MICE OVEREXPRESSING EXTRACELLULAR SUPEROXIDE DISMUTASE HAVE INCREASED RESISTANCE TO FOCAL CEREBRAL ISCHEMIA H. SHENG,* R. D. BART,† T. D. OURY,‡ R. D. PEARLSTEIN,§ J. D. CRAPO¶ and D. S. WARNER*§  Departments of *Anesthesiology, †Pediatrics, ‡Pathology and §Surgery, Duke University Medical Center, Durham, NC 27710, U.S.A. ¶Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206, U.S.A. Abstract––Transgenic mice, which had been transfected with the human extracellular superoxide dismutase gene, causing an approximate five-fold increase in brain parenchymal extracellular superoxide dismutase activity, were used to investigate the role of extracellular superoxide dismutase in ischemic brain injury. Transgenic (n =21) and wild-type (n =19) mice underwent 90 min of intraluminal middle cerebral artery occlusion and 24 h of reperfusion. Severity of resultant hemiparesis and cerebral infarct size were measured. Wild-type mice had larger infarcts (cortex: wild type=3714 mm 3 , transgenic=2713 mm 3 , P = 0.03; subcortex: wild type= 3314 mm 3 , transgenic=2310 mm 3 , P = 0.02). Neurological scores, however, were similar (P = 0.29). Other mice underwent autoradiographic determination of intra-ischemic cerebral blood flow. The volume of tissue at risk of infarction (defined as volume of tissue where blood flow was < 25 ml/100 g/min) was similar between groups (cortex: wild type=5115 mm 3 , transgenic= 479 mm 3 , P = 0.65; subcortex: wild type= 3916 mm 3 , transgenic=3717 mm 3 , P = 0.81). These results indicate that antioxidant scavenging of free radicals by extracellular superoxide dismutase plays an important role in the histological response to a focal ischemic brain insult.  1998 IBRO. Published by Elsevier Science Ltd. Key words: extracellular superoxide dismutase, brain, cerebral blood flow, mouse, transgenic, Circle of Willis. Increased free radical formation is a known conse- quence of cerebral ischemia. 4,21 Natural defenses against oxidative stress include the antioxidant enzymes glutathione peroxidase, catalase and super- oxide dismutase (SOD). Three isoforms of SOD are known to exist in the mammalian brain. 1 Copper– zinc superoxide dismutase (CuZn-SOD) is found in the cytosol and nucleus of cells. Increased activity of CuZn-SOD found in mice carrying the transgene for this isoform results in a reduction of both focal and global ischemic brain injury. 13,24 Conversely, tar- geted disruption of the CuZn-SOD coding sequence causes increased edema and enlarged infarcts after murine middle cerebral artery occlusion (MCAO). 8 The manganese superoxide dismutase (Mn-SOD) isoform, located in mitochondria, has been less thor- oughly characterized. However, a preliminary report indicates that mice deficient in Mn-SOD have enlarged infarcts resulting from MCAO. 12 This information cumulatively implicates a crucial role for intracellular SOD in modulating free radical injury associated with cerebral ischemia. The final isoform of SOD, extracellular superoxide dismutase (EC-SOD), is located in the extracellular matrix. In the brain, EC-SOD is normally found in substantially smaller concentrations than either CuZn-SOD or Mn-SOD. 17 For this reason, little attention has been directed towards understanding the role of this SOD isoform in the response to cerebral ischemia. Consequently, there is also little known about the relative importance of extracellular versus intracellular superoxide in the ischemic brain. This experiment utilized a strain of mice overexpress- ing EC-SOD to investigate the role EC-SOD plays in the pathogenesis of ischemia/reperfusion injury. We hypothesized that an increase in EC-SOD activity would result in an attenuation of injury resulting from an episode of transient focal cerebral ischemia. EXPERIMENTAL PROCEDURES This study was approved by the Duke University Animal Care and Use Committee. The EC-SOD transgenic mice used in this experiment have been described previously. 17 Briefly, transgenic mice were generated by microinjecting nuclei of fertilized oocytes from (C57Bl/6C3H)F 1 female mice with the cDNA of human EC-SOD containing a -actin promoter. Mice carrying the transgene were ident- ified by polymerase chain reaction amplification of the To whom correspondence should be addressed. A bbreviations: CBF, cerebral blood flow; CuZn-SOD, copper–zinc superoxide dismutase; EC-SOD, extracellu- lar superoxide dismutase; MCAO, middle cerebral artery occlusion; Mn-SOD, manganese superoxide dismutase; SOD, superoxide dismutase. Pergamon N euroscience Vol. 88, No. 1, pp. 185–191, 1999 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+ 0.00 PII: S0306-4522(98)00208-5 185