Cold-induced Brain Edema and Infarction Are Reduced in Transgenic Mice Overexpressing CuZn-Superoxide Dismutase P. H. Chan, PhD,*t G. Y. Yang, MD," S. F. Chen, PhD,t E. Carlson, BS,$ and C. J. Epstein, MD$§

It has been proposed that oxygen-derived radicals, superoxide in particular, are involved in the alteration of bloodbrain barrier permeability and the pathogenesis of brain edema following trauma, ischemia, and reperfusion injury. Using transgenic mice that overexpress the human gene for copper-zinc-superoxide dismutase, we studied the role of superoxide radicals in the blood-brain permeability changes, edema development, and delayed infarction resulting from cold-trauma brain injury. At 2 hours after a 30-second cold injury, cerebral water and Evans blue contents were reduced, respectively, from 80 f 0.2% and 132.7 f 12.9 pgigm of dry weight for nontransgenic mice to 78.5 k 0.3% and 87.1 9.9 pg/gm of dry weight for transgenic mice. Infarction, as measured by 2,3,5-triphenyltetraoliumchloride staining, was reduced by 52% in transgenic brains. These data indicate that an increased level of superoxide dismutase activity in the brain reduces the development of vasogenic brain edema and infarction. Superoxide radicals play an important role in the pathogenesis of these lesions in cold-traumatized brain.

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Chan PH, Yang GY, Chen SF, Carlson E, Epstein CJ. Cold-induced brain edema and infarction are reduced in transgenic mice overexpressing CuZn-superoxide dismutase. Ann Neurol 1991;29:482-486

During the past few decades, a large accumulated body of experimental data has indicated that the biological reduction of molecular oxygen can yield dangerously reactive free rddicds [l}. About 2 to 55% of the electron flow in isolated brain mitochondria produces superoxide (0,- ) and hydrogen peroxide (H,O,) {2). These constantly produced oxygen radicals are scavenged by endogenous antioxidants including superoxide dismutases (SODS) and glutathione perixodase. However, pathological insults, including ischemia and trauma, perturb this defense mechanism and result in the overproduction of oxygen radicals and increased levels of lipid peroxidation {3-7). Using a model of brain edema produced by coldinduced injury in rats, we demonstrated an early elevation in the concentrations of superoxide radical, followed by permeability changes in the blood-brain barrier and development of edema in injured brain {S]. Intravenous injection of liposome-entrapped copperzinc-superoxide dismutase ( C a n - S O D ) reduced the brain levels of superoxide radicals and ameliorated permeability changes in the blood-brain barrier and brain edema 18). Although these studies established the role of superoxide radicals in the pathogenesis of vasogenic edema and brain injury, they did not elucidate the site

and the mode of the action of SOD. Furthermore, it is also unclear whether or not the beneficial effects of liposome-entrapped SOD were due to its ability 10 inhibit the inflammatory response of leukocytes and other cells to brain injury. To assess the direct role of SOD in the pathogenesis of vasogenic brain edema and infarction in cold-injured brain, we used human CuZn-SOD transgenic mice overexpressing C a n - S O D activity [9}. The aim of this study was to test the hypothesis that the severity of vasogenic brain edema and subsequent infarction would be reduced by the increased endogenous level of CuZn-SOD enzymatic activity in brain tissue, should superoxide radicals play a major role in their pathogenesis.

From the Departments of 'Neurosurgery, TNeudogy, +"Pediatrics, and §Biochermstry and Biophysics, University of Cahfornia School of Medicine, San Francisco, CA

Address correspondence to Dr Chan, CNS Injury and Edema Research Center, and the Department of Neurology, School of Medicine, University of California, San Francisco, CA 94143-0114

Materials and Methods Transgenic mice of strain TgHS/SF-2 18 carrying the human CuZn-SOD (h-SOD-1)gene, produced as described by Epstein and colleagues @},were used. The genome of this strain carries several copies of the h-SOD-1 gene, presumably in a tandem array. The founder mice have been bred to produce transgenic offspring expressing the h-SOD-1 genes. Transgenic mice were identified by northern blot analysis for the presence of the messenger RNA (rnRNA) of h-SOD-1, as well as by the detection of h-SOD-1 enzymatic activity in

Received Aug 16, 1990, and in revised form Nov 1 Accepted for publication Nov 5 , 1990

482 Copyright 0 1991 by the American Neurological Association

brain tissue using nondenaturing gel electrophoresis followed by nitroblue tetrazolium staining 191. There were no observable phenotypic differences between transgenic mice and nontransgenic littermates.

Determination of h-SOD-1 Activity The expression of h-SOD-1 activity in brain and spinal cord was checked both by nondenaturing gel electrophoresis 193 and by the assay of total C a n - S O D activity as assessed by its ability to inhibit superoxide radical-dependent cytochrome C reduction {lo]. Tissues were homogenized in approximately 10 volumes of 1 mM ethylenediaminetetraacetic acid (EDTA) and centrifuged at 4000 g. The supernatants containing 30 p,g of protein in 5 to 10 yl were analyzed by electrophoresis on a 10% nondenaturing polyacrylarnide gel followed by nitroblue tetrazolium staining. The remaining cytosolic fraction was used for assay of total CuZn-SOD activity [lo]. One unit of SOD is defined as the amount of SOD required to inhibit cytochrome C reduction by 50%. Traama Model Transgenic mice and nontransgenic littermates were subjected to cold-induced injury in a randomized blind fashion. The identity of the mice was withheld from the operator. Individual animals were assigned a code, and the code was not broken until all measurements were completed. The mice, weighing 25 to 35 gm, were anesthetized with 35 mgi 100 gm of body weight of chloral hydrate injected intraperitoneally and placed in a stereotaxic apparatus. A concave probe, 0.5 cm in diameter, attached to a brass cup (20 cm3) filled with dry ice and acetone ( - 50"C), was applied directly to the right side of the bony skull for 30 seconds; the overlying skin wound was closed with a suture after the cold injury. This cold injury model is a highly reproducible method of inducing vasogenic brain edema, as has previously been shown in rats {S, 111. Determinations of Brain Water and Evans B b e Permeability Evans blue, 0.03 ml, 4% in Krebs-Ringer (K-R) solution, was administered through a femoral vein 90 minutes after cold injury. A thoracotomy was performed 30 minutes later. The animal was perfused with 0.9% saline solution at room temperature through the aorta via the left ventricle. A volume of 10 to 20 ml at a pressure of about 80 mm H g was given until the collected perfusate was colorless. The brain was removed quickly, and the cortex, which was stamed by Evans blue, was removed and weighed. An identical portion ofthe contralateral hemisphere was also removed These cortices were dried at 105°C for 24 hours, reweighed, and placed in 1.0 ml of K-R solution for another 24 hours. The samples were then homogenized in the K-R solution and mixed with a vortex for 2 minutes after the addition of 0.5 ml of 60% trichloroacetic acid to precipitate protein. The samples were then cooled for 30 minutes and centrifuged for 20 minutes at 1,000 g, and the supernatants were measured at 610 nm for absorbance of Evans blue using a Hewlett Packard 845 1A DIODE array spectrophotometer (Palo Alto, CA). Evans blue is expressed as &gm of dry weight calculated against a standard curve. Water content is expressed as %H,O, which is calculated as [(wet weight-dry weight)/wet weight) x 100%.

Determination of infarct Size At 24 hours after cold injury, the brains were removed without perfusion. Coronal brain slices, 2 mm thick, were obtained using a brain slicer. The brain slices were then immersed in a 2% 2,3,5-triphenyltetraoliumchloride (TI'C) solution (in Dulbecco phosphate buffer, p H 7 . 4 )as reported previously E12). The infarcted area was quantitated by an image analysis system [13], and the percentage of the infarct area (non-'ITC staining area) with respect to total area was calculated. Statistical Analysis Differences among the groups were assessed by analysis of variance. Differences between individual groups were determined by Student's t test. The minimum level of significance was p 5 0.05. Results are expressed as mean f standard error of mean (SEM).

Results Figure 1 shows the presence of human CuZn-SOD enzymatic activity in cerebral cortex, cerebellum, and spinal cord resulting from the expression of h-SOD- 1 transgenes in mice. In nontransgenic mice, only mouse C a n - S O D activity was expressed, whereas in transgenic mice human SOD homodimer activity as well as the humadmouse SOD heterodimer were found. The actual amount of total CuZn-SOD activity in cerebral cortices, cerebella, and spinal cords of transgenic and nontransgenic mice are shown in the Table. The cortices and cerebella of transgenics had a near threefold increase in CuZn-SOD activity as compared to that of nontransgenics. Transgenic spinal cord CuZn-SOD

Fig 1. Expression of human copper-zific-superoxidedismutase (CuZnSODj enzymatic activity in cwebralcortex, rerebellam,and spinal cord of transgenic mice, Both mouse and human CuZnSOD enzymatic activity were detected using nondenaturing gel electrophoresisfollowed by nitvoblue tetrazoliamstainang. i= transgenic mice; - = nontransgenic mice.

Chan et al: Cold-Induced Brain Injury in Transgenic Mice

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CuZn-SOD Lmels (unitslmg of Protein) in Neural Tissues of Tranqenic and Nontransgenic Mice"

Nontransgenic (N) Transgenic (T) T/N Cerebral cortex 7.9 5 0.5 Cerebellum 9.0 * 2.0 Spinal cord 15.4 t 2.0

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Fig 2. Brain water contents in the ipsilateral cold-lesioned hemzsphere and contralateral (control) hemisphere in nontransgenic (normal, n = 25) and transgenic mice in = 231. Values are mean 2 SEM. "p 5 0.01, compared to contralateral hemisphere. 'p 5 0.05, compared to nontransgenic cold-lesioned hemisphere. Analysis of variance followed by paired t test or unpaired t tests was used, respectively.

activity increased only 2.3-fold, but the nontransgenic level was higher than in other parts of the nervous sys tern. Following the cold injury, the cerebral water content in the nontransgenic mice was significantly elevated from a value of 77.5 -+ 0.1% in the contralateral hemisphere to 80.0 0.2% in the ipsilateral hemisphere (n = 25). However, in transgenic mice, the cerebral water content remained low in the hemisphere with the lesion (78.5 2 0.3%), as compared to the contralateral hemisphere (77.5 k 0.25%) (n = 23) (Fig 2). Similarly, Evans blue permeability was significantly reduced in the cerebral cortex of the cold-injured transgenic mice (Fig 3B). The brain Evans blue content in nontransgenic mice increased from a control value of 23.6 2 2.3 to 130.7 2 12.9 pdgm of dry weight, whereas the levels in transgenic mice increased from 30.7 t 3.8 to 87.1 f 9.9 &gm of dry weight (see Fig 3C). These data demonstrate a twofold increase in blood-brain barrier permeability following cold injury in nontransgenic mice, compared with transgenic mice. The development of cerebral infarction, measured by TTC staining for viable mitochondria1 dehydrogenase activities, was significantly reduced in brain cortex

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484 Annals of Neurology Vol 29 No 5 May 1991

of transgenic mice following cold injury (Fig 4A). The cerebral cortical infarct area was 6.6 k 0.9% for nontransgenic and 3.2 t 0.4% for transgenic mice, a 52% reduction (see Fig 4B).

Discussion Oxygen-derived radicals, superoxide radicals in particular, have been implicated in the pathogenesis of brain edema, microvascular abnormality, and neuronal cell death following cerebral ischemia, trauma, and other neurological disorders [ 3 , 4 , 6, 141. Numerous experimental approaches have been used to identify the superoxide radical reactions, as well as to ameliorate the superoxide radical production associated with neurological dysfunction. Lui and associates successfully used polyethylene glycol (PEG)-conjugated SOD and PEGcatalase to reduce the extent of cerebral cortical infarction following focal cerebral ischemia [ 151. Liposome-entrapped CuZn-SOD has been successfully used by our laboratory to ameliorate cold-induced vasogenic edema and chronic infarction following focal cerebral ischemia IS, 12). Although these studies may provide therapeutic potential in ischemic and traumatized brain injury, alternative experimental approaches are needed to clarify the mode of action of SOD in brain injury. One such experimental model is the use of transgenic mice that overexpress endogenous anrioxidant activity. Epstein and associates [9} developed transgenic mice overexpressing CuZn-SOD [9, 16, 171. The transgenic strain of TgHSISF-218 used in the present study has been well characterized, showing that only the h-SOD-1 genes are the extra genes in the mouse genome [93. Although qualitative cellular expression of h-SOD-1 genes in the central nervous system in situ was not performed in our present study, circumstantial evidence obtained from brain homogenates and from cultured cells has indicated that h-SOD-1 genes are being expressed in all the neural elements including neurons, glia, and endothelial cells. Primary cortical neurons have been successfully developed from these transgenic mice and from their normal nontransgenic littermates. We have shown that glutamate neurotoxicity is significantly reduced in transgenic neurons that contain a 2.5-fold increase in SOD enzymatic activity [18}. Thus, h-SOD-1 transgenic Fig 3. Redzrced Evans blue extraz,fasationin cold-injured brain of transgenic mice. A represmtatitJepicture of Evans blue permeability in cold-injured brain of nontransgenic (A)and transgenic mice (B). (Cj Quantitaticte measurement of Evans blue contents in ipsikzteral and contralateral hemispheres obtained from transgenic in = 23) and nontransgenic mice in = 25j. Values are mean SEM. "p < 0.01, compared t o contralateral hemisphere. 'p < 0.01, compared t o ipsileteral cold-lesioned hemisphere. Analysis of variance jollwed by paired t test fov the same animal or unpaired t test for different animals was used.

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Fig 4. Redmd infarct size in cold-injwed brain of transgenic mice. Infarct size was determined by 2,3,5-triphen.y~tetrazo~ian~ chloride (TTC)staining for mitochondria1dehydrogenase activity. (A)A representativepicture taken aftw TTC staining, indicating the dztively smaller infarct size in transgenicmice. (BI Quantitative anai'ysis of infarct .rize in nontransgenic (n = 1SJ and transgenic mice In = 15: p < 0.01). Vabes are mean f SEM, wing anahsir of variance followed by anpaired t test.

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Chan et al: Cold-Induced Brain lnjury in Transgenic Mice

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mice appear to be a valuable model for studying the role of superoxide radicals in brain injury. We chose cold-induced brain trauma and injury in mice as a model for early development of vasogenic edema (i.e., 1 to 4 hours) and delayed infarction (i.e., 24 hours) for several reasons: First, the time course of the permeability changes in the blood-brain barrier and vasogenic edema have been well characterized in rats in o u r laboratory. Our preliminary and pilot studies also have shown that the temporal sequence for vasogenic edema and infarction were similar in mice. O n this basis, the times for study of both vasogenic edema and infarction were chosen. Second, the pathophysiology and biochemical sequelae of the cold-induced brain injury are similar to traumatic brain injury since vascular compartments are the major targets for free radical attack [3, 8, 111. In addition, this cold-injury model displays focal neuronal and glial damage that is similar to the pathological events following head trauma. The levels of both brain water and Evans blue 2 hours following a 30-second cold injury were significantly reduced in transgenic mice (see Figs 2, 3, and

4). Although we did not directly measure the level of brain superoxide radicals in these studies, the data suggest that the activity of a single form of SOD is responsible for the pathogenesis of vasogenic edema associated with vascular damage following traumatic brain injury. However, a direct cause-and-effect relationship between increased SOD in endothelial cells and reduced vasogenic edema in transgenic mice following cold-induced injury is not clear and requires further elucidation. Furthermore, our studies also demonstrate that delayed infarction, as measured by the lack of 'ITC staining of mitochondria1 dehydrogenase activity, was significantly reduced in transgenic mice 24 hours after injury. These results further suggest that superoxide radicals play a major role in causing brain-cell death following a cold injury. It is not clear, however, which brain cells (neurons, astrocytes, or endothelial cells) are more resistant to the cold-induced injury. It is also not clear whether the protective mechanism of SOD against cortical infarction is related to the reduction in glutamate toxicity observed in the in vitro neuronal culture studies [18). In addition to their relevance to the pathogenesis of vasogenic edema in brain trauma, the human CuZnSOD transgenic mice will be of special importance in assessing the role of superoxide radicals in cerebral ischemia. Our preliminary data have suggested that cerebral infarction following focal cerebral ischemia is significantly reduced in these transgenic animals, further confirming the role of superoxide radicals in the pathogenesis of neuronal death in focal cerebral ischemia [19]. Thus, the transgenic mice overexpressing h-SOD-1 are valuable models for studying the potential role of free radicals and CuZn-SOD in neurological 486 Annals of Neurology Vol 29 No 5

May 1991

disorders, including aging, Alzheimer's disease, and other forms of neurodegenerative diseases. This work was supported in part by National Institutes of Health grants HD-17001, AG-08938, NS-14543, and NS-25372. We thank Dr Robert Fishman for his critical comments, J. J. Hollingsworth for preparing the manuscript, and Teodosia Zamora for raising the animals.

References 1. Fredovich I. Biological effects of the superoxide radical. Arch Biochem Biophys 1986;274:1-11 2. Boveris A, Chance B. The mitochondrial generation of hydrogen peroxide. Biochem J 1973;134:707-716 3. Kontos HA. Oxygen radicals in cerebral vascular injury. Circ Res 1985;57:508-516 4. Hall ED, Braughler JM. Central nervous system trauma and stroke. 11. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation. Free Radic Biol Med 1989;6:303-313 5 . Siesjo BK, Ahardh C-D, Bengtsson F. Free radicals and brain damage. Cereb Brain Metab Rev 1989;1:165-211 6. Chan PH. The role of oxygen radicals in brain injury and edema. In: Chow CK, ed. Cellular antioxidant defense mechanisms. Boca Raton, F L CRC Press, 1989;89-109 7. Choi DW. Glutamate neurotoxiciry and diseases of the nervous system. Neuron 1988;1:623-634 8. Chan PH, Longar S, Fishman RA. Protective effects of liposome-entrapped superoxide dismutase on post-traumatic brain edema. Ann Neurol 1987;21:540-547 9. Epstein CJ, Avrabam KB, Lovett M, et al. Transgenic mice with increased CdZn-superoxide dismutase activity: animal model of dosage effects in Down syndrome. Proc Natl Acad Sci USA 1987;84:8041-8048 10. Crapo JD, McCord JM, Fridovich I. Preparation and assay of slrperoxide dismutase. Methods Enzymol 1778;53:382-393 11. Chan PH, Longar S, Fishman RA. Phospholipid degradation and edema development in cold injured rat brain. Brain Res 1983;227:329-337 12. Imaizumi S, Woolworth V, Fishman RA, Chan PH. Liposomeentrapped superoxide dismutase reduces cerebral infarction in focal cerebral ischemia. Stroke 1990;2I :1312- 1317 13. Swanson RA, Morton MT, Wu GT, et al. A semiautomated method for measuring brain infarct volume. J Cereb Blood Flow Metab 1990;10:290-293 14. Choi DW. Methods for antagonizing glutamate neurotoxicity. Cerebrovasc Brain Metab Rev 1990;2:105-147 15. Liu TH, Beckman JS, Freeman BA, et al. Polyethylene glycolconjugated superoxide dismutase and catalase reduce ischemic brain injury. Am J Physiol 1989;256:H589-H593 16. Epstein CJ, Huang 'IT, Chan PH, Carlson E. The molecular biology of Down syndrome. In: Beyreuther K, Schettle G, eds. Proceedings of the International Workshop on the Molecular Mechanisms of Aging. Heidelberg: Springer, 1990~98-109 17. Epstein CJ, Berger CN, Carlson EJ, et al. Models for Down syndrome: chromosome 2 1-specific genes in mice. In: Patterson D, Epstein CJ, eds. Molecular genetics of chromosome 21 and Down syndrome. New York: Wiley-Liss, 1990215-232 18. Chan PH, Chu L, Chen SF, et al. Reducedglutamate neurotoxicity in transgenic mice overexpressing human Can-superoxide clismutase. In: Davis J, ed. 17th Princeton Conference on Cerebrovascular Diseases. Supplement to Stroke, 1990;21:1II-80111-83

19. Kinouchi H , Imdizumi S, Carlson E, et al. Focal cerebral ischemic infarction and brain edema are reduced in transgenic mice overexpressing human superoxide dismutase. SOCNeurosci Abstr 1990;16:276