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Inhibitory Effect of MCI-186, a Free Radical Scavenger, on Cerebral Ischemia Following Rat Middle Cerebral Artery Occlusion
ISSN 0306-3623/98 $19.00 1 .00 PII S0306-3623(97)00311-X All rights reserved
Gen. Pharmac. Vol. 30, No. 4, pp. 575–578, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Inhibitory Effect of MCI-186, a Free Radical Scavenger, on Cerebral Ischemia Following Rat Middle Cerebral Artery Occlusion Atsuhiro Mizuno, Kazuo Umemura* and Mitsuyoshi Nakashima Department of Pharmacology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-31, Japan ABSTRACT. 1. In this study, we investigated the effect of a radical scavenger, MCI-186 (3-methyl1-phenyl-2-pyrazolin-5-one), on cerebral damage induced by rat middle cerebral artery (MCA) occlusion, and further measured the hydroxyl radical level at the ischemic border zone using a microdialysis technique. 2. MCI-186, at a dose of 3 mg/kg per 30 min, was administerated as a continuous infusion two times for 30 min, starting 20 min and then 80 min after Rose Bengal injection. 3. MCI-186 significantly (P,0.05) reduced size of cerebral damage 24 hr after MCA occlusion and significantly (P,0.05) reduced hydroxyl radical level. gen pharmac 30;4:575–578, 1998. 1998 Elsevier Science Inc. KEY WORDS. Hydroxyl radical, 2.3-DHBA, microdialysis, MCI-186, cerebral ischemia
INTRODUCTION Reactive oxygen species are generated within brain tissue during ischemic injury (Demopoulos et al., 1980; Raichle, 1983). However, the sources of reactive oxygen species implicated in ischemic injury have not yet been clearly identified. It has been proposed that the mechanism of oxygen free radical generation may include the stimulation of the xanthine–xanthine oxidase system in cerebral vessels (Betz, 1985) and activation of neutrophils (Matsuo et al., 1995) and arachidonic acid metabolism (Simonian and Coyler, 1996). Free radical-induced lipid peroxidation, oxidation of proteins and nucleic acids may contribute to the neuronal injury after cerebral ischemia. Damage to the cell membrane, for example, may result in dysfunction of essential membrane activities. MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one), a newly synthesized free radical scavenger, exerts beneficial free radical scavenging and antioxidant characteristics (Watanabe et al., 1994) and prevents the peroxidative vascular endothelial cell damage caused by hydroperoxyeicosatetraenoic acid (Watanabe et al., 1988) in in vitro study. Furthermore, MCI-186 has been tested in various different experimental models for evaluatuation of its protective effects in cerebral ischemia/reperfusion (Abe et al., 1988; Nishi et al., 1989; Oishi et al., 1989; Watanabe et al., 1988) and myocardial ischemia/ reperfusion (Minhaz et al., 1996). Thus, it seems that the neuroprotective effects of MCI-186 are attributable to the inhibition of free radical production. In this study, we investigated the effect of MCI-186 on ischemic damage using middle cerebral artery (MCA) thrombosis model in rats, and determined whether its effect might be responsible for hydroxyl radical scavenging action by the salicylate hydroxylation technique using microdialysis, which has been suggested as a chemical trap for potential hydroxyl radical formation (Fig. 1) (Floyd et al., 1984, 1986; Morimoto et al., 1996). *To whom correspondence should be addressed. Received 23 May 1997.
MATERIALS AND METHODS
Animal preparation Male Wistar rats (SLC Experimental Animal Co., Ltd., Shizuoka, Japan), weighing 240–270 g, were anesthetized with a 1.5% halothane and oxygen gas mixture. Body temperature was maintained at 37.58C with a heating pad (K-Module, Model K-20, America Pharmaseal Co.), and a catheter for the administration of Rose Bengal or other agents placed in the femoral vein. MCA occlusion caused by photochemically induced thrombosis in the rat has been described by Umemura et al. (1993, 1994, 1996). In brief, the scalp and temporal muscles were reflected, and a subtemporal craniotomy was performed using a dental drill under an operating microscope. A 3-mm-diameter circular area of the window was illuminated with green light and the entire illuminated segment, including the proximal end of the lenticulostriate branch, became thrombotically occluded. Photoillumination by green light (540-nm wavelength) was achieved by using a xenon lamp (L4887, Hamamatsu Photonics, Japan) with a heat-absorbing filter and a green filter. Irradiation was directed by a 3-mm-diameter optic fiber mounted on a micromanipulator. The head of the optic fiber was placed on the window in the skull base, and Rose Bengal (20 mg/kg) was injected intravenously and photoillumination was performed for 10 min. MCI-186 (3 mg/kg per 30 min) or vehicle was administered for 30 min intravenously, starting at 20 min and then 80 min after Rose Bengal injection in each of seven animals. The incisions were closed after the operation. Animals were killed by decapitation 24 hr after MCA occlusion, the brain was immediately excised under the pentobarbital anesthesia and sectioned in 1-mm-thick slices from 1.5 mm anterior to 4.5 mm posterior to the bregma with a microslicer, and then six consecutive slices were stained with 2,3,5-triphenyltetrazolium chloride (Katayama, Japan). Photographs of the posterior view of the slices were then taken. For the slice, the ratio of area of cerebral damage to the entire area of the corresponding cerebrum was calculated using a computerized image analysis system. The amount of striatum damage in four slices from 0.5 mm anterior to 3.5 mm posterior to the bregma was measured.
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FIGURE 1. Products of the attack by hydroxyl radical on the salicylate molecule. Salicylate metabolites to 2.3- and 2.5-DHBA, and a small amount of catechol by hydroxyl radical. The 2.3DHBA concentration was measured and the hydroxyl radical level was estimated.
Measurement of hydroxyl radical at ischemic border zone The hydroxyl radical at the ischemic border zone was measured as a 2.3-dihydroxybenzoic acid (DHBA) concentration in the dialysate using a microdialysis technique. A microdialysis probe (2-mm dialysis membrane, CMA/12, Stockholm, Sweden) was stereotaxically implanted in the right dorsolateral parietal cortex (0.5 mm posterior to the bregma, 4 mm lateral to the midline and 3 mm ventral to the cortex surface). This probe inserted into the ischemic border zone, which was reported in our previous studies (Umemura et al., 1996). The probe was continuously perfused with Ringer’s solution composed of 147 mM NaCl, 4 mM KCl, 2.3 mM CaCl2 and 5 mM sodium salicylate at a rate of 2 ml/min, starting at 30 min before implantation until the end of the experiment, using a microinfusion pump (CMA/102). MCA occlusion was performed 90 min after implantation of the microdialysis probe, dialysates were collected at 15-min intervals during the 180 min after MCA occlusion. MCI186 (3 mg/kg per 30 min) or vehicle was administered for 30 min intravenously, starting 20 min and then 80 min after Rose Bengal injection in each of five animals.
HPLC system for determination of 2.3-DHBA Salicylate metabolites to 2.3- and 2.5-DHBA, and a small amount of catechol by hydroxyl radical. 2.5-DHBA is generated via the cytochrome P-450 system (Halliwell et al., 1991; Ingelman-Sundberg et al., 1991), thus we measured the 2.3-DHBA concentration and estimated the hydroxyl radical level. 2.3-DHBA was analyzed by the HPLC system (Shimadzu, Tokyo, Japan) customized with coulometric detector (ESA, Bedford, MA). The guard cell and detector 1 or 2 of analytical cell were operated at potentials of 10.35 V, 20.1 V and 10.30 V, respectively. The separation of different compounds was accomplished by an analytical column (TSK Gel 80-Ts; 15034.6-mm inner diameter, Tosoh, Tokyo, Japan) with a 5-mm particle size. The column was used at 408C with a mobile phase consisting of 30 mM disodium hydrogen phosphate, 30 mM phosphoric acid, 10 mg ethylenediamine tetraacetic acid disodium salt and 1 mg 1-pentanesulfonic acid sodium salt to a total volume of 1 l. The flow rate was 1.0 ml/min. Calibration curves were run daily with 2.3DHBA standard. From peak height of the external standards, dialysate concentration was calculated.
Statistical analysis Data are reported as means6SE. Statistical analysis was made with an unpaired Student’s t-test or two-way ANOVA for comparison between two groups. P,0.05 was considered significant. RESULTS
Amount of cerebral damage The dorsolateral cortex and striatum were damaged in the control group. MCI-186 (3 mg/kg per 30 min) significantly (P,0.05) re-
FIGURE 2. Protective effect of MCI-186 (3 mg/kg for 30 min) (open column) on cerebral damage in seven animals compared with the control group (solid column). MCI-186 was administered as continuous infusion two times (each for 30 min), starting 20 min and then 80 min after Rose Bengal injection. Data represent mean6SE of seven animals. *P,0.05 vs. control group.
duced the amount of cerebral damage in the cortex but not in the striatum (Fig. 2).
Measurement of 2.3-DHBA and inhibitory effect of MCI-186 The mean preischemic values for 2.3-DHBA concentration were 35.7466.80 and 40.6562.93 pmol/ml in the control group and the group treated with MCI-186, respectively, and there was no significant difference between the groups. After the MCA occlusion, the concentration of 2.3-DHBA increased by 1.7-fold at 30 min compared with baseline levels and remained high throughout the 180min observation period. MCI-186 (3 mg/kg per 30 min) significantly (P,0.05) suppressed the 2.3-DHBA concentration that was increased by the MCA occlusion (Fig. 3). DISCUSSION In this study, thrombotic occlusion of the MCA was induced by photochemical reaction between Rose Bengal and green light, which caused endothelial injury followed by platelet adhesion, aggregation and formation of a platelet and fibrin-rich thrombus at the site of the photochemical reaction (Saniabadi et al., 1995). Using this model, we investigated if focal cerebral ischemia induced by the MCA occlusion results in an increase of hydroxyl radical generation in the ischemic border zone. The concentration of 2.3-DHBA, which indicates hydroxyl radical generation, increased by 1.7-fold at 30 min compared with the baseline levels and remained high during the 180-min observation period. MCI-186 reduced the 2.3-DHBA concentration that was increased by ischemia, and thus it is likely that the reduction in the amount of cerebral damage is attributable to the decrease in hydroxyl radical concentration in this study. Plasma concentration of MCI-186 after intravenous injection decreased rapidly and biphasically. Furthermore, the changes in brain MCI-186 concentration was in parallel with the plasma concentration and the blood–brain barrier permeability of this drug was about 60%. Based on our observations and the present findings, MCI-186 may cause a reduction in hydroxyl radical within 3 hr after the ischemia. In addition, an increase of hydroxyl radical in the early phase
Inhibitory Effect of MCI-186
577 SUMMARY 1. In this study, we investigated the effect of a radical scavenger, MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one), on cerebral damage induced by rat middle cerebral artery (MCA) occlusion, and also measured the hydroxyl radical level at the ischemic border zone using a microdialysis technique. 2. The rat MCA was occluded by a thrombus induced by a photochemical reaction. The hydroxyl radical level at the ischemic border zone was determined as a concentration of 2.3-dihydroxybenzoic acid metabolized from salicylate, which was perfused in the microdialysis probe. 3. MCI-186, at a dose of 3 mg/kg for 30 min, was administered as a continuous infusion two times, starting 20 min and then 80 min after Rose Bengal injection. 4. MCI-186 significantly (P,0.05) reduced cortex damage 24 hr after MCA occlusion. 5. At the ischemic border zone, the hydroxyl radical increased by 1.7-fold following ischemia. MCI-186 significantly (P,0.05) reduced the hydroxyl radical level. 6. The reduction in the amount of cerebral damage by MCI-186 may be attributable to the inhibition of hydroxyl radical level at the ischemic border zone.
FIGURE 3. Effect of MCI-186 on a concentration of 2.3-DHBA as formation of hydroxyl radical induced by MCA occlusion. Data represent mean6SE values of 2.3-DHBA concentrations in five animals treated with saline (solid circle) and five animals treated with MCI-186 (open circles). Ringer’s solution containing 5 mM sodium salicylate was perfused through a microdialysis probe during 180 min after MCA occlusion. may play a key role in the development of cerebral damage after MCA occlusion in rats. Production of free radicals during hypoxic or ischemic conditions is widely known (Braughler and Hall, 1989; Hall and Braughler, 1989). In global ischemia, radicals are formed predominantly during the period of postischemic reperfusion (Althaus et al., 1993; Cao et al., 1988; Oliver et al., 1990; Sakamoto et al., 1991). In focal ischemia, the participation of free radical generation by ischemia/reperfusion injury was suggested by the effectiveness of free radical scavenging drugs (Abe et al., 1988; Johshita et al., 1989; Oh and Betz, 1991; Watanabe et al., 1994,) and superoxide dismutase (Kinouchi et al., 1991; Liu et al., 1989; Matsumiya et al., 1991). However, direct measurement of free radical generation was not performed in those studies. In the present study, we measured 2.3-DHBA concentration in vivo at the ischemic border zone following thrombotic occlusion of the MCA, and confirmed the increase during the early ischemic phase. It has been reported that 2.3- and 2.5-DHBA concentrations increased during both the ischemic and reperfusion phases, whereas local cerebral blood flow during ischemia at a site adjacent to the microdialysis probe was about 25% of preischemic baseline (Morimoto et al., 1996), suggesting that this residual flow was apparently able to deliver enough oxygen to the tissue to generate free radicals. Furthermore, Demopoulos et al. (1980) demonstrated that free radical was generated even when tissue oxygen level was only 5% of normal level. In this study, 2.3-DHBA concentration at the ischemic core did not increase hydroxyl radical (data not shown). Based on our observations and other reported studies, hydroxyl radical may be generated at the ischemic border zone. In conclusion, hydroxyl radical may play a key role in the development of cerebral damage at an early phase. The reduction in the amount of cerebral damage ion by MCI-186 may be attributable to the decrease in hydroxyl radical concentration at the ischemic border zone.
References Abe K., Yuki S. and Kogure K. (1988) Strong attenuation of ischemic and postischemic brain edema in rats by a novel free radical scavenger. Stroke 19, 480–485. Althaus J. S., Andrus P. K., Williams C. M., Von Voigtlander P. F., Cazers A. R. and Hall E. D. (1993) The use of salicylate hydroxylation to detect hydroxy radical generation in ischemic and traumatic brain injury. Reversal by tirilazad mesylate (U-74006F). Mol. Chem. Neuropathol. 20, 147–162. Betz A. L. (1985) Identification of hypoxanthine transport and xanthine oxidase activity in brain capillaries. J. Neurochem. 44, 574–579. Braughler J. M. and Hall E. D. (1989) Central nervous system trauma and stroke. I. Biochemical consideration for oxygen radical formation and lipid peroxidation. Free Rad. Biol. Med. 6, 289–294. Cao W., Carney J. M., Duchon A., Floyd R. A. and Chevion M. (1988) Oxygen free radical involvement in ischemia and reperfusion injury to brain. Neurosci. Lett. 88, 233–238. Demopoulos H. B., Flamm E. S., Pietronigro D. D. and Seligman M. L. (1980) The free radical pathology and the microcirculation in the major central nervous system disorders. Acta Physiol. Scand. 492(Suppl.), 91– 119. Floyd R. A., Watson J. J. and Wong P. K. (1984) Sensitive assay of hydroxy radical formation utilizing high pressure liquid chromatography with electrochemical detection phenol and salicylate hydroxylation products. J. Biochem. Biophys. Meth. 10, 221–235. Floyd R. A., Henderson R., Watson J. J. and Wong P. K. (1986) Use of salicylate with high pressure liquid chromatography and electrochemical detection (LCED) as a sensitive measure of hydroxyl free radicals in adriamycin treated rats. J. Free Rad. Biol. Med. 2, 13–18. Hall E. D. and Braughler J. M. (1989) Central nervous system trauma and stroke. II. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation. Free Rad. Biol. Med. 6, 303– 313. Halliwell B., Kaur H. and Ingelman-Sundberg M. (1991) Hydroxylation of salicylate as an assay for hydroxyl radicals: Cautionary note. Free Rad. Biol. Med. 10, 439–441. Ingelman-Sundberg M., Kaur H., Terelius Y., Persson J. O. and Halliwell B. (1991) Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Lack of production of 2.3-dihydroxybenzoate unless hydroxyl radical formation is permitted. Biochem. J. 276, 753–757. Johshita H., Asano T., Hanamura T. and Takakura K. (1989) Effect of indomethacin and a free radical scavenger on cerebral blood flow and edema after cerebral artery occlusion in cats. Stroke 20, 788–794. Kinouchi H., Epstein C. J., Mizui T., Carlson E., Chen S. F. and Chan P. H. (1991) Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase. Proc. Natl. Acad. Sci. USA 88, 11158–11162. Liu T. H., Beckman J. S., Freeman B. A., Hogan E. L. and Hsu C. Y. (1989)
578 Polyethylene glycol-conjugate superoxide dismutase and catalase reduces ischemic brain injury. Am. J. Physiol. 256, H589–H593. Matsumiya N., Koehler R. C., Kirsch J. R. and Traystman R. J. (1991) Conjugated superoxide dismutase reduces extent of caudate injury after transient focal ischemia in cats. Stroke 22, 1193–1200. Matsuo Y., Kihara T., Ikeda M., Ninomiya M., Onodera H. and Kogure K. (1995) Role of neutrophils in radical production during ischemia and reperfusion of the rat brain: Effect of neutrophil depletion on extracellular ascorbyl radical formation. J. Cereb. Blood Flow Metab. 15, 941–947. Minhaz U., Tanaka M., Tsukamoto H., Watanabe K., Koide S., Shohtsu A. and Nakazawa H. (1996) Effect of MCI-186 on postischemic reperfusion injury in isolated rat heart. Free Rad. Res. 24, 361–367. Morimoto T., Globus M. Y.-T., Busto R., Martinez E. and Ginsberg M. D. (1996) Simultaneous measurement of salicylate hydroxylation and glutamate release in the penumbral cortex following transient middle cerebral artery occlusion in rats. J. Cereb. Blood Flow Metab. 16, 92–99. Nishi H., Watanabe T., Sakurai H., Yuki S. and Ishibashi A. (1989) Effect of MCI-186 on brain edema in rats. Stroke 20, 1236–1240. Oh S. M. and Betz A. L. (1991) Interaction between free radicals and excitatory amino acids in the formation of ischemic brain edema in rats. Stroke 22, 915–921. Oishi R., Itoh Y., Nishibori M., Watanabe T., Nishi H. and Saeki K. (1989) Effect of MCI-186 on ischemia-induced changes in monoamine metabolism in rat brain. Stroke 20, 1557–1564. Oliver C. N., Starke-Reed P. E., Stadtman E. R., Liu G. J., Carney J. M. and Floyd R. A.(1990) Oxidative damage to brain proteins, loss of glutamine synthetase activity, and production of free radicals during ischemic/re-
A. Mizuno et al. perfusion-induced injury to gerbil brain. Proc. Natl. Acad. Sci. USA 87, 5144–5147. Raichle M. E. (1983) The pathophysiology of brain ischemia. Ann. Neurol. 13, 2–10. Sakamoto A, Ohnishi S. T., Ohnishi T. and Ogawa R. (1991) Relationship between free radical production and lipid peroxidation during ischemiareperfusion injury in rat brain. Brain Res. 554, 186–192. Saniabadi A. R., Umemura K., Matsumoto N., Sakuma S. and Nakashima M. (1995)Vessel wall injury and arterial thrombosis induced by a photochemical reaction. Thromb. Haemost. 73, 868–872. Simonian N. A., Coyle J. T. (1996) Oxidative stress in neurodegenerative diseases. Ann. Rev. Pharmac. Toxicol. 36, 83–106. Umemura K., Gemba T., Mizuno A. and Nakashima M. (1996) Inhibitory effect of MS-153 on elevation brain glutamate level induced by rat middle cerebral artery occlusion. Stroke 27, 1624–1628. Umemura K., Wada K., Uematsu T., Mizuno A. and Nakashima M. (1994) Effect of 21-aminosteroid lipid peroxidation inhibitor, U74006F, in the rat middle cerebral artery occlusion model. Eur. J. Pharmac. 251, 69-74. Umemura K., Wada K., Uematsu T. and Nakashima M. (1993) Evaluation of the combination of tissue type plasminogen activator, SUN9216, and thromboxane A2 receptor antagonist, vapiprost, in rat middle cerebral artery thrombosis model. Stroke 24, 1077–1081. Watanabe T., Morita I., Nishi H. and Murota S. (1988) Preventive effect of MCI-186 on 15-HPETE induced vascular endothelial cell injury in vitro. Prostagland. Leuk. Essential Fatty Acids 33, 81–87. Watanabe T., Yuki S., Egawa M. and Nishi H. (1994) Protective effect of MCI-186 on cerebral ischemia: Possible involvement of free radical scavenging and antioxidant action. J. Pharm. Exp. Ther. 268, 1597–1604.
Gen. Pharmac. Vol. 30, No. 4, pp. 575–578, 1998 Copyright 1998 Elsevier Science Inc. Printed in the USA.
Inhibitory Effect of MCI-186, a Free Radical Scavenger, on Cerebral Ischemia Following Rat Middle Cerebral Artery Occlusion Atsuhiro Mizuno, Kazuo Umemura* and Mitsuyoshi Nakashima Department of Pharmacology, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-31, Japan ABSTRACT. 1. In this study, we investigated the effect of a radical scavenger, MCI-186 (3-methyl1-phenyl-2-pyrazolin-5-one), on cerebral damage induced by rat middle cerebral artery (MCA) occlusion, and further measured the hydroxyl radical level at the ischemic border zone using a microdialysis technique. 2. MCI-186, at a dose of 3 mg/kg per 30 min, was administerated as a continuous infusion two times for 30 min, starting 20 min and then 80 min after Rose Bengal injection. 3. MCI-186 significantly (P,0.05) reduced size of cerebral damage 24 hr after MCA occlusion and significantly (P,0.05) reduced hydroxyl radical level. gen pharmac 30;4:575–578, 1998. 1998 Elsevier Science Inc. KEY WORDS. Hydroxyl radical, 2.3-DHBA, microdialysis, MCI-186, cerebral ischemia
INTRODUCTION Reactive oxygen species are generated within brain tissue during ischemic injury (Demopoulos et al., 1980; Raichle, 1983). However, the sources of reactive oxygen species implicated in ischemic injury have not yet been clearly identified. It has been proposed that the mechanism of oxygen free radical generation may include the stimulation of the xanthine–xanthine oxidase system in cerebral vessels (Betz, 1985) and activation of neutrophils (Matsuo et al., 1995) and arachidonic acid metabolism (Simonian and Coyler, 1996). Free radical-induced lipid peroxidation, oxidation of proteins and nucleic acids may contribute to the neuronal injury after cerebral ischemia. Damage to the cell membrane, for example, may result in dysfunction of essential membrane activities. MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one), a newly synthesized free radical scavenger, exerts beneficial free radical scavenging and antioxidant characteristics (Watanabe et al., 1994) and prevents the peroxidative vascular endothelial cell damage caused by hydroperoxyeicosatetraenoic acid (Watanabe et al., 1988) in in vitro study. Furthermore, MCI-186 has been tested in various different experimental models for evaluatuation of its protective effects in cerebral ischemia/reperfusion (Abe et al., 1988; Nishi et al., 1989; Oishi et al., 1989; Watanabe et al., 1988) and myocardial ischemia/ reperfusion (Minhaz et al., 1996). Thus, it seems that the neuroprotective effects of MCI-186 are attributable to the inhibition of free radical production. In this study, we investigated the effect of MCI-186 on ischemic damage using middle cerebral artery (MCA) thrombosis model in rats, and determined whether its effect might be responsible for hydroxyl radical scavenging action by the salicylate hydroxylation technique using microdialysis, which has been suggested as a chemical trap for potential hydroxyl radical formation (Fig. 1) (Floyd et al., 1984, 1986; Morimoto et al., 1996). *To whom correspondence should be addressed. Received 23 May 1997.
MATERIALS AND METHODS
Animal preparation Male Wistar rats (SLC Experimental Animal Co., Ltd., Shizuoka, Japan), weighing 240–270 g, were anesthetized with a 1.5% halothane and oxygen gas mixture. Body temperature was maintained at 37.58C with a heating pad (K-Module, Model K-20, America Pharmaseal Co.), and a catheter for the administration of Rose Bengal or other agents placed in the femoral vein. MCA occlusion caused by photochemically induced thrombosis in the rat has been described by Umemura et al. (1993, 1994, 1996). In brief, the scalp and temporal muscles were reflected, and a subtemporal craniotomy was performed using a dental drill under an operating microscope. A 3-mm-diameter circular area of the window was illuminated with green light and the entire illuminated segment, including the proximal end of the lenticulostriate branch, became thrombotically occluded. Photoillumination by green light (540-nm wavelength) was achieved by using a xenon lamp (L4887, Hamamatsu Photonics, Japan) with a heat-absorbing filter and a green filter. Irradiation was directed by a 3-mm-diameter optic fiber mounted on a micromanipulator. The head of the optic fiber was placed on the window in the skull base, and Rose Bengal (20 mg/kg) was injected intravenously and photoillumination was performed for 10 min. MCI-186 (3 mg/kg per 30 min) or vehicle was administered for 30 min intravenously, starting at 20 min and then 80 min after Rose Bengal injection in each of seven animals. The incisions were closed after the operation. Animals were killed by decapitation 24 hr after MCA occlusion, the brain was immediately excised under the pentobarbital anesthesia and sectioned in 1-mm-thick slices from 1.5 mm anterior to 4.5 mm posterior to the bregma with a microslicer, and then six consecutive slices were stained with 2,3,5-triphenyltetrazolium chloride (Katayama, Japan). Photographs of the posterior view of the slices were then taken. For the slice, the ratio of area of cerebral damage to the entire area of the corresponding cerebrum was calculated using a computerized image analysis system. The amount of striatum damage in four slices from 0.5 mm anterior to 3.5 mm posterior to the bregma was measured.
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FIGURE 1. Products of the attack by hydroxyl radical on the salicylate molecule. Salicylate metabolites to 2.3- and 2.5-DHBA, and a small amount of catechol by hydroxyl radical. The 2.3DHBA concentration was measured and the hydroxyl radical level was estimated.
Measurement of hydroxyl radical at ischemic border zone The hydroxyl radical at the ischemic border zone was measured as a 2.3-dihydroxybenzoic acid (DHBA) concentration in the dialysate using a microdialysis technique. A microdialysis probe (2-mm dialysis membrane, CMA/12, Stockholm, Sweden) was stereotaxically implanted in the right dorsolateral parietal cortex (0.5 mm posterior to the bregma, 4 mm lateral to the midline and 3 mm ventral to the cortex surface). This probe inserted into the ischemic border zone, which was reported in our previous studies (Umemura et al., 1996). The probe was continuously perfused with Ringer’s solution composed of 147 mM NaCl, 4 mM KCl, 2.3 mM CaCl2 and 5 mM sodium salicylate at a rate of 2 ml/min, starting at 30 min before implantation until the end of the experiment, using a microinfusion pump (CMA/102). MCA occlusion was performed 90 min after implantation of the microdialysis probe, dialysates were collected at 15-min intervals during the 180 min after MCA occlusion. MCI186 (3 mg/kg per 30 min) or vehicle was administered for 30 min intravenously, starting 20 min and then 80 min after Rose Bengal injection in each of five animals.
HPLC system for determination of 2.3-DHBA Salicylate metabolites to 2.3- and 2.5-DHBA, and a small amount of catechol by hydroxyl radical. 2.5-DHBA is generated via the cytochrome P-450 system (Halliwell et al., 1991; Ingelman-Sundberg et al., 1991), thus we measured the 2.3-DHBA concentration and estimated the hydroxyl radical level. 2.3-DHBA was analyzed by the HPLC system (Shimadzu, Tokyo, Japan) customized with coulometric detector (ESA, Bedford, MA). The guard cell and detector 1 or 2 of analytical cell were operated at potentials of 10.35 V, 20.1 V and 10.30 V, respectively. The separation of different compounds was accomplished by an analytical column (TSK Gel 80-Ts; 15034.6-mm inner diameter, Tosoh, Tokyo, Japan) with a 5-mm particle size. The column was used at 408C with a mobile phase consisting of 30 mM disodium hydrogen phosphate, 30 mM phosphoric acid, 10 mg ethylenediamine tetraacetic acid disodium salt and 1 mg 1-pentanesulfonic acid sodium salt to a total volume of 1 l. The flow rate was 1.0 ml/min. Calibration curves were run daily with 2.3DHBA standard. From peak height of the external standards, dialysate concentration was calculated.
Statistical analysis Data are reported as means6SE. Statistical analysis was made with an unpaired Student’s t-test or two-way ANOVA for comparison between two groups. P,0.05 was considered significant. RESULTS
Amount of cerebral damage The dorsolateral cortex and striatum were damaged in the control group. MCI-186 (3 mg/kg per 30 min) significantly (P,0.05) re-
FIGURE 2. Protective effect of MCI-186 (3 mg/kg for 30 min) (open column) on cerebral damage in seven animals compared with the control group (solid column). MCI-186 was administered as continuous infusion two times (each for 30 min), starting 20 min and then 80 min after Rose Bengal injection. Data represent mean6SE of seven animals. *P,0.05 vs. control group.
duced the amount of cerebral damage in the cortex but not in the striatum (Fig. 2).
Measurement of 2.3-DHBA and inhibitory effect of MCI-186 The mean preischemic values for 2.3-DHBA concentration were 35.7466.80 and 40.6562.93 pmol/ml in the control group and the group treated with MCI-186, respectively, and there was no significant difference between the groups. After the MCA occlusion, the concentration of 2.3-DHBA increased by 1.7-fold at 30 min compared with baseline levels and remained high throughout the 180min observation period. MCI-186 (3 mg/kg per 30 min) significantly (P,0.05) suppressed the 2.3-DHBA concentration that was increased by the MCA occlusion (Fig. 3). DISCUSSION In this study, thrombotic occlusion of the MCA was induced by photochemical reaction between Rose Bengal and green light, which caused endothelial injury followed by platelet adhesion, aggregation and formation of a platelet and fibrin-rich thrombus at the site of the photochemical reaction (Saniabadi et al., 1995). Using this model, we investigated if focal cerebral ischemia induced by the MCA occlusion results in an increase of hydroxyl radical generation in the ischemic border zone. The concentration of 2.3-DHBA, which indicates hydroxyl radical generation, increased by 1.7-fold at 30 min compared with the baseline levels and remained high during the 180-min observation period. MCI-186 reduced the 2.3-DHBA concentration that was increased by ischemia, and thus it is likely that the reduction in the amount of cerebral damage is attributable to the decrease in hydroxyl radical concentration in this study. Plasma concentration of MCI-186 after intravenous injection decreased rapidly and biphasically. Furthermore, the changes in brain MCI-186 concentration was in parallel with the plasma concentration and the blood–brain barrier permeability of this drug was about 60%. Based on our observations and the present findings, MCI-186 may cause a reduction in hydroxyl radical within 3 hr after the ischemia. In addition, an increase of hydroxyl radical in the early phase
Inhibitory Effect of MCI-186
577 SUMMARY 1. In this study, we investigated the effect of a radical scavenger, MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one), on cerebral damage induced by rat middle cerebral artery (MCA) occlusion, and also measured the hydroxyl radical level at the ischemic border zone using a microdialysis technique. 2. The rat MCA was occluded by a thrombus induced by a photochemical reaction. The hydroxyl radical level at the ischemic border zone was determined as a concentration of 2.3-dihydroxybenzoic acid metabolized from salicylate, which was perfused in the microdialysis probe. 3. MCI-186, at a dose of 3 mg/kg for 30 min, was administered as a continuous infusion two times, starting 20 min and then 80 min after Rose Bengal injection. 4. MCI-186 significantly (P,0.05) reduced cortex damage 24 hr after MCA occlusion. 5. At the ischemic border zone, the hydroxyl radical increased by 1.7-fold following ischemia. MCI-186 significantly (P,0.05) reduced the hydroxyl radical level. 6. The reduction in the amount of cerebral damage by MCI-186 may be attributable to the inhibition of hydroxyl radical level at the ischemic border zone.
FIGURE 3. Effect of MCI-186 on a concentration of 2.3-DHBA as formation of hydroxyl radical induced by MCA occlusion. Data represent mean6SE values of 2.3-DHBA concentrations in five animals treated with saline (solid circle) and five animals treated with MCI-186 (open circles). Ringer’s solution containing 5 mM sodium salicylate was perfused through a microdialysis probe during 180 min after MCA occlusion. may play a key role in the development of cerebral damage after MCA occlusion in rats. Production of free radicals during hypoxic or ischemic conditions is widely known (Braughler and Hall, 1989; Hall and Braughler, 1989). In global ischemia, radicals are formed predominantly during the period of postischemic reperfusion (Althaus et al., 1993; Cao et al., 1988; Oliver et al., 1990; Sakamoto et al., 1991). In focal ischemia, the participation of free radical generation by ischemia/reperfusion injury was suggested by the effectiveness of free radical scavenging drugs (Abe et al., 1988; Johshita et al., 1989; Oh and Betz, 1991; Watanabe et al., 1994,) and superoxide dismutase (Kinouchi et al., 1991; Liu et al., 1989; Matsumiya et al., 1991). However, direct measurement of free radical generation was not performed in those studies. In the present study, we measured 2.3-DHBA concentration in vivo at the ischemic border zone following thrombotic occlusion of the MCA, and confirmed the increase during the early ischemic phase. It has been reported that 2.3- and 2.5-DHBA concentrations increased during both the ischemic and reperfusion phases, whereas local cerebral blood flow during ischemia at a site adjacent to the microdialysis probe was about 25% of preischemic baseline (Morimoto et al., 1996), suggesting that this residual flow was apparently able to deliver enough oxygen to the tissue to generate free radicals. Furthermore, Demopoulos et al. (1980) demonstrated that free radical was generated even when tissue oxygen level was only 5% of normal level. In this study, 2.3-DHBA concentration at the ischemic core did not increase hydroxyl radical (data not shown). Based on our observations and other reported studies, hydroxyl radical may be generated at the ischemic border zone. In conclusion, hydroxyl radical may play a key role in the development of cerebral damage at an early phase. The reduction in the amount of cerebral damage ion by MCI-186 may be attributable to the decrease in hydroxyl radical concentration at the ischemic border zone.
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