Cell death after exposure to subarachnoid hemolysate correlates inversely with expression of CuZn-superoxide dismutase.

BACKGROUND AND PURPOSE: Subarachnoid hemolysate (SAH) has been associated with oxidative brain injury, cell death, and apoptosis. We hypothesized that over-expression of CuZn-superoxide dismutase (CuZn-SOD) would protect against injury after SAH, whereas reduction of its expression would exacerbate injury. METHODS: Saline (n=16) or hemolysate (n=50) was injected into transgenic mice overexpressing CuZn-SOD (SOD1-Tg), CuZn-SOD heterozygous knockout mutants (SOD1+/-), and wild-type littermates (Wt). Mice were killed at 24 hours. Stress gene induction was evaluated by immunocytochemistry and Western blotting for hemeoxygenase-1 and heat shock protein 70. Apoptosis was evaluated by 3'-OH nick end-labeling and DNA gel electrophoresis. Cell death was quantified through histological assessment after cresyl violet staining. RESULTS: Histological assessment demonstrated neocortical cell death in regions adjacent to the blood injection. Overall cell death was reduced 43% in SOD1-Tg mutants (n=6) compared with Wt littermates (n=6; P<0.02). In contrast, cell death was increased >40% in SOD1+/- mutants (n=6; P<0.05). Both hemeoxygenase-1 and heat shock protein 70 were induced after SAH. Apoptosis was also present after SAH, as evidenced by 3'-OH end-labeling and DNA laddering. However, the degree of stress gene induction and apoptosis did not vary between Wt, SOD1-Tg, and SOD1+/- mice. CONCLUSIONS: The extent of CuZn-SOD expression in the cytosol correlates with cell death after exposure to SAH in a manner separate from apoptosis. Overexpression of CuZn-SOD may potentially be an avenue for therapeutic intervention.

Matrix metalloproteinase inhibition prevents oxidative stress-associated blood-brain barrier disruption after transient focal cerebral ischemia.

Oxidative stress generated during stroke is a critical event leading to blood-brain barrier (BBB) disruption with secondary vasogenic edema and hemorrhagic transformation of infarcted brain tissue, restricting the benefit of thrombolytic reperfusion. In this study, the authors demonstrate that ischemia-reperfusion-induced BBB disruption in mice deficient in copper/zinc-superoxide dismutase (SOD1) was reduced by 88% ( P < 0.0001) and 73% ( P < 0.01), respectively, after 3 and 7 hours of reperfusion occurring after 1 hour of ischemia by the inhibition of matrix metalloproteinases. Accordingly, the authors show that local metalloproteinase-generated proteolytic imbalance is more intense in ischemic regions of SOD1 mice than in wild-type litter mates. Moreover, active in situ proteolysis is, for the first time, demonstrated in ischemic leaking capillaries that produce reactive oxygen species. By showing that oxidative stress mediates BBB disruption through metalloproteinase activation in experimental ischemic stroke, this study provides a new target for future therapeutic strategies to prevent BBB disruption and potentially reperfusion-triggered intracerebral hemorrhage.

Overexpression of copper and zinc superoxide dismutase in transgenic mice prevents the induction and activation of matrix metalloproteinases after cold injury-induced brain trauma.

Matrix metalloproteinases (MMPs), a family of proteolytic enzymes which degrade the extracellular matrix, are implicated in blood-brain barrier disruption, which is a critical event leading to vasogenic edema. To investigate the role of reactive oxygen species (ROS) in the expression of MMPs in vasogenic edema, the authors measured gelatinase activities before and after cold injury (CI) using transgenic mice that overexpress superoxide dismutase-l. A marked induction of pro-gelatinase B (pro-MMP-9) was seen 2 hours after CI and was maximized at 12 hours in wild-type mice. The pro-MMP-9 level was significantly lower in transgenic mice 4 hours (P < 0.001) and 12 hours (P < 0.05) after CI compared to wild-type mice. The activated MMP-9 was detected from 6 to 24 hours after injury. A mild induction of pro-gelatinase A (pro-MMP-2) was seen at 6 hours and was sustained until 7 days. In contrast. the activated form of MMP-2 appeared at 24 hours, was maximized at 7 days, and was absent in transgenic mice. Western blot analysis showed that the tissue inhibitors of metalloproteinases were not modified after CI. The results suggest that ROS production after CI may contribute to the induction and/or activation of MMPs and could thereby exacerbate endothelial cell injury and the development of vasogenic edema after injury. Key Words: Metalloproteinases-Brain-Vasogenic edema-Reactive oxygen species-Superoxide dismutase.

Oxidative stress-dependent release of mitochondrial cytochrome c after traumatic brain injury.

Mitochondrial cytochrome c translocation to the cytosol initiates the mitochondrial-dependent apoptotic pathway. This event has not been previously reported in traumatic brain injury (TBI). The authors determined the expression of cytochrome c in cytosolic and mitochondrial fractions after severe TBI produced by the controlled cortical impact model in the mouse. One hour after trauma there was an increase in cytosolic cytochrome c immunoreactivity. The increases in cytosolic cytochrome c preceded DNA fragmentation, which started at 4 hours. Western blots of mitochondrial and cytosolic fractions confirmed that there was a translocation of cytochrome c from the mitochondria after TBI. Mice deficient in manganese superoxide dismutase (MnSOD) showed an increased loss of mitochondrial cytochrome c after trauma, but less apoptotic cell death 4 and 24 hours after injury compared with wild-type control mice. However, the overall cell death was increased in MnSOD mice, as illustrated by a larger cortical lesion in these animals. The results show that cytochrome c is released from the mitochondria after severe TBI partly by a free radical-dependent mechanism, and that massive mitochondrial cytochrome c release is a predictor of necrotic cell death rather than apoptosis.

Early appearance of activated matrix metalloproteinase-9 after focal cerebral ischemia in mice: a possible role in blood-brain barrier dysfunction.

During cerebral ischemia blood-brain barrier (BBB) disruption is a critical event leading to vasogenic edema and secondary brain injury. Gelatinases A and B are matrix metalloproteinases (MMP) able to open the BBB. The current study analyzes by zymography the early gelatinases expression and activation during permanent ischemia in mice (n = 15). ProMMP-9 expression was significantly (P < 0.001) increased in ischemic regions compared with corresponding contralateral regions after 2 hours of ischemia (mean 694.7 arbitrary units [AU], SD +/- 238.4 versus mean 107.6 AU, SD +/- 15.6) and remained elevated until 24 hours (mean 745.7 AU, SD +/- 157.4). Moreover, activated MMP-9 was observed 4 hours after the initiation of ischemia. At the same time as the appearance of activated MMP-9, we detected by the Evan's blue extravasation method a clear increase of BBB permeability. Tissue inhibitor of metalloproteinase-1 was not modified during permanent ischemia at any time. The ProMMP-2 was significantly (P < 0.05) increased only after 24 hours of permanent ischemia (mean 213.2 AU, SD +/- 60.6 versus mean 94.6 AU, SD +/- 13.3), and no activated form was observed. The appearance of activated MMP-9 after 4 hours of ischemia in correlation with BBB permeability alterations suggests that MMP-9 may play an active role in early vasogenic edema development after stroke.

Excitotoxicity is required for induction of oxidative stress and apoptosis in mouse striatum by the mitochondrial toxin, 3-nitropropionic acid.

Excitotoxicity is implicated in the pathogenesis of several neurologic diseases, such as chronic neurodegenerative diseases and stroke. Recently, it was reported that excitotoxicity has a relationship to apoptotic neuronal death, and that the mitochondrial toxin, 3-nitropropionic acid (3-NP), could induce apoptosis in the striatum. Although striatal lesions produced by 3-NP could develop through an excitotoxic mechanism, the exact relationship between apoptosis induction and excitotoxicity after 3-NP treatment is still not clear. The authors investigated the role of excitotoxicity and oxidative stress on apoptosis induction within the striatum after intraperitoneal injection of 3-NP. The authors demonstrated that removal of the corticostriatal glutamate pathway reduced superoxide production and apoptosis induction in the denervated striatum of decorticated mice after 3-NP treatment. Also, the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, prevented apoptosis in the striatum after 3-NP treatment for 5 days, whereas the non-NMDA receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline, was ineffective. The authors also evaluated the initial type of neuronal death by 3-NP treatment for different durations from 1 to 5 days. In early striatal damage, apoptotic neuronal death initially occurred after 3-NP treatment. Our data show that excitotoxicity related to oxidative stress initially induces apoptotic neuronal death in mouse striatum after treatment with 3-NP.

Differing effects of copper,zinc superoxide dismutase overexpression on neurotoxicity elicited by nitric oxide, reactive oxygen species, and excitotoxins.

Overexpression of Cu,Zn superoxide dismutase (SOD1) reduces ischemic injury in some stroke models but exacerbates injury in a neonatal stroke model and in other settings. The current study used a SOD1 transgenic (SOD1-Tg) murine cortical culture system, derived from the same mouse strain previously used for the stroke models, to identify conditions that determine whether SOD1 overexpression in neurons is protective or detrimental. The nitric oxide (NO) donors S-nitroso-N-acetylpenicillamine, spermine-NONOate, and diethylamine-NONOate produced less death in SOD1-Tg neurons than in wild-type neurons (p < 0.01). Also, NO produced markedly less 3-nitrotyosine in SOD1-Tg cells. In contrast, the superoxide generator menadione produced significantly greater death and nearly twice as much 2'7'-dichlorofluorescein fluorescence in SOD1-Tg neurons than in wild-type neurons, suggesting increased peroxide formation in the SOD1-Tg cells. No significant difference was observed in the vulnerability of the two cell types to H2O2, the product of the SOD reaction. Overexpression of SOD1 also had no effect on neuronal vulnerability to glutamate, N-methyl-D-aspartate, or kainate. These observations suggest that SOD1 overexpression can reduce neuronal death under conditions where peroxynitrite formation is a significant factor, but may exacerbate neuronal death under conditions of rapid intracellular superoxide formation or impaired H2O2 disposal.

Overexpression of copper/zinc superoxide dismutase does not prevent neonatal lethality in mutant mice that lack manganese superoxide dismutase.

There are two types of intracellular superoxide dismutases: the mitochondrial manganese SOD (MnSOD) and the cytoplasmic copper/zinc SOD (CuZnSOD). Mutant mice that lack MnSOD die shortly after birth because of cardiomyopathy and mitochondrial injury. In order to verify if CuZnSOD could compensate for MnSOD deficiency, a new mutant mouse that overexpresses CuZnSOD but is deficient in MnSOD was generated by crossing MnSOD knockout mice with CuZnSOD transgenic mice. CuZnSOD activity was significantly increased in the blood, brain, liver, and heart of MnSOD knockout, CuZnSOD transgenic mice when compared with nontransgenic mice. However, overexpression of CuZnSOD did not prevent neonatal lethality in mice that lack MnSOD, nor did it prevent oxidative aconitase inactivation, nor did it rescue MnSOD-deficient astrocytes in culture. Based on our findings, which emphasize the strong enzymatic compartmentalization of CuZnSOD and MnSOD, therapeutic antioxidant strategies should consider the final intracellular localization of the antioxidant used, especially when those strategies are directed against mitochondrial diseases.

Paternal alcohol exposure: developmental and behavioral effects on the offspring of rats.

The effect of paternal alcohol exposure on neurochemical and behavioral parameters was investigated using as a model system glial cells derived from newborn rat brain and cultured for 4 weeks. The total brain neurochemical parameters from rats born to mothers sired by an alcohol treated father were also investigated. Enzymatic markers of nerve cell development (enolase isoenzymes and glutamine synthetase) and the defense system (superoxide dismutase) against free radicals formed during alcohol degradation were measured in order to evaluate nerve cell damage. Behavioral locomotor tests (open-field, novelty-seeking, light/dark) were carried out to show long-lasting effects of paternal alcoholization on the offspring. Behavioral and developmental alterations were found until 1 year of age in the offspring and a significant growth retardation was observed in the males. Our results suggest that paternal alcohol exposure produces developmental and behavioral effects in the offspring. The consequence of either alcohol withdrawal during stage one spermatogenesis, or maternal diet supplementation with manganese during pregnancy were investigated. It was observed that some of the effects of paternal alcohol exposure on the offspring may be reversed by these treatments.

Spreading depression-induced expression of c-fos and cyclooxygenase-2 in transgenic mice that overexpress human copper/zinc-superoxide dismutase.

Spreading depression (SD) is a wave of sustained depolarization challenging the energy metabolism of cells without causing irreversible damage. SD is a major mechanism of gene induction that takes place in cortical injury, including ischemia. We studied the role of oxygen radicals in SD-induced c-fos and cyclooxygenase-2 (COX-2) induction using transgenic (Tg) mice that overexpress copper/zinc-superoxide dismutase (SOD1). The frequency, amplitude and duration of SD waves were similar in the Tg mice and wild-type littermates. c-fos and COX-2 mRNAs were strongly induced 1 and 4 h after SD. The induction of both genes was slightly but significantly less at 4 h in the Tg mice. The results indicate that even a mild, noninjurious metabolic stimulation increases the concentration of oxygen radicals to the level that contributes to gene expression.

Oxygen deprivation but not a combination of oxygen, glucose, and serum deprivation induces DNA degradation in mouse cortical neurons in vitro: attenuation by transgenic overexpression of CuZn-superoxide dismutase.

The present work was designed to study the possible implication of apoptosis in ischemic neuronal death, a phenomenon that has been suggested to be involved in neurodegeneration following focal as well as global ischemia. In this study, mouse cortical neurons in primary culture were subjected to oxygen deprivation or oxygen, glucose, and serum deprivation to simulate hypoxia and "ischemia-like" conditions; also, cellular viability as well as DNA degradation were investigated. The results showed that DNA degradation occurred in neurons subjected to oxygen deprivation but not to oxygen and substrate deprivation together. This DNA degradation, resulting in a laddering by agarose gel electrophoresis, could be prevented by cycloheximide and actinomycin-D treatments, although these inhibitors were unable to reduce neuronal death. To investigate if DNA degradation could be elicited by an intracellular free radical generation during reoxygenation, transgenic neurons overexpressing copper-zinc superoxide dismutase were subjected to 9 h of oxygen deprivation and analyzed after 24 h of reoxygenation. The results showed a significant attenuation of DNA degradation in these cells and confirmed a possible relationship between reactive oxygen species and neuronal apoptosis. This study opens the way to further investigations regarding the involvement of an apoptotic process in necrotic neuronal death, and provides some new insights into the mechanisms underlying selective sensitivity of neuronal cells to oxygen and glucose deprivation.

Targeted expression of human CuZn superoxide dismutase gene in mouse central nervous system.

Copper zinc superoxide dismutase (CuZnSOD) is an important enzyme for the detoxification of reactive oxygen species. Particularly in the central nervous system (CNS), reactive oxygen species are often associated with acute brain injuries and chronic neurodegeneration. It has been demonstrated in vivo that there is an inverse correlation between CuZnSOD activity and neuronal death after acute brain injury. To further understand the protective role of CuZnSOD upon neurons, we have generated transgenic mouse lines with targeted expression of the human CuZnSOD gene (SOD1) that is driven by a rat neuron-specific enolase gene promoter in neurons of the CNS. The transgenic SOD1 expression was restricted to the CNS identified by reverse transcriptase polymerase chain reaction and SOD gel electrophoresis assays. The CuZnSOD activity was significantly increased in the brain stem of the transgenic mice. Immunostaining of human CuZnSOD activity showed that Purkinje cells in the cerebellar cortex were the most intensely stained neurons in the CNS of the transgenic mice.

Trolox and 6,7-dinitroquinoxaline-2,3-dione prevent necrosis but not apoptosis in cultured neurons subjected to oxygen deprivation.

There is a growing body of evidence suggesting that apoptosis is involved in ischemic brain injury. Recent studies suggest that a rapid necrosis masked a more subtle apoptotic death in neurons subjected to oxygen deprivation in culture. To test this hypothesis, we treated cultured neurons with potential antinecrotic drugs during and after oxygen deprivation. The results show that 6, 7-dinitroquinoxaline-2,3-dione (DNQX) and 6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid (Trolox), which interfered with kainate receptor activation and lipid peroxidation respectively, prevented necrosis but allowed neurons to undergo apoptosis. Flow cytometric analysis of DNA degradation and hydrogen peroxide generation, as well as fluorescent microscopy of nuclear fragmentation revealed that apoptotic activity was higher in 6, 7-dinitroquinoxaline-2,3-dione-treated cells than in Trolox-treated cells. This difference in occurrence of apoptosis may be due to the difference in oxidative stress generated from these two different agents.

Reduced mitochondrial manganese-superoxide dismutase activity exacerbates glutamate toxicity in cultured mouse cortical neurons.

Studies of neuronal injury and death after cerebral ischemia and various neurodegenerative diseases have increasingly focused on the interactions between mitochondrial function, reactive oxygen species (ROS) production and glutamate neurotoxicity. Recent findings suggest that increased mitochondrial ROS production precedes neuronal death after glutamate treatment. It is hypothesized that under pathological conditions when mitochondrial function is compromised, extracellular glutamate may exacerbate neuronal injury. In the present study, we focus on the relationship between mitochondrial superoxide production and glutamate neurotoxicity in cultured cortical neurons with normal or reduced levels of manganese-superoxide dismutase (MnSOD) activity. Our results demonstrate that neurons with reduced MnSOD activity are significantly more sensitive to transient exposure to extracellular glutamate. The increased sensitivity of cultured cortical neurons with reduced MnSOD activity is characteristically subject only to treatment by glutamate but not to other glutamate receptor agonists, such as N-methyl-d-aspartate, kainate and quisqualate. We suggest that the reduced MnSOD activity in neurons may exacerbate glutamate neurotoxicity via a mechanism independent of receptor activation.

Apoptosis in the striatum of rats following intraperitoneal injection of 3-nitropropionic acid.

The present study investigated the mechanism of cellular degeneration within the striatum following administration of the mitochondrial toxin, 3-nitropropionic (3-NP) acid. Internucleosomal fragmentation typical of apoptosis was present in the DNA of cells from the striatum of 3-NP-treated rats. DNA fragmentation was also evident in this region by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling. The data suggest that striatal cells die by apoptosis following administration of 3-NP.

Decreased expression of bcl-2 and bcl-x mRNA coincides with apoptosis following intracerebral administration of 3-nitropropionic acid.

The mitochondrial toxin, 3-nitropropionic acid (3-NP), is an irreversible inhibitor of succinate dehydrogenase that induces apoptosis in vitro and in vivo. We injected 3-NP into the striatum of rats to examine the potential role of Bcl-2 or Bcl-x, proteins that can inhibit apoptosis, in brain injury due to 3-NP. Electrophoretic examination of striatal tissue indicated that 3-NP induced internucleosomal fragmentation typical of apoptosis. There was also histologic evidence of apoptosis based on staining by the terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) method. Apoptosis was first observed 6 h after injection, was maximal at 1 day, and was still observed on day 7. Expression of bcl-2, bcl-x, and c-jun mRNA expression was evaluated 1, 3, 6, and 12 h and 1, 3, 5, and 7 days after injection using in situ hybridization. Both bcl-2 and bcl-x mRNA expression in the striatum decreased starting at 6 h and continued to 5 days after injection. This was in contrast to an apparent increase in c-jun expression. The similarity in the time course of apoptosis to that of suppression of bcl-2 and bcl-x mRNA suggests that changes in expression of these genes may contribute to apoptosis following 3-NP injection.

Neuroprotective effects of an antioxidant in cortical cerebral ischemia: prevention of early reduction of the apurinic/apyrimidinic endonuclease DNA repair enzyme.

We examined the effects of the free radical scavenger, 21-aminosteroid, on apurinic/apyrimidinic endonuclease (APE/Ref-1) protein expression and subsequent infarction volume after photothrombotic cortical cerebral ischemia in mice. Immunohistochemistry and Western blot analysis showed a significant reduction in APE/Ref-1 expression 6 and 24 h after ischemia in untreated animals, whereas in drug-treated animals the reduction was much less at the same time points. The administration of 21-aminosteroid significantly decreased subsequent infarction volume 3 days after ischemia. These data suggest that 21-aminosteroid prevents the early decrease of APE/Ref-1 expression, thereby reducing cortical infarction after photothrombotic cerebral ischemia.

Blood-brain barrier disruption, HSP70 expression and apoptosis due to 3-nitropropionic acid, a mitochondrial toxin.

3-Nitropropionic acid (3-NP), a mitochondrial toxin, induces apoptosis in the striatum. We wanted to determine if there was a relationship between mitochondrial dysfunction, disruption of the blood-brain barrier (BBB), and apoptosis. BBB disruption following intrastriatal injection of 3-NP was assessed by Evans blue leakage, brain water content, and by the expression of the 70 kDa heat shock protein (HSP70) and mRNA. Apoptosis was assessed by in situ terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) and gel electrophoresis to detect internucleosomal DNA fragmentation. Microscopic evidence of Evans blue leakage due to 3-NP was present only 3 hr after injection. Both internucleosomal DNA fragmentation and TUNEL-labeling did not appear until 24 hr after injection. HSP70 (protein and mRNA) was also elevated by 24 hr. There was a quantitative increase in Evans blue leakage and brain water content due to 3-NP by 3 days after injection. Our results suggest that BBB disruption is an early event followed by increased HSP70 expression and apoptosis. We speculate that 3-NP damages endothelial cells, leading to vasogenic edema and apoptosis.

[Morphology and physiology of the blood-brain barrier]. / Morphologie et physiologie de la barrière hématoencéphalique.

The blood-brain barrier (BBB) is a complex biological system that consists of endothelial cells, pericytes and astrocytes, which are involved in the induction and maintenance of its physiological and ultrastructural characteristics. The BBB plays a primordial role in isolating the cerebral parenchyma as well as in controlling brain homeostasis by its selective permeability to nutriments and other molecules flowing through the cerebral microcapillaries. A better knowledge of this system is crucial in order to improve the efficiency of brain penetration by drugs, and in order to prevent BBB opening, leading to brain edema, in physiopathological situations such as brain ischemia, trauma or inflammatory processes.

[Blood-brain barrier pathophysiology and ischaemic brain oedema]. / Physiopathologie de la barrière hématoencéphalique et oedème cérébral d'origine ischémique.

Cerebral oedema is a potentially lethal complication of brain infarction. Ischemia, by altering membrane ionic pump function, induces cell swelling and cytotoxic oedema. It also initiates early oxidative and inflammatory cascades leading to blood-brain barrier disruption, vasogenic oedema and haemorrhagic transformation. The mechanisms of blood-brain barrier disruption involve endothelial cell activation and endothelial basal membrane degradation by matrix metalloproteinases. Reperfusion by tissue plasminogen activators is the only treatment improving stroke prognosis. This treatment also increases vasogenic oedema and the risk of symptomatic haemorrhagic transformation, reducing the benefit of reperfusion. Experimental studies suggest that the inhibition of blood-brain barrier proteolysis reduces vasogenic oedema and the risk of haemorrhage. This recent progress in the understanding of blood-brain barrier disruption during ischaemia brings forward new therapeutic strategies using agents capable of interfering with the ischaemic cascade in order to increase the therapeutic window between the onset of ischaemia and thrombolytic reperfusion.