Miki Fujimura

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.

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.

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.

Cytosolic Redistribution of Cytochrome C after Transient Focal Cerebral Ischemia in Rats

Recent in vitro cell-free studies have shown that cytochrome c release from mitochondria is a critical step in the apoptotic process. The present study examined the expression of cytochrome c protein after transient focal cerebral ischemia in rats, in which apoptosis was assumed to contribute to the expansion of the ischemic lesion. In situ labeling of DNA breaks in frozen sections after 90 minutes of middle cerebral artery (MCA) occlusion showed a significant number of striatal and cortical neurons, which were maximized at 24 hours after ischemia, exhibiting chromatin condensation, nuclear segmentation, and apoptotic bodies. Cytosolic localization of cytochrome c was detected immunohistochemically in the ischemic area as early as 4 hours after 90 minutes of MCA occlusion. Western blot analysis of the cytosolic fraction revealed a strong single 15-kDa band, characteristic of cytochrome c, only in the samples from the ischemic hemisphere. Western blot analysis of the mitochondrial fraction showed a significant amount of mitochondrial cytochrome c in nonischemic brain, which was decreased in ischemic brain 24 hours after ischemia. These results provide the first evidence that cytochrome c is being released from mitochondria to the cytosol after transient focal ischemia. Although further evaluation is necessary to elucidate its correlation with DNA fragmentation, our results suggest the possibility that cytochrome c release may play a role in DNA-damaged neuronal cell death after transient focal cerebral ischemia in rats.

Early appearance of activated matrix metalloproteinase-9 and blood-brain barrier disruption in mice after focal cerebral ischemia and reperfusion.

Blood-brain barrier (BBB) disruption is thought to play a critical role in the pathophysiology of ischemia/reperfusion. Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that can degrade all the components of the extracellular matrix when they are activated. Gelatinase A (MMP-2) and gelatinase B (MMP-9) are able to digest the endothelial basal lamina, which plays a major role in maintaining BBB impermeability. The present study examined the expression and activation of gelatinases before and after transient focal cerebral ischemia (FCI) in mice. Adult male CD1 mice were subjected to 60 min FCI and reperfusion. Zymography was performed from 1 to 23 h after reperfusion using the protein extraction method with detergent extraction and affinity-support purification. MMP-9 expression was also examined by both immunohistochemistry and Western blot analysis, and tissue inhibitors to metalloproteinase-1 was measured by reverse zymography. The BBB opening was evaluated by the Evans blue extravasation method. The 88-kDa activated MMP-9 was absent from the control specimens, while it appeared 3 h after transient ischemia by zymography. At this time point, the BBB permeability alteration was detected in the ischemic brain. Both pro-MMP-9 (96 kDa) and pro-MMP-2 (72 kDa) were seen in the control specimens, and were markedly increased after FCI. A significant induction of MMP-9 was confirmed by both immunohistochemistry and Western blot analysis. The early appearance of activated MMP-9, associated with evidence of BBB permeability alteration, suggests that activation of MMP-9 contributes to the early formation of vasogenic edema after transient FCI.

Neuronal Death/Survival Signaling Pathways in Cerebral Ischemia

SummaryCumulative evidence suggests that apoptosis plays a pivotal role in cell deathin vitro after hypoxia. Apoptotic cell death pathways have also been implicated in ischemic cerebral injury inin vivo ischemia models. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and the numerous reports suggest the involvement of cell survival/death signaling pathways in the pathogenesis of apoptotic cell death in ischemic lesions. In these models, reoxygenation during reperfusion provides a substrate for numerous enzymatic oxidation reactions. Oxygen radicals damage cellular lipids, proteins and nucleic acids, and initiate cell signaling pathways after cerebral ischemia. Genetic manipulation of intrinsic antioxidants and factors in the signaling pathways has provided substantial understanding of the mechanisms involved in cell death/survival signaling pathways and the role of oxygen radicals in ischemic cerebral injury. Future studies of these pathways may provide novel therapeutic strategies in clinical stroke.

Mild Hypothermia Attenuates Cytochrome C Release but Does Not Alter Bcl-2 Expression or Caspase Activation After Experimental Stroke

Mild hypothermia protects the brain from ischemia, but the underlying mechanisms of this effect are not well known. The authors previously found that hypothermia reduces the density of apoptotic cells, but it is not certain whether temperature alters associated biochemical events. Mitochondrial release of cytochrome c has recently been shown to be a key trigger in caspase activation and apoptosis via the intrinsic pathway. Using a model of transient focal cerebral ischemia, the authors determined whether mild hypothermia altered expression of Bcl-2 family proteins, mitochondrial release of cytochrome c, and caspase activation. Mild hypothermia significantly decreased the amount of cytochrome c release 5 hours after the onset of ischemia, but mitochondrial translocation of Bax was not observed until 24 hours. Mild hypothermia did not alter Bcl-2 and Bax expression, and caspase activation was not observed. The present study provides the first evidence that intraischemic mild hypothermia attenuates the release of cytochrome c in the brain, but does not appear to affect other biochemical aspects of the intrinsic apoptotic pathway. They conclude that necrotic processes may have been interrupted to prevent cytochrome c release, and that the ameliorative effect of mild hypothermia may be a result of maintaining mitochondrial integrity. Furthermore, the authors show it is unlikely that mild hypothermia alters the intrinsic apoptotic pathway.

SOD1 Down-Regulates NF-κB and c-Myc Expression in Mice after Transient Focal Cerebral Ischemia

Reactive oxygen species (ROS) are implicated in reperfusion injury after focal cerebral ischemia (FCI). Reactive oxygen species regulate activity of transcription factors like NF-κB. The authors investigated the role of ROS in NF-κB activity after FCI using transgenic mice that overexpressed human copper/zinc-superoxide dismutase (SOD1) and that had reduced infarction volume after FCI. Superoxide dismutase transgenic and wild-type mice were subjected to 1 hour of middle cerebral artery occlusion (MCAO) and subsequent reperfusion. Immunohistochemistry showed SOD1 overexpression attenuated ischemia-induced NF-κB p65 immunoreactivity. Colocalization of NF-κB and the neuronal marker, micro-tubule-associated proteins (MAPs), showed that NF-κB was up-regulated in neurons after FCI. Electrophoretic mobility shift assays showed that SOD1 overexpression reduced ischemia-induced NF-κB DNA binding activity. Supershift assays showed that DNA–protein complexes contained p65 and p50 subunits. Immunoreactivity of c-myc, an NF-κB downstream gene, was increased in the ischemic cortex and colocalized with NF-κB. Western blotting showed that SOD1 overexpression reduced NF-κB and c-Myc protein levels in the ischemic brain. Colocalization of c-Myc and TUNEL staining was observed 24 hours after FCI. The current findings provide the first evidence that SOD1 overexpression attenuates activation of NF-κB after transient FCI in mice and that preventing this early activation may block expression of downstream deleterious genes like c-myc, thereby reducing ischemic damage.

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-1. 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.