Proc. Natl. Acad. Sci. USA Vol. 88, pp. 11158-11162, December 1991 Neurobiology
Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase (middle cerebral artery occlusion/brain edema/superoxide radicals)
HIROYUKI KINOUCHI*, CHARLES J. EPSTEINtt, TAKUJI MIZUI*, ELAINE CARLSONt, SYLVIA F. CHEN*, AND PAK H. CHAN*§1 Departments of *Neurology, §Neurosurgery, tPediatrics, and *Biochemistry and Biophysics, University of California, San Francisco, CA 94143
Communicated by John A. Clements, September 16, 1991 (receivedfor review June 28, 1991)
Oxygen-derived free radicals have been imABSTRACT plicated in the pathogenesis of vasogenic edema and infarction caused by ischeria and reperfusion injury. In earlier studies, exogenously supplied liposome-entrapped CuZn superoxide dismutase (CuZn-SOD) ameliorated ischemic brain edema and infarction in rats following focal cerebral ischemla. To ascertain directly the role of SOD in the protection against superoxide radical-induced injury, we measured infarct size and water content 24 hr following focal cerebral ischemia in nontransgenic mice and in transgeic mice bearing the human SODI gene. These transgenic mice have 3.1-fold higher cellular CuZn-SOD activity in the brain than do their nontransgenic littermates. We also'measured antioxidant levels (reduced glutathione and reduced ascorbate) of contralateral cortex, infarct cortex, surrounding cortex, and striatum. Infarct size and brain edema were s ntly decreased in trenic mice compared with nontransgenic mice. Reduced glutathione and reduced ascorbate levels decreased in the ischemlc hemisphere, -but levels- in surrounding cortex and striaturm were gficantly higher in trasgenic'mice than in nontransgenic mice. These rEsults indicate that increased endogenous SOD activity in brain reduces the level of ischemic damage and support the concept that superoxide radicals play an important role in the- pathogenesis of infarction and edema following focal cerebral ischemia.
The role of oxygen free radicals in the pathogenesis of infarction and edema following cerebral ischemia has been intensively investigated since the report of Flamm et al. (1). Because of technical 4ffficulties in the measurement of free radicals'in brain tissues the part played by free radicals in the pathogenesis of cerebral ischemia still remains unclear (2, 3). However, reports of decreased levels of lipid-soluble and water-soluble endogenous antioxidants and of increased levels of conjugated dienes and lipid peroxidation (4-10) support the notion that free radicals are involved in ischemic brain
chemia in the gerbil brain (13). Liposome-entrapped CuZnSOD, which has a half life of 4.2 hr (11), reduces the severity of traumatic and ischemic injuries (14, 15). Although these studies provide potential therapeutic precedents for the management of brain injury, alternative experimental approaches are needed to address the issues of the role of oxygen free radicals and the mode of action ofSOD in ischemic brain injury. To investigate directly the role of increased brain CuZn-SOD in the pathogenesis of brain injuries presumed to involve superoxide radicals, we have used transgenic (Tg) mice carrying the human CuZn-SOD gene. These mice have increased levels ofendogenous CuZnSOD activity in brain and other organs (16, 17). Our aim in this study was to examine the hypothesis that superoxide radicals play a role in the pathogenesis of cerebral ischemia and, therefore, that ischemic brain damage would be reduced by the presence of increased endogenous levels of CuZnSOD activity in brain parenchyma.
MATERIALS AND METHODS Tg Mice. Tg mice of strain TgHS/SF-218 carrying human SOD) genes were derived from the founder stock described by Epstein et al. (16). The founder mice had been bred with CD-1 mice to produce Tg offspring carrying the SOD) gene. Tg mice were identified by qualitative demonstration of human CuZn-SOD, using nondenaturing gel electrophoresis followed by nitro blue tetrazolium staining (16). There were no observable phenotypic differences between Tg mice and nontransgenic (nTg) normal littermates. Focal Cerebral Ischemia. Male Tg and nTg mice were subjected to focal cerebral ischemia in a randomized blind fashion. The mice, weighing 30-35 g, were anesthetized with 2% halothane followed by 0.5% halothane and chloral hydrate (200 mg/kg, i.p.). In the studies of physiological parameters, a right femoral arterial catheter was placed for blood pressure recording and blood sampling for gas:"Ad pH analysis. Body temperature was maintained at 370C with a heating pad controlled by a rectal probe. Focal cerebral ischemia was produced by a method described by Chen et al. (18) and by Imaizumi et al. (15). The left middle cerebral artery (MCA) was occluded by electrical coagulation just proximal to the pyriform branch. Immediately following occlusion of the MCA, the left common carotid artery was ligated and the right common carotid artery was occluded for 1 hr with a microaneurysmal clip.
injury.
Indirect methods using specific antioxidants [e.g., superoxide dismutase (SOD) and catalase] have traditionally been used to implicate oxygen free radicals in physiological or pathological processes. Unfortunately, the half-life of CuZnSOD in circulating blood is extremely short (6 min), and it is unable to pass the blood-brain barrier. Therefore, use has been made of chemically modified enzymes for work on cerebral injury (11), and it has been found that polyethylene glycol-conjugated CuZn-SOD (PEG-SOD) and PEG-catalase reduce the degree of cortical infarction resulting from focal cerebral ischemia (12). Similarly, CuZn-SOD conjugated with divinyl ether/maleic acid copolymer ameliorated delayed hippocampal neuronal death after global cerebral is-
Abbreviations: SOD, superoxide dismutase; Tg, transgenic; nTg, nontransgenic; MCA, middle cerebral artery; ACA, anterior cerebral artery; GSH, reduced glutathione; AH2, reduced ascorbate. ITo whom reprint requests should be addressed at: CNS Injury and Edema Research Center, Departments of Neurology and Neurosurgery, Box 0114, School of Medicine, University of California, San Francisco, CA 94143.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 11158
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Measurement of Infarct Size. At 24 hr after ischemia, the brains were removed and the forebrain was cut coronally 1 mm (plane A), 3 mm (plane B), 5 mm (plane C), and 7 mm (plane D) distal from the frontal pole by using the Mouse Brain Matrix (Harvard Apparatus). The brain slices were then stained with 2% (wt/vol) 2,3,5-triphenyltetrazolium chloride in Dulbecco's phosphate buffer (pH 7.4) at 370C (15). Triphenyltetrazolium chloride, which stains mitochondrial dehydrogenase, accurately delineates the infarct area as compared with measurement using traditional staining (14, 19, 20). The infarcted area and the bilateral hemispheric area were quantitated by an image-analysis system (21). Total infarct volume and total hemispheric volume were calculated according to Liu et al. (12). Water Content and Biochemical Analysis. Animals were decapitated while under deep anesthesia. Brains were quickly removed and dissected into right (contralateral) cerebral cortex, left infarct cortex fed mainly by MCA (MCA cortex), left surrounding cerebral cortex fed mainly by anterior cerebral artery (ACA cortex), and left striatum in slices from frontal pole to 7 mm posterior. The dissected brains were immediately assayed for water content. Water content was measured by drying the brain tissue at 105'C for >48 hr and is expressed as percent water. For the measurement of reduced glutathione (GSH) and reduced ascorbate (AH2), the dissected brains were immediately frozen in liquid nitrogen and homogenized with 10 volumes of ice-cold 6% perchloric acid containing 1 mM EDTA. Protein was removed by centrifugation at 10,000 X g for 10 min, and the supernatants were assayed for GSH and AH2. GSH was measured by determining nonprotein sufhydryls according to the method of Ellman (22), with the assumption that the majority of brain nonprotein sulfhydryls are GSH (23). AH2 was measured using ascorbate oxidase as described by Cooper et al. (2). Tissue protein was measured according to Lowry et al. (24), with bovine serum albumin (Sigma) as the standard.
RESULTS Physiological Measurements. There were no significant differences between Tg and nTg mice in mean arterial blood pressure, partial pressure of 02 and C02, and pH (data not shown).
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FIG. 2. Infarct size (mm2) in Tg and nTg mice. Infarct areas determined by staining with triphenyltetrazolium chloride were reduced in Tg mice relative to nTg mice following 24 hr of ischemia. There were significant differences in the coronal slices 3 and 5 mm distal from the frontal pole. Number of determinations was 15 in each group. *, P < 0.01; **, P < 0.05 (versus nTg, by Student's t test).
Infarct Sizes and Hemispheric Enlargement. The method for inducing focal cerebral ischemia described above yielded reproducible cortical infarcts in the MCA territory of the mouse brain as measured by mitochondrial dehydrogenase staining with triphenyltetrazolium chloride (Figs. 1 and 2). As is shown in Fig. 2, these infarcts were significantly smaller in Tg mice than in nTg mice in planes B and C [nTg, 7.83 + 0.76 mm2 (22.6 ± 1.5% of the hemisphere; mean ± SEM) and 8.73 + 0.61 mm2 (23.7 ± 1.4%); Tg, 5.41 ± 0.32 mm2 (18.9 ± 0.8%), P < 0.05, and 5.12 ± 0.39 mm2 (15.3 ± 1.2%), P < 0.01, respectively]. The ratios of left to right hemispheric areas, measures of cerebral edema, are shown in Fig. 3. Although there were no significant differences between Tg and nTg in the crosssectional sizes of the right hemispheres, the sizes of left hemispheres of Tg mice were significantly smaller than those of nTg mice in planes A, B, and C. The total infarct volumes, hemispheric volumes, infarct percentages, and left/right hemisphere ratios of Tg and nTg mice are shown in Table 1. Water Content. Water content in the left hemisphere was increased 24 hr after ischemia in both groups. However, the increases in water content of MCA cortex and ACA cortex were significantly less in Tg mice than in nTg mice (Fig. 4). Water content in striatum increased significantly at 24 hr of ischemia in both groups, but there was no significant difference between Tg and nTg mice.
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FIG. 1. Illustration of cortical infarction (black area) in brains of Tg and nTg mice as shown by staining with triphenyltetrazolium chloride following 24 hr of ischemia. The sizes of cortical infarction in Tg mice were significantly smaller than in nTg mice. See Materials and Methods for definition of planes A-D.
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FIG. 3. Reduced hemispheric enlargement (brain edema) in Tg mice following MCA occlusion. Data represent the ratios of left to right hemispheric areas in coronal slices 1, 3, 5, and 7 mm distal from the frontal pole. The ratios in Tg mice are significantly smaller than those in nTg mice in three of the slices. Number of determinations was 15 in each group. *, P < 0.01; **, P < 0.05 (versus nTg, by Student's t test).
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Proc. Natl. Acad. Sci. USA 88 (1991)
Table 1. Total infarcted volume and hemispheric volume in Tg and nTg mice at 24 hr of MCA occlusion nTg Tg Volume, mm3 Left hemisphere 212.3 ± 5.2 189.4 ± 4.3* Right hemisphere 179.5 ± 2.5 176.6 ± 3.9* Infarction 39.9 ± 4.0 25.6 ± 2.1* Percent of infarction 18.4 ± 1.3 13.7 ± 0.8* Left/right ratio 1.18 ± 0.02 1.07 ± 0.01* Total volumes of infarction and of the left and the right hemispheres were calculated according Liu et al. (12). Each value is mean + SEM of 15 animals. *, P < 0.001 (versus nTg, by Student's t test).
Antioxidant Levels. There were no significant differences in GSH levels either among the areas of the brain or between Tg and nTg mice before ischemia (Fig. 5). The GSH level in the left hemisphere decreased significantly from the level of preischemia. MCA cortex showed the most decreased level, but there was no significant difference between Tg (9.4 ± 0.7 nmol/mg of protein) and nTg (9.2 ± 0.6 nmol/mg of protein) mice at 24 hr of ischemia. However, the GSH levels of ACA cortex and striatum in Tg mice (15.8 ± 0.4 and 16.5 ± 0.6 nmol/mg of protein, respectively) were significantly higher than in nTg mice [14.4 ± 0.4 (P < 0.05) and 13.1 ± 0.4 (P < 0.01) nmol/mg of protein, respectively]. AH2 levels showed changes similar to those of GSH levels (Fig. 6). AH2 levels also decreased in each area of the left hemisphere in both Tg and nTg mice. Although there were no significant differences in the AH2 level in MCA cortex, the levels in ACA cortex and striatum of Tg mice (19.3 ± 0.7 and 15.9 ± 0.7 nmol/mg of protein, respectively) were significantly higher than in nTg mice [16.3 ± 0.5 (P < 0.01) and 13.2 ± 0.6 (P < 0.05) nmol/mg of protein, respectively] at 24 hr of ischemia.
DISCUSSION Tg mice of strain TgHS/SF-218 overexpressing CuZn-SOD have a 3.1-fold increased level of endogenous CuZn-SOD activity in brain (16, 25). Evidence from brain homogenates and cultured cells indicates that the CuZn-SOD genes are being expressed in all the nervous elements, including neurons, glia, and endothelial cells (26). Therefore, the complicating and confounding issues regarding the permeability of the blood-brain barrier to the enzyme, its half-life in blood, and potential systemic side effects of exogenously supplied enzyme could be eliminated in studies of ischemic brain
injury.
Although focal ischemia is dense (or complete) in the "focus," which usually compromises a large part of the
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adjacent areas and the overlying cortex, there is a perifocal "penumbra" zone with a more moderate degree of ischemia (5, 27). This area, which is initially viable, albeit with a critical low perfusion, later suffers neuronal death, glial necrosis, and infarction. Very probably, any therapy that proves to decrease infarct size does so by preventing recruitment of the penumbra zone in the infarction process (5). The mechanism for the decrease of GSH in ischemic brain is unknown at present. One metabolic route is the GSH-GSH peroxidase system. This enzymatic system is accompanied by the concomitant formation of oxidized glutathione (GSSG). However, several studies have reported that GSH content decreases after ischemia and/or reperfusion in rat brain without a concomitant increase in GSSG (2, 3, 28). Other studies have suggested that GSH in the absence of GSH peroxidase can act as a free radical scavenger (29-31). From these results, it is suggested that the decrease in GSH levels following cerebral ischemia is caused by GSH not only serving as a substrate for GSH peroxidase but also scavenging free radicals. Recently, we have shown (32) that the endogenous GSH in the brain is a crucial factor in the defense
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FIG. 5. GSH contents in right cerebral cortex, left MCA cortex (infarct cortex), left ACA cortex, and left striatum before and 24 hr after MCA occlusion. GSH levels in left hemisphere decreased significantly in both Tg and nTg mice at 24 hr of ischemia. However, the level in ACA cortex and striatum remained significantly higher in Tg than in nTg mice. Number ofdeterminations was 11 in each group. *, P < 0.01; **, P < 0.05 (versus nTg, by Student's t test). P, preischemia; I, ischemia.
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FIG. 4. Water content in the cortex of Tg and nTg mice before and 24 hr after MCA occlusion. Water content of both MCA cortex and ACA cortex increased significantly from control value (preischemic value). Number of determinations was 8 in each group. *, P < 0.01; **, P < 0.05 (versus nTg by Student's t test).
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FIG. 6. AH2 contents in right cerebral cortex, left MCA cortex (infarct cortex), left ACA cortex, and left striatum before and 24 hr after MCA occlusion. AH2 levels showed similar changes to GSH levels. There was no significant difference in MCA cortex between Tg and nTg mice. However, the levels in ACA cortex and striatum were significantly higher in Tg mice than in nTg mice. Number of determinations was 13 in each group. *, P < 0.01; **, P < 0.05 (versus nTg, by Student's t test). P, preischemia; I, ischemia.
Neurobiology: Kinouchi et al. against ischemic injury, since depletion of brain GSH with buthionine sulfoximine, a selective inhibitor for -t-glutamylcysteine synthetase, increased postischemic infarct size and edema. As in other tissues, ascorbic acid exists almost exclusively in its reduced form in brain (>99%) (2, 8, 33). It has also been reported that AH2 decreases significantly following cerebral ischemia and head injury. Ascorbic acid is an efficient scavenger of a variety of oxygen-containing radicals (34, 35). However, it can switch from anti- to prooxidant activity, depending on its concentration and the presence of free transition metal irons (36). Furthermore, despite its nature as a relatively polar lipid-insoluble molecule, several investigators have demonstrated that ascorbate interacts directly not only with components of the phospholipid bilayer but also with superoxide anion as a radical scavenger and that it terminates the propagative formation of free radicals (37). Our studies showed that the penumbra area was spared from cerebral infarction in Tg mice. Although we have not measured the superoxide radicals in the penumbra area because of assay difficulties, we assayed the antioxidant levels (GSH and AH2) in Tg and nTg brains. Given these facts, our data that the levels of GSH and AH2 in the penumbra area remained significantly higher in Tg animals than in nTg animals suggests the possibility that the high level of endogenous CuZn-SOD protects the penumbra area from ischemic damage both by directly scavenging superoxide radicals and by reducing oxidative stress through modulating other antioxidant levels. Furthermore, lipid peroxidation is reduced in trisomy 16 mouse brain, which has a 1.5-fold increased level of CuZn-SOD activity because of a gene dose effect (38). Overall, these observations suggest that increased endogenous SOD activity in Tg mice alters the antioxidant system so as to favor the protection of the brain against ischemic injury. Other important metabolic events in the penumbra are the depolarization and ensuing "calcium overload" of cells by excitatory amino acids (39). It has been shown that excitatory amino acids may promote enzymatic reactions through the activation of N-methyl-D-aspartate receptors leading to cytosolic free Ca21 overload, activation of arachidonic acid cascade turnover, and free radical production (40, 41). Furthermore, brain production of free radicals during ischemia causes simultaneously an increased output of the excitatory amino acids, specifically glutamate (42). Interrupting this vicious cycle by SOD may thus provide protection against ischemic brain injury induced by the arachidonic acidglutamate cascade in the penumbra area. Our results suggest that oxidative stress may be an important factor in amplifying N-methyl-D-aspartate receptor-mediated, Ca2+-dependent biochemical events that lead to neuronal cell death. Further studies are warranted to elucidate these possibilities. Besides the cytoprotective mechanism, one could argue that the differences in cerebral blood flow between Tg and nTg mice may account for the observed differences in neuroprotection. However, our preliminary studies suggest that there were no differences in cerebral blood flow measured by laser doppler, a semiquantitative method, between Tg and nTg mice, nor were any morphological and anatomical differences noted in the vascular supply in the Tg mice. Therefore we conclude that the reduced oxidative stress appears to be the major mechanism underlying the cytoprotection in the Tg mice. Nevertheless, our studies also demonstrated that the GSH levels and the infarct sizes in ischemic brain were closely related to the duration of clipping of the contralateral common carotid artery (32), indicating that the collateral flow and some degree of reperfusion may play an important role contributing to the demonstrated difference in infarct size and edema between Tg and nTg animals.
Proc. Natl. Acad. Sci. USA 88 (1991)
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We have recently demonstrated (17) that vasogenic edema caused by cold injury is reduced in Tg mice overexpressing CuZn-SOD, suggesting that this type ofedema is mediated by superoxide radicals. The finding in the present study that the increase in water content was significantly reduced in the Tg mice following 24 hr of ischemia also supports the involvement of superoxide radicals in the pathogenesis of ischemic brain edema. We speculate that endothelial cells may be the major source of the superoxide radicals that cause brain edema, since endothelial cells have a 3.7-fold greater level of xanthine oxidase activity than do homogenates of cerebral cortex (43). However, a direct correlation between the increased CuZn-SOD in endothelial cells and the reduced edema in Tg mice following focal cerebral ischemic injury cannot presently be established and requires further investigation. We thank Dr. Robert Fishman for his critical comments and thank Teodosia Zamora and Liza Reola for assistance in breeding the mice. This work was supported in part by National Institutes of Health Grants AG08938, NS14543, and NS25372. 1. Flamm, E. S., Demopoulus, H. B., Seligman, M. L., Roser, R. G. & Ransohoff, J. (1978) Stroke 9, 445-447. 2. Cooper, A. J. L., Pulsinelli, W. A. & Duffy, T. E. (1980) J. Neurochem. 35, 1242-1245. 3. Rehncrona, S., Folbergrova, J., Smith, D. S. & Siesjo, B. K. (1980) J. Neurochem. 34, 477-486. 4. Kontos, H. A. & Wei, E. P. (1986) J. Neurosurg. 64, 803-807. 5. Siesjo, B. K., Agardh, C.-D. & Bengtsson, F. (1989) Cereb. Brain Metab. Rev. 1, 165-211. 6. Chan, P. H. (1989) in Cellular Antioxidant Defense Mechanisms, ed. Chow, C. K. (CRC, Boca Raton, FL), pp. 89-109. 7. Hall, E. D. & Braughler, J. M. (1989) Free Rad. Biol. 6, 303-313. 8. Kinuta, Y., Kikuchi, H., Ishikawa, M., Kimura, M. & Itokawa, Y. (1989) J. Neurosurg. 71, 421-429. 9. Abe, K., Yoshida, S., Watson, B., Busto, R., Kogure, K. & Ginsberg, M. D. (1983) Brain Res. 273, 166-169. 10. Watson, B. D., Busto, R., Goldberg, W. J., Santiso, M., Yoshida, S. & Ginsberg, M. D. (1984) J. Neurochem. 43, 268-274. 11. Turrens, J. F., Crapo, J. P. & Freeman, B. A. (1984) J. Clin. Invest. 73, 87-95. 12. Liu, T. H., Beckman, J. S., Freeman, B. A., Hogan, E. L. & Hsu, C. Y. (1989) Am. J. Physiol. 256, H589-H593. 13. Kitagawa, K., Matsumoto, M., Oda, T., Niinobe, M., Hata, R., Handa, N., Fukunaga, R., Isaka, Y., Kimura, K., Maeda, H., Mikoshiba, K. & Kamada, T. (1990) Neuroscience 35, 551-558. 14. Chan, P. H., Longar, S. & Fishman, R. A. (1987) Ann. Neurol. 21, 540-547. 15. Imaizumi, S., Woolworth, V., Fishman, R. A. & Chan, P. H.
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Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase (middle cerebral artery occlusion/brain edema/superoxide radicals)
HIROYUKI KINOUCHI*, CHARLES J. EPSTEINtt, TAKUJI MIZUI*, ELAINE CARLSONt, SYLVIA F. CHEN*, AND PAK H. CHAN*§1 Departments of *Neurology, §Neurosurgery, tPediatrics, and *Biochemistry and Biophysics, University of California, San Francisco, CA 94143
Communicated by John A. Clements, September 16, 1991 (receivedfor review June 28, 1991)
Oxygen-derived free radicals have been imABSTRACT plicated in the pathogenesis of vasogenic edema and infarction caused by ischeria and reperfusion injury. In earlier studies, exogenously supplied liposome-entrapped CuZn superoxide dismutase (CuZn-SOD) ameliorated ischemic brain edema and infarction in rats following focal cerebral ischemla. To ascertain directly the role of SOD in the protection against superoxide radical-induced injury, we measured infarct size and water content 24 hr following focal cerebral ischemia in nontransgenic mice and in transgeic mice bearing the human SODI gene. These transgenic mice have 3.1-fold higher cellular CuZn-SOD activity in the brain than do their nontransgenic littermates. We also'measured antioxidant levels (reduced glutathione and reduced ascorbate) of contralateral cortex, infarct cortex, surrounding cortex, and striatum. Infarct size and brain edema were s ntly decreased in trenic mice compared with nontransgenic mice. Reduced glutathione and reduced ascorbate levels decreased in the ischemlc hemisphere, -but levels- in surrounding cortex and striaturm were gficantly higher in trasgenic'mice than in nontransgenic mice. These rEsults indicate that increased endogenous SOD activity in brain reduces the level of ischemic damage and support the concept that superoxide radicals play an important role in the- pathogenesis of infarction and edema following focal cerebral ischemia.
The role of oxygen free radicals in the pathogenesis of infarction and edema following cerebral ischemia has been intensively investigated since the report of Flamm et al. (1). Because of technical 4ffficulties in the measurement of free radicals'in brain tissues the part played by free radicals in the pathogenesis of cerebral ischemia still remains unclear (2, 3). However, reports of decreased levels of lipid-soluble and water-soluble endogenous antioxidants and of increased levels of conjugated dienes and lipid peroxidation (4-10) support the notion that free radicals are involved in ischemic brain
chemia in the gerbil brain (13). Liposome-entrapped CuZnSOD, which has a half life of 4.2 hr (11), reduces the severity of traumatic and ischemic injuries (14, 15). Although these studies provide potential therapeutic precedents for the management of brain injury, alternative experimental approaches are needed to address the issues of the role of oxygen free radicals and the mode of action ofSOD in ischemic brain injury. To investigate directly the role of increased brain CuZn-SOD in the pathogenesis of brain injuries presumed to involve superoxide radicals, we have used transgenic (Tg) mice carrying the human CuZn-SOD gene. These mice have increased levels ofendogenous CuZnSOD activity in brain and other organs (16, 17). Our aim in this study was to examine the hypothesis that superoxide radicals play a role in the pathogenesis of cerebral ischemia and, therefore, that ischemic brain damage would be reduced by the presence of increased endogenous levels of CuZnSOD activity in brain parenchyma.
MATERIALS AND METHODS Tg Mice. Tg mice of strain TgHS/SF-218 carrying human SOD) genes were derived from the founder stock described by Epstein et al. (16). The founder mice had been bred with CD-1 mice to produce Tg offspring carrying the SOD) gene. Tg mice were identified by qualitative demonstration of human CuZn-SOD, using nondenaturing gel electrophoresis followed by nitro blue tetrazolium staining (16). There were no observable phenotypic differences between Tg mice and nontransgenic (nTg) normal littermates. Focal Cerebral Ischemia. Male Tg and nTg mice were subjected to focal cerebral ischemia in a randomized blind fashion. The mice, weighing 30-35 g, were anesthetized with 2% halothane followed by 0.5% halothane and chloral hydrate (200 mg/kg, i.p.). In the studies of physiological parameters, a right femoral arterial catheter was placed for blood pressure recording and blood sampling for gas:"Ad pH analysis. Body temperature was maintained at 370C with a heating pad controlled by a rectal probe. Focal cerebral ischemia was produced by a method described by Chen et al. (18) and by Imaizumi et al. (15). The left middle cerebral artery (MCA) was occluded by electrical coagulation just proximal to the pyriform branch. Immediately following occlusion of the MCA, the left common carotid artery was ligated and the right common carotid artery was occluded for 1 hr with a microaneurysmal clip.
injury.
Indirect methods using specific antioxidants [e.g., superoxide dismutase (SOD) and catalase] have traditionally been used to implicate oxygen free radicals in physiological or pathological processes. Unfortunately, the half-life of CuZnSOD in circulating blood is extremely short (6 min), and it is unable to pass the blood-brain barrier. Therefore, use has been made of chemically modified enzymes for work on cerebral injury (11), and it has been found that polyethylene glycol-conjugated CuZn-SOD (PEG-SOD) and PEG-catalase reduce the degree of cortical infarction resulting from focal cerebral ischemia (12). Similarly, CuZn-SOD conjugated with divinyl ether/maleic acid copolymer ameliorated delayed hippocampal neuronal death after global cerebral is-
Abbreviations: SOD, superoxide dismutase; Tg, transgenic; nTg, nontransgenic; MCA, middle cerebral artery; ACA, anterior cerebral artery; GSH, reduced glutathione; AH2, reduced ascorbate. ITo whom reprint requests should be addressed at: CNS Injury and Edema Research Center, Departments of Neurology and Neurosurgery, Box 0114, School of Medicine, University of California, San Francisco, CA 94143.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 11158
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Measurement of Infarct Size. At 24 hr after ischemia, the brains were removed and the forebrain was cut coronally 1 mm (plane A), 3 mm (plane B), 5 mm (plane C), and 7 mm (plane D) distal from the frontal pole by using the Mouse Brain Matrix (Harvard Apparatus). The brain slices were then stained with 2% (wt/vol) 2,3,5-triphenyltetrazolium chloride in Dulbecco's phosphate buffer (pH 7.4) at 370C (15). Triphenyltetrazolium chloride, which stains mitochondrial dehydrogenase, accurately delineates the infarct area as compared with measurement using traditional staining (14, 19, 20). The infarcted area and the bilateral hemispheric area were quantitated by an image-analysis system (21). Total infarct volume and total hemispheric volume were calculated according to Liu et al. (12). Water Content and Biochemical Analysis. Animals were decapitated while under deep anesthesia. Brains were quickly removed and dissected into right (contralateral) cerebral cortex, left infarct cortex fed mainly by MCA (MCA cortex), left surrounding cerebral cortex fed mainly by anterior cerebral artery (ACA cortex), and left striatum in slices from frontal pole to 7 mm posterior. The dissected brains were immediately assayed for water content. Water content was measured by drying the brain tissue at 105'C for >48 hr and is expressed as percent water. For the measurement of reduced glutathione (GSH) and reduced ascorbate (AH2), the dissected brains were immediately frozen in liquid nitrogen and homogenized with 10 volumes of ice-cold 6% perchloric acid containing 1 mM EDTA. Protein was removed by centrifugation at 10,000 X g for 10 min, and the supernatants were assayed for GSH and AH2. GSH was measured by determining nonprotein sufhydryls according to the method of Ellman (22), with the assumption that the majority of brain nonprotein sulfhydryls are GSH (23). AH2 was measured using ascorbate oxidase as described by Cooper et al. (2). Tissue protein was measured according to Lowry et al. (24), with bovine serum albumin (Sigma) as the standard.
RESULTS Physiological Measurements. There were no significant differences between Tg and nTg mice in mean arterial blood pressure, partial pressure of 02 and C02, and pH (data not shown).
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FIG. 2. Infarct size (mm2) in Tg and nTg mice. Infarct areas determined by staining with triphenyltetrazolium chloride were reduced in Tg mice relative to nTg mice following 24 hr of ischemia. There were significant differences in the coronal slices 3 and 5 mm distal from the frontal pole. Number of determinations was 15 in each group. *, P < 0.01; **, P < 0.05 (versus nTg, by Student's t test).
Infarct Sizes and Hemispheric Enlargement. The method for inducing focal cerebral ischemia described above yielded reproducible cortical infarcts in the MCA territory of the mouse brain as measured by mitochondrial dehydrogenase staining with triphenyltetrazolium chloride (Figs. 1 and 2). As is shown in Fig. 2, these infarcts were significantly smaller in Tg mice than in nTg mice in planes B and C [nTg, 7.83 + 0.76 mm2 (22.6 ± 1.5% of the hemisphere; mean ± SEM) and 8.73 + 0.61 mm2 (23.7 ± 1.4%); Tg, 5.41 ± 0.32 mm2 (18.9 ± 0.8%), P < 0.05, and 5.12 ± 0.39 mm2 (15.3 ± 1.2%), P < 0.01, respectively]. The ratios of left to right hemispheric areas, measures of cerebral edema, are shown in Fig. 3. Although there were no significant differences between Tg and nTg in the crosssectional sizes of the right hemispheres, the sizes of left hemispheres of Tg mice were significantly smaller than those of nTg mice in planes A, B, and C. The total infarct volumes, hemispheric volumes, infarct percentages, and left/right hemisphere ratios of Tg and nTg mice are shown in Table 1. Water Content. Water content in the left hemisphere was increased 24 hr after ischemia in both groups. However, the increases in water content of MCA cortex and ACA cortex were significantly less in Tg mice than in nTg mice (Fig. 4). Water content in striatum increased significantly at 24 hr of ischemia in both groups, but there was no significant difference between Tg and nTg mice.
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FIG. 3. Reduced hemispheric enlargement (brain edema) in Tg mice following MCA occlusion. Data represent the ratios of left to right hemispheric areas in coronal slices 1, 3, 5, and 7 mm distal from the frontal pole. The ratios in Tg mice are significantly smaller than those in nTg mice in three of the slices. Number of determinations was 15 in each group. *, P < 0.01; **, P < 0.05 (versus nTg, by Student's t test).
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Proc. Natl. Acad. Sci. USA 88 (1991)
Table 1. Total infarcted volume and hemispheric volume in Tg and nTg mice at 24 hr of MCA occlusion nTg Tg Volume, mm3 Left hemisphere 212.3 ± 5.2 189.4 ± 4.3* Right hemisphere 179.5 ± 2.5 176.6 ± 3.9* Infarction 39.9 ± 4.0 25.6 ± 2.1* Percent of infarction 18.4 ± 1.3 13.7 ± 0.8* Left/right ratio 1.18 ± 0.02 1.07 ± 0.01* Total volumes of infarction and of the left and the right hemispheres were calculated according Liu et al. (12). Each value is mean + SEM of 15 animals. *, P < 0.001 (versus nTg, by Student's t test).
Antioxidant Levels. There were no significant differences in GSH levels either among the areas of the brain or between Tg and nTg mice before ischemia (Fig. 5). The GSH level in the left hemisphere decreased significantly from the level of preischemia. MCA cortex showed the most decreased level, but there was no significant difference between Tg (9.4 ± 0.7 nmol/mg of protein) and nTg (9.2 ± 0.6 nmol/mg of protein) mice at 24 hr of ischemia. However, the GSH levels of ACA cortex and striatum in Tg mice (15.8 ± 0.4 and 16.5 ± 0.6 nmol/mg of protein, respectively) were significantly higher than in nTg mice [14.4 ± 0.4 (P < 0.05) and 13.1 ± 0.4 (P < 0.01) nmol/mg of protein, respectively]. AH2 levels showed changes similar to those of GSH levels (Fig. 6). AH2 levels also decreased in each area of the left hemisphere in both Tg and nTg mice. Although there were no significant differences in the AH2 level in MCA cortex, the levels in ACA cortex and striatum of Tg mice (19.3 ± 0.7 and 15.9 ± 0.7 nmol/mg of protein, respectively) were significantly higher than in nTg mice [16.3 ± 0.5 (P < 0.01) and 13.2 ± 0.6 (P < 0.05) nmol/mg of protein, respectively] at 24 hr of ischemia.
DISCUSSION Tg mice of strain TgHS/SF-218 overexpressing CuZn-SOD have a 3.1-fold increased level of endogenous CuZn-SOD activity in brain (16, 25). Evidence from brain homogenates and cultured cells indicates that the CuZn-SOD genes are being expressed in all the nervous elements, including neurons, glia, and endothelial cells (26). Therefore, the complicating and confounding issues regarding the permeability of the blood-brain barrier to the enzyme, its half-life in blood, and potential systemic side effects of exogenously supplied enzyme could be eliminated in studies of ischemic brain
injury.
Although focal ischemia is dense (or complete) in the "focus," which usually compromises a large part of the
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adjacent areas and the overlying cortex, there is a perifocal "penumbra" zone with a more moderate degree of ischemia (5, 27). This area, which is initially viable, albeit with a critical low perfusion, later suffers neuronal death, glial necrosis, and infarction. Very probably, any therapy that proves to decrease infarct size does so by preventing recruitment of the penumbra zone in the infarction process (5). The mechanism for the decrease of GSH in ischemic brain is unknown at present. One metabolic route is the GSH-GSH peroxidase system. This enzymatic system is accompanied by the concomitant formation of oxidized glutathione (GSSG). However, several studies have reported that GSH content decreases after ischemia and/or reperfusion in rat brain without a concomitant increase in GSSG (2, 3, 28). Other studies have suggested that GSH in the absence of GSH peroxidase can act as a free radical scavenger (29-31). From these results, it is suggested that the decrease in GSH levels following cerebral ischemia is caused by GSH not only serving as a substrate for GSH peroxidase but also scavenging free radicals. Recently, we have shown (32) that the endogenous GSH in the brain is a crucial factor in the defense
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FIG. 5. GSH contents in right cerebral cortex, left MCA cortex (infarct cortex), left ACA cortex, and left striatum before and 24 hr after MCA occlusion. GSH levels in left hemisphere decreased significantly in both Tg and nTg mice at 24 hr of ischemia. However, the level in ACA cortex and striatum remained significantly higher in Tg than in nTg mice. Number ofdeterminations was 11 in each group. *, P < 0.01; **, P < 0.05 (versus nTg, by Student's t test). P, preischemia; I, ischemia.
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FIG. 6. AH2 contents in right cerebral cortex, left MCA cortex (infarct cortex), left ACA cortex, and left striatum before and 24 hr after MCA occlusion. AH2 levels showed similar changes to GSH levels. There was no significant difference in MCA cortex between Tg and nTg mice. However, the levels in ACA cortex and striatum were significantly higher in Tg mice than in nTg mice. Number of determinations was 13 in each group. *, P < 0.01; **, P < 0.05 (versus nTg, by Student's t test). P, preischemia; I, ischemia.
Neurobiology: Kinouchi et al. against ischemic injury, since depletion of brain GSH with buthionine sulfoximine, a selective inhibitor for -t-glutamylcysteine synthetase, increased postischemic infarct size and edema. As in other tissues, ascorbic acid exists almost exclusively in its reduced form in brain (>99%) (2, 8, 33). It has also been reported that AH2 decreases significantly following cerebral ischemia and head injury. Ascorbic acid is an efficient scavenger of a variety of oxygen-containing radicals (34, 35). However, it can switch from anti- to prooxidant activity, depending on its concentration and the presence of free transition metal irons (36). Furthermore, despite its nature as a relatively polar lipid-insoluble molecule, several investigators have demonstrated that ascorbate interacts directly not only with components of the phospholipid bilayer but also with superoxide anion as a radical scavenger and that it terminates the propagative formation of free radicals (37). Our studies showed that the penumbra area was spared from cerebral infarction in Tg mice. Although we have not measured the superoxide radicals in the penumbra area because of assay difficulties, we assayed the antioxidant levels (GSH and AH2) in Tg and nTg brains. Given these facts, our data that the levels of GSH and AH2 in the penumbra area remained significantly higher in Tg animals than in nTg animals suggests the possibility that the high level of endogenous CuZn-SOD protects the penumbra area from ischemic damage both by directly scavenging superoxide radicals and by reducing oxidative stress through modulating other antioxidant levels. Furthermore, lipid peroxidation is reduced in trisomy 16 mouse brain, which has a 1.5-fold increased level of CuZn-SOD activity because of a gene dose effect (38). Overall, these observations suggest that increased endogenous SOD activity in Tg mice alters the antioxidant system so as to favor the protection of the brain against ischemic injury. Other important metabolic events in the penumbra are the depolarization and ensuing "calcium overload" of cells by excitatory amino acids (39). It has been shown that excitatory amino acids may promote enzymatic reactions through the activation of N-methyl-D-aspartate receptors leading to cytosolic free Ca21 overload, activation of arachidonic acid cascade turnover, and free radical production (40, 41). Furthermore, brain production of free radicals during ischemia causes simultaneously an increased output of the excitatory amino acids, specifically glutamate (42). Interrupting this vicious cycle by SOD may thus provide protection against ischemic brain injury induced by the arachidonic acidglutamate cascade in the penumbra area. Our results suggest that oxidative stress may be an important factor in amplifying N-methyl-D-aspartate receptor-mediated, Ca2+-dependent biochemical events that lead to neuronal cell death. Further studies are warranted to elucidate these possibilities. Besides the cytoprotective mechanism, one could argue that the differences in cerebral blood flow between Tg and nTg mice may account for the observed differences in neuroprotection. However, our preliminary studies suggest that there were no differences in cerebral blood flow measured by laser doppler, a semiquantitative method, between Tg and nTg mice, nor were any morphological and anatomical differences noted in the vascular supply in the Tg mice. Therefore we conclude that the reduced oxidative stress appears to be the major mechanism underlying the cytoprotection in the Tg mice. Nevertheless, our studies also demonstrated that the GSH levels and the infarct sizes in ischemic brain were closely related to the duration of clipping of the contralateral common carotid artery (32), indicating that the collateral flow and some degree of reperfusion may play an important role contributing to the demonstrated difference in infarct size and edema between Tg and nTg animals.
Proc. Natl. Acad. Sci. USA 88 (1991)
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We have recently demonstrated (17) that vasogenic edema caused by cold injury is reduced in Tg mice overexpressing CuZn-SOD, suggesting that this type ofedema is mediated by superoxide radicals. The finding in the present study that the increase in water content was significantly reduced in the Tg mice following 24 hr of ischemia also supports the involvement of superoxide radicals in the pathogenesis of ischemic brain edema. We speculate that endothelial cells may be the major source of the superoxide radicals that cause brain edema, since endothelial cells have a 3.7-fold greater level of xanthine oxidase activity than do homogenates of cerebral cortex (43). However, a direct correlation between the increased CuZn-SOD in endothelial cells and the reduced edema in Tg mice following focal cerebral ischemic injury cannot presently be established and requires further investigation. We thank Dr. Robert Fishman for his critical comments and thank Teodosia Zamora and Liza Reola for assistance in breeding the mice. This work was supported in part by National Institutes of Health Grants AG08938, NS14543, and NS25372. 1. Flamm, E. S., Demopoulus, H. B., Seligman, M. L., Roser, R. G. & Ransohoff, J. (1978) Stroke 9, 445-447. 2. Cooper, A. J. L., Pulsinelli, W. A. & Duffy, T. E. (1980) J. Neurochem. 35, 1242-1245. 3. Rehncrona, S., Folbergrova, J., Smith, D. S. & Siesjo, B. K. (1980) J. Neurochem. 34, 477-486. 4. Kontos, H. A. & Wei, E. P. (1986) J. Neurosurg. 64, 803-807. 5. Siesjo, B. K., Agardh, C.-D. & Bengtsson, F. (1989) Cereb. Brain Metab. Rev. 1, 165-211. 6. Chan, P. H. (1989) in Cellular Antioxidant Defense Mechanisms, ed. Chow, C. K. (CRC, Boca Raton, FL), pp. 89-109. 7. Hall, E. D. & Braughler, J. M. (1989) Free Rad. Biol. 6, 303-313. 8. Kinuta, Y., Kikuchi, H., Ishikawa, M., Kimura, M. & Itokawa, Y. (1989) J. Neurosurg. 71, 421-429. 9. Abe, K., Yoshida, S., Watson, B., Busto, R., Kogure, K. & Ginsberg, M. D. (1983) Brain Res. 273, 166-169. 10. Watson, B. D., Busto, R., Goldberg, W. J., Santiso, M., Yoshida, S. & Ginsberg, M. D. (1984) J. Neurochem. 43, 268-274. 11. Turrens, J. F., Crapo, J. P. & Freeman, B. A. (1984) J. Clin. Invest. 73, 87-95. 12. Liu, T. H., Beckman, J. S., Freeman, B. A., Hogan, E. L. & Hsu, C. Y. (1989) Am. J. Physiol. 256, H589-H593. 13. Kitagawa, K., Matsumoto, M., Oda, T., Niinobe, M., Hata, R., Handa, N., Fukunaga, R., Isaka, Y., Kimura, K., Maeda, H., Mikoshiba, K. & Kamada, T. (1990) Neuroscience 35, 551-558. 14. Chan, P. H., Longar, S. & Fishman, R. A. (1987) Ann. Neurol. 21, 540-547. 15. Imaizumi, S., Woolworth, V., Fishman, R. A. & Chan, P. H.
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