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Perinatal cerebral hypoxia-ischemia, resulting from compromised placental or pulmonary gas exchange, is a major cause of acute perinatal brain injury, leading ultimately to neurologic damage such as cerebral palsy, mental retardation, and epilepsy(1). To maintain adequate brain metabolism during hypoxia, vasodilation occurs. If asphyxia persists, however, blood flow to the brain is ultimately reduced due to secondary hypotension and bradycardia, resulting in cerebral ischemia and necrosis(2).

During cerebral hypoxia-ischemia, a number of metabolic changes occur that result in the production of oxygen free radicals, most notably superoxide. The accumulation of oxygen free radicals can cause neuronal death through reactions involving lipid peroxidation(3, 4), protein oxidation(5, 6), and DNA damage(7, 8).

CuZn/SOD is an endogenous antioxidant enzyme that removes free radicals from neurons in the brain by converting superoxide anion to hydrogen peroxide. Glutathione peroxidase and catalase are then able to catalyze the disposal of hydrogen peroxide to H2O and O2. Although CuZn/SOD has been shown to reduce infarction severity induced by certain types of injury in adult animal models(9–15), its neuroprotective role in the developing perinatal brain to hypoxic-ischemic injury has yet to be studied.

In perinatal animal models, in vivo studies have demonstrated that the hydroxyl radical scavengers allopurinol and oxypurinol reduce infarction severity due to hypoxic-ischemic injury implying that oxygen free radicals are partly responsible for the damage caused during perinatal cerebral hypoxia-ischemia(16, 17). Because CuZn/SOD is a catalyst of free radical turnover, it might also lessen brain damage during perinatal hypoxia-ischemia, as do the oxygen free radical scavengers allopurinol and oxypurinol.

We used a strain of Tg mice whose heterozygotes express a 3.1-fold increase in human SOD-1 activity in all brain regions compared with their nTg litter mates(18). By using a model of hypoxic-ischemic encephalopathy and comparing hypoxic-ischemic brain damage in CuZn/SOD Tg mice and nTg mice, we sought to determine the effect of overexpression of the CuZn/SOD gene on perinatal mice undergoing hypoxic-ischemic insult.

METHODS

Tg mice. The mice used for these experiments were bred as follows. Tg mice of strain TgHS/SF-218/3 carrying human SOD1 genes were derived from the founder stock described by Epstein et al.(18). A linear 14.5-kb EcoRI-BamHI fragment containing the entire hCuZnSOD gene, was excised from the recombinant plasmid pHGSOD-SV neo and separated from plasmid sequences before microinjection. Approximately 500 copies of the purified fragment were microinjected into the male pronuclei. The founder mice had been bred with CD-1 mice to produce Tg offspring carrying the SOD1 gene. Tg mice were identified by qualitative demonstration of human CuZn/SOD, using nondenaturing gel electrophoresis followed by nitro blue tetrazolium staining as previously described(18). There were no observable phenotypic differences between Tg mice and nTg littermates.

Hypoxic-ischemic injury. We used a perinatal hypoxic-ischemic model developed by Rice et al.(1) that is based on the Levine(19) procedure(20). All animal research was approved by the University of California San Francisco Committee on Animal Research and was performed with the highest standards of humane care as set forth in the Guide for the Care and Use of Laboratory Animals, U.S. Department of Health and Human Services, Publication No. 85-23, 1985.

Briefly, 92 mice at postnatal d 7 were anesthetized with 2.5% halothane, 15% N2O, balance O2, and the right common carotid artery was exposed and occluded by electrical coagulation. The incision was sutured, and the pups were returned to their dams immediately after the surgery for at least 2 h to recover and feed. They were then placed in 1000-mL airtight containers partially submerged in a 37°C water bath through which a humidified atmosphere of 8% O2 and 92% N2 was introduced via inlet and outlet tubing. The pups remained in the hypoxic container for 90 min, and those that survived were returned to their dams.

Preparation and histologic scoring of brains. Five days after the hypoxic-ischemic procedure, the animals were anesthetized with 50 mg/kg pentobarbital intraperitoneally and perfused through the left ventricle with cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brains were immediately removed and postfixed in the same paraformaldehyde solution for 1-3 h and then transferred to cold 30% sucrose in 0.1 M phosphate buffer until sectioning. Coronal forebrain sections were cut at 50-μm intervals using a vibratome. Sections were stained with cresyl violet and then scored for degree of damage blindly as follows: 0, no detectable neuronal cell loss; 1, scattered neuronal cell loss with occasional astroglia; 2, columnar damage in the cortex involving predominantly layers II through IV; and 3, cystic infarction and gliosis.

Brain infarction. The extent of brain infarction was determined by measuring the area of surviving cortex with a video image analysis system using National Institutes of Health Image 1.47. The left and right cortices(contra- and ipsilateral) of three coronal sections from each brain were measured at the level of the anterior hippocampus by tracing the image which was calculated as mm2. Data are expressed as percent viable ipsilateral cortex over contralateral cortex, × 100.

Statistics. Mann-Whitney U test was used to analyze nonparametric ordinal data, and coded χ2 analysis was used for categorical data.

RESULTS

Survival data. Sixty-seven percent of the pups survived the procedure (Table 1). There was no difference in the survival of the Tg and nTg mice to the hypoxic-ischemic procedure (p= 0.602, χ2 test). Three Tg mice and three nTg mice who survived the procedure but were killed by their dams or died naturally before the time of sacrifice were considered to have survived the insult.

Table 1 Survival of transgenic and wild-type mice after hypoxic-ischemic insult
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Histopathology. Figure 1 shows the damage that was observed in the Tg and nTg mice 5 d after the hypoxic-ischemic injury. Brain injury ranged from no detectable damage to extensive cystic infarction. The hippocampus was the most sensitive region to neuronal loss during hypoxia-ischemia in the mice regardless of the presence of the transgene. Only 17% of the Tg mice were free of histologic damage (score of 0), compared with 37% of the nTg mice. In addition, 48% of the Tg mice suffered cystic infarct (score of 3) compared with only 22% of the nTg mice(Table 2). Cystic infarctions varied in severity from destruction limited to portions of the cortex along the middle cerebral artery distribution to infarction that extended to the striatum and the thalamus, virtually destroying the affected hemisphere. Overall, the CuZn/SOD Tg mice had more severe histologic damage scores than their nTg litter-mates(p = 0.0463, Mann-Whitney U test).

Figure 1
figure 1

Histopathologic damage by severity score. Coronal sections of mouse brain through the anterior hippocampus show increasing degrees of histopathologic damage by assigning a numerical value for the degree of damage observed under light microscopy. (A) Cresyl violet-stained section shows no neuronal loss (score 0); B, minimal neuronal cell loss and astroglia (arrow) (score 1); C, columnar infarction (score 2); D, cystic infarction and gliosis(score 3). Scale bar = 200 μm.

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Table 2 Distribution of histopathologic scores in CuZn/SOD Tg and nTg mice after hypoxic-ischemic insult
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Brain infarction. In both Tg and nTg mice, there was a significant difference between the coronal area of the cortex contralateral to the common carotid artery occlusion and the area of remaining ipsilateral cortex. Because the contralateral area is assumed to be approximately equal to cortical area in nonischemic brains, the contralateral areas were used to express the degree of damage due to the hypoxia-ischemia. When areas are expressed as percentages (ipsilateral over contralateral) to minimize the effect of individual variation in brain size (Table 3), Tg mice had more extensive infarction than nTg mice (p = 0.0274, Mann-Whitney).

Table 3 Cortical areas of CuZn/SOD transgenic and non-transgenic mice after hypoxic-ischemic injury
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DISCUSSION

It was our goal to examine the role of superoxide radical formation in the pathogenesis of hypoxic-ischemic injury in 7-d-old mice. Our results demonstrate that CuZn/SOD Tg mice expressing three times normal amounts of CuZn/SOD suffered brain infarction more frequently than did nTg mice. Furthermore, the Tg mice had higher histologic severity scores than the nTg mice, indicating that they suffered more extensive brain damage. These findings suggest that brain injury in 7-d-old mice caused by hypoxia-ischemia is mediated in part by hydrogen peroxide production. The Tg animals did not die more frequently than the control littermates, suggesting that mortality in this model is related more to hemodynamic and cardiogenic compromise which was not evident in either group.

Hydrogen peroxide may account for the damage observed in our Tg mice by its accumulation in the brain due to elevated concentrations of the CuZn/SOD with normal levels of glutathione peroxidase. In the brains of uninjured nTg mice at postnatal d 7, CuZn/SOD mRNA and activity levels are reported to be four times greater than glutathione peroxidase levels(21). Although these activities are measurements obtained in vitro under pharmacologic, not physiologic, conditions, it is possible that this ratio may be increased under conditions of oxidative stress in vivo. The relative discrepancies in enzyme activities may result in an accumulation of hydrogen peroxide. This imbalance leading to hydrogen peroxide accumulation may account for some of the damage observed in the nTg mice. Increasing the activity of the CuZn/SOD, as in the Tg mice, without increasing GSH activity, would increase the accumulation of hydrogen peroxide and lead to greater damage. An accumulation of hydrogen peroxide may cause cellular damage by reacting with the superoxide radical in the presence of Fe2+ or Cu2+ to produce the hydroxyl radical, which is able to attack almost all cell constituents and damage cell membranes by initiating lipid peroxidation(22).

The above mechanism assumes that elevated levels of CuZn/SOD and/or hydrogen peroxide do not induce increased expression of GSH, leading to an increased detoxification of superoxide. Our results imply that induction does not occur, as the Tg mice exhibited more brain injury than the nTg mice instead of less injury as we would have expected if induction occurred. Glutathione concentrations are no different in the CuZn/SOD Tg mice than in nTg mice at P7 but during hypoxia-ischemia there is more consumption of GSH in Tg brains (Ferriero DM, Chan PH, unpublished observations). Similarly, Ceballos-Picot et al.(23) demonstrated that, at 2 mo, 12 mo, and 28 mo, glutathione peroxidase activity is not increased in Tg mice overexpressing CuZn/SOD when compared with the corresponding age group control. However, in the adult animals of the strain used in these experiments, glutathione peroxidase concentrations have been shown to rise in proportion to CuZn/SOD concentrations(24). In murine fibroblasts transfected with CuZn/SOD expression vectors, GSH peroxidase rises after exposure to the superoxide generator, paraquat(25). Further study is needed to determine whether CuZn/SOD and glutathione peroxidase are independently regulated during insults such as hypoxia-ischemia in the immature nervous system.

The outcome of ischemic injury may be different in the perinatal and adult brain due to changing proportions of superoxide detoxifying enzymes. When Chanet al.(26) subjected adults of this Tg strain to permanent middle cerebral artery occlusion, they found no differences between the Tg mice and their nTg littermates in infarct volume, local cerebral blood flow, or neurologic deficits. The difference between these results and our results suggests that enzymes responsible for the pathogenesis of brain injury may be differentially expressed in the perinatal and adult brain. Hothersall et al.(27) found that the activity of glutathione peroxidase was approximately 25% lower in uninjured 1-20-d-old rats compared with 4-mo-old rats, and about 40% lower compared with 12-mo-old rats. Glutathione reductase activity, on the other hand, rapidly increased between d 1 and 5, at which point the activity approximated the adult values(27). The exacerbation of damage observed in our 7-d-old mice compared with the lack of effect of CuZn/SOD overexpression in the adult mice of Chan et al. may have resulted from a decreased ratio of glutathione peroxidase to glutathione reductase in the perinatal mice.

There are many possibilities for the observed increased damage in the Tg brains. Another mechanism which may account for the increased damage observed in our Tg mice is increased toxicity from nitric oxide radical production. Nitric oxide radical (NO˙) is formed in the brain by NOS, a calcium/calmodulin-dependent enzyme. Calcium influx triggered byN-methyl-D-aspartic acid receptor activation during hypoxia-ischemia activates neuronal NOS, producing a significant local elevation in NO˙ production. This NO˙ may combine with the superoxide produced during hypoxia-ischemia to produce peroxynitrite. At physiologic pH, peroxynitrite degrades instantly to hydroxyl radical and nitrogen dioxide(NO2)(28). Although there is increased scavenging of superoxide in the CuZn/SOD Tg mice, which would decrease peroxynitrite, other mechanisms of nitric oxide-induced brain injury exist. There is an iron-independent reaction between NO˙ and hydrogen peroxide that leads to increase production of hydroxyl radical(29). In addition, at subphysiologic levels of intracellular L-arginine or tetrabiopterin, activation of brain NOS by calcium leads to formation of other reactive oxygen species instead of NO, especially hydrogen peroxide(30).

Further support of the hypothesis that NOS is involved in the pathogenesis of injury in these mice comes from the work of Oury et al.(31) who found that Tg adult mice overexpressing human extracellular SOD displayed increased sensitivity to CNS oxygen toxicity in an hyperbaric environment compared with nTg littermates. Inhibition of extracellular SOD and CuZn/SOD reduced the oxygen toxicity and more importantly, in the presence of a NOS inhibitor, the Tg mice no longer displayed any increased sensitivity to CNS oxygen toxicity compared with nTg littermates(31).

In summary, we have found that overexpression of SOD exacerbates perinatal hypoxic-ischemic brain injury. These data suggest that the neonatal brain may be unable to effectively detoxify hydrogen peroxide produced during periods of oxidative stress or that excess NO is produced in this setting. Further studies are underway to determine whether reactive oxygen species are critical determinants of injury after hypoxia-ischemia in the developing nervous system so that effective therapies aimed against their accumulation could prove beneficial.