy-, and methoxycarbonyl-PROXYL solutions were evaluated with an osmometer (OM 801, Vogel Gmbh, Giessen Germany) to be 298, 283, and 298 mOsm/kg, respectively. SOD (from bovine erythrocytes), catalase (from bovine liver), 1,3-dimethyl-2-thiourea (DMTU), and 2,3,5triphenyletrazolium chloride (TTC) were purchased from Wako Pure Chemical Industries (Osaka, Japan). 3-Methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) and egg yolk lecithin were donated by Mitsubishi Pharma Corporation (Tokyo, Japan) and NOF Corporation, respectively. SOD and catalase were once dissolved in saline at 5000 units/ ml, kept on ice bath, diluted with saline, and then administered into rats on the same day. The inactivated SOD was prepared by the method of Wetscher et al. [37]. All of the other reagents used were of the highest purity commercially available. Animals Male Wistar rats that weighed 180 –200 g were purchased from CLEA Japan (Tokyo, Japan). The rats were housed in a temperature- and humidity-controlled room, and fed commercial diet (MF; Oriental Yeast Co., Ltd., Tokyo, Japan) and water ad libitum. Reversible focal cerebral ischemia was induced with an intraluminal suture according to the model of MCA occlusion. In brief, anesthesia was induced with 3% halothane (Takeda Chemical Industries Ltd., Osaka, Japan) in air, and maintained with 1.6% halothane using a facemask. A 2 cm

ROS generation in MCAO rat

midline incision was made on the anterior neck, and the right common carotid artery (CCA), extra carotid artery (ECA), and internal carotid artery (ICA) were exposed. The CCA and ECA were ligated and a suture was placed around the ICA for ligation. An embolus was made by inserting a 4-0 nylon surgical thread into the ICA via a small incision. The MCA was occluded by advancing the embolus into the internal carotid artery to block the origin of the MCA, and the left common carotid artery was occluded with a clip to complete the MCAO. After 1 h of MCAO, the MCA was reperfused by withdrawing the embolus and clip. Rats in the sham-operated group were treated with the clip, but the nylon thread was not inserted. All of the procedures and animal care protocols were approved by the Committee on Ethics in Animal Experiments, Graduate School of Pharmaceutical Sciences, Kyushu University, and were conducted according to the Guidelines for Animal Experiments of the Graduate School of Pharmaceutical Sciences, Kyushu University. ESR measurements For the in vivo ESR measurements, 200 ␮l of an isotonic PROXYL solution (100 mM) was administered intra-arterially through a cannula in the left external carotid artery. The common carotid artery was occluded (using a clip) only during the ESR measurement, to minimize the effects of blood flow. ESR spectra were taken at regular intervals at the head domain using a 300-MHz ESR spectrometer (JEOL Co. Ltd., Akishima, Japan) with a loop-gap resonator (70 mm i.d. and 5 mm in length), as reported previously [25,28]. The power of the 300 MHz microwave was 14 dB (about 1.19 mW). The amplitude of the 100 kHz field modulation was 0.1 mT. The signal decay rate was determined from the semilogarithmic plot of signal intensity versus time after probe injection. SOD (1 or 10 units/rat), catalase (10 units/rat), or DMTU (100 mM) were administered simultaneously with the probe to confirm the relationship between the signal decay of the nitroxyl probe and ROS generation. MCI-186 (3 mg/kg dissolved in saline) was administered intravenously immediately after reperfusion. The in vitro ESR measurements were carried out at room temperature with an X-band ESR spectrometer (JES-1X; JEOL Co. Ltd.). The power of the 9.4 GHz microwave was 5.0 mW, and the amplitude of the 100 kHz field modulation was 0.1 mT. Detection of the spin probes in brain tissue The animals were killed by transcardiac perfusion of heparinized saline 15 min after intra-arterial injection of the spin probes. The brain was removed, and the isch-

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emic hemisphere was homogenized in ice-cold phosphate buffer. The homogenates were mixed with phosphate buffer and 1 mM potassium ferricyanide for determinations of intact nitroxyl radical and total nitroxyl radical, respectively. The homogenate was then assayed with an X-band ESR spectrometer. The levels of nitroxyl radical were determined from the ESR signal intensity by calibrating the signal intensity with that of Mn2⫹, which was used as the standard. Histological and pathological experiments Immediately after the rats were sacrificed by cervical dislocation, the brain was removed and 2 mm thick sections were sliced, starting 4 mm from the frontal pole. The sections were put in a plastic dish that contained 2% TTC, and incubated at 37°C for 30 min. The stained sections were photographed within 1 h. For hematoxylin and eosin (HE) staining, the brain was fixed by immersion in a 4% paraformaldehyde solution, and coronal blocks were then cut, dehydrated, and embedded in paraffin. The sections were stained with hematoxylin and eosin. Edema was estimated from the water content of the brain. The brain was immediately removed and the cerebellum and medulla oblongata were cut away and discarded. The brain was divided into ipsilateral and contralateral hemispheres. The wet weights of the tissue segments were measured. After drying in a desiccating oven for 24 h at 100°C, the dry weights of the segments were measured, and the percentage water content of the wet tissue was calculated. BBB disruption was estimated using Evans blue leakage. After ischemia, a 4% Evans blue solution (2.5 ␮l/g tissue in saline) was injected intravenously, the animals were killed by transcardiac perfusion of heparinized saline, and the Evans blue in the cerebral ischemic hemispheres was extracted with formamide at 37°C for 72 h. The Evans blue content of the extract was measured at 630 nm, and estimated in micrograms of Evans blue/ hemisphere by comparing it with the standard solution. Determination of the location of the spin probes in/out of membranes The location of the nitroxyl probes in or out of membranes was determined with ESR measurement of the probes in liposomal membranes and in mouse brain. Liposomes were prepared as described previously [38,39]. Various amounts of egg yolk lecithin were suspended in PBS containing 1 mM of the probes, and then observed with X-band ESR spectroscopy. For in vivo ESR measurement, isotonic solutions of the probes were intravenously injected to mice, and then in vivo spectra

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Fig. 1. Photographs of coronal sections after various periods of reperfusion, which followed 1 h of MCAO. MCAO model rats were prepared by inserting a 4-0 nylon surgical thread into the internal carotid artery via a small incision. For TTC staining, the rat brains were dissected after 2 min (A), 30 min (B), 3 h (C), 6 h (D), 12 h (E), and 24 h (F) of reperfusion, which followed 1 h of MCAO. Two-mm thick sections were obtained 4 mm from the frontal pole using a brain slicer, and stained with TTC. The unstained areas represent infarcted tissues. No discernible infarction was observed in the reperfusion period between 2 min and 3 h. In contrast, severe and widespread infarctions were observed after 12 h of reperfusion.

were recorded at head of mice as described previously [31]. Statistical analysis All of the results are shown as the mean ⫾ SD. Statistical significance was analyzed using either the two-tailed Student’s t-test or Dunnett’s test. A probability value of .05 was set as the minimum level of statistical significance. RESULTS

Brain damage in transient MCAO rats Brain damage was evaluated in the transient MCAO model based on the areas of infarction in the rat brain tissues at 2 min, 30 min, 3 h, 6 h, 12 h, and 24 h after reperfusion following the 1 h MCAO. Figure 1 illustrates the typical TTC-staining patterns of the rat brain tissues. The cerebral infarction region, which was unstained or faintly red, was clearly evident around the middle cerebral artery after 12 h and 24 h of reperfusion, whereas no discernible infarction was observed after 2 min and 30 min of reperfusion. The brain tissues in the sham-operated group showed no discernible areas of infarction (data not shown). HE-stained brain tissue after 30 min and 24 h of reperfusion after 1 h of ischemia are shown in Fig. 2A and B, respectively. After 24 h of reperfusion, the tissues were spongy, and neuronal cell necrosis (but not gliosis) was observed. The number of necrotic cells was higher in the cortex of the ischemic hemisphere than in that of the contralateral hemisphere. The brain section obtained 30 min after ischemia reperfusion seemed normal, with no evidence of leukocyte infiltration or damage. The histo-

logical results clearly demonstrate that distinct cerebral ischemia reperfusion damage does not occur until 30 min after reperfusion following MCAO. ESR spectra and location of spin probes in the rat brain after transient MCAO ROS generation in the rat brain was evaluated based on the in vivo ESR spectral changes of three different spin probes (Table 1). As reported previously, carboxyand methoxycarbonyl-PROXYL gave different ESR images and tissue distributions in the brain after intravenous administration [31–33]. Carboxy-PROXYL was present only in the cerebrovascular lumen, whereas methoxycarbonyl-PROXYL was found in both the lumen and cells. As shown in Table 1, carbamoyl-PROXYL has a partition coefficient that is intermediate between those of carboxy- and methoxycarbonyl-PROXYL, which indicates that cell membranes have low permeability for carbamoyl-PROXYL. Figure 3A shows typical ESR spectra for the three spin probes in the head regions of the rats after intraarterial administration. The ESR spectra of carboxy- and carbamoyl-PROXYL consisted of sharp triplet lines with identical peak heights and 1.65 mT of hyperfine splitting (hfs). The characteristics of carboxy- and carbamoylPROXYL were identical to that of the spin probe dissolved in saline, which indicates that these two spin probes remain within the cerebrovascular lumen. On the other hand, the peak heights of the methoxycarbonylPROXYL triplet lines were not the same, and the lines at lower and higher magnetic fields were slightly smaller than that at the medium magnetic field. Previously, we reported that methoxycarbonyl-PROXYL gave hydrophilic and lipophilic components at lower and higher

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Fig. 2. HE-staining patterns of rat coronal sections after 30 min (A) and 24 h (B) of reperfusion, following MCAO. The MCAO model rats were prepared as described in the legend to Fig. 1. The cellular morphology of the neurons at 30 min of reperfusion (A) is typical of normal neurons, but the coronal section of the 24 h reperfusion rat shows necrotic features, such as cellular swelling (B). Scale bar: 50 ␮m.

magnetic fields in the ESR spectra of mice after intravenous injection, because the probe partitioned into the aqueous and lipidic phases [31,33]. In the present experiment, a large modulation width was used in the ESR measurement due to the lower level of sensitivity in rat experiments. The larger modulation width was expected to decrease spectral resolution and distort the lines at lower and higher magnetic fields without splitting. To confirm the location of the nitroxyl probes in or out of membranes, the following 2 experiments were carried out, (i) in vitro measurement of the probes with liposomes, and (ii) in vivo measurement of the probes at mouse brain. Figure 4A, B, and C demonstrate typical X-band ESR spectra of the probes in PBS and in liposomes, and the expanded one in liposomes, respectively. All three probes gave isotropic triplet lines having the same hfs of 1.71 mT in PBS, and carboxyl-PROXYL in liposomes gave the same spectrum as that in PBS. Methoxycarbonyl-PROXYL in liposomes had two components, aquatic and lipidic, and the latter, hfs of which was mT, was clearly separated from the former at higher magnetic field (Fig. 4B, C). Observation of two components in liposomes was reported by Shimshick and McConnell [40]. The ratio of lipidic to aquatic components increased as increase of liposomal lipid, indicating that the lipidic signal should be due to the probes in membranes. The lipidic signal was clearly observed in in vivo spectrum of methoxycarbonyl-PROXYL after intravenous injection into mice (Fig. 4D). Carbamoyl-PROXYL, which has a partition coefficient that is intermediate between carboxy- and methoxycarbonyl-PROXYL, is expected to have intermedi-

ate-level membrane permeabilizing activity, and thus may penetrate cerebrovascular cell membranes with low efficiency. In fact, the spectrum of carbamoyl-PROXYL in liposomes had a shoulder at higher magnetic field, as indicated with arrows in Fig. 4B and C. The shoulder peak of carbamoyl-PROXYL was much less separated from the parent peak than that of lipidic signal of methoxycarbonyl-PROXYL (Fig. 4C), which made difficult to determine hfs of the shoulder peak. The intensity of the shoulder peak clearly depended on the amount of lipid, indicating that trace amount of carbamoylPROXYL can penetrate into membrane. In vivo spectrum of carbamoyl-PROXYL at mice head after intravenous injection was similar to that of carboxy-PROXYL, but the ratio of peak at higher magnetic field to that at center was 0.82 in carbamoylPROXYL and 0.89 in carboxy-PROXYL. The ratio of methoxycarbonyl-PROXYL was calculated to be ca. 0.65, with the aquatic peak at higher magnetic field to that at center. The intermediate ratio of carbamoylPROXYL implies that the probes may locate at the interface between aquatic and lipidic phase, probably at the interface of the cerebrovascular cell membranes. The locations of the three spin probes in the cerebrovascular lumen and brain cells were also investigated by determining probe recovery in the brain after transcardiac perfusion. Table 1 demonstrates the recovery percentages of the intact and total (intact and reduced form) spin probes in the brain after transcardiac perfusion. The total amounts of carboxy-, carbamoyl-, and methoxycarbonyl-PROXYL recovered from sham-operated brains were below the detection limit, 0.05% and 0.1% of the

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Fig. 3. Typical ESR spectra of spin probes at the head domain of a rat (A), and the signal decay curves in sham and transient MCAO rats (B). The spin probe (100 mM in a 200 ␮l volume) was administered intra-arterially to rats after 30 min of reperfusion, which followed a 1 h MCAO. Immediately after the injection, the ESR spectra at the head domain were measured at regular intervals using a 300 MHz ESR spectrometer (JEOL, Akishima, Japan) with a loop-gap resonator (70 mm i.d. and 5 mm in length).

injection dose, respectively. In a previous study, we confirmed that more than 3% of methoxycarbonylPROXYL was recovered from nonperfused brain 3 min after intravenous injection, and that the ESR-CT image gave a distinct image of methoxycarbonyl-PROXY in the brain [32]. The results shown in Table 1 and Fig. 4 strongly indicate that all three spin probes appear predominantly in the cerebrovascular lumen, and that carbamoyl-PROXYL penetrate cerebrovascular cell membranes with low efficiency, whereas methoxycarbonylPROXY readily crosses the membrane. The 30 min reperfusion period after MCAO caused slight increases in the total recovery percentages of carbamoyl- and methoxycarbonyl-PROXYL from 0.049% to 0.054%, and from 0.1% to 0.3% of the injection doses, respectively. These findings indicate that the characteristic locations of the three probes were retained in the brain for at least 30 min after reperfusion following MCAO. In vivo ROS generation after transient MCAO in rats The in vivo ESR spectra of the three probes in the head region decreased gradually with time after intra-

arterial injection. Figure 3B shows the semilogarithmic plots of the signal intensities as a function of time after the probes were injected in sham-operated (open circle) and transient MCAO (closed circle) rats. The signal decay rate of carbamoyl-PROXYL was clearly enhanced in transient MCAO rats as compared with control rats, whereas no differences in the signal decay rates were observed for carboxy-PROXYL. MethoxycarbonylPROXYL produced similar declines in the semilogarithmic plot for the transient MCAO and sham-operated rats, although a lag-time was noted for the transient MCAO rats. We reported previously that signal decay was enhanced in some oxidative injury models, such as ironinduced liver damage [27], diesel exhaust particle-induced lung damage [21], streptozotocin-induced diabetics [25,26], and ammonium-induced stomach injury [28]. In this study, the effect of antioxidants on the enhanced decay rate was used to evaluate in vivo ROS generation. Figure 5 demonstrates the signal decay rates of three spin probes in the sham-operated and transient MCAO (1 h ischemia and 30 min reperfusion) groups. Interestingly, distinct enhancement of the signal decay was observed with carbamoyl-PROXYL when the rat

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Fig. 4. Typical ESR spectra of the three nitroxyl probe, carboxy-, carbamoyl-, and methoxycarbonyl-PROXYL, in PBS (A), in liposomes (B), the expanded one in liposomes (C), and at the head domain of a mouse after intravenous injection (D). One hundred to 400 mM of egg yolk lecithin was suspended in PBS containing 1 mM of the probes, and then observed with X-band ESR spectroscopy as described previously [38,39]. For in vivo ESR measurement, isotonic solutions of the probes were intravenously injected to mice, and then in vivo spectra were obtained at head of mice as described previously [31]. The arrows indicate the lipidic component.

groups were compared; the signal decay rate of the transient MCAO group was increased ca. 1.7-fold more than that of the sham-operated group. Neither carboxyPROXYL nor methoxycarbonyl-PROXYL gave significant increases in the signal decay rate in either group, although methoxycarbonyl-PROXYL gave the highest decay rate in both groups, probably due to reduction by cellular reductants and/or redox enzymes [41– 44]. The signal decay rate of methoxycarbonyl-PROXYL tended to decrease in transient MCAO group. It might be due to less activity of cellular reductants and/or redox enzymes. The in vivo ESR measurements of enhanced signal decay were performed in the presence of antioxidants to confirm the generation of ROS in the transient MCAO rats. The simultaneous administration of Cu/Zn-SOD (10 units/rat) with the spin probe significantly suppressed the enhanced signal decay rate, whereas inactivated SOD did not have this effect (data not shown). One unit/rat of

Fig. 5. Signal decay rates of the three PROXYLs that were injected intra-arterially after the 30 min of reperfusion that followed 1 h of MCAO. The signal decay rates after 30 min of reperfusion following a 1 h MCAO (closed column) and the sham group (open column) were obtained from the slope of the semilogarithmic plot of signal intensity vs. the time after intra-arterial injection, as shown in Fig. 3. Each value represents the mean ⫾ SD. The values in parenthesis are the numbers of animals. * p ⬍ .005.

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Fig. 7. The effects of the spin probes on edema formation. The spin probes (50 mg/kg) were injected intravenously 0 and 90 min after reperfusion following MCAO, and the water content of the ischemic hemisphere was measured to evaluate edema formation. Each value represents the mean ⫾ SD. The values in parenthesis are the numbers of animals. * p ⬍ .01 and ** p ⬍ .005 for comparisons of the sham (open column) and MCAO groups (closed column); and # p ⬍ .05 and ## p ⬍ .005 for comparisons of the MCAO and antioxidant groups. Fig. 6. The effects of SOD, catalase, and DMTU on the enhanced signal decay rates. The signal decay rates were determined after 30 min of reperfusion, which followed 1 h of MCAO, as described in the legend to Fig. 5. The indicated dosages of SOD (A), catalase (B), and DMTU (C) were injected simultaneously with carbamoyl-PROXYL. Each value represents the mean ⫾ SD. The values in parenthesis are the numbers of animals. * p ⬍ .05, ** p ⬍ .01, and *** p ⬍ .005 for comparisons of the sham (open column) and MCAO groups (closed column); and # p ⬍ .05 for comparisons of the MCAO and antioxidant groups.

Cu/Zn-SOD increased the signal decay rate, but the increment was not significant. The in vivo effect of SOD on free radical reactions may be still obscure. Neither catalase (10 units/rat) nor 100 mM DMTU (•OH scavenger) suppressed the enhanced signal decay rate (Fig. 6). It should be noted that enhanced signal decay was completely suppressed by the •OH scavenger in dieselexhaust particle-induced lung damage [21,22]. The enhanced signal decay rate in the iron-induced liver damage model was suppressed by Trolox and an iron chelator, but not by the •OH scavenger [27]. None of the inhibitors had any effect on the signal decay rate in the sham-operated group. The results of this study suggest that the enhanced signal decay observed in MCAO rats is caused, not by the Fenton reaction, but through a reaction that involves SOD. Of the three probes used, only carbamoyl-PROXYL gave enhanced signal decay, implying that SOD-related ROS are produced in vivo at the interface of the cerebrovascular cell membrane in the MCAO rat. Involvement of spin probes in transient MCAO injury Nitroxyl radicals are known to act as antioxidants that reduce oxidative damage. Therefore, the presence of

carbamoyl-PROXYL should reduce the degree of transient MCAO damage, assuming that the enhanced signal decay of carbamoyl-PROXYL, which was used to confirm in vivo ROS generation, is related pathologically to the transient MCAO injuries. Figure 7 demonstrates the suppression of edema formation with carboxy-, carbamoyl-, and methoxycarbonyl-PROXYL. Only carbamoyl-PROXYL, which was injected intravenously at 0 and 90 min after reperfusion following MCAO, significantly suppressed edema formation to the control level, whereas methoxycarbonyl- and carboxy-PROXYL had partial or null effects. These findings confirm that ROS generation, which was monitored with carbamoylPROXYL at the interface of the cerebrovascular cell membrane in vivo, causes edema formation in MCAO rats. Kinetics of brain damage and ROS generation in transient MCAO rats A time-dependent evaluation of in vivo ROS generation was used to clarify the relationship between ROS generation and oxidative damage. Figure 8 demonstrates the time-dependent relationships between cerebrovascular ROS generation, edema formation, and vascular permeability. There was a distinct time lag in the process of oxidative damage. BBB dysfunction, which was evaluated by Evans blue leakage, was not apparent after MCAO until the rats were reperfused for 3 h, whereas leakage increased at 6 h, and significant leakage was obvious after 24 h of reperfusion (Fig. 8A). Edema formation, which was calculated as the water content of the ischemic hemisphere, was unchanged at 2 min and 30 min after reperfusion, whereas there was a significant

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Fig. 8. Kinetics of brain damage and ROS generation. BBB dysfunction (A), edema of the ischemic hemisphere (B), and ROS generation (C) were evaluated from the analyses of Evans blue leakage, water content, and enhanced signal decay rate of carbamoyl-PROXYL, respectively, which were determined after various periods of reperfusion that followed the 1 h MCAO. Each value represents the mean ⫾ SD. The values in parenthesis are the numbers of animals. * p ⬍ .05, ** p ⬍ .01, and *** p ⬍ .005 for comparisons of the sham (open column) and MCAO groups (closed column).

increase in water content after 3 h of reperfusion (Fig. 8B). The BBB dysfunction and edema formation data strongly suggest that distinct histological damage did not occur until 30 min after reperfusion following MCAO. On the other hand, the signal decay rate of carbamoylPROXYL was significantly enhanced after 30 min of reperfusion following MCAO, whereas histological damage did not occur until 3 h of reperfusion. Therefore, the characteristic occurrence of carbamoyl-PROXYL at the interface of the cerebrovascular cell membrane was apparently maintained during the measurements at 30 min of reperfusion after MCAO, which indicates that ROS generation at the interface of the cerebrovascular cell membrane induces brain damage. The significantly enhanced signal decay of carbamoyl-PROXYL persisted during 24 h of reperfusion (Fig. 8C). Signal reduction is reportedly enhanced by intracellular antioxidants [42,43] and/or redox enzymes [41,44]. These mechanisms may also participate in the enhancement of the signal decay at 12 and 24 h after reperfusion, because the spin probe seems to penetrate

into the cytoplasm through the damaged cerebrovascular barrier. MCI-186 (edaravone) suppresses in vivo ROS generation in transient MCAO rats MCI-186 was reported to decrease cerebral infarction in the transient MCAO model [34], and was found to block brain edema [45] by inhibiting lipid peroxidation [46,47]. Figure 9A shows the in vitro ESR spectra of carbamoyl-PROXYL with and without MCI-186. The presence of MCI-186 (3 mg/ml) did not change the shape or intensity of the carbamoyl-PROXYL signal after a 20 min incubation, which confirms that MCI-186 does not react directly with carbamoyl-PROXYL. Intravenous injection of MCI-186 (3 mg/kg) immediately after reperfusion completely suppressed cerebrovascular ROS generation in the transient MCAO rats (Fig. 9B). It is noteworthy that the therapeutic dose of MCI-186 (3 mg/kg), which is much lower than the dose used in the in vitro experiment, distinctly suppressed ROS generation in the transient MCAO rats. SOD is

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Fig. 9. The effects of MCI-186 on the in vitro (A) and in vivo (B) signal decay rates of carbamoyl-PROXYL. For the in vitro measurements, 2 mM of carbamoyl-PROXYL was incubated with or without MCI-186 (3 mg/ml) for 20 min, and the ESR spectra were obtained with a 9.4 GHz ESR spectrometer. For the in vivo signal decay measurements, MCI-186 (3 mg/kg) was administered intravenously immediately after reperfusion, and the signal decay rates of carbamoyl-PROXYL were evaluated as described in the legend to Fig. 5, using a 300 MHz ESR spectrometer. MCI-186 suppressed ROS generation at 30 min of reperfusion after the 1 h MCAO. Each value represents the mean ⫾ SD. The values in parenthesis are the numbers of animals. * p ⬍ .05 and # p ⬍ .05 for comparisons of the sham (open column) and MCAO groups (closed column), and for comparisons of the MCAO and spin probetreated groups.

reported to suppress the formation of lipid-derived radicals [48,49]. The ROS generation that was detected with carbamoyl-PROXYL was also suppressed by SOD (Fig. 6). These findings indicate that the ROS that are generated at the interface of the cerebrovascular cell membrane are in fact, lipid-derived radicals. DISCUSSION

We are the first to apply the in vivo ESR/spin probe technique to transient MCAO rats, thereby providing the first direct evidence of in vivo ROS generation at the interface of the cerebrovascular cell membrane in the transient MCAO model rat. The in vivo ESR measurements with the nitroxyl probe are noninvasive and provide evidence for the generation of ROS in the form of enhanced signal decay [21,25–28]. The enhanced signal decay induced by ROS has also been confirmed in in vitro experiments [22,29,30]. In the present study, ROS generation was evaluated using three different spin probes: carboxy-, carbamoyl-, and methoxycarbonyl-PROXYL (Table 1). As reported previously [31–33] and confirmed in this

article, carboxy-PROXYL localizes only to the cerebrovascular lumen, and methoxycarbonyl-PROXY passes through cerebrovascular cell membranes. CarbamoylPROXYL, which has an intermediate character with respect to membrane permeability, penetrates the cerebrovascular cell membrane with low efficiency. The membrane structure of synaptosomes was reported to be changed by transient ischemia [50]. Oxidative damage also changed the structure of LDL [51]. Such structural changes of bio-membrane may induce the enhancement of membrane permeability [39,52,53]. If the membrane permeability of the probes is changed during in vivo ESR observation, direct reduction by the redox enzymes in cells may enhance the in vivo signal decay rate of the nitroxyl probes. Any significant leakage of Evans blue was, however, not observed in the brain after 30 min of post-MCAO reperfusion (Fig. 8), indicating that the barrier capability was maintained in cerebrovascular cell membranes during in vivo ESR measurements. The recovery percentages of the probes in the transcardiac-perfused brain also confirmed that the characteristic locations of the three probes were retained in the brain after 30 min of post-MCAO reperfusion (Table 1). These data suggest that the enhanced signal decays observed with carbamoyl-PROXYL in the brain after 30 min of post-MCAO reperfusion should not be due to the change of membrane permeability of the probes. Of the three spin probes, only carbamoyl-PROXYL showed enhanced signal decay after 30 min of postMCAO reperfusion in rats (Fig. 5), and this enhanced signal decay was suppressed significantly by the administration of SOD (Fig. 6), which suggests that SODrelated ROS are produced at the interfaces of cerebrovascular cell membranes. SOD has been reported to catalyze O2⫺ dismutation [54] and to suppress the formation of lipid-derived radicals [48,49], which suggests that the ROS that were detected as the enhanced signal decay of carbamoyl-PROXYL are lipid-derived radicals. Quite recently, the Ministry of Health, Labor, and Welfare of Japan (the Japanese equivalent of the FDA) approved MCI-186 as a cerebral function-improving drug. MCI-186 decreased cerebral infarction in the transient MCAO model [34] and blocked brain edema [45] by inhibiting lipid peroxidation, which is reportedly one of the major causes of cerebral ischemic damage [46,47]. The present study demonstrates that the clinical dosage of MCI-186 suppresses cerebrovascular ROS generation in transient MCAO model rats, which re-confirms that the generated ROS are lipid-derived. Nitroxyl radicals were reported to protect the brain from injuries caused by ischemia-reperfusion through several processes, including radical scavenging [55–57]. In the present paper, carbamoyl-PROXYL reduced significantly edema formation after 3 h of reperfusion fol-

ROS generation in MCAO rat

lowing transient MCAO, whereas carboxy- and methoxycarbonyl-PROXYL showed little and slight protective effects, respectively. The solutions of all three probes examined were isotonic, indicating that the suppression of edema formation by carbamoyl-PROXYL was not due to the osmotic effect of the probe. The effect of carbamoyl-PROXYL on infarct formation was also reported in TTC-staining of the brain after transient MCAO [58]. The ROS that are generated at the interfaces of cerebrovascular cell membranes may be involved in brain injury, because carbamoyl-PROXYL penetrates the cerebrovascular cell membranes, albeit with low efficiency. Cerebral infarction was recognized after 12 h of reperfusion following MCAO. This time-course of infarction was consistent with that described in a previous report [34], in which it was also reported that after a 1 h ischemic insult, reperfusion for more than 24 h was needed for the infarction to clear, and that the infarction was diminished by treatment with antioxidants, such as PBN. The appearance of apoptotic cells in the hippocampal neurons also required a long period of reperfusion after 30 min of ischemia; in this case, apoptotic cells appeared after 6 d of reperfusion [59]. These results suggest that cerebral injuries, such as infarction, edema, and apoptosis, require longer reperfusion periods than the dozen or so hours in the MCAO rat model, although there have been few reports on the relationship between the progression of injury and ROS generation in transient MCAO. An important finding in this study was the observed time lag between in vivo ROS generation and brain injury (Figs. 1 and 8). The reperfusion period required for cerebrovascular ROS generation was much shorter than that required for brain damage, as estimated by TTC staining and edema formation. It has been suggested that oxidative injury plays a role in cerebrovascular damage after ischemia reperfusion, thus predisposing the brain tissue to damage [60]. In the clinical trial, MCI-186 was more effective when administered within 24 h. The clinical dose of MCI-186 suppressed cerebrovascular ROS generation in the transient MCAO model rats. The time lag between cerebrovascular ROS generation and brain injury demonstrated in this study may be related to the therapeutic window for antioxidant treatment in stroke victims. Acknowledgements — This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan and the Takeda Science Foundation. We thank Prof. Ryozo Oishi, Prof. Toru Iwaki, and Dr. Tomomi Ide of Kyushu University School of Medicine for helpful discussions, Mitsubishi Pharma Corporation and NOF Corporation for donating MCI-186 (edaravone) and egg yolk lecithin, respectively.

1629 REFERENCES

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1631 ABBREVIATIONS

Carbamoyl-PROXYL—3-carbamoyl-2,2,5,5,5-tetramethylpyrrolidine-l-oxyl Carboxy-PROXYL—3-carboxy-2,2,5,5,5-tetramethylpyrrolidine-l-oxyl DMTU—1,3-dimethyl-2-thiourea ESR— electron spin resonance HE— hematoxylin and eosin IR—ischemia-reperfusion MCAO—middle cerebral artery occlusion MCI-186 —3-methyl-1-phenyl-2-pyrazolin-5-one Methoxycarbonyl-PROXYL—3-methoxycarbonyl2,2,5,5,5-tetramethylpyrrolidine-l-oxyl ROS—reactive oxygen species SOD—superoxide dismutase TTC—2,3,5-triphenyltetrazolium chloride