Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Although the mechanisms of ischemic brain damage have not been clearly determined, accumulated experimental evidence suggests that production of free radicals is possibly one of the major factors involved (Flamm et al., 1978; Siesjo et al., 1989; Siesjo, 1992). Several reports have demonstrated that free radicals generated during ischemia play an important role in the development of neuronal damage (Chan, 1992; Kinouchi et al., 1991; Kitagawa et al., 1990; Cao and Phillis, 1994).

Free radical species of potential importance in cerebral ischemia include superoxide and hydroxyl radicals. The hydroxyl radical is highly reactive among oxygen radicals. Once excessive hydroxyl radicals are released, lipid peroxidation, which causes changes in the fluidity and permeability of membranes, is induced (Schmidley, 1990). Recently, oxygen radical formation was confirmed in the penumbral cortex during middle cerebral artery occlusion by the use of salicylate hydroxylation (Morimoto et al., 1996; Ginsberg et al., 1994).

MCI-186 is a potent scavenger of hydroxyl radicals inhibiting not only hydroxyl radicals but iron-induced peroxidative injuries (Murota et al., 1990; Watanabe et al., 1988). Furthermore, MCI-186 has been reported to have protective effects in both hemispheric embolization and transient cerebral ischemia in rats (Abe et al., 1988; Nishi et al., 1989; Oishi et al., 1989; Watanabe et al., 1994a,b). Recently, it has been proved that MCI-186 changes into OPB after the reaction with peroxy radicals in vitro (fig. 1) (Yamamoto et al., 1996). Thus, we are particularly interested in evaluating the effects of MCI-186 on ischemic brain damage and detecting this OPB in the penumbral area of the ischemic brain to emphasize the involvement of the reaction with radicals in its potent antiischemic effects.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Products of the attack by oxygen radicals on MCI-186. MCI-186 reacts with peroxyl radicals into OPB.

In the present study, the effects of MCI-186 on infarct areas, neurological deficits and rCBF in a rat thrombotic dMCA occlusion model was investigated. In addition, we have measured the oxidation products of MCI-186 in the penumbral cortex of a thrombotic dMCA occlusion model.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Animal preparation. Male Sprague-Dawley rats weighing 250 to 350 g were used. For the microdialysis study, the animals were anesthetized by inhalation of 2% of halothane in 70% nitrous oxide and 30% oxygen and spontaneous respiration. Body temperature was maintained at 37.5°C with a heating pad (K-module Model K-20, American Pharmaseal Company, Valencia, CA) during the operation. Head temperature was monitored and maintained at 37.0°C throughout the experiment by a warming lamp placed above the head. CCA occlusion was performed according to the method of Chen et al. (1986). The MCA thrombosis model in the rat was described previously (Umemura et al., 1993). A catheter for the administration of rose bengal was placed in the femoral vein. The left CCA was occluded permanently with a 3-0 suture. After the occlusion of the left CCA, the scalp and temporalis muscle were folded over and a subtemporal craniotomy was performed with a dental drill under an operating microscope to open an elliptical bone window (4 mm in major axis).

Photoirradiation. The window was irradiated with green light; and the entire irradiated segment, including the Y-shaped juncture of the frontal and parietal branches of the left MCA, became thrombotically occluded. Photoirradiation with green light (wave length, 540 nm) was achieved by use of a xenon lamp (L4887: Hamamatsu Photonics, Hamamatsu, Japan) with a heat-absorbing filter and a green filter. The light source was a short-arc type, Super-Quiet xenon lamp. The xenon lamp was chosen for the high brightness and stable light intensity necessary for irradiating a small area. The light was prefiltered and concentrated by an elliptical reflector that had a special coating for efficient absorption of infrared and ultraviolet radiations. The remaining visible light was modified spectrally by filters to produce pure green light with a bandwidth of 80 nm, centered at 540 nm. Elimination of infrared and ultraviolet radiations was essential, because they can heat up and damage biological tissues. The irradiation was directed by an optic fiber 3 mm in diameter mounted on a micromanipulator. The head of the optic fiber was placed on the window in the skull base at a distance 2 mm above the vessel, and it provided an irradiation dose of 0.62 W/cm². The local brain temperature in the brain was not modified by the light irradiation. Rose bengal (20 mg/kg, Wako, Osaka, Japan) was injected intravenously. The complete MCA photothrombotic occlusion was observed under a microscope approximately 4 min after i.v. injection of rose bengal. Photoirradiation was continued for a further 10 min. The drugs were administered i.v. 5 min and 35 min after dMCA occlusion. The photochemical reaction between rose bengal and the green light caused endothelial cell injury followed by platelet adhesion and the formation of a platelet-rich thrombus. The neck and head wounds were closed, the catheter was removed and the animal was returned to its home cage when fully awake. In the sham operation group, each animal's vessel was identified but was not occluded.

Behavioral testing. For behavioral testing and histological studies, four groups of rats were studied: sham-operated animals, vehicle-administered controls and MCI-186-treated animals at doses of 1 and 3 mg/kg. Each group had 10 animals. According to the method of Bederson et al. (1986), neurological deficits such as hemiplegia were evaluated in a posture test 24 h after the occlusion. This evaluation was carried out in a blind fashion, the behavioral evaluations and experiments being performed by different investigators.

Histological procedures. After the neurological examination, the brain was removed immediately and cut into six coronal slices (1 mm in thickness) by a rat brain slicer (Zivic-Miller Lab., Inc., Allison Park, PA). For determining the infarction area, each section was stained with the TTC (Wako, Osaka, Japan) solution. Infarcted areas were not stained by TTC (Isayama et al., 1991). The size of the infarcted area was measured by a computerized image analysis system (Nexus, Tokyo, Japan). Infarct volumes were derived at the position between slice 1 and slice 6 by means of sequentially numbered areas.

Measurement of rCBF. Measurement of rCBF was determined by the IAP quantitative autoradiographic technique described by Sakurada et al. (1978). Under anesthesia with ether, cannulas were implanted into the femoral artery and vein, and the animals were placed in an apparatus for fixation 1 day after dMCA occlusion. Two hours or more after recovery from anesthesia, 740 kBq of IAP (specific activity, 20 GBq/mmol, New England Nuclear Corp., Boston, MA) was infused i.v. over a period of 45 s, during which time repeated blood samples were taken from the femoral artery catheter. The blood samples were collected in drops onto filter papers disks (1.5 cm square). At 45 s rats were decapitated, and the brain was rapidly removed from the skull and frozen in powder dry ice. The radioactivity in the brain was measured by autoradiography. The brains were cut into sections, 20 µm in thickness, by means of a cryomicrotome (Bright 5030, UK) at 20°C. A series of five serial sections were prepared approximately every 500 µm from 1.2 mm anterior to bregma, and numbered from the front side. The autoradiographic sections were then attached to radiographic film (IX150, Fuji Film, Tokyo, Japan), which was exposed for 2 weeks, together with a set of standard film (RPA504L, Amersham, Arlington Heights, IL). The filter paper disks containing the blood samples were transferred to scintillation counter vials and suspended in 10 ml of scintillator solution (ACS-II, Amersham) followed by measurement of blood radioactivity in a liquid scintillation counter (TRI-CARB, Packard Instrument Co., Rockville, MD).

View larger version (136K): [in this window] [in a new window]   Fig. 5.   The image of [14C]IAP autoradiograms at a place 0.7 mm from bregma in rat 1 day after thrombotic dMCA occlusion. (a) frontoparietal cortex, motor area; (b) frontoparietal cortex, somatosensory area.

Drug administration methods. MCI-186 was synthesized by Mitsubishi Chemical Corporation (Tokyo, Japan). MCI-186 was dissolved in 1 N NaOH and titrated to pH 7.4 with 1 N HCl to prepare concentrations of 10 and 30 mg/ml. Drugs used in this study were physiological saline (vehicle, 1 ml/kg) and MCI-186 (1 or 3 mg/ml/kg, bolus injection). The drugs were administered i.v. 5 min and 35 min after dMCA occlusion.

Preparation of rat brain homogenates. Five male Sprague-Dawley rats weighing 250 to 350 g were decapitated after transcardiac perfusion with 50 ml of saline at 100 mm Hg while under pentobarbital anesthesia. The cerebrum was removed and homogenized in 9 volumes of 67 mM phosphate buffer (pH 7.4) in an ice-cold bath. The homogenate was centrifuged at 900 × g for 10 min. The supernatant was incubated with 14.8 to 20.6 µM [¹⁴C]MCI-186 at 37°C for 30 min. Five separate experiments were carried out with use of five rats.

Measurement of [¹⁴C]MCI-186 and radical reaction products. To 500 µl of sample in a glass tube was added 200 µl of 4-methyl-MCI-186 acetonitrile solution (1 mg/ml) which was used for an antioxidant, 100 µl of MCI-186 acetonitrile solution (1 mg/ml) and 3.7 ml of acetonitrile. The tube was placed on a vortex mixer for 1 min. The supernatant and precipitation were separated by centrifugation at 1600 × g for 5 min. The supernatant was evaporated to dryness in rotary evaporator at 50°C, the residue was dissolved in 400 µl of HPLC solvent A. The solution was filtered through a millipore filter (0.45 µm), and 250 µl of aliquot was applied in HPLC. To the precipitation in a glass tube was added 2 ml of 2 N NaOH. The tube was heated at 90°C for 10 min in a water bath, and titrated to pH 7.5 with 1 N HCl, which was then extracted with 10 ml of butanol for 10 min and centrifuged at 1600 × g for 5 min. The organic phase was evaporated to dryness in rotary evaporator at 50°C, the residue was dissolved in 400 µl of HPLC solvent A. The solution was filtered through a millipore filter (0.45 µm), and 250 to 300 µl of aliquot was injected in HPLC.

High performance liquid chromatography. The apparatus was composed of a pump (L-6200, Hitachi, Tokyo, Japan), an injector (7161, Rheodyne, Cotati, CA), a column oven (TCM, Waters Associates, Milford, MA), a radioactive detector (Ramona-5-LS, Raytest, Germany) a fraction collector (model 202, Gilson, Middleton, WI) and a UV detector (SPD-6A, Shimadzu, Kyoto, Japan). The analysis was done with an L-column ODS (4.6 mm internal diameter × 250 mm, Chemicals Inspection and Testing Institute, Tokyo, Japan). The column temperature was 50°C. The analysis required gradient elusion. The eluents were: Solvent A, methanol-100 mM phosphate buffer (pH 5.7) (30:70, v/v) with 5 mM TBA; solvent B, methanol-100 mM phosphate buffer (pH 5.7) (60:40, v/v) with 5 mM TBA. The gradient used was 3 min of 95% solvent A, linear programming during 17 min to 100% solvent B, 15 min of 100% solvent B, reequilibration to 95% solvent A. The flow rate was 1 ml/min, and the elute was monitored radioactivity and UV absorption at 254 nm.

Microdialysis study. A craniectomy for microdialysis was made above the left frontoparietal cortex with a dental drill. A small incision was made in the dura and pia-arachnoid with a 27-gauge needle to permit implantation of a microdialysis probe without a possible compression injury to the cortex. A microdialysis probe (2-mm dialysis membrane, CMA/11; Carnegie Medicin, Stockholm, Sweden) was stereotaxically implanted in the left dorsolateral parietal cortex. We positioned the microdialysis probe proximate to a motor area. The probe was continuously perfused with Ringer's solution (147 mM NaCl, 4 mM KCl, 1.3 mM CaCl₂) at a flow rate of 2 µl/min by means of a microinfusion pump (CMA100; Carnegie Medicin, Stockholm, Sweden). The perfusion fluid was switched to [¹⁴C]MCI-186 (9.1-11.3 mM) in Ringer's solution 10 min after dMCA occlusion. The MCI-186 perfusion was continued for a further 90 min. The perfusate sample was collected. In the sham-operated group; MCI-186 was perfused by the same method as in the dMCA occlusion group. After the [¹⁴C]MCI-186 perfusion, the rats were decapitated. The brains were quickly removed, and 400 µl of water, 200 µl of 4-methyl-MCI-186 acetonitrile solution (1 mg/ml), 100 µl of MCI-186 acetonitrile solution (1 mg/ml) and 2.9 ml of acetonitrile were added to the left cortex. The mixture was homogenized with a Polytoron (Kinematica, Switzerland). The supernatant and precipitation were separated by centrifugation at 1600 × g for 5 min.

Statistical analysis. Statistical analysis of the infarction size was conducted by analysis of variance, and paired comparisons with a control group were performed by Dunnett's test. Neurological deficits comparisons with a control group were analyzed by nonparametric Dunnett's test. The OPB levels were statistically analyzed by the student's t test. The statistical analysis was performed by Super ANOVA and Yukms. Data were expressed as mean ± S.E.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

The preliminary experiments revealed that MCI-186 did not exert any influence on the brain or body temperature during the early stage of the drug treatment, compared with the vehicle-treated control (data not shown).

Influence of MCI-186 on infarct volume, neurological deficits and rCBF. There were no areas of ischemia in the cerebral hemispheres of the sham-operated animals. The infarction areas were predominantly located in the frontal and sensorimotor regions of the cortex. Figure 2 demonstrates a typical example of a cerebral infarction in this study. Infarct volume in the high dose of MCI-186 was significantly (P < .05) reduced compared with the vehicle-treated group (fig. 3).

View larger version (92K): [in this window] [in a new window]   Fig. 2.   Coronal sections of the TTC-stained rat cerebrum 1 day after thrombotic dMCA occlusion. Each section was cut out coronally into 1-mm-thick slices from the frontal lobe. The order of numbers indicates the position from the frontal lobe. The upper was receiving vehicle; the lower was receiving MCI-186 (3 mg/kg).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of MCI-186 on infarct volume at 1 day after dMCA occlusion. Results are mean ± S.E. of 10 animals. *P < .05 in comparison with the corresponding vehicle-treated group (Dunnett's test).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Effect of MCI-186 on neurological deficits at 1 day after dMCA occlusion. Results are mean ± S.E. of 10 animals. *P < .05 in comparison with the corresponding vehicle-treated group (Nonparametric Dunnett's test).

Formation of the radical reaction products of MCI-186 in brain homogenates. [¹⁴C]MCI-186 was incubated with rat brain homogenates for 30 min at 37°C. Table 2 shows the extraction rate of radioactivity and the composition of extracted radioactivity on HPLC. The recovery ratio of radioactivity to acetonitrile/water supernatant from the preincubation sample and the reaction sample were about 100% and 60%, respectively. About 70% of radioactivity in precipitation from the reaction sample was extracted to butanol after alkaline hydrolysis. Figure 6a shows a typical chromatogram of standards of MCI-186 and OPB dissolved in HPLC solvent A. The peaks of MCI-186 and OPB can be separated. As shown in figure 6b, except for the peak of [¹⁴C]MCI-186, no peak was observed in a preincubation sample, which demonstrated that MCI-186 was not reacted during sample preparation. Figure 6c shows a chromatogram of a supernatant from the reaction sample. The compositions of MCI-186 and OPB were about 70% and 6%, respectively. As shown in figure 6d, the fraction of radioactivity was mostly OPB in the extract of the precipitation from the reaction sample. The composition of OPB was 90%, and MCI-186 was not detected. Figure 6e shows a typical chromatogram of the precipitation sample spiked with the standard of OPB, which was monitored by UV absorption. Retention time for OPB was shifted in every precipitation sample, so monitoring the retention time for OPB were carried out by spiked the standard of OPB.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Identification of OPB in rat brain homogenate. (a) Chromatogram of the standards of MCI-186 and OPB which were detected by UV absorption (254 nm); (b) chromatogram of a supernatant of a preincubation mixture of [14C]MCI-186 and rat brain homogenate which was monitored by radioactivity; (c) chromatogram of a supernatant of a reaction mixture of [14C]MCI-186 and rat brain homogenate which was monitored by radioactivity; (d) chromatogram of an extract of a precipitation from a reaction mixture of [14C]MCI-186 and rat brain homogenates which was monitored by radioactivity; (e) Chromatogram of an extract of a precipitation from a reaction mixture of [14C]MCI-186 and rat brain homogenates spiked with the standard of OPB which was monitored by UV absorption (254 nm).

Formation of the radical reaction products of MCI-186 in ischemic penumbra. After the perfusion of [¹⁴C]MCI-186 to the ischemic penumbral region by the microdialysis method, the radioactivity of the brain cortex was extracted to acetonitorile and butanol and was analyzed by HPLC. The OPB were not detected in the microdialysis perfusate taken from both the sham-operated and dMCA occlusion cortex (data not shown). The recovery ratio of the radioactivities to the supernatant from the rat cortex/acetonitrile/water mixture were 87.1 ± 0.9%, 94.2 ± 1.4% in the sham-operated and dMCA occlusion group, respectively. The fraction of radioactivity was mostly MCI-186. On the other hand, OPB was detected mainly from the extract after alkaline hydrolysis of the precipitation in both groups (fig. 7). The extraction ratio of radioactivity to butanol from the precipitation were 62.3 ± 3.3% and 53.9 ± 4.8% in the sham and dMCA occlusion group, respectively. Contents of OPB in the extraction was shown in table 3. The OPB significantly elevated throughout thrombotic dMCA occlusion (P < .05).

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Typical chromatograms of an extract of a precipitation from a rat cerebral cortex perfused with [14C]MCI-186. The preparation of an extract sample is described in the text.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The thrombotic occlusion of dMCA was induced by the photochemical reaction between rose bengal and green light, which causes endothelial injury followed by platelet adhesion and formation of a platelet and fibrin-rich thrombus without affecting dura mater (Umemura et al., 1993; Matsuno et al., 1993). The infarction of this model was observed mainly in the cortical area ipsilateral to the occluded MCA 1 day after the thrombus formation. Chen et al. (1986) and Markgraf et al. (1993) reported that the occlusion of dMCA by electrocoagulation induced the infarction limited to the cortex after MCA occlusion. The site of the infarction in our model was similar to the site in these models.

The present results showed that MCI-186 improved both the neurological deficits and the infarction area. This was supported by the observation that appearance of neurological deficits in the posture test has a good correlation with infarct area (Bederson et al., 1986). On the contrary, MCI-186 had no effect in rCBF 1 day after dMCA occlusion. This suggests that it would be difficult for the histological outcome to correlate with the changes in rCBF 1 day after dMCA occlusion. Hakim et al. (1992) have also reported that there is no predictable correlation between the time-dependent changes affecting rCBF and the histological changes 2 days after MCA occlusion. Recently, it was demonstrated that the relationship between local cerebral glucose utilization and rCBF was disturbed in the acute focal ischemic penumbra and led to irreversible ischemic damage (Back et al., 1995). MCI-186 might affect the metabolism-flow uncoupling during the first hours after dMCA occlusion. Further investigations in acute stage are needed.

The cerebral ischemia causes the NO synthase induction of endothelium, monocyte and macrophage. In the ischemic neurons, the excessive activation of the N-methyl-D-aspartate receptor also causes the Ca⁺⁺-dependent stimulation of NO synthase (Dawson et al., 1991; Garthwaite, 1991; Synder, 1992). NO rapidly reacts with superoxide in aqueous solution to form peroxynitrite. Under physiological conditions, peroxynitrite is easily photonated to form peroxynitrous acid. After photonation, peroxynitrous acid is decomposed to the hydroxyl radical (Lipton et al., 1993). MCI-186 is thought to react with the hydroxyl radical which was induced by NO. Therefore, MCI-186 might reduce the neuronal damage by NO production during ischemia. On the other hand, it is not known that MCI-186 might alter NO synthase and might react with NO itself. MCI-186 might not affect rCBF increase by NO.

Zaleska and Floyd (1985) reported that there is hydroxyl radical-dependent and iron-dependent lipid peroxidation in rat brain homogenates. Additionally, MCI-186 has been reported to prevent lipid peroxidation in rat brain homogenates (Watanabe et al., 1994a), and MCI-186 changes into OPB after the reaction with peroxy radicals (Yamamoto et al., 1996). In the present study, we demonstrated that OPB was isolated from the reaction mixture, including rat brain homogenates and MCI-186. This finding suggests that MCI-186 reacts with free radicals in OPB inhibiting the lipid peroxidation in rat homogenates and that OPB could also be isolated from rat brain homogenates.

In the present study, OPB was increased in the cortical penumbra of the rat ischemic brain after 90 min of MCI-186 perfusion in this area. These findings demonstrate that MCI-186 can react with oxygen radical species in the penumbra area of the ischemic brain and therefore might show the protective effect on the ischemic brain damage.

Recently, Morimoto et al. (1996) have reported that hydroxyl radical formation was increased in the penumbral cortex during the MCA occlusion by use of the salicylate hydroxylation method. They positioned the microdialysis probe proximate to the penumbra which was defined as having rCBF of 20 to 40% of control (Takagi et al., 1993; Back et al., 1995). We positioned the microdialysis probe more proximate to the motor area in which rCBF was less than 44% of control. Thus, there is no apparent discrepancy of the ischemic insult in the penumbra between our model and Morimoto's. Taking these findings and Morimoto's into consideration, there seems to be no doubt that the oxygen radical species, including the hydroxyl radical, is increased in the penumbra of the permanent ischemic brain, where peroxidative attack by those toxic radical species may accelerate ischemic injury.

In focal ischemia, the participation of free radicals in the production of ischemia or reperfusion injury was suggested by the effectiveness of free radical scavenging drugs (Cao and Phillis, 1994, Murota et al., 1990; Nishi et al., 1989; Oh and Betz, 1991) and superoxide dismutase (Imaizumi et al., 1990; Kinouchi et al., 1991). However, direct measurement of free radicals in the ischemic penumbral cortex was only performed by use of the salicylate hydroxylation method which could detect hydroxyl radicals (Morimoto et al., 1996; Ginsberg et al., 1994). In the present study, the OPB content was elevated in the ischemic penumbral cortex after thrombotic dMCA occlusion, which suggests that MCI-186 reacts with free radicals in vivo.

In conclusion, MCI-186 improved neurological deficits and reduced infarct area 1 day after occlusion when it was administered after thrombotic occlusion of the dMCA. In the penumbra, MCI-186 mainly changed into OPB. Our results may suggest that the reactive oxygen species contribute to brain injury after permanent focal ischemia and the treatment with MCI-186 in permanent focal ischemia would be beneficial.