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Stroke. 1997;28:1797-1804

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(Stroke. 1997;28:1797-1804.)
© 1997 American Heart Association, Inc.

Articles

Overexpression of CuZn-Superoxide Dismutase Reduces Hippocampal Injury After Global Ischemia in Transgenic Mice

Kensuke Murakami, MD; Takeo Kondo, MD; Charles J. Epstein, MD; Pak H. Chan, PhD

From the CNS Injury and Edema Research Center, Departments of Neurological Surgery and Neurology (K.M., T.K., P.H.C.) and Department of Pediatrics (C.J.E.), School of Medicine, University of California, San Francisco, Calif.

Correspondence to Pak H. Chan, PhD, Department of Neurosurgery, Stanford University School of Medicine, 701B Welch Rd, Stanford, CA 94305-5784. E-mail phchan{at}itsa.ucsf.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Background and Purpose The role of copper, zinc-superoxide dismutase (CuZn-SOD) in hippocampal injury after transient global ischemia was studied using transgenic (Tg) mice and wild-type littermates.

Methods Global ischemia was induced by bilateral common carotid artery occlusion. The hemisphere with the hypoplastic posterior communicating artery was determined and then the hippocampus in this hemisphere was evaluated qualitatively using a score of 0 to 4 and quantitatively using an image analyzer.

Results Hippocampal injury was reduced in Tg mice after both 5 and 10 minutes of ischemia. In the 5-minute ischemia group, the mean score of the injury was significantly lower in Tg than nontransgenic (nTg) mice at 3 days. In the 10-minute group, the hippocampal injury was reduced more in Tg than nTg mice at 1 day. Quantitative evaluation by an image analyzer confirmed the qualitative data. Neurons with fragmented DNA were also studied in the hippocampal injury. In the 5-minute group, despite the reduction of the injury in Tg mice, their neurons with fragmented DNA were relatively increased at 1 day. In the 10-minute group, this ratio was almost the same in both nTg and Tg mice.

Conclusions CuZn-SOD plays a protective role in the pathogenesis of selective hippocampal injury after brief ischemia, whether the insult is relatively mild or intense. Furthermore, CuZn-SOD may reduce both necrotic and DNA fragmented neuronal death after global ischemia.


Key Words: cerebral ischemia, global • superoxide dismutase • hippocampus • mice


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Oxygen free radicals or oxidants have been proposed to be involved in the development of many neurological disorders and brain dysfunctions.1 2 One role of oxygen free radicals in brain injury appears to involve reperfusion after cerebral ischemia. Reperfusion supplies oxygen to the ischemic region of the brain, but the oxygen could be the substrate for oxidative reaction producing oxygen radicals. Although several antioxidant enzymes (including SOD, glutathione peroxidase, and catalase) process those oxygen radicals, overproduced oxygen radicals exceeding the capacity of the endogenous antioxidant enzymes cause oxidative stress or injury of brain cells in pathological conditions such as ischemia. We have demonstrated that the overexpression of CuZn-SOD, a cytosolic isoenzyme of SODs specific for scavenging superoxide radicals in Tg mice plays a protective role in the pathogenesis of superoxide radical-mediated brain injury, including cold injury-induced brain edema,3 focal transient ischemia/reperfusion injury,4 5 traumatic brain injury,6 and hypoxic and excitotoxic neuronal injury in cultures.7 8 In CuZn-SOD Tg mice, cerebral infarction and brain edema after transient focal ischemia followed by reperfusion were highly reduced. Furthermore, neurological deficit outcome was also improved in Tg mice versus nTg mice. However, a significant reduction of infarct size after permanent focal ischemia could not be seen in the Tg mice.9 Therefore, overexpressed CuZn-SOD is mainly neuroprotective during reperfusion after ischemia.

The hippocampus, particularly the CA1 subregion, is known to be one of the most vulnerable regions in the brain to transient global ischemia. Neuronal loss in the CA1 subregion of the hippocampus has been shown to occur in a delayed fashion after transient global cerebral ischemia,10 11 although its detailed mechanism is still unclear. Recent investigations demonstrated that apoptotic neuronal death, which has different morphological and biochemical features from those of necrosis, or passive cell death, contributes to neuronal injury after various brain insults, including ischemia,12 13 trauma,14 and excitotoxicity.15 16 17 Hippocampal injury after transient global ischemia also has been shown to result from the apoptotic mechanism.18 19 Although the mechanism of apoptosis induction is likely to be multifactorial, reactive oxygen species have been suggested as a major mediator for apoptosis.20 21 22 23

The present study in CuZn-SOD Tg mice was designed to clarify whether CuZn-SOD plays a protective role in the pathogenesis of hippocampal injury after transient global ischemia and if so which of the two pathways to neuronal cell death, apoptosis or necrosis, is ameliorated in hippocampal injury after transient global ischemia.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Global Cerebral Ischemia
CuZn-SOD Tg HS/SF-218/3 mice and nTg littermates (male, 3 months old, 35 to 45 g) that overexpressed a threefold increase in CuZn-SOD activity in brain cells24 were subjected to global ischemia. Global ischemia was induced by BCCAO under controlled ventilation as detailed below. All animals were treated in accordance with the University of California, San Francisco, guidelines, The Treatment of Laboratory Animals, and an animal protocol approved by the Animal Care Committee of the University of California.

Mice were anesthetized with chloral hydrate (350 mg/kg IP) and xylazine (4 mg/kg IP). Skin incision was performed on the midline of the ventral neck, and the endotracheal tube was intubated under a surgical microscope. Respiration was controlled by using an animal ventilator (rodent ventilator model 683) with inspiratory stroke volume of 0.5 mL and a respiratory rate of 120 breaths per minute. Rectal temperature was maintained at 37°C with a homeothermic blanket. BCCA bifurcations were exposed and temporary clips (Zen temporary clip) were applied to the bifurcation to occlude both the external and internal carotid arteries. After 5 or 10 minutes of BCCAO, the temporary clips were removed and the restoration of blood flow visually observed. Skin incisions were sutured, and the experimental animals were cared for in individual cages at 20°C.

Evaluation of Physiological Condition
Four each of the nTg and Tg mice were used to assess physiological conditions. Before BCCAO was induced, the left femoral artery was cannulated to measure MABP, pH, PCO2, and PO2. MABP values were measured an average of 30 seconds to 1 minute before ischemia, during ischemia, and 1 minute after blood flow restoration. Arterial blood samples for blood gas analysis were taken after postischemia MABP values were measured.

Carbon Black Evaluation of Plasticity of the Posterior Communicating Arteries
Since the plasticity of the PcomAs would influence the outcome of hippocampal injury after transient global ischemia, we assessed the plasticity of the PcomAs before the histological evaluation of the hippocampal injury. The experimental animals were anesthetized with ketamine (200 mg/kg) and xylazine (10 mg/kg). After thoracotomy was performed, a cannula was introduced into the ascending aorta through the left ventricle. Transcardial perfusion fixation was performed with 200 mL of 10 U/mL heparin in saline and 200 mL of 3.7% formaldehyde. Carbon black (KOH-I-NOOR) in an equal volume of 20% gelatin in ddH2O (0.2 mL) was injected through the cannula. The removed brains were fixed in 3.7% formaldehyde for 24 hours and stored in PBS. Plasticity of the PcomAs in both hemispheres was assessed by a blinded investigator using a dissecting microscope (Stemi 2000C). The PcomA in each hemisphere was examined independently and graded on a scale of 0 to 3 (Fig 1Down). Group 0 indicated no connection between anterior and posterior circulation; group 1, anastomosis in capillary phase; group 2, small truncal PcomA; group 3, truncal PcomA. Groups 0 and 1 were classified as containing hypoplastic PcomAs, and groups 2 and 3 as containing normal PcomAs. The arteries with scores of 0 and 1 were classified as the hypoplastic PcomA group [PcomA(-)] and those with scores of 2 and 3 were classified as the normal PcomA group [PcomA(+)].



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  		Figure 1. Scheme showing various levels of plasticity of the PcomA (A through D) and representative photomicrographs of the PcomA injected with carbon black (E, F). A indicates Group 0; B, group 1; C, group 2; D, group 3. Groups 0 and 1 contained hypoplastic PcomAs and groups 2 and 3 contained normal PcomAs. E indicates the hemisphere graded as Group 0; F, the hemisphere with a patent PcomA (arrowhead) graded as Group 3. Note that the posterior cerebral artery (PCA) (open arrow) originates from the internal carotid artery (ICA) and the PcomA connects the PCA and the superior cerebellar artery (SCA) (arrow) in mice. BA indicates basilar artery.

Histological Analysis of Hippocampal Injury
Brain samples, which were fixed with 3.7% formaldehyde and evaluated for PcomA plasticity, were also used for evaluation of the hippocampal injury. The hippocampus in the hemisphere with the hypoplastic PcomA was chosen for the histological analysis of hippocampal injury on the basis that complete ischemia might be induced without a supply of collateral blood flow from posterior circulation. Coronal sections 50-µm thick were taken from the brain (including the hippocampus) using a vibratome. The brain sections were stained with cresyl violet. Neuronal damage in the hippocampus was qualitatively and quantitatively evaluated by a blinded investigator. The qualitative evaluation was based on a scoring system of 0 to 4, as suggested by Møller et al25 (Fig 2Down). Using an image analysis system (MCID) quantitative evaluation was done by measuring the length of CA1 neuronal loss in the brain section at the level of the posterior commissure. The present system was created to evaluate infarct size in focal stroke by measuring the areas of stained sections with optical densities exceeding a threshold value26 and was originally applied to measure the neuronal loss in CA1 after global ischemia without sampling error and observer bias. The ratio of CA1 neuronal loss was calculated as (length of CA1 neuronal loss/length of total CA1 subregion)x100%.



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  		Figure 2. Representative photomicrographs showing the typical injury of the hippocampus in each grade. A and F indicate no damage to any hippocampal subregion (Grade 0); B and G, scattered ischemic neurons in CA1 and/or hilus subregions (Grade 2); C and H, moderate ischemic neuronal loss in CA1 subregion (Grade 3); D and I, severe pyramidal cell loss within the CA1 subregion (Grade 4); E and J, extensive cell loss in all hippocampal subregions (Grade 5). Scale bar=1 mm (A through E), 500 µm (F through J).

In Situ Detection and Quantification of Neurons with DNA Fragmentation in Hippocampal Injury
DNA-fragmented neurons were evaluated using TUNEL staining. Brain samples for the detection of DNA fragmentation, a possible indication of apoptosis in hippocampal injury, were prepared separately. Brains were frozen in 2-methyl butane at -20°C immediately after decapitation. Brain sections at the level of the posterior commissure were taken using a cryostat and stored at -80°C.

Slides were fixed with 3.7% formaldehyde in PBS for 45 minutes. Endogenous peroxidase was inactivated with 2% hydrogen peroxide and 100 mmol/L sodium azide for 30 minutes. After slides were washed with PBS, they were immersed in TdT buffer (Gibco BRL) at room temperature for 15 minutes and incubated with TdT and biotin-16-uridine-5'-triphosphate (Boehringer Mannheim) at 37°C for 60 minutes. The reaction was stopped by washing the slides with 6 mmol/L sodium citrate and 60 mmol/L sodium chloride for 30 minutes; the slides then were incubated with 2% bovine serum albumin in PBS. After the sections were washed with PBS, they were incubated with avidin peroxidase for 30 minutes at room temperature and staining was visualized with 3 mmol/L 3,3'-diaminobenzidine tetrahydrochloride and 18 mmol/L hydrogen peroxide in PBS. These brain sections were also stained with methyl green.

On the basis that TUNEL-positive cells have extensively damaged DNA and are probably undergoing apoptotic cell death, these cells in the CA1 subregion were quantified with a light microscope by a blinded investigator. A 5x5-mm grid was located approximately at the center of the hippocampal lesion in the CA1 subregion. The numbers of TUNEL-positive and total injured neurons within the grid were counted at a magnification of x400. The ratio of the number of TUNEL-positive neurons to the total number of the injured neurons was calculated and expressed as percent of TUNEL-positive cells in each group.

Statistical Analysis
We used the Student's t test for data comparison between two groups. In the histological analysis, the statistical significance of differences between each time course and mice group was evaluated by Kruskal-Wallis followed by intergroup Mann-Whitney U test in qualitative analysis and by ANOVA in quantitative analysis. The quantitative analysis of TUNEL-positive cells was evaluated using ANOVA between each group. Significance between groups was assigned at the level of <5% probability (P<.05).


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Physiological Conditions and Mortality in Global Ischemia
As shown in Table 1, physiological data showed no significant difference between nTg and Tg mice. MABPs were slightly lower than those in mice anesthetized with chloral hydrate, as we previously reported.27 However, blood gas measurements showed slightly higher PO2 and lower PCO2 compared with the previous data reported.

The mortality of each group is shown in Table 2. There was no statistically significant difference in these mortality data. In the 5-minute ischemia group, the mortality was higher in nTg mice than in Tg mice at both 1 and 3 days. However, the mortality was slightly greater in Tg than in nTg animals after 10 minutes ischemia.

Plasticity of the PcomA
No significant difference was seen between nTg and Tg mice in the plasticity of the PcomA (Fig 3Down). Mice with patent PcomAs on either side were most frequently observed in both the nTg and Tg groups. The mean score of PcomA plasticity was 1.17 in nTg (n=92) and 1.46 in Tg (n=96) mice, and there was no significant difference between the two groups (Mann-Whitney U test).



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  		Figure 3. Plasticity of the posterior communicating artery (PcomA) in nTg and Tg mice. White and black dots show scores of each evaluated hemisphere, and the bars show the mean score of PcomA in nTg and Tg mice, respectively. There was no significant difference in the plasticity of PcomA between nTg and Tg mice.

Hippocampal Injury After Global Ischemia
Injured neurons in the hippocampus showed various levels of severity after global ischemia. The hemisphere with the hypoplastic PcomA was therefore used to normalize the anatomic background, which could affect the ischemic condition induced by BCCAO. Several types of damaged neurons were observed in the hippocampal injury. One of those neurons displayed the slightly condensed nucleus. The most frequently observed neurons in the injured hippocampus displayed an oval or triangular nucleus (Fig 4ADown). These neurons, the neurons with an oval-shaped nucleus in particular, occasionally displayed small particles that appeared to be fragmented nuclei. These compact neurons were observed to coexist in the same lesion of the hippocampus. On the other hand, hippocampal neurons with different features of morphology displayed a swollen cell body or cell lysis (Fig 4CDown).



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  		Figure 4. Representative photomicrographs showing morphological features of injured hippocampal neurons by cresyl violet staining (A and C) and DNA fragmentation by TUNEL staining (B and D) at 1 day after 10 minutes ischemia in nTg mice and at 3 days after 5 minutes ischemia in Tg mice, respectively. Hippocampal neurons with condensed nuclei were partly labeled by TUNEL staining (A and B), indicating no relationship of morphological and biochemical features in those neurons. However, most of morphologically necrotic neurons, displaying swollen cell bodies or nucleolysis were not labeled, and if labeled, showed nonspecific staining (B and D). Scale bar=100 µm.

The hippocampal injury with hypoplastic PcomA in the ipsilateral hemisphere was evaluated both qualitatively and quantitatively. The qualitative evaluation is shown in Fig 5Down. These data demonstrate that hippocampal injury was reduced in Tg mice versus nTg mice. In addition to the reduction of injury in Tg mice, hippocampal injury progressed from 1 to 3 days after ischemia in the 5-minute ischemia group, although the statistical difference was not seen between 1 and 3 days. The mean score was 1.71 in nTg (n=7) and 0.86 in Tg (n=7) mice at 1 day, and no significant difference was seen between nTg and Tg mice. At 3 days, however, the hippocampal injury score of Tg mice was significantly lower than that of nTg mice (P<.05, Mann-Whitney U test). The mean score was 2.09 in nTg (n=22) and 1.17 in Tg (n=18) mice at 3 days.



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  		Figure 5. Qualitative analysis of neuronal damage of the hippocampal injury in nTg and Tg mice at 1 and 3 days after global ischemia. White and black dots show the score of the injury in the hemisphere used in nTg and Tg mice, respectively, and each column shows mean score of the hippocampal injury of each group. *P<.05, Mann-Whitney U test. Hippocampal injury was ameliorated in Tg mice in the 5- and 10-minute ischemia groups, and at both 1 and 3 days. The injury was significantly milder in Tg than nTg mice at 3 days. However, this injury was near the peak at 1 day after 10 minutes ischemia in nTg mice, although it progressed at 3 days in Tg mice. In the 10-minute group, a significant difference was obtained at 1 day rather than 3 days.

In the 10-minute ischemia group, hippocampal injury was also reduced in Tg mice. The hippocampal injury at 1 day was also significantly more severe in nTg than in Tg mice. Mean scores were 3.60 in nTg (n=10) and 2.43 in Tg (n=7) mice at 1 day, and hippocampal injury was significantly reduced in Tg than nTg mice (P<.05, Mann-Whitney U test). Hippocampal injury was likely to progress only in Tg mice and maximize at 1 day in nTg mice, although statistical significance was not obtained. Scores were 3.13 in nTg (n=15) and 2.91 in Tg (n=11) mice at 3 days after ischemia.

The quantitative analysis data, measured using an image analysis system, are shown in Fig 6Down and demonstrated the tendency of hippocampal injury after global ischemia to be reduced in Tg mice versus nTg mice, which supports the results obtained from the qualitative analysis. In the 5-minute ischemia group, the development of hippocampal injury was obtained in a similar manner by use of qualitative analysis. At 1 day, nTg and Tg mice had 11.8±20.0% (n=7) and 0% (n=7) neuronal loss, respectively. At 3 days after ischemia, neuronal loss in the CA1 subregion had progressed, and 24.3±35.6% (n=22) and 12.2±28.4% (n=18) neurons were damaged in nTg and Tg mice, respectively. In the 10-minute group, the development of neuronal loss was not seen as time-progressed, but neuronal loss in the CA1 subregion was milder in Tg than nTg mice at both 1 and 3 days after ischemia. At 1 day, neuronal loss was 48.2±37.4% in nTg (n=10) and 29.8±37.3% in Tg (n=7) mice. These data were 48.1±38.0% (n=15) and 33.6±32.2% (n=11), respectively, at 3 days after ischemia. In the quantitative analysis, no statistically significant difference could be seen between nTg and Tg mice and between 1 and 3 days in either the 5- or 10-minute ischemia groups.



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  		Figure 6. Quantitative analysis of neuronal loss in the CA1 subregion in nTg and Tg mice at 1 and 3 days after global ischemia. Each column shows mean±SD. At any time point both 5 and 10 minutes after ischemia, neuronal loss was reduced in Tg mice, although no significance was seen. In the 5-minute ischemia group, neuronal loss tended to progress from 1 to 3 days, whereas 10 minutes of ischemia maximized the hippocampal injury at 1 day.

DNA Fragmentation in Hippocampal Neurons After Global Ischemia
Histological analysis demonstrates several types of ischemic neurons in the CA1 subregion of the hippocampus. However, biochemical features in TUNEL staining for the in situ detection of degraded DNA are not consistent with the characteristic morphological features. Compact neurons with a condensed nucleus, which occasionally displayed small particles and appeared to be apoptotic neurons, were partly labeled by TUNEL staining. However, this specific labeling could not be observed in all of these neurons (Fig 4BUp). The swollen or lysed neuron, displaying morphologically necrotic features, was not stained specifically (Fig 4DUp). Therefore, the neurons intensely labeled by TUNEL staining did not display uniform morphological features indicating apoptosis, cell shrinkage, nuclei condensation, and nuclei fragmentation.

On the basis of these biochemical and morphological features of ischemic neurons in the hippocampal CA1 subregion, the ratio of apoptotic neurons in total ischemic neurons was investigated. Fig 7Down shows percent of TUNEL-positive neurons in the hippocampal neuronal injury at 1 or 3 days after global ischemia. In the 5-minute ischemia group, the ratio was significantly lower in the nTg versus the Tg mice at 1 day (P<.05, ANOVA). These apoptotic neurons were 21.9±11.4% (n=4) and 44.2±20.0% (n=5) of total injured neurons in nTg and Tg mice, respectively. At 3 days, after 5 minutes of ischemia, this ratio was 34.1±9.2% in nTg and 42.5±2.1% in Tg mice. In the 10-minute group, this ratio was 38.6±20.5% in nTg (n=7) and 35.0±24.2% in Tg (n=4) mice at 1 day and 38.2±20.9% in nTg (n=5) and 31.1±20.4% in Tg (n=2) mice at 3 days, respectively.



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  		Figure 7. The ratio of TUNEL-positive neurons in hippocampal injury. Values are mean±SD. *P<.05 by ANOVA. In the 5-minute ischemia group, the ratio was significantly increased in Tg mice at 1 day, despite the reduction of total hippocampal injury. However, this ratio was at the same level between the nTg and Tg mice in the 10-minute ischemia group.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study demonstrated that endogenously overexpressed CuZn-SOD plays a protective role in the development of hippocampal injury after transient global ischemia. Furthermore, this protective role was obtained both in acute injury after relatively intense ischemia and with progressive injury after relatively mild ischemia. In situ detection of DNA fragmentation by TUNEL staining demonstrated that apoptotic neuronal death partly contributes to hippocampal injury after transient global ischemia in mice. Furthermore, neuroprotective action by overexpressed CuZn-SOD is also involved in the apoptotic process after global ischemia.

In the study of global as well as focal ischemia, both physiological and anatomic backgrounds were important factors affecting the outcome of neuronal injury, especially in the experiments in which genetically different animals were used. In this study, physiological conditions, including MABP and arterial blood gas, were not significantly different between nTg and Tg mice. The other factors, including the plasticity of the PcomA, are probably the most important components of the anatomic background affecting the outcome in global ischemia.28 Because of the lack of a PcomA as a connection between anterior and posterior circulation, gerbils have been widely used for global ischemia studies on the basis that BCCAO can induce almost complete forebrain ischemia without the reduction of collateral blood flow from posterior circulation, including the hemorrhagic hypotension,29 bilateral vertebral artery occlusion,10 and basilar artery occlusion, seen in rats.30 A reliable model of mouse global ischemia has been recently established by Moskowitz's group using a three-vessel occlusion strategy.31 The relatively high mortality associated with the occlusion of the basilar artery and the complications of surgery render this model somewhat difficult for stroke researchers when smaller animals such as mice are used. Also, the higher mortality may be inconvenient, because the availability of transgenic animals is usually a limiting factor. On the other hand, several other models, which induce a global ischemia-like condition, including hypoxia,32 unilateral common carotid artery occlusion with systemic hypoxia,33 and decapitation,34 35 have also been proposed. We previously tried to apply these rat models to mice but were unsuccessful because of high mortality rates and frequent complications. We previously reported that the residual regional cortical blood flow is correlated with the plasticity of the PcomA in BCCAO.36 Furthermore, hippocampal injury was occasionally observed after brief ischemia, usually in either hemisphere. Therefore, we evaluated the plasticity of the PcomA, and the hippocampus with the hypoplastic PcomA in the ipsilateral hemisphere was selectively used on the basis that complete ischemia was induced. This method might be useful for the investigation of global ischemia in mice.

Hippocampal injury was determined both qualitatively and quantitatively after relatively mild and intense global ischemia following 5 and 10 minutes BCCAO, respectively. In the quantitative evaluation, an image analysis system was used to detect neuronal loss in the CA1 subregion. Since it is rather difficult to count the number of ischemic neurons or neuronal loss due to phagocytosis in the thick brain sections used herein (50 µm) and we generally note a close correlation of the lost number per a certain length of the CA1 region, we selected the indirect method.

However, some types of neuronal injury, scattered or mild neuronal injury in particular, could not be detected because of the lack of difference in the density of staining and the limited sensitivity of the present system. Furthermore, since only the section at the posterior commissure was used in this analysis, hippocampal injury in the other parts could not be evaluated. Therefore, the combination of quantitative and qualitative evaluation was used in the present study. Qualitative evaluation clearly demonstrated the protective role of CuZn-SOD in global ischemia. The development and occurrence of hippocampal injury appeared to depend on the duration of ischemia, which is strongly correlated with the intensity of the ischemic insult. Hippocampal injury after 5 minutes ischemia tended to progress slowly and matured at a later time, although statistical evidence was not achieved. The injury was reduced in Tg mice at both 1 and 3 days after global ischemia, and it was significant at 3 days. On the other hand, in the 10-minute ischemia group, the hippocampal injury was maximized as early as 1 day after ischemia in nTg mice; the mean score was higher at 1 than at 3 days. This discrepancy may be due to the fact that the severely injured mice died 1 to 3 days after ischemia and thus were excluded from the evaluation. However, the hippocampal injury tended to progress from 1 to 3 days in Tg mice. At 1 day after ischemia, the mean score was significantly lower in Tg than nTg mice, although it was at the same level at 3 days. Quantitative evaluation supported these observations, although the significance could not be seen because of the large standard deviation of the data due to the limited sensitivity of this method. However, despite the limitation in this method, it was demonstrated that overexpression of CuZn-SOD protects hippocampal injury from global ischemia. The delayed development of injury was also demonstrated in the 5-minute ischemia group, as is true with the qualitative evaluation.

Delayed neuronal cell death and selective vulnerability in hippocampal neurons (the CA1 neuron in particular) after transient global ischemia have been investigated in great detail since first reported.10 11 These phenomena have been explained by many hypotheses, including glutamate toxicity,37 protein synthesis inhibition,38 39 40 41 42 neurotrophic factor,43 44 45 46 oxygen radicals,47 48 49 50 and mitochondrial dysfunction.51 These mechanisms are likely to contribute partly to hippocampal neuronal injury after brief ischemia. Recent studies have demonstrated that apoptosis is also involved in hippocampal injury after global ischemia.18 19 Therefore, we also determined by TUNEL staining the role of overexpressed CuZn-SOD in DNA fragmentation, a possible process preceding to apoptosis, after mild and intense global ischemia. The present study demonstrated that DNA fragmentation also contributes to hippocampal injury after global ischemia in mice. Hippocampal injury displayed several types of morphological features in ischemic neurons. These neurons were observed in both ischemia groups in both groups of mice. Not all of the neurons displaying the morphological features of apoptosis were labeled by TUNEL staining. Therefore, in the present study, the contribution of DNA fragmented neurons was evaluated as the ratio of TUNEL-positive neurons in the hippocampal injury. In the 5-minute ischemia group, the percent of TUNEL-positive neurons was increased at 1 day in Tg mice relative to nTg mice, although the total injury to the hippocampus was reduced in Tg mice. However, in the 10-minute ischemia group, this ratio was almost the same between nTg and Tg mice. One possible explanation for this result is the difference in the time course of the apoptotic process. Since apoptosis is a relatively short-lived process, it is possible that more extensive apoptosis might occur in nTg mice at an earlier time period (0 to 12 hours), which could lead to a relative reduction in TUNEL-positive cells at later time points. Furthermore, it is also possible that this discrepancy of the ratio of TUNEL-positive neurons between the 5- and 10-minute ischemia groups might result from a difference of intensity of the ischemic insult. Decrease of TUNEL-positive neurons means that TUNEL-negative cells, which might also include necrotic cells, were increased in nTg mice after 5 minutes ischemia. Thus, the increased TUNEL-positive neurons in Tg mice after 5 minutes ischemia may reflect more TUNEL-positive neurons that were unmasked by necrotic neurons. It has been demonstrated that excitotoxicity mediated by the N-methyl-D-aspartate receptor induces calcium overload and generates superoxide anion causing neuronal injury, and two distinct pathways to neuronal death, apoptosis or necrosis, have been demonstrated depending on the intensity of the insult.52 We have demonstrated that oxygen deprivation, but not a combination with substrate induces DNA degradation in cortical neurons.7 Considering these findings together with the present results, overexpressed CuZn-SOD effectively detoxifies abnormally overproduced superoxide anions and reduces oxidative stress to neurons, which might also alter the pathways to neuronal death, apoptosis or necrosis, in Tg mice. On the other hand, after intense ischemia by 10 minutes BCCAO, the ratio was not altered by overexpressed CuZn-SOD in Tg mice, while oxidative stress might be partly detoxified by overexpressed CuZn-SOD and the hippocampal injury reduced in Tg mice as shown in the histological analysis. The fact that we no longer observe significant differences in cell death at 3 days in Tg mice suggests that increased levels of CuZn-SOD may only delay the maturation of ischemic injury, but does not offer long lasting protection in this global cerebral ischemia model. This question can be addressed by studying a longer period of reperfusion after relatively mild ischemia (ie, <5 minutes). These studies are now being undertaken in our laboratory.

We conclude that overexpressed CuZn-SOD plays a protective role in the pathogenesis of hippocampal injury either in an acute or progressive fashion. Furthermore, we postulate that the alternation of the pathway to neuronal death might depend on the intensity of oxidative stress and that CuZn-SOD possibly ameliorates both types of neuronal death in hippocampal injury after transient global ischemia.


*    Selected Abbreviations and Acronyms
 
BCCA = bilateral common carotid artery
BCCAO = bilateral common carotid artery occlusion
(CuZn)-SOD = copper/zinc SOD; cytosolic isoenzyme of SODs specific for scavenging superoxide radicals
MABP = mean arterial blood pressure
NTg = nontransgenic
PcomAs = posterior communicating arteries
PcomA(-) = arteries with scores of 0 and 1; the hypoplastic PcomA group
PcomA(+) = arteries with scores of 2 and 3; the normal PcomA group
SOD = superoxide dismutase
TdT = terminal deoxynucleotidyl transferase buffer
Tg = transgenic
TUNEL = terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling


*    Acknowledgments
 
This study was supported by National Institutes of Health grants P-50-NS-14543, NS-25372, P-01-AG-68938, and N-01-NS-52334. The authors thank Sylvia Chen, Liza Reola, and Bernard Calagui for their technical assistance and Cheryl Christensen for her editorial assistance.

Received January 22, 1997; revision received May 16, 1997; accepted May 28, 1997.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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