Pak H. Chan

Reactive Oxygen Radicals in Signaling and Damage in the Ischemic Brain

Reactive oxygen species have been implicated in brain injury after ischemic stroke. These oxidants can react and damage the cellular macromolecules by virtue of the reactivity that leads to cell injury and necrosis. Oxidants are also mediators in signaling involving mitochondria, DNA repair enzymes, and transcription factors that may lead to apoptosis after cerebral ischemia. Transgenic or knockout mice with cell- or site-specific prooxidant and antioxidant enzymes provide useful tools in dissecting the events involving oxidative stress in signaling and damage in ischemic brain injury.

Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke.

Cerebral edema contributes significantly to morbidity and death associated with many common neurological disorders. However, current treatment options are limited to hyperosmolar agents and surgical decompression, therapies introduced more than 70 years ago. Here we show that mice deficient in aquaporin-4 (AQP4), a glial membrane water channel, have much better survival than wild-type mice in a model of brain edema caused by acute water intoxication. Brain tissue water content and swelling of pericapillary astrocytic foot processes in AQP4-deficient mice were significantly reduced. In another model of brain edema, focal ischemic stroke produced by middle cerebral artery occlusion, AQP4-deficient mice had improved neurological outcome. Cerebral edema, as measured by percentage of hemispheric enlargement at 24 h, was decreased by 35% in AQP4-deficient mice. These results implicate a key role for AQP4 in modulating brain water transport, and suggest that AQP4 inhibition may provide a new therapeutic option for reducing brain edema in a wide variety of cerebral disorders.

Role of Oxidants in Ischemic Brain Damage

BACKGROUND AND PURPOSE Oxygen free radicals or oxidants have been proposed to be involved in acute central nervous system injury that is produced by cerebral ischemia and reperfusion. Because of the transient nature of oxygen radicals and the technical difficulties inherent in accurately measuring their levels in the brain, experimental strategies have been focused on the use of pharmacological agents and antioxidants to seek a correlation between the exogenously supplied specific radical scavengers (ie, superoxide dismutase and catalase) and the subsequent protection of cerebral tissues from ischemic injury. However, this strategy entails problems (hemodynamic, pharmacokinetic, toxicity, blood-brain barrier permeability, etc) that may cloud the data interpretation. This mini-review will focus on the oxidant mechanisms in cerebral ischemic brain injury by using transgenic and knockout mice as an alternative approach. METHODS Transgenic and knockout mutants that either overexpress or are deficient in antioxidant enzyme/protein levels have been successfully produced. The availability of these genetically modified animals has made it possible to investigate the role of certain oxidants in ischemic brain cell damage in molecular fashion. RESULTS It has been shown that an increased level of CuZn-superoxide dismutase and antiapoptotic protein Bcl-2 in the brains of transgenic mice protects neurons from ischemic/reperfusion injury, whereas a deficiency in CuZn-superoxide dismutase or mitochondrial Mn-superoxide dismutase exacerbates ischemic brain damage. Target disruption of neuronal nitric oxide synthase in mice also provides neuronal protection against permanent and transient focal cerebral ischemia. CONCLUSIONS I conclude that molecular genetic approaches in modifying antioxidant levels in the brain offer a unique tool for understanding the role of oxidants in ischemic brain damage.

Human copper-zinc superoxide dismutase transgenic mice are highly resistant to reperfusion injury after focal cerebral ischemia.

Background and Purpose We have demonstrated in a previous study that superoxide radicals play a role in the pathogenesis of cerebral infarction, using a transgenic mouse model of distal middle cerebral artery occlusion, permanent ipsilateral cerebral carotid artery occlusion, and 1-hour contralateral cerebral carotid artery occlusion that produced infarction only in the cortex. However, the role of superoxide radicals in reperfusion injury in transgenic mice overexpressing superoxide dismutase (SOD) is unknown. Using a mouse model of intraluminal blockade of middle cerebral artery that produced both cortical and striatal infarction, we now further examined the role of superoxide radicals in ischemic cerebral infarction after reperfusion in transgenic mice overexpressing human CuZn-SOD activity. Methods Transgenic mice of strain Tg HS/SF-218, carrying human SOD-1 genes, and nontransgenic littermates were anesthetized with chloral hydrate (350 mg/kg IP) and xylazine (4 mg/kg IP). Physiological parameters were maintained at a normal range using a 30% O2/70% N2O gas mixture inserted via an inhalation mask. Body temperature was maintained at 37±0.5°C by using a heating pad throughout the studies. The middle cerebral artery occlusion was achieved with a 5-0 rounded nylon suture placed within the internal cerebral artery for 3 hours followed by the removal of the suture to allow reperfusion for another 3 hours. Cerebral infarct size in brain slices and infarct volume, neurological deficit, cortical blood flow, and glutathione levels were measured in both transgenic and nontransgenic mice. Results Compared with the nontransgenic mice, the infarcted areas were significantly decreased in coronal slices from transgenic mice. The infarct volume (in cubic millimeters) was reduced by 26% in transgenic mice after ischemia and reperfusion. This decrease in the infarct volume in transgenic mice closely paralleled the reduced neurological deficits. Introduction of the suture to block blood supply to the middle cerebral artery territory produced a rapid decrease in the relative surface blood flow in the ipsilateral core and the peri-ischemic (penumbra) areas. There were no significant differences in the local cerebral blood flow in the ischemic core or the penumbra areas between the transgenic and nontransgenic groups. However, the level of reduced glutathione in the penumbra area was significantly higher in transgenic mice than in nontransgenic mice, whereas there was no difference in the reduced glutathione levels in the ischemic core between these two groups. Conclusions Our study demonstrated that superoxide radicals play a major role in the pathogenesis of cerebral infarction in reperfusion injury after a focal stroke. The reduction in infarct volume and neurological deficits is not dependent on the changes in cerebral blood flow but rather correlate with reduced oxidative stress in the ischemic brain tissue, which was indicated by the relatively high levels of endogenous reduced glutathione in transgenic mice.

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 Evans 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.

NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation

Neuronal NMDA receptor (NMDAR) activation leads to the formation of superoxide, which normally acts in cell signaling. With extensive NMDAR activation, the resulting superoxide production leads to neuronal death. It is widely held that NMDA-induced superoxide production originates from the mitochondria, but definitive evidence for this is lacking. We evaluated the role of the cytoplasmic enzyme NADPH oxidase in NMDA-induced superoxide production. Neurons in culture and in mouse hippocampus responded to NMDA with a rapid increase in superoxide production, followed by neuronal death. These events were blocked by the NADPH oxidase inhibitor apocynin and in neurons lacking the p47phox subunit, which is required for NADPH oxidase assembly. Superoxide production was also blocked by inhibiting the hexose monophosphate shunt, which regenerates the NADPH substrate, and by inhibiting protein kinase C zeta, which activates the NADPH oxidase complex. These findings identify NADPH oxidase as the primary source of NMDA-induced superoxide production.

Brain injury, edema, and vascular permeability changes induced by oxygen‐derived free radicals

We studied the cerebral effects of oxygen-derived free radicals generated from the xanthine oxidase/ hypoxanthine/ADP-Fej3+ system. Xanthine oxidase/hypoxanthine/ADP-Fe3+ solution (0.1 ml) was infused into caudate putamen, and brain was frozen rapidly in situ. Brain water and sodium content increased concomitant with decreased potassium content at 24 hours and 48 hours after the infusion. The degree of brain edema and injury depended on the dose of xanthine oxidase. Spongy neuropil and neuronal cytoplasmic vacuoles were seen at 2 hours, with an infiltration by polymorphonuclear leukocytes at 24 hours, followed by lipid-laden macrophages and reactive astrocytes. Leakage of fluorescent dye into neuropil was seen at 2 hours, but not later. These data suggest that oxygen-derived free radicals damage endothelial cells of the blood-brain barrier; the brain injury is characterized by edema and by structural damage of neurons and giia.

Oxidative Stress in Ischemic Brain Damage: Mechanisms of Cell Death and Potential Molecular Targets for Neuroprotection

Significant amounts of oxygen free radicals (oxidants) are generated during cerebral ischemia/reperfusion, and oxidative stress plays an important role in brain damage after stroke. In addition to oxidizing macromolecules, leading to cell injury, oxidants are also involved in cell death/survival signal pathways and cause mitochondrial dysfunction. Experimental data from laboratory animals that either overexpress (transgenic) or are deficient in (knock-out) antioxidant proteins, mainly superoxide dismutase, have provided strong evidence of the role of oxidative stress in ischemic brain damage. In addition to mitochondria, recent reports demonstrate that NADPH oxidase (NOX), an important pro-oxidant enzyme, is also involved in the generation of oxidants in the brain after stroke. Inhibition of NOX is neuroprotective against cerebral ischemia. We propose that superoxide dismutase and NOX activity in the brain is a major determinant for ischemic damage/repair and that these major anti- and pro-oxidant enzymes are potential endogenous molecular targets for stroke therapy.

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.

Manganese superoxide dismutase mediates the early release of mitochondrial cytochrome C and subsequent DNA fragmentation after permanent focal cerebral ischemia in mice.

Recent studies have shown that release of mitochondrial cytochrome c is a critical step in the apoptosis process. We have reported that cytosolic redistribution of cytochrome c in vivooccurred after transient focal cerebral ischemia (FCI) in rats and preceded the peak of DNA fragmentation. Although the involvement of reactive oxygen species in the cytosolic redistribution of cytochrome cin vitro has been suggested, the detailed mechanism by which cytochrome c release is mediated in vivo has not yet been established. Also, the role of mitochondrial oxidative stress in cytochrome c release is unknown. These issues can be addressed using knock-out mutants that are deficient in the level of the mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD). In this study we examined the subcellular distribution of the cytochrome c protein in both wild-type mice and heterozygous knock-outs of the Mn-SOD gene (Sod2 −/+) after permanent FCI, in which apoptosis is assumed to participate. Cytosolic cytochrome c was detected as early as 1 hr after ischemia, and correspondingly, mitochondrial cytochrome c showed a significant reduction 2 hr after ischemia (p< 0.01). Cytosolic accumulation of cytochrome c was significantly higher in Sod2 −/+ mice compared with wild-type animals (p < 0.05).N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone (z-VAD.FMK), a nonselective caspase inhibitor, did not affect cytochrome c release after ischemia. A significant amount of DNA laddering was detected 24 hr after ischemia and increased in Sod2 −/+ mice. These data suggest that Mn-SOD blocks cytosolic release of cytochrome c and could thereby reduce apoptosis after permanent FCI.