Nobuo Noshita

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.

Temporal Profile of Angiogenesis and Expression of Related Genes in the Brain after Ischemia

Angiogenesis is an intricately regulated phenomenon. Its mechanisms in the ischemic brain have not been clearly elucidated. The authors investigated expression of angiogenesis-related genes using a complementary DNA (cDNA) array method as well as Western blotting and immunohistochemistry, and compared these studies with a temporal profile of angiogenesis in mouse brains after ischemia. The number of vessels significantly increased 3 days after injury, and proliferating endothelial cells increased as early as 1 day. This means that angiogenesis occurs immediately after the injury. Ninety-six genes implicated in angiogenesis were investigated with a cDNA array study. It was found that 42, 29, and 13 genes were increased at 1 hour, 1 day, and 21 days, respectively. Most of the well-known angiogenic factors increased as early as 1 hour. Vessel-stabilizing factors such as thrombospondins also increased. At 1 day, however, thrombospondins decreased to lower levels than in the control, indicating a shift from vascular protection to angiogenesis. At 21 days, many genes were decreased, but some involved in tissue repair were newly increased. Western blotting and immunohistochemistry showed findings compatible with the cDNA array study. Many molecules act in an orchestrated fashion in the brain after ischemia and should be taken into account for therapeutic angiogenesis for stroke.

Evidence of Phosphorylation of Akt and Neuronal Survival after Transient Focal Cerebral Ischemia in Mice

The serine-threonine kinase, Akt, prevents apoptosis by phosphorylation at serine-473 in several cell systems. After phosphorylation, activated Akt inactivates other apoptogenic factors, such as Bad or caspase-9, thereby inhibiting cell death. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after transient focal cerebral ischemia in mice subjected to 60 minutes of focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Phospho-Akt was analyzed by immunohistochemistry and Western blot analysis. The DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end-labeling (TUNEL). Immunohistochemistry showed the expression of phospho-Akt was markedly increased in the middle cerebral artery territory cortex at 4 hours of reperfusion compared with the control, whereas it was decreased by 24 hours. Western blot analysis showed a significant increase of phospho-Akt 4 hours after focal cerebral ischemia in the cortex, whereas phospho-Akt was decreased in the ischemic core. Double staining with phospho-Akt and TUNEL showed different cellular distributions of phospho-Akt and TUNEL-positive staining. Phosphorylation of Akt was prevented after focal cerebral ischemia by LY294002, a phosphatidylinositol 3-kinase inhibitor, which facilitated subsequent DNA fragmentation. These results suggest that phosphorylation of Akt may be involved in determining cell survival or cell death after transient focal cerebral ischemia.

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.

Effects of global ischemia duration on neuronal, astroglial, oligodendroglial, and microglial reactions in the vulnerable hippocampal CA1 subregion in rats

The hippocampal CA1 neurons are selectively vulnerable to global ischemia, and neuronal death occurs in a delayed manner. The threshold of global ischemia duration that induces neuronal death has been studied, but the relationship between ischemia duration and glial death in the hippocampal CA1 area has not been fully studied. We examined neuronal/glial viability and morphological changes in the CA1 subregion after different durations of global ischemia. Global ischemia was induced in Sprague-Dawley rats by 10, 5, and 3 min of bilateral common carotid artery occlusion and hypotension. At 1-56 days after ischemia, the morphological reactions of neurons, astrocytes, oligodendrocytes, and microglia were immunohistochemically evaluated. Most of the hippocampal CA1 pyramidal neurons underwent delayed death at 3 days after 10/5 min of ischemia, but not after 3 min of ischemia. The number of astrocytes gradually declined after 10/5 min of ischemia, and viable astrocytes showed characteristic staged morphological reactions. Oligodendrocytes also showed morphological changes in their processes after 10/5 min of ischemia. Microglia transformed into a reactive form at 5 days only after 10/5 min of ischemia. These data suggest that some morphological changes in glial cells were not dependent on neuronal cell death, but their own reactions to the different severity of ischemia.

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.

Manganese Superoxide Dismutase Affects Cytochrome c Release and Caspase-9 Activation after Transient Focal Cerebral Ischemia in Mice

Release of cytochrome c from mitochondria to cytosol is a critical step in the mitochondrial-dependent signaling pathways of apoptosis. The authors have reported that manganese superoxide dismutase (Mn-SOD) attenuated cytochrome c release and apoptotic cell death after focal cerebral ischemia (FCI). To investigate downstream to the cytochrome c-dependent pathway, the authors examined caspase-9 activation after transient FCI by immunohistochemistry and Western blotting in both wild-type and Sod2 −/+ mice. Mice were subjected to 60 minutes of middle cerebral artery occlusion followed by 1, 2, 4, or 24 hours of reperfusion. Two hours after reperfusion, cytochrome c and caspase-9 were observed in the cytosol and significantly increased in Sod2 −/+ mutants compared with wild-type mice as shown by Western blotting. Immunofluorescent double labeling for cytochrome c and caspase-9 showed cytosolic cytochrome c 1 hour after transient FCI. Cleaved caspase-9 first appeared in the cytosol at 2 hours and colocalized with cytochrome c. Terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick and labeling (TUNEL) showed significant increase of positive cells in Sod2 −/+ mice compared with the wild-type in the cortex, but not in the caudate putamen. The current study revealed Mn-SOD might affect cytochrome c translocation and downstream caspase activation in the mitochondrial-dependent cell death pathway after transient FCI.

Copper-Zinc Superoxide Dismutase Affects Akt Activation After Transient Focal Cerebral Ischemia in Mice

Background and Purpose— The serine-threonine kinase Akt is activated by phosphorylation at serine-473. After phosphorylation, activated Akt inactivates BAD or caspase-9 or other apoptogenic components, thereby inhibiting cell death. In this study we examined the relationship between Akt phosphorylation and oxidative stress after transient focal cerebral ischemia (FCI) using copper-zinc superoxide dismutase (SOD1) transgenic (Tg) mice. Methods— The mice were subjected to 60 minutes of middle cerebral artery occlusion by intraluminal suture blockade followed by 1, 4, and 24 hours of reperfusion. Phospho-Akt expression was examined by immunohistochemistry and Western blot analysis. Production of superoxide anion was assessed by the hydroethidine method in both wild-type mice and SOD1 Tg mice. DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase–mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL). Results— Immunohistochemistry demonstrated that phospho-Akt was constitutively expressed and was decreased in the ischemic core as early as 1 hour after reperfusion, whereas it was temporally increased in the cortex at 4 hours. Phospho-Akt expression was enhanced in the SOD1 Tg mice. Western blot analysis showed that phospho-Akt was maximized 4 hours after reperfusion in the wild-type mice, whereas phospho-Akt was increased as early as 1 hour after ischemia in the SOD1 Tg mice. There was a significant decrease in TUNEL-positive cells in the SOD1 Tg mice compared with the wild-type mice. Conclusions— The present study suggests that SOD1 may contribute to the early activation of the Akt cell survival signaling pathway and may attenuate subsequent DNA damage after transient FCI.

Early Decrease in DNA Repair Proteins, Ku70 and Ku86, and Subsequent DNA Fragmentation After Transient Focal Cerebral Ischemia in Mice

Background and Purpose— Ku70 and Ku86, multifunctional DNA repair proteins, bind to broken DNA ends, including double-strand breaks, and trigger a DNA repair pathway. To investigate the involvement of these proteins in DNA fragmentation after ischemia/reperfusion, Ku protein expression was examined before and after transient focal cerebral ischemia (FCI) in mice. Methods— Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal suture blockade of the middle cerebral artery. Ku protein expression was studied by immunohistochemistry and Western blot analysis. DNA fragmentation was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL). The spatial relationship between Ku expression and DNA fragmentation was examined by double labeling with Ku and TUNEL after reperfusion. Results— Immunohistochemistry showed constitutive expression of Ku proteins in control brains. The number of Ku-expressing cells was decreased in the entire middle cerebral artery territory as early as 4 hours after reperfusion and remained reduced until 24 hours. Western blot analyses confirmed the significant reduction of these proteins (59.4% and 57.7% reduction in optical density at 4 hours of reperfusion from the normal level of Ku70 and Ku86 bands, respectively;P <0.001). DNA gel electrophoresis demonstrated DNA laddering 24 hours after reperfusion, but not at 4 hours. Double staining with Ku and TUNEL showed a concomitant loss of Ku immunoreactivity and TUNEL-positive staining. Conclusions— These results suggest that the early reduction of Ku proteins and the loss of defense against DNA damage may underlie the mechanism of DNA fragmentation after FCI.

Overexpression of human copper/zinc-superoxide dismutase in transgenic animals attenuates the reduction of apurinic/ apyrimidinic endonuclease expression in neurons after in vitro ischemia and after transient global cerebral ischemia

Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity. Apurinic/apyrimidinic endonuclease (APE) is a multifunctional protein in the DNA base excision repair pathway which repairs apurinic/apyrimidinic sites in DNA. We investigated the involvement of oxidative stress and expression of APE in neurons after oxygen–glucose deprivation and after global cerebral ischemia. Our results suggest that overexpression of human copper/zinc‐superoxide dismutase reduced oxidative stress with a subsequent decrease in APE expression. Production of oxygen free radicals and inhibition of the base excision repair pathway may play pivotal roles in the cell death pathway after ischemia.