Bio

Academic Appointments


Administrative Appointments


  • Director of Research, Department of Neurosurgery, Stanford University School of Medicine (1997 - 2012)

Honors & Awards


  • James R. Doty Professor of Neurosurgery and Neurosciences, Stanford University (2007)
  • Mencius Type A & Type B Scholarships, Chinese University of Hong Kong (1960-1961)
  • University Scientific Achievement Scholarship, Chinese University of Hong Kong (1962-1963)
  • Jacob Javits Neuroscience Investigator Award, National Institutes of Health (1997-2004)
  • Bugher Foundation Award, American Heart Association (2001-2004)
  • Chairman, 22nd Princeton Conference on Cerebrovascular Disease (2000)
  • President, National Neurotrauma Society (2000-2001)
  • Member, Neurology A Study Section (1985-1989)
  • Member, NINDS Neurological Disorders Program Project Review A Committee (1996-2000)
  • Co-Chair, Neurovascular Protective Mechanisms, NINDS Stroke Progress Review Group (July 15-18, 2001)
  • Member, NINDS Stroke Progress Review Group (January 6, 2003)
  • Member, NIMH Board of Scientific Counselors (April 27-28, 2003)
  • Invited Speaker and Participant, Spinal Cord Injury Workshop, National Academy of Sciences USA (May 24, 2004)
  • Member, External Scientific Review Committee, Canadian Stroke Network (2004-present)
  • Thomas Willis Award, American Stroke Association (2008)

Professional Education


  • BS, Chinese University of Hong Kong, Biology (1964)
  • MA, UCLA, Biochemistry (1970)
  • PhD, UCLA, Biology (1972)

Research & Scholarship

Current Research and Scholarly Interests


My primary research interest is to understand the molecular and cellular mechanisms of cell death in the CNS following acute injuries such as ischemia and trauma and chronic neurodegenerative diseases. We focus on the role of oxidative stress, mitochondrial dysfunction, DNA damage and repair, various gene expressions (gene family of HSP72, Bcl-2, c-fos and COX-2) and various transcription factors (NF-kappaB, AP1, p53) in the pathogenesis of necrosis and/or apoptosis. Various transgenic and knockout mutant mice of CuZn-superoxide dismutase (SOD1) and mitochondrial manganese SOD (SOD2) have been generated to address the role of superoxide radicals in these cell death processes. In addition, human SOD1 transgenic rats have been established as a model for the study of oxidative mechanisms and acute and chronic CNS injuries. The long-term goal of our research is to derive therapeutic strategies at the cellular and molecular level to ameliorate cell death in CNS injuries.

Teaching

2013-14 Courses


Graduate and Fellowship Programs


Publications

Journal Articles


  • The role of PARL and HtrA2 in striatal neuronal injury after transient global cerebral ischemia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Yoshioka, H., Katsu, M., Sakata, H., Okami, N., Wakai, T., Kinouchi, H., Chan, P. H. 2013; 33 (11): 1658-1665

    Abstract

    The presenilin-associated rhomboid-like (PARL) protein and high temperature requirement factor A2 (HtrA2) are key regulators of mitochondrial integrity and play pivotal roles in apoptosis. However, their roles after cerebral ischemia have not been thoroughly elucidated. To clarify these roles, mice were subjected to transient global cerebral ischemia, and striatal neuronal injury was assessed. Western blot and coimmunoprecipitation analyses revealed that PARL and processed HtrA2 localized to mitochondria, and that PARL was bound to HtrA2 in sham animals. Expression of PARL and processed HtrA2 in mitochondria significantly decreased 6 to 72 hours after ischemia, and the binding of PARL to HtrA2 disappeared after ischemia. In contrast, expression of processed HtrA2 increased 24 hours after ischemia in the cytosol, where HtrA2 was bound to X chromosome-linked inhibitor-of-apoptosis protein (XIAP). Administration of PARL small interfering RNA inhibited HtrA2 processing and worsened ischemic neuronal injury. Our results show that downregulation of PARL after ischemia is a key step in ischemic neuronal injury, and that it decreases HtrA2 processing and increases neuronal vulnerability. In addition, processed HtrA2 released into the cytosol after ischemia contributes to neuronal injury via inhibition of XIAP.

    View details for DOI 10.1038/jcbfm.2013.139

    View details for Web of Science ID 000326559300002

    View details for PubMedID 23921894

  • Prevention of JNK phosphorylation as a mechanism for rosiglitazone in neuroprotection after transient cerebral ischemia: activation of dual specificity phosphatase JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Okami, N., Narasimhan, P., Yoshioka, H., Sakata, H., Kim, G. S., Jung, J. E., Maier, C. M., Chan, P. H. 2013; 33 (1): 106-114

    Abstract

    Rosiglitazone, a synthetic peroxisome proliferator-activated receptor-? (PPAR?) agonist, prevents cell death after cerebral ischemia in animal models, but the underlying mechanism has not been clarified. In this study, we examined how rosiglitazone protects neurons against ischemia. Mice treated with rosiglitazone were subjected to 60 minutes of focal ischemia followed by reperfusion. Rosiglitazone reduced infarct volume after ischemia and reperfusion. We show that this neuroprotective effect was reversed with a PPAR? antagonist. Western blot analysis showed a significant increase in expression of phosphorylated stress-activated protein kinases (c-Jun N-terminal kinase (JNK) and p38) in ischemic brain tissue. Rosiglitazone blocked this increase. Furthermore, we observed that rosiglitazone increased expression of the dual-specificity phosphatase 8 (DUSP8) protein and messenger RNA in ischemic brain tissue. Dual-specificity phosphatase 8 is a mitogen-activated protein kinase phosphatase that can dephosphorylate JNK and p38. Another key finding of the present study was that knockdown of DUSP8 in primary cultured cortical neurons that were subjected to oxygen-glucose deprivation diminished rosiglitazone's effect on downregulation of JNK phosphorylation. Thus, rosiglitazone's neuroprotective effect after ischemia is mediated by blocking JNK phosphorylation induced by ischemia via DUSP8 upregulation.

    View details for DOI 10.1038/jcbfm.2012.138

    View details for Web of Science ID 000313224600014

    View details for PubMedID 23032483

  • Interleukin 6-preconditioned neural stem cells reduce ischaemic injury in stroke mice BRAIN Sakata, H., Narasimhan, P., Niizuma, K., Maier, C. M., Wakai, T., Chan, P. H. 2012; 135: 3298-3310

    Abstract

    Transplantation of neural stem cells provides a promising therapy for stroke. Its efficacy, however, might be limited because of massive grafted-cell death after transplantation, and its insufficient capability for tissue repair. Interleukin 6 is a pro-inflammatory cytokine involved in the pathogenesis of various neurological disorders. Paradoxically, interleukin 6 promotes a pro-survival signalling pathway through activation of signal transducer and activator of transcription 3. In this study, we investigated whether cellular reprogramming of neural stem cells with interleukin 6 facilitates the effectiveness of cell transplantation therapy in ischaemic stroke. Neural stem cells harvested from the subventricular zone of foetal mice were preconditioned with interleukin 6 in vitro and transplanted into mouse brains 6 h or 7 days after transient middle cerebral artery occlusion. Interleukin 6 preconditioning protected the grafted neural stem cells from ischaemic reperfusion injury through signal transducer and activator of transcription 3-mediated upregulation of manganese superoxide dismutase, a primary mitochondrial antioxidant enzyme. In addition, interleukin 6 preconditioning induced secretion of vascular endothelial growth factor from the neural stem cells through activation of signal transducer and activator of transcription 3, resulting in promotion of angiogenesis in the ischaemic brain. Furthermore, transplantation of interleukin 6-preconditioned neural stem cells significantly attenuated infarct size and improved neurological performance compared with non-preconditioned neural stem cells. This interleukin 6-induced amelioration of ischaemic insults was abolished by transfecting the neural stem cells with signal transducer and activator of transcription 3 small interfering RNA before transplantation. These results indicate that preconditioning with interleukin 6, which reprograms neural stem cells to tolerate oxidative stress after ischaemic reperfusion injury and to induce angiogenesis through activation of signal transducer and activator of transcription 3, is a simple and beneficial approach for enhancing the effectiveness of cell transplantation therapy in ischaemic stroke.

    View details for DOI 10.1093/brain/aws259

    View details for Web of Science ID 000311644800016

    View details for PubMedID 23169920

  • Involvement of Mitogen-Activated Protein Kinase Pathways in Expression of the Water Channel Protein Aquaporin-4 after Ischemia in Rat Cortical Astrocytes JOURNAL OF NEUROTRAUMA Nito, C., Kamada, H., Endo, H., Narasimhan, P., Lee, Y., Chan, P. H. 2012; 29 (14): 2404-2412

    Abstract

    Brain edema after ischemic brain injury is a key determinant of morbidity and mortality. Aquaporin-4 (AQP4) plays an important role in water transport in the central nervous system and is highly expressed in brain astrocytes. However, the AQP4 regulatory mechanisms are poorly understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs), which are involved in changes in osmolality, might mediate AQP4 expression in models of rat cortical astrocytes after ischemia. Increased levels of AQP4 in primary cultured astrocytes subjected to oxygen-glucose deprivation (OGD) and 2 h of reoxygenation were observed, after which they immediately decreased at 0?h of reoxygenation. Astrocytes exposed to OGD injury had significantly increased phosphorylation of three kinds of MAPKs. Treatment with SB203580, a selective p38 MAPK inhibitor, or SP600125, a selective c-Jun N-terminal kinase inhibitor, significantly attenuated the return of AQP4 to its normal level, and SB203580, but not SP600125, significantly decreased cell death. In an in vivo study, AQP4 expression was upregulated 1-3 days after reperfusion, which was consistent with the time course of p38 phosphorylation and activation, and decreased by the p38 inhibition after transient middle cerebral artery occlusion (MCAO). These results suggest that p38 MAPK may regulate AQP4 expression in cortical astrocytes after ischemic injury.

    View details for DOI 10.1089/neu.2012.2430

    View details for Web of Science ID 000308701400004

    View details for PubMedID 22676888

  • Neural Stem Cells Genetically Modified to Overexpress Cu/Zn-Superoxide Dismutase Enhance Amelioration of Ischemic Stroke in Mice STROKE Sakata, H., Niizuma, K., Wakai, T., Narasimhan, P., Maier, C. M., Chan, P. H. 2012; 43 (9): 2423-?

    Abstract

    The harsh host brain microenvironment caused by production of reactive oxygen species after ischemic reperfusion injury offers a significant challenge to survival of transplanted neural stem cells (NSCs) after ischemic stroke. Copper/zinc-superoxide dismutase (SOD1) is a specific antioxidant enzyme that counteracts superoxide anions. We have investigated whether genetic manipulation to overexpress SOD1 enhances survival of grafted stem cells and accelerates amelioration of ischemic stroke.NSCs genetically modified to overexpress or downexpress SOD1 were administered intracerebrally 2 days after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from Days 0 to 28 after stroke.Overexpression of SOD1 suppressed production of superoxide anions after ischemic reperfusion injury and reduced NSC death after transplantation. In contrast, downexpression of SOD1 promoted superoxide generation and increased oxidative stress-mediated NSC death. Transplantation of SOD1-overexpressing NSCs enhanced angiogenesis in the ischemic border zone through upregulation of vascular endothelial growth factor. Moreover, grafted SOD1-overexpressing NSCs reduced infarct size and improved behavioral performance compared with NSCs that were not genetically modified.Our findings reveal a strong involvement of SOD1 expression in NSC survival after ischemic reperfusion injury. We propose that conferring antioxidant properties on NSCs by genetic manipulation of SOD1 is a potential approach for enhancing the effectiveness of cell transplantation therapy in ischemic stroke.

    View details for DOI 10.1161/STROKEAHA.112.656900

    View details for Web of Science ID 000308416300040

    View details for PubMedID 22713489

  • Release of mitochondrial apoptogenic factors and cell death are mediated by CK2 and NADPH oxidase JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Kim, G. S., Jung, J. E., Narasimhan, P., Sakata, H., Yoshioka, H., Song, Y. S., Okami, N., Chan, P. H. 2012; 32 (4): 720-730

    Abstract

    Activation of the NADPH oxidase subunit, NOX2, and increased oxidative stress are associated with neuronal death after cerebral ischemia and reperfusion. Inhibition of NOX2 by casein kinase 2 (CK2) leads to neuronal survival, but the mechanism is unknown. In this study, we show that in copper/zinc-superoxide dismutase transgenic (SOD1 Tg) mice, degradation of CK2? and CK2?' and dephosphorylation of CK2? against oxidative stress were markedly reduced compared with wild-type (WT) mice that underwent middle cerebral artery occlusion. Inhibition of CK2 pharmacologically or by ischemic reperfusion facilitated accumulation of poly(ADP-ribose) polymers, the translocation of apoptosis-inducing factor (AIF), and cytochrome c release from mitochondria after ischemic injury. The eventual enhancement of CK2 inhibition under ischemic injury strongly increased 8-hydroxy-2'-deoxyguanosine and phosphorylation of H2A.X. Furthermore, CK2 inhibition by tetrabromocinnamic acid (TBCA) in SOD1 Tg and gp91 knockout (KO) mice after ischemia reperfusion induced less release of AIF and cytochrome c than in TBCA-treated WT mice. Inhibition of CK2 in gp91 KO mice subjected to ischemia reperfusion did not increase brain infarction compared with TBCA-treated WT mice. These results strongly suggest that NOX2 activation releases reactive oxygen species after CK2 inhibition, triggering release of apoptogenic factors from mitochondria and inducing DNA damage after ischemic brain injury.

    View details for DOI 10.1038/jcbfm.2011.176

    View details for Web of Science ID 000302472100013

    View details for PubMedID 22146192

  • Minocycline-Preconditioned Neural Stem Cells Enhance Neuroprotection after Ischemic Stroke in Rats JOURNAL OF NEUROSCIENCE Sakata, H., Niizuma, K., Yoshioka, H., Kim, G. S., Jung, J. E., Katsu, M., Narasimhan, P., Maier, C. M., Nishiyama, Y., Chan, P. H. 2012; 32 (10): 3462-3473

    Abstract

    Transplantation of neural stem cells (NSCs) offers a novel therapeutic strategy for stroke; however, massive grafted cell death following transplantation, possibly due to a hostile host brain environment, lessens the effectiveness of this approach. Here, we have investigated whether reprogramming NSCs with minocycline, a broadly used antibiotic also known to possess cytoprotective properties, enhances survival of grafted cells and promotes neuroprotection in ischemic stroke. NSCs harvested from the subventricular zone of fetal rats were preconditioned with minocycline in vitro and transplanted into rat brains 6 h after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from days 0-28 after stroke. For in vitro experiments, NSCs were subjected to oxygen-glucose deprivation and reoxygenation. Cell viability and antioxidant gene expression were analyzed. Minocycline preconditioning protected the grafted NSCs from ischemic reperfusion injury via upregulation of Nrf2 and Nrf2-regulated antioxidant genes. Additionally, preconditioning with minocycline induced the NSCs to release paracrine factors, including brain-derived neurotrophic factor, nerve growth factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor. Moreover, transplantation of the minocycline-preconditioned NSCs significantly attenuated infarct size and improved neurological performance, compared with non-preconditioned NSCs. Minocycline-induced neuroprotection was abolished by transfecting the NSCs with Nrf2-small interfering RNA before transplantation. Thus, preconditioning with minocycline, which reprograms NSCs to tolerate oxidative stress after ischemic reperfusion injury and express higher levels of paracrine factors through Nrf2 up-regulation, is a simple and safe approach to enhance the effectiveness of transplantation therapy in ischemic stroke.

    View details for DOI 10.1523/JNEUROSCI.5686-11.2012

    View details for Web of Science ID 000301295300022

    View details for PubMedID 22399769

  • Neuroprotection by Interleukin-6 Is Mediated by Signal Transducer and Activator of Transcription 3 and Antioxidative Signaling in Ischemic Stroke STROKE Jung, J. E., Kim, G. S., Chan, P. H. 2011; 42 (12): 3574-U371

    Abstract

    Interleukin-6 (IL-6) has been shown to have a neuroprotective effect in brain ischemic injury. However, its molecular mechanisms are still poorly understood. In this study, we investigated the neuroprotective role of the IL-6 receptor (IL-6R) by IL-6 in the reactive oxygen species defense system after transient focal cerebral ischemia (tFCI).IL-6 was injected in mice before and after middle cerebral artery occlusion. Coimmunoprecipitation assays were performed for analysis of an IL-6R association after tFCI. Primary mouse cerebral cortical neurons were transfected with small interfering RNA probes targeted to IL-6R? or gp130 and were used for chromatin-immunoprecipitation assay, luciferase promoter assay, and cell viability assay. Reduction in infarct volumes by IL-6 was measured after tFCI.IL-6R was disrupted through a disassembly between IL-6R? and gp130 associated by protein oxidation after reperfusion after tFCI. This suppressed phosphorylation of signal transducer and activator of transcription 3 (STAT3) and finally induced neuronal cell death through a decrease in manganese-superoxide dismutase. However, IL-6 injections prevented disruption of IL-6R against reperfusion after tFCI, consequently restoring activity of STAT3 through recovery of the binding of STAT3 to gp130. Moreover, IL-6 injections restored the transcriptional activity of the manganese-superoxide dismutase promoter through recovery of the recruitment of STAT3 to the manganese-superoxide dismutase promoter and reduced infarct volume after tFCI.This study demonstrates that IL-6 has a neuroprotective effect against cerebral ischemic injury through IL-6R-mediated STAT3 activation and manganese-superoxide dismutase expression.

    View details for DOI 10.1161/STROKEAHA.111.626648

    View details for Web of Science ID 000297941500049

    View details for PubMedID 21940958

  • NADPH oxidase is involved in post-ischemic brain inflammation NEUROBIOLOGY OF DISEASE Chen, H., Kim, G. S., Okami, N., Narasimhan, P., Chan, P. H. 2011; 42 (3): 341-348

    Abstract

    Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is widely expressed in brain tissue including neurons, glia, and endothelia in neurovascular units. It is a major source of oxidants in the post-ischemic brain and significantly contributes to ischemic brain damage. Inflammation occurs after brain ischemia and is known to be associated with post-ischemic oxidative stress. Post-ischemic inflammation also causes progressive brain injury. In this study we investigated the role of NOX2 in post-ischemic cerebral inflammation using a transient middle cerebral artery occlusion model in mice. We demonstrate that mice with NOX2 subunit gp91(phox) knockout (gp91 KO) showed 35-44% less brain infarction at 1 and 3 days of reperfusion compared with wild-type (WT) mice. Minocycline further reduced brain damage in the gp91 KO mice at 3 days of reperfusion. The gp91 KO mice exhibited less severe post-ischemic inflammation in the brain, as evidenced by reduced microglial activation and decreased upregulation of inflammation mediators, including interleukin-1? (IL-1?), tumor necrosis factor-?, inducible nitric oxide synthases, CC-chemokine ligand 2, and CC-chemokine ligand 3. Finally, we demonstrated that an intraventricular injection of IL-1? enhanced ischemia- and reperfusion-mediated brain damage in the WT mice (double the infarction volume), whereas, it failed to aggravate brain infarction in the gp91 KO mice. Taken together, these results demonstrate the involvement of NOX2 in post-ischemic neuroinflammation and that NOX2 inhibition provides neuroprotection against inflammatory cytokine-mediated brain damage.

    View details for DOI 10.1016/j.nbd.2011.01.027

    View details for Web of Science ID 000290081700014

    View details for PubMedID 21303700

  • Oxidative Stress in Ischemic Brain Damage: Mechanisms of Cell Death and Potential Molecular Targets for Neuroprotection ANTIOXIDANTS & REDOX SIGNALING Chen, H., Yoshioka, H., Kim, G. S., Jung, J. E., Okami, N., Sakata, H., Maier, C. M., Narasimhan, P., Goeders, C. E., Chan, P. H. 2011; 14 (8): 1505-1517

    Abstract

    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.

    View details for DOI 10.1089/ars.2010.3576

    View details for Web of Science ID 000288851000011

    View details for PubMedID 20812869

  • Consistent Injury to Medium Spiny Neurons and White Matter in the Mouse Striatum after Prolonged Transient Global Cerebral Ischemia JOURNAL OF NEUROTRAUMA Yoshioka, H., Niizuma, K., Katsu, M., Sakata, H., Okami, N., Chan, P. H. 2011; 28 (4): 649-660

    Abstract

    A reproducible transient global cerebral ischemia (tGCI) mouse model has not been fully established. Although striatal neurons and white matter are recognized to be vulnerable to ischemia, their injury after tGCI in mice has not been elucidated. The purpose of this study was to evaluate injuries to striatal neurons and white matter after tGCI in C57BL/6 mice, and to develop a reproducible tGCI model. Male C57BL/6 mice were subjected to tGCI by bilateral common carotid artery occlusion (BCCAO). Mice whose cortical cerebral blood flow after BCCAO decreased to less than 13% of the pre-ischemic value were used. Histological analysis showed that at 3 days after 22 min of BCCAO, striatal neurons were injured more consistently than those in other brain regions. Quantitative analysis of cytochrome c release into the cytosol and DNA fragmentation in the striatum showed consistent injury to the striatum. Immunohistochemistry and Western blot analysis revealed that DARPP-32-positive medium spiny neurons, the majority of striatal neurons, were the most vulnerable among the striatal neuronal subpopulations. The striatum (especially medium spiny neurons) was susceptible to oxidative stress after tGCI, which is probably one of the mechanisms of vulnerability. SMI-32 immunostaining showed that white matter in the striatum was also consistently injured 3 days after 22 min of BCCAO. We thus suggest that this is a tGCI model using C57BL/6 mice that consistently produces neuronal and white matter injury in the striatum by a simple technique. This model can be highly applicable for elucidating molecular mechanisms in the brain after global ischemia.

    View details for DOI 10.1089/neu.2010.1662

    View details for Web of Science ID 000289240200013

    View details for PubMedID 21309724

  • NADPH oxidase mediates striatal neuronal injury after transient global cerebral ischemia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Yoshioka, H., Niizuma, K., Katsu, M., Okami, N., Sakata, H., Kim, G. S., Narasimhan, P., Chan, P. H. 2011; 31 (3): 868-880

    Abstract

    Medium spiny neurons (MSNs) constitute most of the striatal neurons and are known to be vulnerable to ischemia; however, the mechanisms of the vulnerability remain unclear. Activated forms of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (NOX), which require interaction between cytosolic and membrane-bound subunits, are among the major sources of superoxide in the central nervous system. Although increasing evidence suggests that NOX has important roles in neurodegenerative diseases, its roles in MSN injury after transient global cerebral ischemia (tGCI) have not been elucidated. To clarify this issue, C57BL/6 mice were subjected to tGCI by bilateral common carotid artery occlusion for 22?minutes. Western blot analysis revealed upregulation of NOX subunits and recruitment of cytosolic subunits to the cell membrane at early (3 to 6?hours) and late (72?hours) phases after tGCI. Taken together with immunofluorescent studies, this activation arose in MSNs and endothelial cells at the early phase, and in reactive microglia at the late phase. Pharmacological and genetic inhibition of NOX attenuated oxidative injury, microglial activation, and MSN death after tGCI. These findings suggest that NOX has pivotal roles in MSN injury after tGCI and could be a therapeutic target for brain ischemia.

    View details for DOI 10.1038/jcbfm.2010.166

    View details for Web of Science ID 000287842700009

    View details for PubMedID 20859296

  • Hemoglobin-induced oxidative stress contributes to matrix metalloproteinase activation and blood-brain barrier dysfunction in vivo JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Katsu, M., Niizuma, K., Yoshioka, H., Okami, N., Sakata, H., Chan, P. H. 2010; 30 (12): 1939-1950

    Abstract

    Hemoglobin (Hb) released from extravasated erythrocytes is implicated in brain edema after intracerebral hemorrhage (ICH). Hemoglobin is a major component of blood and a potent mediator of oxidative stress after ICH. Oxidative stress and matrix metalloproteinases (MMPs) are associated with blood-brain barrier (BBB) dysfunction. This study was designed to elucidate whether Hb-induced oxidative stress contributes to MMP-9 activation and BBB dysfunction in vivo. An intracerebral injection of Hb into rat striata induced increased hydroethidine (HEt) signals in parallel with MMP-9 levels. In situ gelatinolytic activity colocalized with oxidized HEt signals in vessel walls, accompanied by immunoglobulin G leakage and a decrease in immunoactivity of endothelial barrier antigen, a marker of endothelial integrity. Administration of a nonselective MMP inhibitor prevented MMP-9 levels and albumin leakage in injured striata. Moreover, reduction in oxidative stress by copper/zinc-superoxide dismutase (SOD1) overexpression reduced oxidative stress, MMP-9 levels, albumin leakage, and subsequent apoptosis compared with wild-type littermates. We speculate that Hb-induced oxidative stress may contribute to early BBB dysfunction and subsequent apoptosis, partly through MMP activation, and that SOD1 overexpression may reduce Hb-induced oxidative stress, BBB dysfunction, and apoptotic cell death.

    View details for DOI 10.1038/jcbfm.2010.45

    View details for Web of Science ID 000284825900006

    View details for PubMedID 20354546

  • Oxidative stress increases phosphorylation of I kappa B kinase-alpha by enhancing NF-kappa B-inducing kinase after transient focal cerebral ischemia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Song, Y. S., Kim, M., Kim, H., Jung, B., Yang, J., Narasimhan, P., Kim, G. S., Jung, J. E., Park, E., Chan, P. H. 2010; 30 (7): 1265-1274

    Abstract

    The IkappaB kinase (IKK) complex is a central component in the classic activation of the nuclear factor-kappaB (NF-kappaB) pathway. It has been reported to function in physiologic responses, including cell death and inflammation. We have shown that IKK is regulated by oxidative status after transient focal cerebral ischemia (tFCI) in mice. However, the mechanism by which oxidative stress influences IKKs after tFCI is largely unknown. Nuclear accumulation and phosphorylation of IKKalpha (pIKKalpha) were observed 1 h after 30 mins of tFCI in mice. In copper/zinc-superoxide dismutase knockout mice, levels of NF-kappaB-inducing kinase (NIK) (an upstream kinase of IKKalpha), pIKKalpha, and phosphorylation of histone H3 (pH3) on Ser10 were increased after tFCI and were higher than in wild-type mice. Immunohistochemistry showed nuclear accumulation and pIKKalpha in mouse brain endothelial cells after tFCI. Nuclear factor-kappaB-inducing kinase was increased, and it enhanced pH3 by inducing pIKKalpha after oxygen-glucose deprivation (OGD) in mouse brain endothelial cells. Both NIK and pH3 interactions with IKKalpha were confirmed by coimmunoprecipitation. Treatment with IKKalpha small interfering RNA significantly reduced cell death after OGD. These results suggest that augmentation of NIK, IKKalpha, and pH3 in response to oxidative stress is involved in cell death after cerebral ischemia (or stroke).

    View details for DOI 10.1038/jcbfm.2010.6

    View details for Web of Science ID 000279408000002

    View details for PubMedID 20125184

  • Reperfusion and Neurovascular Dysfunction in Stroke: from Basic Mechanisms to Potential Strategies for Neuroprotection MOLECULAR NEUROBIOLOGY Jung, J. E., Kim, G. S., Chen, H., Maier, C. M., Narasimhan, P., Song, Y. S., Niizuma, K., Katsu, M., Okami, N., Yoshioka, H., Sakata, H., Goeders, C. E., Chan, P. H. 2010; 41 (2-3): 172-179

    Abstract

    Effective stroke therapies require recanalization of occluded cerebral blood vessels. However, reperfusion can cause neurovascular injury, leading to cerebral edema, brain hemorrhage, and neuronal death by apoptosis/necrosis. These complications, which result from excess production of reactive oxygen species in mitochondria, significantly limit the benefits of stroke therapies. We have developed a focal stroke model using mice deficient in mitochondrial manganese-superoxide dismutase (SOD2-/+) to investigate neurovascular endothelial damage that occurs during reperfusion. Following focal stroke and reperfusion, SOD2-/+ mice had delayed blood-brain barrier breakdown, associated with activation of matrix metalloproteinase and high brain hemorrhage rates, whereas a decrease in apoptosis and hemorrhage was observed in SOD2 overexpressors. Thus, induction and activation of SOD2 is a novel strategy for neurovascular protection after ischemia/reperfusion. Our recent study identified the signal transducer and activator of transcription 3 (STAT3) as a transcription factor of the mouse SOD2 gene. During reperfusion, activation of STAT3 and its recruitment into the SOD2 gene were blocked, resulting in increased oxidative stress and neuronal apoptosis. In contrast, pharmacological activation of STAT3 induced SOD2 expression, which limits ischemic neuronal death. Our studies point to antioxidant-based neurovascular protective strategies as potential treatments to expand the therapeutic window of currently approved therapies.

    View details for DOI 10.1007/s12035-010-8102-z

    View details for Web of Science ID 000278095800012

    View details for PubMedID 20157789

  • Mitochondrial and apoptotic neuronal death signaling pathways in cerebral ischemia BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE Niizuma, K., Yoshioka, H., Chen, H., Kim, G. S., Jung, J. E., Katsu, M., Okami, N., Chan, P. H. 2010; 1802 (1): 92-99

    Abstract

    Mitochondria play important roles as the powerhouse of the cell. After cerebral ischemia, mitochondria overproduce reactive oxygen species (ROS), which have been thoroughly studied with the use of superoxide dismutase transgenic or knockout animals. ROS directly damage lipids, proteins, and nucleic acids in the cell. Moreover, ROS activate various molecular signaling pathways. Apoptosis-related signals return to mitochondria, then mitochondria induce cell death through the release of pro-apoptotic proteins such as cytochrome c or apoptosis-inducing factor. Although the mechanisms of cell death after cerebral ischemia remain unclear, mitochondria obviously play a role by activating signaling pathways through ROS production and by regulating mitochondria-dependent apoptosis pathways.

    View details for DOI 10.1016/j.bbadis.2009.09.002

    View details for Web of Science ID 000273138500011

    View details for PubMedID 19751828

  • CK2 Is a Novel Negative Regulator of NADPH Oxidase and a Neuroprotectant in Mice after Cerebral Ischemia JOURNAL OF NEUROSCIENCE Kim, G. S., Jung, J. E., Niizuma, K., Chan, P. H. 2009; 29 (47): 14779-14789

    Abstract

    NADPH oxidase is a major complex that produces reactive oxygen species (ROSs) during the ischemic period and aggravates brain damage and cell death after ischemic injury. Although many approaches have been tested for preventing production of ROSs by NADPH oxidase in ischemic brain injury, the regulatory mechanisms of NADPH oxidase activity after cerebral ischemia are still unclear. In this study, we identified casein kinase 2 (CK2) as a critical modulator of NADPH oxidase and elucidated the role of CK2 as a neuroprotectant after oxidative insults to the brain. We found that the protein levels of the catalytic subunits CK2alpha and CK2alpha', as well as the total activity of CK2, are significantly reduced after transient focal cerebral ischemia (tFCI). We also found this deactivation of CK2 caused by ischemia/reperfusion increases expression of Nox2 and translocation of p67(phox) and Rac1 to the membrane after tFCI. Interestingly, we found that the inactive status of Rac1 was captured by the catalytic subunit CK2alpha under normal conditions. However, binding between CK2alpha and Rac1 was immediately diminished after tFCI, and Rac1 activity was markedly increased after CK2 inhibition. Moreover, we found that deactivation of CK2 in the mouse brain enhances production of ROSs and neuronal cell death via increased NADPH oxidase activity. The increased brain infarct volume caused by CK2 inhibition was restored by apocynin, a NADPH oxidase inhibitor. This study suggests that CK2 can be a direct molecular target for modulation of NADPH oxidase activity after ischemic brain injury.

    View details for DOI 10.1523/JNEUROSCI.4161-09.2009

    View details for Web of Science ID 000272185100010

    View details for PubMedID 19940173

  • Inhibition of NADPH oxidase is neuroprotective after ischemia-reperfusion JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Chen, H., Song, Y. S., Chan, P. H. 2009; 29 (7): 1262-1272

    Abstract

    Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is well known as a major source for superoxide radical generation in leukocytes. Superoxide radicals play a significant role in brain ischemia-reperfusion (I/R) injury. Recent data have also shown expression of NOX in the brain. However, the manner by which NOX is involved in pathologic processes after cerebral ischemia remains unknown. Therefore, we subjected mice deficient in the NOX subunit, gp91(phox) (gp91(phox)-/-), those treated with the NOX inhibitor, apocynin, and wild-type (WT) mice to 75 mins of focal ischemia followed by reperfusion. At 24 h of reperfusion, the gp91(phox)-/- and apocynin-treated mice showed 50% less brain infarction and 70% less cleaved spectrin compared with WT mice. The levels of 4-hydroxy-2-nonenal, malondialdehyde, and 8-hydroxy-2'-deoxyguanosine increased significantly after I/R, indicating oxidative brain injury. NADPH oxidase inhibition reduced biomarker generation. Furthermore, NOX was involved in postischemic inflammation in the brains, as less intercellular adhesion molecule-1 upregulation and less neutrophil infiltration were found in the NOX-inhibited mice after I/R. Moreover, gp91(phox) expression increased after ischemia, and was further aggravated by genetic copper/zinc-superoxide dismutase (SOD1) ablation, but ameliorated in SOD1-overexpressing mice. This study suggests that NOX plays a role in oxidative stress and inflammation, thus contributing to ischemic brain injury.

    View details for DOI 10.1038/jcbfm.2009.47

    View details for Web of Science ID 000267485800004

    View details for PubMedID 19417757

  • Faster Recovery of Cerebral Perfusion in SOD1-Overexpressed Rats After Cardiac Arrest and Resuscitation STROKE Xu, Y., Liachenko, S. M., Tang, P., Chan, P. H. 2009; 40 (7): 2512-2518

    Abstract

    Protracted hypoperfusion is one of the hallmarks of secondary cerebral derangement after cardiac arrest and resuscitation (CAR), and reactive oxygen species have been implicated in reperfusion abnormalities.Using transgenic (Tg) rats overexpressing copper zinc superoxide dismutase (SOD1), we investigated the role of this intrinsic antioxidant in the restoration of cerebral blood flow (CBF) after CAR. Nine Tg and 11 wild-type (WT) rats were subjected to a nominal 15-minute cardiac arrest, and CBF was measured using the noninvasive arterial spin labeling MRI method before and during cardiac arrest, and 0 to 2 hours and 1 to 5 days after resuscitation.The SOD1-Tg rats showed rapid normalization of CBF 1 day after the insult, whereas CBF in WT animals remained abnormal for at least 5 days, showing a progressive increase in CBF from hypo- to hyperperfusion on postresuscitation days 1 to 5. The long-term outcome, as measured by survival time, change in body weight, and mapping of apparent diffusion coefficient (ADC) for ion/water homeostasis, was significantly better in the SOD1-Tg rats.Our results support the notion that reactive oxygen species are at least partially responsible for microvascular reperfusion disorders.

    View details for DOI 10.1161/STROKEAHA.109.548453

    View details for Web of Science ID 000267467900037

    View details for PubMedID 19461023

  • Regulation of Mn-Superoxide Dismutase Activity and Neuroprotection by STAT3 in Mice after Cerebral Ischemia JOURNAL OF NEUROSCIENCE Jung, J. E., Kim, G. S., Narasimhan, P., Song, Y. S., Chan, P. H. 2009; 29 (21): 7003-7014

    Abstract

    Cerebral ischemia and reperfusion increase superoxide anions (O(2)(*-)) in brain mitochondria. Manganese superoxide dismutase (Mn-SOD; SOD2), a primary mitochondrial antioxidant enzyme, scavenges superoxide radicals and its overexpression provides neuroprotection. However, the regulatory mechanism of Mn-SOD expression during cerebral ischemia and reperfusion is still unclear. In this study, we identified the signal transducer and activator of transcription 3 (STAT3) as a transcription factor of the mouse Mn-SOD gene, and elucidated the mechanism of O(2)(*-) overproduction after transient focal cerebral ischemia (tFCI). We found that Mn-SOD expression is significantly reduced by reperfusion in the cerebral ischemic brain. We also found that activated STAT3 is usually recruited into the mouse Mn-SOD promoter and upregulates transcription of the mouse Mn-SOD gene in the normal brain. However, at early postreperfusion periods after tFCI, STAT3 was rapidly downregulated, and its recruitment into the Mn-SOD promoter was completely blocked. In addition, transcriptional activity of the mouse Mn-SOD gene was significantly reduced by STAT3 inhibition in primary cortical neurons. Moreover, we found that STAT3 deactivated by reperfusion induces accumulation of O(2)(*-) in mitochondria. The loss of STAT3 activity induced neuronal cell death by reducing Mn-SOD expression. Using SOD2-/+ heterozygous knock-out mice, we found that Mn-SOD is a direct target of STAT3 in reperfusion-induced neuronal cell death. Our study demonstrates that STAT3 is a novel transcription factor of the mouse Mn-SOD gene and plays a crucial role as a neuroprotectant in regulating levels of reactive oxygen species in the mouse brain.

    View details for DOI 10.1523/JNEUROSCI.1110-09.2009

    View details for Web of Science ID 000266438800026

    View details for PubMedID 19474327

  • Oxidative stress and mitochondrial dysfunction as determinants of ischemic neuronal death and survival JOURNAL OF NEUROCHEMISTRY Niizuma, K., Endo, H., Chan, P. H. 2009; 109: 133-138

    Abstract

    Mitochondria are the powerhouse of the cell. Their primary physiological function is to generate adenosine triphosphate through oxidative phosphorylation via the electron transport chain. Reactive oxygen species generated from mitochondria have been implicated in acute brain injuries such as stroke and neurodegeneration. Recent studies have shown that mitochondrially-formed oxidants are mediators of molecular signaling, which is implicated in the mitochondria-dependent apoptotic pathway that involves pro- and antiapoptotic protein binding, the release of cytochrome c, and transcription-independent p53 signaling, leading to neuronal death. Oxidative stress and the redox state of ischemic neurons are also implicated in the signaling pathway that involves phosphatidylinositol 3-kinase/Akt and downstream signaling, which lead to neuronal survival. Genetically modified mice or rats that over-express or are deficient in superoxide dismutase have provided strong evidence in support of the role of mitochondrial dysfunction and oxidative stress as determinants of neuronal death/survival after stroke and neurodegeneration.

    View details for DOI 10.1111/j.1471-4159.2009.05897.x

    View details for Web of Science ID 000265013900019

    View details for PubMedID 19393019

  • VEGF Stimulates the ERK 1/2 Signaling Pathway and Apoptosis in Cerebral Endothelial Cells After Ischemic Conditions STROKE Narasimhan, P., Liu, J., Song, Y. S., Massengale, J. L., Chan, P. H. 2009; 40 (4): 1467-1473

    Abstract

    Cerebral endothelial cells that line microvessels play an important role in maintaining blood flow homeostasis within the brain-forming part of the blood-brain barrier. These cells are injured by hypoxia-induced reperfusion, leading to blood-brain barrier breakdown and exacerbation of ischemic injury. We investigated the roles of vascular endothelial growth factor (VEGF) and the downstream extracellular signal-regulated kinase (ERK) protein after oxygen-glucose deprivation (OGD) in primary endothelial cells.Primary mouse endothelial cells were isolated and subjected to OGD. Western analysis of VEGF and ERK 1/2 protein levels was performed. Cells were transfected with VEGF small interference RNA. A terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) assay and DNA fragmentation assay were used on mouse endothelial cells that overexpress copper/zinc-superoxide dismutase (SOD1).VEGF protein expression was induced and its receptor, Flk-1, was stimulated by OGD. Phosphorylation of ERK 1/2 protein levels was upregulated. Inhibition of phosphorylated ERK (pERK) expression by U0126 reduced endothelial cell death by OGD. Transfection of small interfering RNA for VEGF also inhibited an increase in pERK, suggesting that VEGF acts via ERK. The TUNEL and DNA fragmentation assays showed a significant decrease in TUNEL-positivity in the SOD1-overexpressing endothelial cells compared with wild-type cells after OGD.Our data suggest that OGD induces VEGF signaling via its receptor, Flk-1, and activates ERK via oxidative-stress-dependent mechanisms. Our study shows that in cerebral endothelial cells the ERK 1/2 signaling pathway plays a significant role in cell injury after OGD.

    View details for DOI 10.1161/STROKEAHA.108.534644

    View details for Web of Science ID 000264709500068

    View details for PubMedID 19228841

  • Potential Role of PUMA in Delayed Death of Hippocampal CA1 Neurons After Transient Global Cerebral Ischemia STROKE Niizuma, K., Endo, H., Nito, C., Myer, D. J., Chan, P. H. 2009; 40 (2): 618-625

    Abstract

    p53-upregulated modulator of apoptosis (PUMA), a BH3-only member of the Bcl-2 protein family, is required for p53-dependent and -independent forms of apoptosis. PUMA localizes to mitochondria and interacts with antiapoptotic Bcl-2 and Bcl-X(L) or proapoptotic Bax in response to death stimuli. Although studies have shown that PUMA is associated with pathomechanisms of cerebral ischemia, clearly defined roles for PUMA in ischemic neuronal death remain unclear. The purpose of this study was to determine potential roles for PUMA in cerebral ischemia.Five minutes of transient global cerebral ischemia (tGCI) were induced by bilateral common carotid artery occlusion combined with hypotension.PUMA was upregulated in vulnerable hippocampal CA1 neurons after tGCI as shown by immunohistochemistry. In Western blot and coimmunoprecipitation analyses, PUMA localized to mitochondria and was bound to Bcl-X(L) and Bax in the hippocampal CA1 subregion after tGCI. PUMA upregulation was inhibited by pifithrin-alpha, a specific inhibitor of p53, suggesting that PUMA is partly controlled by the p53 transcriptional pathway after tGCI. Furthermore, reduction in oxidative stress by overexpression of copper/zinc superoxide dismutase, which is known to be protective of vulnerable ischemic hippocampal neurons, inhibited PUMA upregulation and subsequent hippocampal CA1 neuronal death after tGCI.These results imply a potential role for PUMA in delayed CA1 neuronal death after tGCI and that it could be a molecular target for therapy.

    View details for DOI 10.1161/STROKEAHA.108.524447

    View details for Web of Science ID 000262784900044

    View details for PubMedID 19095966

  • The role of Akt signaling in oxidative stress mediates NF-kappa B activation in mild transient focal cerebral ischemia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Song, Y. S., Narasimhan, P., Kim, G. S., Jung, J. E., Park, E., Chan, P. H. 2008; 28 (12): 1917-1926

    Abstract

    Reactive oxygen species, derived from hypoxia and reoxygenation during transient focal cerebral ischemia (tFCI), are associated with the signaling pathway that leads to neuronal survival or death, depending on the severity and duration of the ischemic insult. The Akt survival signaling pathway is regulated by oxidative stress and is implicated in activation of nuclear factor-kappaB (NF-kappaB). Mild cerebral ischemia in mice was used to induce increased levels of Akt phosphorylation in the cortex and striatum. To clarify the role of Akt activation by NF-kappaB after tFCI, we injected the specific Akt inhibitor IV that inhibits Akt phosphorylation/activation. Inhibition of Akt phosphorylation induced decreases in sequential NF-kappaB signaling after 30 mins of tFCI at 1 h. Furthermore, the downstream survival signals of the Akt pathway were also decreased. Akt inhibitor IV increased ischemic infarct volume and apoptotic-related DNA fragmentation. Superoxide production in the ischemic brains of mice pretreated with the Akt inhibitor was higher than in vehicle-treated mice. In addition, those pretreated mice showed a reduction of approximately 33% in copper/zinc-superoxide dismutase expression. We propose that Akt signaling exerts its neuroprotective role by NF-kappaB activation in oxidative cerebral ischemia in mice.

    View details for DOI 10.1038/jcbfm.2008.80

    View details for Web of Science ID 000261127000005

    View details for PubMedID 18628779

  • The PIDDosome mediates delayed death of hippocampal CA1 neurons after transient global cerebral ischemia in rats PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Niizuma, K., Endo, H., Nito, C., Myer, D. J., Kim, G. S., Chan, P. H. 2008; 105 (42): 16368-16373

    Abstract

    A brief period of global brain ischemia, such as that induced by cardiac arrest or cardiopulmonary bypass surgery, causes cell death in vulnerable hippocampal CA1 pyramidal neurons days after reperfusion. Although numerous factors have been suggested to account for this phenomenon, the mechanisms underlying it are poorly understood. We describe a cell death signal called the PIDDosome, a protein complex of p53-induced protein with a death domain (PIDD), receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD), and procaspase-2. We induced 5 min of transient global cerebral ischemia (tGCI) using bilateral common carotid artery occlusion with hypotension. Western blot analysis showed that expression of twice-cleaved fragment of PIDD (PIDD-CC) increased in the cytosolic fraction of the hippocampal CA1 subregion and preceded procaspase-2 activation after tGCI. Caspase-2 cleaved Bid in brain homogenates. Co-immunoprecipitation and immunofluorescent studies demonstrated that PIDD-CC, RAIDD, and procaspase-2 were co-localized and bound directly, which indicates the formation of the PIDD death domain complex. Furthermore, we tested inhibition of PIDD expression by using small interfering RNA (siRNA) treatment that was initiated 48 h before tGCI. Administration of siRNA against PIDD decreased not only expression of PIDD-CC, but also activation of procaspase-2 and Bid, resulting in a decrease in histological neuronal damage and DNA fragmentation in the hippocampal CA1 subregion after tGCI. These results imply that PIDD plays an important role in procaspase-2 activation and delayed CA1 neuronal death after tGCI. We propose that PIDD is a hypothetical molecular target for therapy against neuronal death after tGCI.

    View details for DOI 10.1073/pnas.0806222105

    View details for Web of Science ID 000260597400054

    View details for PubMedID 18845684

  • Role of the p38 mitogen-activated protein kinase/cytosolic phospholipase A(2) signaling pathway in blood-brain barrier disruption after focal cerebral ischemia and reperfusion JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Nito, C., Kamada, H., Endo, H., Niizuma, K., Myer, D. J., Chan, P. H. 2008; 28 (10): 1686-1696

    Abstract

    Cytosolic phospholipase A(2) (cPLA(2)) is a key enzyme that mediates arachidonic acid metabolism, which causes cerebral ischemia-induced oxidative injury, blood-brain barrier (BBB) dysfunction, and edema. Recent reports have shown that p38 mitogen-activated protein kinase (MAPK) is related to phosphorylation and activation of cPLA(2) and release of arachidonic acid. However, involvement of the p38 MAPK pathway in cPLA(2) activation and of reactive oxygen species in expression of p38 MAPK/cPLA(2) after ischemia-reperfusion injury in the brain remains unclear. To address these issues, we used a model of transient focal cerebral ischemia (tFCI) in rats. Western blot analysis showed a significant increase in expression of phospho-p38 MAPK and phospho-cPLA(2) in rat brain cortex after tFCI. Activity assays showed that both p38 MAPK and cPLA(2) activation markedly increased 1 day after reperfusion. Intraventricular administration of SB203580 significantly suppressed activation and phosphorylation of cPLA(2) and attenuated BBB extravasation and subsequent edema. Moreover, overexpression of copper/zinc-superoxide dismutase remarkably diminished activation and phosphorylation of both p38 MAPK and cPLA(2) after reperfusion. These findings suggest that the p38 MAPK/cPLA(2) pathway may promote BBB disruption with secondary vasogenic edema and that superoxide anions can stimulate this pathway after ischemia-reperfusion injury.

    View details for DOI 10.1038/jcbfm.2008.60

    View details for Web of Science ID 000259445100006

    View details for PubMedID 18545259

  • Delayed matrix metalloproteinase inhibition reduces intracerebral hemorrhage after embolic stroke in rats EXPERIMENTAL NEUROLOGY Copin, J., Merlani, P., Sugawara, T., Chan, P. H., Gasche, Y. 2008; 213 (1): 196-201

    Abstract

    Hemorrhagic transformation (HT) and brain edema are life-threatening complications of recombinant tissue plasminogen activator (rt-PA)-induced reperfusion after ischemic stroke. The risk of HT limits the therapeutic window for reperfusion to 3 h after stroke onset. Pre-treatment with matrix metalloproteinase (MMP) inhibitors reduces HT and cerebral edema in experimental stroke. However, whether a delayed therapeutic intervention would be beneficial is unknown. In this study, 215 male Sprague-Dawley rats were subjected to embolic stroke and 75 rats were included in the final analysis. The animals were treated with the MMP inhibitor p-aminobenzoyl-gly-pro-D-leu-D-ala-hydroxamate before or after 3 or 6 h of ischemia. Animals were monitored for reperfusion and received rt-PA 6 h after ischemia onset. The results at 24 h showed that MMP inhibition 3 h after ischemia significantly decreased the degree of brain edema (17% of hemispheric enlargement in the treated group versus 24% in controls, P=0.018), reduced the risk (OR=0.163; 95% CI: 0.029 to 0.953) and gravity (0.09 versus 0.19 mg of parenchymal hemoglobin, P=0.02) of intracerebral hemorrhage, and improved neurological outcome (20% of the treated animals had a slight deficit; all of the controls had a bad outcome, P<0.05). Delaying MMP inhibition to 6 h after ischemia restricted the beneficial role of the treatment to a reduction in the risk of parenchymal hemorrhage (OR=0.242; 95% CI: 0.060 to 0.989). Our results confirm the involvement of MMPs in HT and support the possibility of extending the therapeutic window for thrombolysis in stroke by administering a broad-spectrum MMP inhibitor after the onset of ischemia.

    View details for DOI 10.1016/j.expneurol.2008.05.022

    View details for Web of Science ID 000258943500024

    View details for PubMedID 18590727

  • Deleterious role of superoxide dismutase in the mitochondrial intermembrane space JOURNAL OF BIOLOGICAL CHEMISTRY Goldsteins, G., Keksa-Goldsteine, V., Ahtoniemi, T., Jaronen, M., Arens, E., Akerman, K., Chan, P. H., Koistinaho, J. 2008; 283 (13): 8446-8452

    Abstract

    This work demonstrates how increased activity of copper-zinc superoxide dismutase (SOD1) paradoxically boosts production of toxic reactive oxygen species (ROS) in the intermembrane space (IMS) of mitochondria. Even though SOD1 is a cytosolic enzyme, a fraction of it is found in the IMS, where it is thought to provide protection against oxidative damage. We found that SOD1 controls cytochrome c-catalyzed peroxidation in vitro when superoxide is available. The presence of SOD1 significantly increased the rate of ROS production in mitoplasts, which are devoid of outer membrane and IMS. In response to inhibition of respiration with antimycin A, isolated mouse wild-type mitochondria increased ROS production, but the mitochondria from mice lacking SOD1 (SOD1(-/-)) did not. Also, lymphocytes isolated from SOD1(-/-) mice produced significantly less ROS than did wild-type cells and were more resistant to apoptosis induced by inhibition of respiration. Moreover, an increased amount of the toxic mutant G93A SOD1 in the IMS increased ROS production. The mitochondrial dysfunction and cell damage paradoxically induced by SOD1-mediated ROS production may be implicated in chronic degenerative diseases.

    View details for DOI 10.1074/jbc.M706111200

    View details for Web of Science ID 000254288000042

    View details for PubMedID 18171673

  • Induction of MMP-9 expression and endothelial injury by oxidative stress after spinal cord injury JOURNAL OF NEUROTRAUMA Yu, F., Kamada, H., Niizuma, K., Endo, H., Chan, P. H. 2008; 25 (3): 184-195

    Abstract

    Matrix metalloproteinase-9 (MMP-9) activation plays an important role in blood-brain barrier (BBB) dysfunction after central nervous system injury. Oxidative stress is also implicated in the pathogenesis after cerebral ischemia and spinal cord injury (SCI), but the relationship between MMP-9 activation and oxidative stress after SCI has not yet been clarified. We examined MMP-9 expression after SCI using copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) rats. Our results show that MMP-9 activity significantly increased after SCI in both SOD1 Tg rats and their wild-type (Wt) littermates, although the increase was less in the SOD1 Tg rats. This pattern of MMP-9 expression was further confirmed by immunostaining and Western blot analysis. In situ zymography showed that gelatinolytic activity increased after SCI in the Wt rats, while the increase was less in the Tg rats. Evans blue extravasation increased in both the Wt and Tg rats, but was less in the SOD1 Tg rats. Inhibitor studies showed that, with an intrathecal injection of SB-3CT (a selective MMP-2/MMP-9 inhibitor), the MMP activity, Evans blue extravasation, and apoptotic cell death decreased after SCI. We conclude that increased oxidative stress after SCI leads to MMP-9 upregulation, BBB disruption, and apoptosis, and that overexpression of SOD1 in Tg rats decreases oxidative stress and further attenuates MMP-9 mediated BBB disruption.

    View details for DOI 10.1089/neu.2007.0438

    View details for Web of Science ID 000254579400002

    View details for PubMedID 18352832

  • Increased expression of a proline-rich Akt substrate (PRAS40) in human copper/zinc-superoxide dismutase transgenic rats protects motor neurons from death after spinal cord injury JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Yu, F., Narasimhan, P., Saito, A., Liu, J., Chan, P. H. 2008; 28 (1): 44-52

    Abstract

    The serine-threonine kinase, Akt, plays an important role in the cell survival signaling pathway. A proline-rich Akt substrate, PRAS40, has been characterized, and an increase in phospho-PRAS40 (pPRAS40) is neuroprotective after transient focal cerebral ischemia. However, the involvement of PRAS40 in the cell death/survival pathway after spinal cord injury (SCI) is unclear. Liposome-mediated PRAS40 transfection was performed to study whether overexpression of pPRAS40 is neuroprotective. We further examined the expression of pPRAS40 after SCI by immunohistochemistry and Western blot using copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) rats and wild-type (Wt) littermates. We then examined the relationship between PRAS40 and Akt by injection of LY294002, a phosphatidylinositol 3-kinase (PI3K) pathway inhibitor, or Akt inhibitor IV, a compound that inhibits Akt activation after SCI. Our data demonstrated that increased pPRAS40 resulted in survival of more motor neurons compared with control complementary DNA transfection. Phosphorylated PRAS40 increased in the Wt rats after SCI, whereas there was a greater and prolonged increase in the SOD1 Tg rats. Coimmunoprecipitation showed that binding of pPRAS40 with 14-3-3 increased 1 day after SCI in the Wt rats, whereas there was a significant increase in the Tg rats. The inhibitor studies showed that phospho-Akt and pPRAS40 were decreased after injection of LY294002 or Akt inhibitor IV. We conclude that an increase in pPRAS40 by transfection after SCI results in survival of motor neurons, and overexpression of SOD1 in the Tg rats results in an increase in endogenous pPRAS40 and a decrease in motor neuron death through the PI3K/Akt pathway.

    View details for DOI 10.1038/sj.jcbfm.9600501

    View details for Web of Science ID 000251820400005

    View details for PubMedID 17457363

  • Reduction in oxidative stress by superoxide dismutase overexpression attenuates acute brain injury after subarachnoid hemorrhage via activation of Akt/glycogen synthase kinase-3 beta survival signaling JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Endo, H., Nito, C., Kamada, H., Yu, F., Chan, P. H. 2007; 27 (5): 975-982

    Abstract

    Recent studies have revealed that oxidative stress has detrimental effects in several models of neurodegenerative diseases, including subarachnoid hemorrhage (SAH). However, how oxidative stress affects acute brain injury after SAH remains unknown. We have previously reported that overexpression of copper/zinc-superoxide dismutase (SOD1) reduces oxidative stress and subsequent neuronal injury after cerebral ischemia. In this study, we investigated the relationship between oxidative stress and acute brain injury after SAH using SOD1 transgenic (Tg) rats. SAH was produced by endovascular perforation in wild-type (Wt) and SOD1 Tg rats. Apoptotic cell death at 24 h, detected by a cell death assay, was significantly decreased in the cerebral cortex of the SOD1 Tg rats compared with the Wt rats. The mortality rate at 24 h was also significantly decreased in the SOD1 Tg rats. A hydroethidine study demonstrated that superoxide anion production after SAH was reduced in the cerebral cortex of the SOD1 Tg rats. Moreover, phosphorylation of Akt and glycogen synthase kinase-3beta (GSK3beta), which are survival signals in apoptotic cell death, was more enhanced in the cerebral cortex of the SOD1 Tg rats after SAH using Western blot analysis and immunohistochemistry. We conclude that reduction in oxidative stress by SOD1 overexpression may attenuate acute brain injury after SAH via activation of Akt/GSK3beta survival signaling.

    View details for DOI 10.1038/sj.jcbfm.9600399

    View details for Web of Science ID 000245999300009

    View details for PubMedID 16969382

  • Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase JOURNAL OF CLINICAL INVESTIGATION Suh, S. W., Gum, E. T., Hamby, A. M., Chan, P. H., Swanson, R. A. 2007; 117 (4): 910-918

    Abstract

    Hypoglycemic coma and brain injury are potential complications of insulin therapy. Certain neurons in the hippocampus and cerebral cortex are uniquely vulnerable to hypoglycemic cell death, and oxidative stress is a key event in this cell death process. Here we show that hypoglycemia-induced oxidative stress and neuronal death are attributable primarily to the activation of neuronal NADPH oxidase during glucose reperfusion. Superoxide production and neuronal death were blocked by the NADPH oxidase inhibitor apocynin in both cell culture and in vivo models of insulin-induced hypoglycemia. Superoxide production and neuronal death were also blocked in studies using mice or cultured neurons deficient in the p47(phox) subunit of NADPH oxidase. Chelation of zinc with calcium disodium EDTA blocked both the assembly of the neuronal NADPH oxidase complex and superoxide production. Inhibition of the hexose monophosphate shunt, which utilizes glucose to regenerate NADPH, also prevented superoxide formation and neuronal death, suggesting a mechanism linking glucose reperfusion to superoxide formation. Moreover, the degree of superoxide production and neuronal death increased with increasing glucose concentrations during the reperfusion period. These results suggest that high blood glucose concentrations following hypoglycemic coma can initiate neuronal death by a mechanism involving extracellular zinc release and activation of neuronal NADPH oxidase.

    View details for DOI 10.1172/JCI30077

    View details for Web of Science ID 000245451700013

    View details for PubMedID 17404617

  • Reduced oxidative stress promotes NF-kappa B-mediated neuroprotective gene expression after transient focal cerebral ischemia: lymphocytotrophic cytokines and antiapoptotic factors JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Song, Y. S., Lee, Y., Narasimhan, P., Chan, P. H. 2007; 27 (4): 764-775

    Abstract

    Nuclear factor-kappa B (NF-kappaB) is activated by oxidative stress such as that induced by transient focal cerebral ischemia (tFCI). Whether NF-kappaB has a role in cell survival or death in stroke is a matter of debate. We proposed that the status of oxidative stress may determine its role in cell death or survival after focal ischemia. To characterize the coordinated expression of genes in NF-kappaB signaling after mild cerebral ischemia, we investigated the temporal profile of a NF-kappaB-pathway-focused DNA array after 30 mins of tFCI in wild-type (WT) mice and human copper/zinc-superoxide dismutase transgenic (SOD1 Tg) mice that had a significantly reduced level of superoxide. Differentially expressed genes among 96 NF-kappaB-related genes were further confirmed and compared in the WT and SOD1 Tg mice using quantitative polymerase chain reaction, Western blotting, and immunohistochemistry. Persistent upregulation of NF-kappaB seen at 7 days in the WT mice was decreased in the SOD1 Tg mice. Lymphocytotrophic cytokine genes such as interleukin-2, interleukin-12, and interferon-alpha1 were increased in the SOD1 Tg mice compared with the WT mice after tFCI. In addition, antiapoptosis factors bcl-2 and tumor necrosis factor receptor-associated factor 1 rapidly increased in the SOD1 Tg mice compared with the WT mice. This study indicates that reduced oxidative stress by SOD1 overexpression increased NF-kappaB-related rapid defenses, such as immune response and antiapoptosis factors, and prevented brain damage after tFCI-induced oxidative stress.

    View details for DOI 10.1038/sj.jcbfm.9600379

    View details for Web of Science ID 000245358800011

    View details for PubMedID 16868554

  • Bad as a converging signaling molecule between survival PI3-K/Akt and death JNK in neurons after transient focal cerebral ischemia in rats JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Kamada, H., Nito, C., Endo, H., Chan, P. H. 2007; 27 (3): 521-533

    Abstract

    Bad, a proapoptotic Bcl-2 family protein, plays a critical role in determining cell death/survival. The phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the c-Jun N-terminal kinase (JNK) pathway are thought to be involved in regulation of Bad. Therefore, the present study was performed to clarify the role of Bad as a common target of the PI3-K/Akt and JNK pathways after transient focal cerebral ischemia (tFCI) in rats. We found that Akt activity increased at 3 h and then decreased, whereas JNK activity increased 7 to 24 h in the peripheral area after tFCI. Administration of LY294002, a PI3-K-specific inhibitor, exacerbated DNA fragmentation, whereas administration of SP600125, a JNK-specific inhibitor, attenuated it. Inhibited by LY294002, phospho-Bad (Ser136) expression increased in the peripheral area 3 h after tFCI, with suppression of Akt activity. Furthermore, phospho-Bad (Ser136) and phospho-Akt (Ser473) were colocalized. Decreases in phospho-Bad (Ser136) and Bad/14-3-3 dimerization and increases in Bcl-X(L)/Bad or Bcl-2/Bad dimerization observed 7 to 24 h after tFCI, were prevented by SP600125 administration, with inhibition of JNK activity. The present study indicates that signal predominance varies from PI3-K/Akt-mediated survival signaling to JNK-mediated death signaling with the development of neuronal damage in the peripheral area after tFCI. This study also suggests that PI3-K/Akt has a role in Bad inactivation, whereas the JNK pathway is involved in Bad activation. We conclude that Bad may be an integrated checkpoint of PI3-K/Akt-mediated survival signaling and JNK-mediated death signaling and that it contributes to cell fate in the peripheral area after cerebral ischemia.

    View details for DOI 10.1038/sj.jcbfm.9600367

    View details for Web of Science ID 000244387100009

    View details for PubMedID 16820799

  • Influence of hyperglycemia on oxidative stress and matrix metalloproteinase-9 activation after focal cerebral ischemia/reperfusion in rats - Relation to blood-brain barrier dysfunction STROKE Kamada, H., Yu, F., Nito, C., Chan, P. H. 2007; 38 (3): 1044-1049

    Abstract

    Hyperglycemia is linked to a worse outcome after ischemic stroke. Among the manifestations of brain damage caused by ischemia are blood-brain barrier (BBB) disruption and edema formation. Oxidative stress and matrix metalloproteinase-9 (MMP-9) activation are implicated in BBB dysfunction after ischemia/reperfusion injury. Our present study was designed to clarify the relation among hyperglycemia, oxidative stress, and MMP-9 activation associated with BBB dysfunction after transient focal cerebral ischemia (tFCI).We used a model of 60 minutes of middle cerebral artery occlusion on the following animals: normoglycemic wild-type rats, wild-type rats with hyperglycemia induced by streptozotocin, and human copper/zinc superoxide dismutase (SOD1) transgenic rats with streptozotocin-induced hyperglycemia. We evaluated edema volume, Evans blue leakage, and oxidative stress, such as the carbonyl groups and oxidized hydroethidine (HEt), SOD activity, and gelatinolytic activity, including MMP-9.Hyperglycemia significantly increased edema volume and Evans blue leakage. Moreover, it enhanced the levels of the carbonyl groups, the oxidized HEt signals, and MMP-9 activity after tFCI without alteration in SOD activity. Gelatinolytic activity and oxidized HEt signals had a clear spatial relation in the hyperglycemic rats. SOD1 overexpression reduced the hyperglycemia-enhanced Evans blue leakage and MMP-9 activation after tFCI.Hyperglycemia increases oxidative stress and MMP-9 activity, exacerbating BBB dysfunction after ischemia/reperfusion injury. Superoxide overproduction may be a causal link among hyperglycemia, MMP-9 activation, and BBB dysfunction.

    View details for DOI 10.1161/01.STR.0000258041.75739.cb

    View details for Web of Science ID 000244482500044

    View details for PubMedID 17272778

  • Mitochondrial translocation of p53 underlies the selective death of hippocampal CA1 neurons after global cerebral ischaemia BIOCHEMICAL SOCIETY TRANSACTIONS Endo, H., Saito, A., Chan, P. H. 2006; 34: 1283-1286

    Abstract

    p53, a tumour suppressor, is involved in DNA repair and cell death processes and mediates apoptosis in response to death stimuli by transcriptional activation of pro-apoptotic genes and by transcription-independent mechanisms. In the latter process, p53 induces permeabilization of the outer mitochondrial membrane by forming an inhibitory complex with a protective Bcl-2 family protein, resulting in cytochrome c release in several cell line systems. However, it is unclear how the mitochondrial p53 pathway mediates neuronal apoptosis after cerebral ischaemia. We examined interaction between the mitochondrial p53 pathway and vulnerable hippocampal CA1 neurons using a tGCI (transient global cerebral ischaemia) rat model. We showed mitochondrial translocation of p53 and its binding to Bcl-X(L). Mitochondrial p53 translocation, interaction between p53 and Bcl-X(L), and cytochrome c release from mitochondria and subsequent CA1 neuronal death were prevented by pifithrin-alpha, a p53-specific inhibitor. These results suggest that the mitochondrial p53 pathway plays a role in delayed CA1 neuronal death after tGCI.

    View details for Web of Science ID 000243013500067

    View details for PubMedID 17073802

  • A new approach for the investigation of reperfusion-related brain injury BIOCHEMICAL SOCIETY TRANSACTIONS Maier, C. M., Hsieh, L., Crandall, T., Narasinnhan, P., Chan, P. H. 2006; 34: 1366-1369

    Abstract

    Effective stroke therapies require recanalization of occluded cerebral blood vessels; however, early reperfusion can cause BBB (blood-brain barrier) injury, leading to cerebral oedema and/or devastating brain haemorrhage. These complications of early reperfusion, which result from excess production of ROS (reactive oxygen species), significantly limit the benefits of stroke therapies. Here, we summarize some of the findings that lead to the development of a novel animal model that facilitates identification of specific free radical-associated components of the reperfusion injury process and allows therapeutic interventions to be assessed. In this model, KO (knockout) mice containing 50% activity of the mitochondrial antioxidant manganese-SOD (superoxide dismutase) (SOD2-KO) undergo transient focal ischaemia followed by reperfusion. These animals have delayed (>24 h) BBB breakdown associated with activation of matrix metalloproteinase-9, inflammation and a high brain haemorrhage rate. These adverse consequences are absent from wild-type littermates, SOD2 overexpressors and minocycline-treated SOD2-KO animals. In addition, using microvessel isolations following in vivo ischaemia/reperfusion, we were able to show that the tight junction membrane protein, occludin, is an early and specific target in ROS-mediated microvascular injury. This new model is ideal for studying ischaemia/reperfusion-induced vascular injury and secondary brain damage and offers a unique opportunity to evaluate free radical-based neurovascular protective strategies.

    View details for Web of Science ID 000243013500085

    View details for PubMedID 17073820

  • Activation of the Akt/GSK3 beta signaling pathway mediates survival of vulnerable hippocampal neurons after transient global cerebral ischemia in rats JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Endo, H., Nito, C., Kamada, H., Nishi, T., Chan, P. H. 2006; 26 (12): 1479-1489

    Abstract

    Recent studies have revealed that the phosphatidylinositol 3-kinase (PI3-K) pathway is involved in apoptotic cell death after experimental cerebral ischemia. The serine-threonine kinase, Akt, functions in the PI3-K pathway and prevents apoptosis by phosphorylation at Ser473 after a variety of cell death stimuli. After phosphorylation, activated Akt inactivates other apoptogenic factors, including glycogen synthase kinase-3beta (GSK3beta), thereby inhibiting cell death. However, the role of Akt/GSK3beta signaling in the delayed death of hippocampal neurons in the CA1 subregion after transient global cerebral ischemia (tGCI) has not been clarified. Transient global cerebral ischemia for 5 mins was induced by bilateral common carotid artery occlusion combined with hypotension. Western blot analysis showed a significant increase in phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) in the hippocampal CA1 subregion after tGCI. Immunohistochemistry showed that expression of phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) was markedly increased in the vulnerable CA1 subregion, but not in the ischemic-tolerant CA3 subregion. Double staining with phospho-GSK3beta (Ser9) and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed different cellular distributions in the CA1 subregion 3 days after tGCI. Phosphorylation of Akt and GSK3beta was prevented by LY294002, a PI3-K inhibitor, which facilitated subsequent DNA fragmentation 3 days after tGCI. Moreover, transgenic rats that overexpress copper/zinc-superoxide dismutase, which is known to be neuroprotective against delayed hippocampal CA1 injury after tGCI, had enhanced and persistent phosphorylation of both Akt and GSK3beta after tGCI. These findings suggest that activation of the Akt/GSK3beta signaling pathway may mediate survival of vulnerable hippocampal CA1 neurons after tGCI.

    View details for DOI 10.1038/sj.jcbfm.9600303

    View details for Web of Science ID 000242441700002

    View details for PubMedID 16538228

  • Akt/GSK3 beta survival signaling is involved in acute brain injury after subarachnoid hemorrhage in rats STROKE Endo, H., Nito, C., Kamada, H., Yu, F., Chan, P. H. 2006; 37 (8): 2140-2146

    Abstract

    Apoptotic cell death is associated with acute brain injury after subarachnoid hemorrhage (SAH). The Akt/glycogen synthase kinase-3beta (GSK3beta) pathway plays an important role in the cell death/survival pathway after a variety of cell death stimuli. However, its role in acute brain injury after SAH remains unknown.We used an endovascular perforation model of SAH in rats. Phospho-Akt and phospho-GSK3beta expression was examined by Western blot analysis and immunohistochemistry. Terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) and a cell death assay were used for detection of apoptosis. We administered LY294002 to examine the role of the Akt/GSK3beta pathway in the phosphoinositide 3-kinase pathway after SAH.Phosphorylation of Akt and GSK3beta was accelerated after SAH. In the cerebral cortex, where acute brain injury was the most severe, phosphorylation of these proteins was observed in the early phase after SAH. Cortical neurons with continuous Akt phosphorylation did not colocalize with TUNEL-positive cells at 24 hours. LY294002 reduced Akt and GSK3beta phosphorylation and increased brain injury after SAH.The present study suggests that the Akt/GSK3beta pathway might be involved in neuronal survival in acute brain injury after SAH.

    View details for DOI 10.1161/01.STR.0000229888.55078.72

    View details for Web of Science ID 000239459000039

    View details for PubMedID 16794215

  • Biphasic role of nuclear factor-kappa B on cell survival and COX-2 expression in SOD1 Tg astrocytes after oxygen glucose deprivation JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Lee, Y., Song, Y. S., Giffard, R. G., Chan, P. H. 2006; 26 (8): 1076-1088

    Abstract

    In cytoplasm, nuclear factor-kappaB (NF-kappaB) is associated with the inhibitory protein, IkappaBalpha. On activation by H2O2, IkappaBalpha is phosphorylated and degraded, exposing the nuclear localization signals on the NF-kappaB heterodimer. Cyclooxygenase-2 (COX-2), which mediates prostaglandin synthesis during inflammation, is induced by oxidative stress mediated by NF-kappaB. We investigated whether the NF-kappaB signaling pathway affected cell death and COX-2 expression after hypoxia-induced oxidative stress in wild-type (WT) and copper/zinc-superoxide dismutase transgenic (SOD1 Tg) astrocytes. In WT astrocytes, phospho-IkappaBalpha was highly expressed after oxygen-glucose deprivation (OGD) and 2 h of reperfusion, concomitant with the decrease in IkappaBalpha. The NF-kappaB p50 level increased similarly in WT and SOD1 Tg astrocytes (1.2-/1.4-fold) after OGD. Electrophoretic mobility shift assay showed higher DNA-binding activity of NF-kappaB p50 in WT than in SOD1 Tg astrocytes 6 h after 4 h of OGD. The COX-2 level was induced by 2.7- and 1.3-fold after OGD in WT and SOD1 Tg astrocytes, and an antioxidant protected both groups against OGD injury. Superoxide dismutase transgenic cells were 23% more protective against OGD injury than WTs when assessed by lactate dehydrogenase release. However, transfection of NF-kappaB small interfering RNAs in SOD1 Tg astrocytes aggravated cell death and increased COX-2 expression. These results suggest that the NF-kappaB signaling pathway induced COX-2 expression and promoted cell death in WTs after OGD injury; however, NF-kappaB activation protected cells and decreased COX-2 expression in SOD1 Tg astrocytes. This biphasic role of NF-kappaB might be coordinately regulated by reactive oxygen species levels in astrocytes, thereby functioning as a regulator of cell death/survival.

    View details for DOI 10.1038/sj.jcbfm.9600261

    View details for Web of Science ID 000239340700008

    View details for PubMedID 16395278

  • Mitochondrial translocation of p53 mediates release of cytochrome c and hippocampal CA1 neuronal death after transient global cerebral ischemia in rats JOURNAL OF NEUROSCIENCE Endo, H., Kamada, H., Nito, C., Nishi, T., Chan, P. H. 2006; 26 (30): 7974-7983

    Abstract

    Although p53 is a key modulator of cellular stress responses, the mechanism of p53-mediated apoptosis is ambiguous. p53 can mediate apoptosis in response to death stimuli by transcriptional activation of proapoptotic genes and transcriptional-independent mechanisms. Recent studies have shown that the p53 protein can directly induce permeabilization of the outer mitochondrial membrane by forming a inhibitory complex with a protective Bcl-2 family protein, resulting in cytochrome c release. However, how the mitochondrial p53 pathway mediates neuronal apoptosis after cerebral ischemia remains unclear. We examined the interaction between the mitochondrial p53 pathway and vulnerable hippocampal CA1 neurons in rats using a transient global cerebral ischemia (tGCI) model. Western blot analysis and immunofluorescent staining revealed mitochondrial p53 translocation after tGCI in the hippocampal CA1 neurons. Coimmunoprecipitation revealed that translocated p53 bound to Bcl-X(L) in the mitochondrial fraction. To examine the effect of a specific p53 inhibitor on the mitochondrial p53 pathway and apoptotic cell death after tGCI, we intravenously administered pifithrin-alpha (PFT). Mitochondrial p53 translocation and interaction between p53 and Bcl-X(L) were prevented by treatment with PFT. Moreover, cytochrome c release from mitochondria and subsequent apoptotic CA1 neuronal death were decreased with PFT treatment. These results suggest that the mitochondrial p53 pathway is one of the novel mechanisms mediating delayed death of vulnerable hippocampal CA1 neurons after tGCI.

    View details for DOI 10.1523/JNEUROSCI.0897-06.2006

    View details for Web of Science ID 000239361200021

    View details for PubMedID 16870742

  • Evaluating therapeutic targets for reperfusion-related brain hemorrhage ANNALS OF NEUROLOGY Maier, C. M., Hsieh, L., Crandall, T., Narasimhan, P., Chan, P. H. 2006; 59 (6): 929-938

    Abstract

    Early reperfusion after an ischemic stroke can cause blood-brain barrier injury with subsequent cerebral edema and devastating brain hemorrhage. These complications of early reperfusion, which result from excess production of reactive oxygen species, significantly limit the benefits of stroke therapies. In this article, we use a novel animal model that facilitates identification of specific components of the reperfusion injury process, including vascular injury and secondary brain damage, and allows assessment of therapeutic interventions.Knock-out (KO) mice containing 50% manganese-superoxide dismutase activity (SOD2-KO) and transgenic mice overexpressing SOD2 undergo transient focal ischemia and reperfusion followed by assessment of infarct, edema, hemorrhage rates, metalloproteinase activation, and microvascular injury.SOD2-KO mice demonstrate delayed (>24h) blood-brain barrier breakdown associated with activation of matrix metalloproteinases, inflammation, and high brain hemorrhage rates. These adverse consequences are absent in wild-type littermates and minocycline-treated SOD2-KO animals. Increased hemorrhage rates also are absent in SOD2 overexpressors, which have reduced vascular endothelial cell death. Finally, we show that the tight junction membrane protein, occludin, is an early and specific target in oxidative stress-induced microvascular injury.This model is ideal for studying ischemia/reperfusion-induced vascular injury and secondary brain hemorrhage and offers a unique opportunity to evaluate antioxidant-based neurovascular protective strategies as potential adjunct treatments to currently approved stroke therapies such as thrombolysis and endovascular clot retrieval.

    View details for DOI 10.1002/ana.20850

    View details for Web of Science ID 000238054800011

    View details for PubMedID 16673393

  • Overexpression of SOD1 in transgenic rats attenuates nuclear translocation of endonuclease g and apoptosis after spinal cord injury JOURNAL OF NEUROTRAUMA Yu, F., Sugawara, T., Nishi, T., Liu, J., Chan, P. H. 2006; 23 (5): 595-603

    Abstract

    Spinal motor neurons are selectively vulnerable after spinal cord injury (SCI). Recent studies suggest they undergo apoptosis after caspase activation through a mitochondria-dependent apoptosis pathway, and that oxidative stress after SCI is likely to play a role. However, other signaling pathways of apoptosis that involve mitochondria have not been thoroughly studied after SCI. Apoptosis-inducing factor (AIF) and endonuclease G (EndoG) are mitochondrial apoptogenic proteins that are capable of inducing neuronal apoptosis when translocated from mitochondria to nuclei through a caspase-independent pathway. In this study, we examined translocation of these proteins and apoptotic cell death of motor neurons. The role of oxidative stress was also studied using transgenic (Tg) rats that overexpress the intrinsic antioxidant copper/zinc-superoxide dismutase (SOD1). Western blots and an activity assay demonstrated that a greater amount of SOD1 and higher activity of SOD presented in mitochondria of Tg rats compared with wild-type (Wt) rats. Immunohistochemistry and Western blots showed translocation of EndoG and AIF from mitochondria to nuclei in motor neurons 1 day after SCI in both groups of rats. However, there was significantly less translocation of EndoG in the Tg rats compared with the Wt rats. Less apoptotic cell death was detected in the Tg rats than in the Wt rats 3 days after SCI. These results suggest that translocation of EndoG and AIF from mitochondria to nuclei may initiate a caspase-independent pathway of apoptosis. An increased level of SOD1 in mitochondria conceivably reduces oxidative stress, thereby attenuating EndoG translocation, and resulting in reduction of caspase-independent apoptosis.

    View details for Web of Science ID 000237529200001

    View details for PubMedID 16689664

  • Mild hypoxia promotes survival and proliferation of SOD2-deficient astrocytes via c-Myc activation JOURNAL OF NEUROSCIENCE Liu, J., Narasimhan, P., Lee, Y. S., Song, Y. S., Endo, H., Yu, F. S., Chan, P. H. 2006; 26 (16): 4329-4337

    Abstract

    Mouse astrocytes deficient in the mitochondrial form of manganese superoxide dismutase (SOD2) do not survive in culture under atmospheric air with 20% oxygen (O2), which is a common condition for cell cultures. Seeding the cells and maintaining them under mild hypoxic conditions (5% O2) circumvents this problem and allows the cells to grow and become confluent. Previous studies from our laboratory showed that this adaptation of the cells was not attributable to compensation by other enzymes of the antioxidant defense system. We hypothesized that transcriptional activity and upregulation of genes other than those with an antioxidant function are involved. Our present study shows that c-Myc was significantly induced and that it inhibited p21 and induced proteins such as cyclin-dependent kinases, cyclin D, and cyclin E, which are involved in the cell cycle process, along with phosphorylation of the retinoblastoma protein and Cdc2 (cell division cycle 2). These mechanisms contribute to cell proliferation. Small interfering RNA of c-Myc, however, blocked proliferation of SOD2 homozygous (SOD2-/-) astrocytes under mild hypoxia consisting of 5% O2, whereas it did not affect the growth of wild-type astrocytes. Our results indicate that c-Myc plays a critical role in hypoxia-induced proliferation and survival of SOD2-/- astrocytes by overcoming injury caused by oxidative stress.

    View details for DOI 10.1523/JNEUROSCI.0382-06.2006

    View details for Web of Science ID 000236912100019

    View details for PubMedID 16624953

  • Interferon-beta fails to protect in a model of transient focal stroke STROKE Maier, C. M., Yu, F. S., Nishi, T., Lathrop, S. J., Chan, P. H. 2006; 37 (4): 1116-1119

    Abstract

    Compelling evidence supporting the role of inflammation in the development of cerebral infarction has focused attention on the potential of antiinflammatory treatment strategies for stroke. Interferon (IFN)-beta, an immunomodulatory agent approved for treatment of multiple sclerosis, is being evaluated in a phase I clinical trial in acute ischemic stroke. In the present study, we evaluated the effects of wild-type rat IFN-beta and its pegylated counterpart (PEG-IFN-beta) in a model of focal ischemia and reperfusion.After 60 minutes of middle cerebral artery occlusion, rats (n=12/group) were treated with IV tail injections of 8 or 16 mug of IFN-beta in 300 muL of PBS once daily for 3 or 7 days or with IV or SC injections of PEG-IFN-beta for 1 day. The animals were assessed daily for weight and for neurological findings. Additional animals underwent complete hematology and chemistry profiles, as well as complete multiorgan necropsy studies. All of the brain tissue was evaluated for assessment of infarct areas, neutrophil infiltration, and presence of hemorrhagic transformations.IFN-beta and PEG-IFN-beta failed to protect against experimental ischemic brain injury as assessed by histopathology and neurological outcome. Furthermore, IFN-beta treatment was associated with significant weight loss and alterations in hematology and chemistry profiles.Our results suggest that additional preclinical studies are warranted.

    View details for DOI 10.1161/01.STR.0000208214.46093.d5

    View details for Web of Science ID 000236292100042

    View details for PubMedID 16514094

  • Epo protects SOD2-deficient mouse astrocytes from damage by oxidative stress GLIA Liu, J., Narasimhan, P., Song, Y. S., Nishi, T., Yu, F. S., Lee, Y. S., Chan, P. H. 2006; 53 (4): 360-365

    Abstract

    Erythropoietin (Epo) expression, which regulates erythropoiesis, has been shown in rat and mouse brain after hypoxia. A previous study from our laboratory showed that astrocytes from manganese-superoxide dismutase (SOD2) homozygous knockout (SOD2(-/-)) mice can survive under 5% O(2), but not under normal aerobic conditions. However, the mechanism involved is not clear. Our preliminary study using reverse transcriptase-polymerase chain reaction showed increased Epo mRNA expression in astrocytes cultured with 5% hypoxia compared with astrocytes under normal conditions. After administration of anti-sense Epo, protection decreased with time. Dose-dependent administration of Epo to SOD2(-/-) mouse astrocytes improved their survivability under normal conditions. Survivability of heterozygous SOD2(-/+) mutant and wild-type mouse astrocyte cultures was the same under normal conditions but, after administration of 2 mM of paraquat, a reactive oxygen species generator, survivability of the SOD2(-/+) astrocytes decreased remarkably compared with the wild-type cells. Epo administration 24 h before exposure to paraquat significantly improved the survivability of the SOD2(-/+) astrocytes. Western blot studies suggest that Jak-Stat signal transduction pathways are involved in this process. Our study demonstrates an important role for Epo in the protection of astrocytes from reactive oxygen species. We suggest that Epo can compensate in part for the antioxidant properties of mitochondrial SOD2 deficiency.

    View details for DOI 10.1002/glia.20289

    View details for Web of Science ID 000235886400003

    View details for PubMedID 16288465

  • Modulation of proline-rich Akt substrate survival signaling pathways by oxidative stress in mouse brains after transient focal cerebral ischemia STROKE Saito, A., Hayashi, T., Okuno, S., Nishi, T., Chan, P. H. 2006; 37 (2): 513-517

    Abstract

    A proline-rich Akt substrate (PRAS) contributes to the regulation of apoptosis after a variety of cell death stimuli, as well as in an in vivo transient focal cerebral ischemia (tFCI) model. We reported previously that overexpression of copper/zinc-superoxide dismutase (SOD1) reduces apoptotic cell death after tFCI. Our present study was designed to clarify the relationship between the PRAS signaling pathway and oxidative stress in the regulation of apoptosis after tFCI.We used a tFCI model with SOD1 transgenic mice and wild-type littermates to examine the expression of phosphorylated PRAS (pPRAS) by Western blotting and immunohistochemistry and the interaction of pPRAS with phosphorylated Akt (pPRAS/pAkt) or the 14-3-3 protein (pPRAS/14-3-3) by coimmunoprecipitation. Direct oxidation of the carbonyl groups, an indication of oxidative injury to total and individual proteins caused by tFCI, was examined using a 2,4-dinitrophenylhydrazone reaction assay.Expression of pPRAS, pPRAS/pAkt, and pPRAS/14-3-3 decreased 2 hours after tFCI. Oxidized hydroethidine did not colocalize with expression of pPRAS. Individual oxidized carbonyls in pPRAS remarkably increased 2 hours after tFCI but were significantly reduced by SOD1 2 hours after tFCI. Expression of pPRAS, pPRAS/pAkt, and pPRAS/14-3-3 was promoted by SOD1 during the same time course.These results suggest that overexpression of SOD1 may affect the PRAS pathway after tFCI by reducing the direct oxidative reaction to pPRAS after reperfusion injury.

    View details for DOI 10.1161/01.STR.0000198826.56611.a2

    View details for Web of Science ID 000234829800078

    View details for PubMedID 16397181

  • Akt/Bad signaling and motor neuron survival after spinal cord injury NEUROBIOLOGY OF DISEASE Yu, F. S., Sugawara, T., Maier, C. M., Hsieh, L. B., Chan, P. H. 2005; 20 (2): 491-499

    Abstract

    The serine-threonine kinase Akt is a cell survival signaling pathway that inactivates the proapoptotic BCL-2 family protein Bad and promotes cell survival in cerebral ischemia. Involvement of the Akt/Bad signaling pathway after spinal cord injury (SCI) is, however, uncertain. Our results showed that phospho-Akt (serine-473) and phospho-Bad (serine-136) were significantly upregulated at 1 day after SCI. In addition, phospho-Akt and phospho-Bad were colocalized in motor neurons that survived SCI and inhibition of PI3-K reduced expression of phospho-Akt and phospho-Bad. Dimerization of Bad with 14-3-3 in the cytosol was increased whereas Bad/Bcl-XL binding in the mitochondria was decreased after SCI. We further found that reduced oxidative stress by SOD1 overexpression in rats enhanced the expression of phospho-Akt, phospho-Bad, Bad/14-3-3 binding and reduced Bad/Bcl-XL binding after SCI, as compared to wild-type rats. We conclude that oxidative stress may play a role in modulating Akt/Bad signaling and subsequent motor neuron survival after SCI.

    View details for DOI 10.1016/j.nbd.2005.04.004

    View details for Web of Science ID 000233096700032

    View details for PubMedID 15896972

  • Superoxide dismutase 1 overexpression reduces MCP-1 and MIP-1 alpha expression after transient focal cerebral ischemia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Nishi, T., Maier, C. M., Hayashi, T., Saito, A., Chan, P. H. 2005; 25 (10): 1312-1324

    Abstract

    Proinflammatory cytokines and chemokines are quickly upregulated in response to ischemia/reperfusion (I/R) injury; however, the relationship between I/R-induced oxidative stress and cytokine/chemokine expression has not been elucidated. We investigated the temporal profile of cytokine and chemokine gene expression in transient focal cerebral ischemia using complementary DNA array technology. Among 96 genes studied, 10, 4, 11, and 5 genes were increased at 6, 12, 24, and 72 h of reperfusion, respectively, whereas, 4, 11, 8, and 21 genes, respectively, were decreased. To clarify the relationship between chemokines and oxidative stress, we compared the gene and protein expression of monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) in wild-type (WT) mice and copper/zinc-superoxide dismutase (SOD 1) transgenic (Tg) mice. Monocyte chemoattractant protein-1 and MIP-1 alpha mRNA were significantly upregulated at 6 to 12 h of reperfusion. In the SOD 1 Tg mice, however, MCP-1 and MIP-1 alpha mRNA expression was significantly decreased 12 h postinsult. In the WT mice, MCP-1 and MIP-1 alpha protein expression peaked 24 h after onset of reperfusion determined by immunohistochemistry. In the SOD 1 Tg mice, MCP-1 and MIP-1 alpha immunopositive cells were reduced, as were concentrations of these proteins (measured by enzyme-linked immunosorbent assay) at 24 h of reperfusion. Our results suggest that MCP-1 and MIP-1 alpha expression is influenced by I/R-induced oxidative stress after transient focal stroke.

    View details for DOI 10.1038/sj.jcbfm.9600124

    View details for Web of Science ID 000232253800007

    View details for PubMedID 15829914

  • Oxidative stress transiently decreases the IKK complex (IKK alpha, beta, and gamma), an upstream component of NF-kappa B signaling, after transient focal cerebral ischemia in mice JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Song, Y. S., Lee, Y. S., Chan, P. H. 2005; 25 (10): 1301-1311

    Abstract

    Nuclear factor-kappaB (NF-kappaB) has a central role in coordinating the expression of a wide variety of genes that control cerebral ischemia. Although there has been intense research on NF-kappaB, its mechanisms in the ischemic brain have not been clearly elucidated. We investigated the temporal profile of NF-kappaB-related genes using a complementary DNA array method in wild-type mice and human copper/zinc-superoxide dismutase transgenic (SOD 1 Tg) mice that had low-level reactive oxygen species (ROS) by scavenging superoxide. Our DNA array showed that IkappaB kinase (IKK) complex (IKKalpha, beta, and gamma) mRNA in the wild-type mice was decreased as early as 1 h after reperfusion, after 30 mins of transient focal cerebral ischemia (tFCI). In contrast, tFCI in the SOD1 Tg mice caused an increase in the IKK complex. The IKK complex protein levels were also drastically decreased at 1 h in the wild-type mice, but did not change in the SOD 1 Tg mice throughout the 7 days. Electrophoretic mobility shift assay revealed activation of NF-kappaB DNA binding after tFCI in the wild-type mice. Nuclear factor-kappaB activation occurred at the same time, as did the phosphorylation and degradation of the inhibitory protein IkappaBalpha. However, SOD 1 prevented NF-kappaB activation, and phosphorylation and degradation of IkappaBalpha after tFCI. Superoxide production and ubiquitinated protein in the SOD 1 Tg mice were also lower than in the wild-type mice after tFCI. These results suggest that ROS are implicated in transient downregulation of IKKalpha, beta, and gamma in cerebral ischemia.

    View details for DOI 10.1038/sj.jcbfm.9600123

    View details for Web of Science ID 000232253800006

    View details for PubMedID 15829915

  • Neuroprotection by hypoxic preconditioning involves oxidative stress-mediated expression of hypoxia-inducible factor and erythropoietin STROKE Liu, J., Narasimhan, P., Yu, F. S., Chan, P. H. 2005; 36 (6): 1264-1269

    Abstract

    Hypoxic preconditioning is an endogenous protection against subsequent lethal hypoxia, but the mechanism involved is not understood. Hypoxia is followed by reactive oxygen species (ROS) production and induces hypoxia-inducible factor (HIF) and its downstream factor erythropoietin (Epo), which is associated with neuroprotection. We hypothesized that these endogenous processes may contribute to hypoxic preconditioning.We used a mouse neuronal culture model, with 2 hours of hypoxia as preconditioning followed by 15 hours of hypoxic insult, and examined the expression of HIF-1alpha, Epo, and their downstream proteins by Western blotting. Copper/zinc-superoxide dismutase (SOD1) transgenic (Tg) mice were used to detect the effect of ROS. Cell survival and apoptosis were detected by mitogen-activated protein 2 quantification, apoptotic-related DNA fragmentation, and caspase-3 fragmentation. Antisense Epo was used to block endogenously produced Epo.Hypoxic preconditioning was protective in wild-type (Wt) neurons but not in neurons obtained from SOD1 Tg mice. In Wt neurons, HIF-1alpha and Epo expression showed a greater increase after hypoxia compared with Tg neurons and reached a higher level with preconditioned hypoxia, followed by pJak2, pStat5, and nuclear factor kappaB (NF-kappaB) expression. Antisense Epo decreased these downstream proteins and the neuroprotection of hypoxic preconditioning.Hypoxic preconditioning induces ROS, which may downregulate the threshold for production of HIF-1alpha and Epo expression during subsequent lethal hypoxia, thus exerting neuroprotection through the Jak2-Stat5 and NF-kappaB pathways.

    View details for DOI 10.1161/01.STR.0000166180.91042.02

    View details for Web of Science ID 000229324800037

    View details for PubMedID 15890996

  • 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 JOURNAL OF NEUROCHEMISTRY Narasimhan, P., Sugawara, T., Liu, J., Hayashi, T., Noshita, N., Chan, P. H. 2005; 93 (2): 351-358

    Abstract

    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.

    View details for DOI 10.1111/j.1471-4159.2005.03039.x

    View details for Web of Science ID 000228017300010

    View details for PubMedID 15816858

  • Modulation of p53 degradation via MDM2-mediated ubiquitylation and the ubiquitin-proteasome system during reperfusion after stroke: role of oxidative stress JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Saito, A., Hayashi, T., Okuno, S., Nishi, T., Chan, P. H. 2005; 25 (2): 267-280

    Abstract

    The tumor suppressor gene p53 plays an important role in the regulation of apoptosis through transcriptional activation of cell cycle control. Degradation of p53 hinders its role in apoptosis regulation. Recent studies have shown that MDM2-mediated ubiquitylation and the ubiquitin-proteasome system are critical regulating systems of p53 ubiquitylation. However, the mechanism regulating p53-mediated neuronal apoptosis after cerebral ischemia remains unknown. We examined the MDM2 pathway and the ubiquitin-proteasome system using a transient focal cerebral ischemia (tFCI) model and analyzed the interaction between p53 regulation and superoxide using copper/zinc superoxide dismutase (SOD1) transgenic mice after tFCI. p53 degradation and ubiquitylation were detected after tFCI. The accumulation of ubiquitylated p53 was inhibited and p53 degradation was facilitated by SOD1. Nuclear translocation and MDM2/Akt interaction were detected after tFCI and were inhibited by phosphatidylinositol 3-kinase inhibition and promoted by SOD1. Cytosolic translocation of the p53/MDM2 complex was detected after tFCI and was promoted by SOD1. Moreover, accumulation of multiubiquitin chains and direct oxidative injury to a proteasome were detected and inhibited by SOD1 after tFCI. These results suggest that SOD1 promotes the MDM2 pathway and the ubiquitin-proteasome system after tFCI and that production of reactive oxygen species after tFCI prevents p53 degradation by inhibiting both systems.

    View details for Web of Science ID 000226740100012

    View details for PubMedID 15678128

  • Oxidative stress and neuronal death/survival signaling in cerebral ischemia MOLECULAR NEUROBIOLOGY Saito, A., Maier, C. M., Narasimhan, P., Nishi, T., Song, Y. S., Yu, F. S., Liu, L., Lee, Y. S., Nito, C., Kamada, H., Dodd, R. L., Hsieh, L. B., Hassid, B., Kim, E. E., Gonzalez, M., Chan, P. H. 2005; 31 (1-3): 105-116

    Abstract

    It has been demonstrated by numerous studies that apoptotic cell death pathways are implicated in ischemic cerebral injury in ischemia models in vivo. 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 oxygen as a substrate for numerous enzymatic oxidation reactions and for mitochondrial oxidative phosphorylation to produce adenosine triphosphate. Oxygen radicals, the products of these biochemical and physiological reactions, are known to damage cellular lipids, proteins, and nucleic acids and to 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 could provide novel therapeutic strategies in clinical stroke.

    View details for Web of Science ID 000230119900009

    View details for PubMedID 15953815

  • Mitochondrial dysfunction and oxidative stress as determinants of cell death/survival in stroke ROLE OF THE MITOCHONDRIA IN HUMAN AGING AND DISEASE: FROM GENES TO CELL SIGNALING Chan, P. H. 2005; 1042: 203-209

    Abstract

    Mitochondria are the powerhouse of the cell. Their primary physiological function is to generate ATP through oxidative phosphorylation via the electron transport chain. Reactive oxygen radicals generated from mitochondria have been implicated in acute brain injuries, like stroke and neurodegeneration. Recent studies have shown that mitochondrially formed oxidants are mediators of molecular signaling and have implicated mitochondria-dependent apoptosis involving pro- and antiapoptotic protein binding, the release of cytochrome c and Smac, the activation of downstream caspase-9 and -3, and the fragmentation of DNA. Oxidative stress and the redox state are also implicated in the survival signaling pathway that involves phosphatidylinositol 3-kinase (PI3-K)/Akt and downstream signaling molecular bindings like Bad/Bcl-X(L) and phosphorylated Bad/14-3-3. Genetically modified mice (SOD1, SOD2) or rats that overexpress or are deficient in superoxide dismutase have provided strong evidence in support of the role of mitochondrial dysfunction and oxidative stress as determinants of neuronal death/survival after stroke and neurodegeneration.

    View details for DOI 10.1196/annals.1338.022

    View details for Web of Science ID 000230893400025

    View details for PubMedID 15965064

  • Damage to the endoplasmic reticulum and activation of apoptotic machinery by oxidative stress in ischemic neurons JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Hayashi, T., Saito, A., Okuno, S., Ferrand-Duke, M., Dodd, R. L., Chan, P. H. 2005; 25 (1): 41-53

    Abstract

    The endoplasmic reticulum (ER), which plays a role in apoptosis, is susceptible to oxidative stress. Because superoxide is produced in the brain after ischemia/reperfusion, oxidative injury to this organelle may be implicated in ischemic neuronal cell death. Activating transcription factor-4 (ATF-4) and C/EBP-homologous protein (CHOP), both of which are involved in apoptosis, are induced by severe ER stress. Using wild-type and human copper/zinc superoxide dismutase transgenic rats, we observed induction of these molecules in the brain after global cerebral ischemia and compared them with neuronal degeneration. In ischemic, wild-type brains, expression of ATF-4 and CHOP was increased in the hippocampal CA1 neurons that would later undergo apoptosis. Transgenic rats had a mild increase in ATF-4 and CHOP and minimal neuronal degeneration, indicating that superoxide was involved in ER stress-induced cell death. We further confirmed attenuation on induction of these molecules in transgenic mouse brains after focal ischemia. When superoxide was visualized with ethidium, signals for ATF-4 and superoxide overlapped in the same cells. Moreover, lipids in the ER were robustly peroxidized by ischemia but were attenuated in transgenic animals. This indicates that superoxide attacked and damaged the ER, and that oxidative ER damage is implicated in ischemic neuronal cell death.

    View details for DOI 10.1038/sj.jcbfm.9600005

    View details for Web of Science ID 000226360000004

    View details for PubMedID 15678111

  • Future targets and cascades for neuroprotective strategies STROKE Chan, P. H. 2004; 35 (11): 2748-2750

    Abstract

    Cumulative evidence suggests that apoptosis plays a pivotal role in neuronal death after cerebral ischemia in various experimental animal models. The time-dependent molecular and biochemical sequelae that lead to apoptotic cell death after the interruption of cerebral blood flow have been established. Many neuroprotective agents that target cell death pathways have been failures, and alternative strategies need to be considered. One such strategy is to target the neuronal survival signaling pathway, which involves the phosphatidylinositol 3-kinase (PI3-K)/Akt (protein kinase B) pathway. It has been demonstrated that PI3-K/Akt and downstream phosphorylated Bad and proline-rich Akt substrate survival signaling cascades are upregulated in surviving neurons in the ischemic brain that overexpresses copper-zinc superoxide dismutase activity. These studies provide an impetus for novel therapeutic targets in neuroprotective strategies in stroke.

    View details for DOI 10.1161/01.STR.0000143325.25610.ac

    View details for Web of Science ID 000224854800038

    View details for PubMedID 15388904

  • Oxidative stress affects the integrin-linked kinase signaling pathway after transient focal cerebral ischemia STROKE Saito, A., Hayashi, T., Okuno, S., Nishi, T., Chan, P. H. 2004; 35 (11): 2560-2565

    Abstract

    The integrin-linked kinase (ILK) signaling pathway contributes to regulation of cellular adhesion, migration, and differentiation, and to apoptotic cell death after a variety of cell death stimuli. We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduces apoptotic cell death by promoting the phosphatidylinositol 3-kinase (PI3-K)/Akt survival pathway after transient focal cerebral ischemia (tFCI). However, the role of the ILK pathway after tFCI and the role of oxygen free radicals in regulation of apoptosis remain unclear.To clarify these issues, we used an in vivo tFCI model with SOD1 transgenic mice and wild-type mice. We administered the PI3-K inhibitor, LY294002, into mouse brains after tFCI and examined the role of PI3-K in the ILK pathway and expression of the ILK/Akt complex by immunohistochemistry, Western blot analysis, and coimmunoprecipitation.A transient increase in ILK was detected early after tFCI and was prevented by treatment with LY294002, but promoted by SOD1. Coimmunoprecipitation revealed that the direct reaction of ILK/Akt transiently increased concurrent with the increase in ILK after tFCI. Moreover, the ILK/Akt complex was prevented by LY294002, but promoted by SOD1.These results suggest that the ILK pathway mediated by PI3-K is affected by tFCI and by SOD1.

    View details for DOI 10.1161/01.STR.0000144653.32853.ed

    View details for Web of Science ID 000224775000036

    View details for PubMedID 15472100

  • Mitochondria and neuronal death/survival signaling pathways in cerebral ischemia NEUROCHEMICAL RESEARCH Chan, P. H. 2004; 29 (11): 1943-1949

    Abstract

    Apoptotic cell death pathways have been implicated in acute brain injuries, including cerebral ischemia, brain trauma, and spinal cord injury, and in chronic neurodegenerative diseases. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and suggest the involvement of mitochondria and the cell survival/death signaling pathways in cell death/survival cascades. Recent studies have implicated mitochondria-dependent apoptosis involving pro- and antiapoptotic protein binding, the release of cytochrome c and second mitochondria-derived activator of caspase, the activation of downstream caspases-9 and -3, and DNA fragmentation. Reactive oxygen species are known to be significantly generated in the mitochondrial electron transport chain in the dysfunctional mitochondria during reperfusion after ischemia, and are also implicated in the survival signaling pathway that involves phosphatidylinositol-3-kinase (PI3-K), Akt, and downstream signaling molecules, like Bad, 14-3-3, and the proline-rich Akt substrate (PRAS), and their bindings. Further studies of these survival pathways may provide novel therapeutic strategies for clinical stroke.

    View details for Web of Science ID 000225538200003

    View details for PubMedID 15662830

  • The c-Jun N-terminal protein kinase signaling pathway mediates Bax activation and subsequent neuronal apoptosis through interaction with Bim after transient focal cerebral ischemia JOURNAL OF NEUROSCIENCE Okuno, S., Saito, A., Hayashi, T., Chan, P. H. 2004; 24 (36): 7879-7887

    Abstract

    The c-Jun N-terminal protein kinase (JNK) signaling pathway is implicated in neuronal apoptosis. The mechanism by which activated JNK induces neuronal apoptosis is strongly linked to mitochondrial apoptogenic proteins, although the molecular machinery downstream of JNK has not been precisely elucidated. Our study examined the relevance of proapoptotic Bcl-2 family members in JNK-mediated apoptosis after transient focal cerebral ischemia (tFCI), which, when induced by 60 min of middle cerebral artery (MCA) occlusion, elevated levels of JNK activity and phospho-JNK in the MCA territory. Phospho-JNK was primarily expressed in neurons and colocalized with terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL)-positive cells. Inhibition of JNK activity by anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), a selective JNK inhibitor, protected neurons from ischemia-induced apoptosis detected by TUNEL staining and an apoptotic-related DNA fragmentation assay. SP600125 blocked translocation of the cell death effector Bax from the cytosol to the mitochondria after tFCI. BimL (Bim long) was induced and phosphorylated parallel to JNK activity. Coimmunoprecipitation studies consistently revealed increased interaction of JNK with BimL, as well as BimL with Bax, after tFCI. SP600125 blocked these interactions at a dose that significantly inhibited JNK-induced neuronal apoptosis. These results suggest that the JNK signaling pathway is involved in ischemia-induced neuronal apoptosis by stimulation, at least in part, of Bax translocation to the mitochondria, in which BimL is likely regulated by JNK as a downstream substrate for transmission of apoptotic signals to Bax.

    View details for DOI 10.1523/JNEUROSCI.1745-04.2004

    View details for Web of Science ID 000223779200013

    View details for PubMedID 15356200

  • Modulation of the Omi/HtrA2 signaling pathway after transient focal cerebral ischemia in mouse brains that overexpress SOD1 MOLECULAR BRAIN RESEARCH Saito, A., Hayashi, T., Okuno, S., Nishi, T., Chan, P. H. 2004; 127 (1-2): 89-95

    Abstract

    Omi/HtrA2 is a novel protein that contributes to the regulation of mitochondrial apoptosis after a variety of cell death stimuli in vitro and is thought to negatively control the inhibitor-of-apoptosis protein (IAP) family. However, the Omi/HtrA2 pathway remains unknown in apoptotic neuronal cell death in vivo. To examine the role of the Omi/HtrA2 pathway and its relationship to oxidative stress after reperfusion following cerebral ischemia, we used a transient focal cerebral ischemia (tFCI) model in copper/zinc-superoxide dismutase (SOD1) transgenic mice and wild-type mice. We evaluated the link between the Omi/HtrA2 pathway and the caspase cascade reaction after tFCI by administration of a pan-caspase inhibitor, Z-VAD-FMK. We observed the time-dependent expression of Omi/HtrA2 and its binding to X-chromosome-linked IAP (Omi/XIAP) by immunohistochemistry, Western blotting and coimmunoprecipitation. Translocation of Omi/HtrA2 into the cytosolic space was detected during the early period after tFCI and was not affected by Z-VAD-FMK administration, but it was prevented by SOD1 overexpression. Coimmunoprecipitation revealed that Omi/XIAP transiently increased and that it was prevented by SOD1 overexpression. These results suggest that the Omi/HtrA2 pathway may play an important role in the progress of apoptotic neuronal cell death and that overexpression of SOD1 may attenuate this apoptotic cell death by preventing the Omi/HtrA2 cell signaling pathway.

    View details for DOI 10.1016/j.molbrainres.2004.05.012

    View details for Web of Science ID 000223567300009

    View details for PubMedID 15306124

  • Oxidative stress is associated with XIAP and Smac/DIABLO signaling pathways in mouse brains after transient focal cerebral ischemia STROKE Saito, A., Hayashi, T., Okuno, S., Nishi, T., Chan, P. H. 2004; 35 (6): 1443-1448

    Abstract

    The interaction of X chromosome-linked inhibitor-of-apoptosis protein (XIAP) with second mitochondria-derived activator of caspase (Smac)/direct inhibitor-of-apoptosis protein-binding protein with low pI (DIABLO) contributes to regulation of apoptosis after a variety of cell death stimuli, and in our reported in vivo transient focal cerebral ischemia (tFCI) model. We have also reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduces apoptotic cell death after tFCI. Our present study was designed to clarify the relationship between the XIAP signaling pathway and oxidative stress in the regulation of apoptosis after tFCI.We used a tFCI model of SOD1 transgenic mice and wild-type littermates to examine the expression of XIAP and Smac/DIABLO by Western blotting and the interaction of XIAP with Smac/DIABLO (XIAP/Smac) or caspase-9 (XIAP/caspase-9) by coimmunoprecipitation. The direct oxidation of carbonyl groups, an indication of oxidative injury to total and individual proteins caused by tFCI, was examined using a 2,4-dinitrophenylhydrazone reaction assay.Direct oxidative injury to cytosolic and mitochondrial proteins was reduced by SOD1 after tFCI. The individual oxidized carbonyls in XIAP, mitochondrial Smac/DIABLO, and caspase-9 were also reduced by SOD1. Expression of XIAP and XIAP/caspase-9 was promoted, whereas translocation of Smac/DIABLO and XIAP/Smac was reduced, by SOD1 after tFCI.These results suggest that overexpression of SOD1 may affect the XIAP pathway after tFCI by reducing the direct oxidative reaction to XIAP regulators after reperfusion injury.

    View details for DOI 10.1161/01.STR.0000128416.28778.7a

    View details for Web of Science ID 000221676600040

    View details for PubMedID 15118177

  • Matrix metalloproteinase-9 and myeloperoxidase expression - Quantitative analysis by antigen immunohistochemistry in a model of transient focal cerebral ischemia STROKE Maier, C. M., Hsieh, L., Yu, F. S., Bracci, P., Chan, P. H. 2004; 35 (5): 1169-1174

    Abstract

    Expression of matrix metalloproteinases (MMPs), proteolytic enzymes that degrade extracellular proteins, is altered after ischemia/reperfusion injury and may contribute to blood-brain barrier (BBB) breakdown. Neutrophils, a source of reactive oxygen species and MMP-9, infiltrate damaged tissue 6 to 24 hours after ischemia and have also been implicated in delayed secondary tissue damage. Here we examined the spatial-temporal relation between MMP-9 expression and neutrophil infiltration after stroke.Knockout mice containing 50% manganese superoxide dismutase activity (SOD2-KOs), which are more susceptible to ischemic damage than wild-type (WT) littermates, underwent quantitative antigen (MMP-9, myeloperoxidase) immunohistochemistry (24 and 72 hours) analysis and protein expression by Western blotting (6, 12, 24, 48, and 72 hours) after transient focal cerebral ischemia. BBB breakdown was determined by Evans blue extravasation.There was a clear spatial relation between MMP-9 expression and Evans blue extravasation. MMP-9-positive cell and vessel counts for SOD2-KOs (72 hours) were significantly different from SOD2-KO (24 hours, P=0.004), WT (24 hours, P=0.01), and WT (72 hours, P=0.007) mice. In contrast, MMP-9-positive neutrophil counts were comparatively low and did not differ by time or animal type. MMP-9 expression was biphasic in SOD2-KOs but not in WT littermates, with a significant increase observed 6 to 12 hours after ischemic insult and again at 48 to 72 hours. SOD2-KOs showed increased MMP-9 expression compared with WT littermates at all time points studied (P< or =0.05).In this model, neutrophils are not the primary source of MMP-9 protein and thus are unlikely the key contributor to BBB breakdown observed in SOD2-KOs.

    View details for DOI 10.1161/01.STR.0000125861.55804.f2

    View details for Web of Science ID 000220987800028

    View details for PubMedID 15060315

  • Oxidative injury to the endoplasmic reticulum in mouse brains after transient focal ischemia NEUROBIOLOGY OF DISEASE Hayashi, T., Saito, A., Okuno, S., Ferrand-Drake, M., Dodd, R. L., Chan, P. H. 2004; 15 (2): 229-239

    Abstract

    Oxidative damage to the endoplasmic reticulum (ER) could be involved in ischemic neuronal cell death because this organelle is susceptible to reactive oxygen species. Using wild-type mice and copper/zinc-superoxide dismutase (SOD1) transgenic mice, we induced focal cerebral ischemia and compared neuronal degeneration and ER stress, that is, phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) and RNA-dependent protein kinase-like ER eIF2alpha kinase (PERK). We found that neurons with severe and prolonged phosphorylation of eIF2alpha and PERK underwent later degeneration, and that this was partially prevented by SOD1 overexpression. Signals for superoxide production and phospho-PERK were colocalized, which further indicates a pivotal role for superoxide in ER damage. We investigated the molecular mechanisms of oxidative ER stress and found that detachment of glucose-regulated protein 78 from PERK was the key step. We conclude that ER damage is involved in oxidative neuronal injury in the brain after ischemia/reperfusion.

    View details for DOI 10.1016/j.nbd.2003.10.005

    View details for Web of Science ID 000220173800008

    View details for PubMedID 15006693

  • Neuroprotective role of a proline-rich akt substrate in apoptotic neuronal cell death after stroke: Relationships with nerve growth factor JOURNAL OF NEUROSCIENCE Saito, A., Narasimhan, P., Hayashi, T., Okuno, S., Ferrand-Drake, M., Chan, P. H. 2004; 24 (7): 1584-1593

    Abstract

    The Akt signaling pathway contributes to regulation of apoptosis after a variety of cell death stimuli. A novel proline-rich Akt substrate (PRAS) was recently detected and found to be involved in apoptosis. In our study, Akt activation was modulated by growth factors, and treatment with nerve growth factor (NGF) reduced apoptotic cell death after ischemic injury. However, the role of the PRAS pathway in apoptotic neuronal cell death after ischemia remains unknown. Phosphorylated PRAS (pPRAS) and the binding of pPRAS/phosphorylated Akt (pPRAS/pAkt) to 14-3-3 (pPRAS/14-3-3) were detected, and their expression transiently decreased in mouse brains after transient focal cerebral ischemia (tFCI). Liposome-mediated pPRAS cDNA transfection induced overexpression of pPRAS, promoted pPRAS/14-3-3, and inhibited apoptotic neuronal cell death after tFCI. The expression of pPRAS, pPRAS/pAkt, and pPRAS/14-3-3 increased in NGF-treated mice but decreased with inhibition of phosphatidylinositol-3 kinase and the NGF receptor after tFCI. These results suggest that PRAS phosphorylation and its interaction with pAkt and 14-3-3 might play an important role in neuroprotection mediated by NGF in apoptotic neuronal cell death after tFCI.

    View details for DOI 10.1523/JNEUROSCI.5209-03.2004

    View details for Web of Science ID 000189035300007

    View details for PubMedID 14973226

  • Neuronal death/survival signaling pathways in cerebral ischemia. NeuroRx : the journal of the American Society for Experimental NeuroTherapeutics Sugawara, T., Fujimura, M., Noshita, N., Kim, G. W., Saito, A., Hayashi, T., Narasimhan, P., Maier, C. M., Chan, P. H. 2004; 1 (1): 17-25

    Abstract

    Cumulative evidence suggests that apoptosis plays a pivotal role in cell death in vitro after hypoxia. Apoptotic cell death pathways have also been implicated in ischemic cerebral injury in in 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.

    View details for PubMedID 15717004

  • Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia ANTIOXIDANTS & REDOX SIGNALING Sugawara, T., Chan, P. H. 2003; 5 (5): 597-607

    Abstract

    Reactive oxygen species have been implicated in brain injury after cerebral ischemia. These oxidants can damage proteins, lipids, and DNA, and lead to cell injury and necrosis. Oxidants are also initiators in intracellular cell death signaling pathways that may lead to apoptosis. The possible targets of this redox signaling include mitochondria, death membrane receptors, and DNA repair enzymes. Genetic manipulation of intrinsic antioxidants and the factors in the signaling pathways has provided substantial progress in understanding the mechanisms in cell death signaling pathways and involvement of oxygen radicals in ischemic brain injury. Future studies of these pathways may provide novel therapeutic strategies in clinical stroke.

    View details for Web of Science ID 000186101200013

    View details for PubMedID 14580316

  • Oxidative damage to the endoplasmic reticulum is implicated in ischemic neuronal cell death JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Hayashi, T., Saito, A., Okuno, S., Ferrand-Drake, M., Dodd, R. L., Nishi, T., Maier, C. M., Kinouchi, H., Chan, P. H. 2003; 23 (10): 1117-1128

    Abstract

    The endoplasmic reticulum (ER), which plays important roles in apoptosis, is susceptible to oxidative stress. Because reactive oxygen species (ROS) are robustly produced in the ischemic brain, ER damage by ROS may be implicated in ischemic neuronal cell death. We induced global brain ischemia on wild-type and copper/zinc superoxide dismutase (SOD1) transgenic rats and compared ER stress and neuronal damage. Phosphorylated forms of eukaryotic initiation factor 2 alpha (eIF2 alpha) and RNA-dependent protein kinase-like ER eIF2 alpha kinase (PERK), both of which play active roles in apoptosis, were increased in hippocampal CA1 neurons after ischemia but to a lesser degree in the transgenic animals. This finding, together with the finding that the transgenic animals showed decreased neuronal degeneration, indicates that oxidative ER damage is involved in ischemic neuronal cell death. To elucidate the mechanisms of ER damage by ROS, we analyzed glucose-regulated protein 78 (GRP78) binding with PERK and oxidative ER protein modification. The proteins were oxidatively modified and stagnated in the ER lumen, and GRP78 was detached from PERK by ischemia, all of which were attenuated by SOD1 overexpression. We propose that ROS attack and modify ER proteins and elicit ER stress response, which results in neuronal cell death.

    View details for DOI 10.1097/01.WCB.0000089600.87125.AD

    View details for Web of Science ID 000185755500002

    View details for PubMedID 14526222

  • Neurodegeneration in striatum induced by the mitochondrial toxin 3-nitropropionic acid: Role of matrix metalloproteinase-9 in early blood-brain barrier disruption? JOURNAL OF NEUROSCIENCE Kim, G. W., Gasche, Y., Grzeschik, S., Copin, J. C., Maier, C. M., Chan, P. H. 2003; 23 (25): 8733-8742

    Abstract

    Blood-brain barrier (BBB) dysfunction is a potential mechanism involved in progressive striatal damage induced by the mitochondrial excitotoxin, 3-nitropropionic acid (3-NP). After activation by proteases and free radicals, matrix metalloproteinases (MMPs), particularly MMP-9 and -2, can digest the endothelial basal lamina leading to BBB opening. Using CD-1 mice, we show that MMP-9 expression by zymography is increased in the injured striatum compared with the contralateral striatum 2 hr after 3-NP injection [133.50 +/- 57.17 vs 50.25 +/- 13.56; mean +/- SD of optical densities in arbitrary units (A.U.); p < 0.005] and remains elevated until 24 hr (179.33 +/- 78.24 A.U.). After 4 hr, MMP-9 expression and activation are accompanied by an increase in BBB permeability. MMP inhibition attenuates BBB disruption, swelling, and lesion volume compared with vehicle-treated controls. There is a clear spatial relationship between MMP-9 expression and oxidized hydroethidine, indicating reactive oxygen species (ROS) production. Furthermore, transgenic mice that overexpress copper/zinc-superoxide dismutase (SOD1) show decreased lesion size and edema along with decreased immunoreactivity for MMP-9, compared with wild-type littermates (lesion: 38.8 +/- 15.1 and 53.3 +/- 10.3, respectively, p < or = 0.05; edema: 21.8 +/- 11.2 and 35.28 +/- 11, respectively, p < or = 0.05; MMP-9-positive cells: 352 +/- 57 and 510 +/- 45, respectively, p < or = 0.005), whereas knock-out mice deficient in SOD1 display significantly greater swelling (48.65 +/- 17; p < or = 0.05). We conclude that early expression and activation of MMP-9 by ROS may be involved in early BBB disruption and progressive striatal damage after 3-NP treatment.

    View details for Web of Science ID 000185534500013

    View details for PubMedID 14507973

  • Interaction between XIAP and Smac/DIABLO in the mouse brain after transient focal cerebral ischemia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Saito, A., Hayashi, T., Okuno, S., Ferrand-Drake, M., Chan, P. H. 2003; 23 (9): 1010-1019

    Abstract

    The X chromosome-linked inhibitor-of-apoptosis protein (XIAP) contributes to apoptosis regulation after a variety of cell death stimuli. XIAP inhibits the caspase reaction via binding to caspases, and is inhibited via binding to the second mitochondria-derived activator of caspase (Smac)/DIABLO to tightly control apoptotic cell death. However, the interaction among XIAP, Smac/DIABLO, and caspases after in vivo cerebral ischemia is not well known. To clarify this issue, the authors examined time-dependent expression and interaction among XIAP, Smac/DIABLO, and activated caspase-9 by immunohistochemistry, Western blot analysis, and immunoprecipitation using an in vivo transient focal cerebral ischemia model. To examine the relationship of the XIAP pathway to the caspase cascade, a pan-caspase inhibitor was administered. XIAP increased concurrently with the release of Smac/DIABLO and the appearance of activated caspase-9 during the early period after reperfusion injury. The bindings of XIAP to Smac/DIABLO and to caspase-9 and the binding of Smac/DIABLO to caspase-9 reached a peak simultaneously after transient focal cerebral ischemia. Neither XIAP nor Smac/DIABLO expression was affected by caspase inhibition. These results suggest that the XIAP pathway was activated upstream of the caspase cascade and that interaction among XIAP, Smac/DIABLO, and caspase-9 plays an important role in the regulation of apoptotic neuronal cell death after transient focal cerebral ischemia.

    View details for Web of Science ID 000185144800002

    View details for PubMedID 12973017

  • Induction of GRP78 by ischemic preconditioning reduces endoplasmic reticulum stress and prevents delayed neuronal cell death JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Hayashi, T., Saito, A., Okuno, S., Ferrand-Drake, M., Chan, P. H. 2003; 23 (8): 949-961

    Abstract

    Although the endoplasmic reticulum (ER) is implicated in neuronal degeneration in some situations, its role in delayed neuronal cell death (DND) after ischemia remains uncertain. The authors speculated that ER stress is involved in DND, that it is reduced by ischemic preconditioning, and that ER stress reduction by preconditioning is due to ER molecular chaperone induction. The phosphorylation status of eukaryotic initiation factor 2alpha (eIF2alpha) and RNA-dependent protein kinase-like ER eIF2alpha kinase (PERK) was investigated in the rat hippocampus after ischemia with and without preconditioning. PERK is phosphorylated by ER stress, which phosphorylates eIF2alpha. To investigate the role of ER molecular chaperones in preconditioning, the authors examined GRP78 and GRP94 expression, both of which are ER chaperones that inhibit PERK phosphorylation, and compared their induction and ischemic tolerance time windows. Phosphorylation of eIF2alpha and PERK was confirmed after severe ischemia but was inhibited by preconditioning. After preconditioning, GRP78 was increased in the brain with a peak at 2 days, which corresponded with the ischemic tolerance time window. Immunoprecipitation and double staining demonstrated involvement of GRP78 in prevention of PERK phosphorylation. These results suggest that GRP78 induced by preconditioning may reduce ER stress and eventual DND after ischemia.

    View details for DOI 10.1097/01.WCB.0000077641.41248.EA

    View details for Web of Science ID 000184604600008

    View details for PubMedID 12902839

  • Treatment with dihydroethidium reduces infarct size after transient focal cerebral ischemia in mice BRAIN RESEARCH Yu, F. S., Sugawara, T., Chan, P. H. 2003; 978 (1-2): 223-227

    Abstract

    A transient focal ischemia model with C57Bl/6 mice was used to investigate whether dihydroethidium is neuroprotective. Different doses (25, 50, 100 mg/kg body weight) were used for pretreatment and the lowest effective dose was used for delayed treatment 1 and 2 h after reperfusion. Our results demonstrate that all the doses used for treatment reduced infarct volume. We conclude that dihydroethidium is neuroprotective by reducing superoxide in mice after stroke.

    View details for DOI 10.1016/S0006-8993(03)02775-6

    View details for Web of Science ID 000184190500026

    View details for PubMedID 12834917

  • Prevention of NMDA-induced death of cortical neurons by inhibition of protein kinase C zeta JOURNAL OF NEUROCHEMISTRY Koponen, S., Kurkinen, K., Akerman, K. E., Mochly-Rosen, D., Chan, P. H., Koistinaho, J. 2003; 86 (2): 442-450

    Abstract

    Excitotoxicity through stimulation of N-methyl-d-aspartate (NMDA) receptors contributes to neuronal death in brain injuries, including stroke. Several lines of evidence suggest a role for protein kinase C (PKC) isoforms in NMDA excitotoxicity. We have used specific peptide inhibitors of classical PKCs (alpha, beta, and gamma), novel PKCs delta and epsilon, and an atypical PKCzeta in order to delineate which subspecies are involved in NMDA-induced cell death. Neuronal cell cultures were prepared from 15-day-old mouse embryos and plated onto the astrocytic monolayer. After 2 weeks in vitro the neurons were exposed to 100 micro m NMDA for 5 min, and 24 h later the cell viability was examined by measuring the lactate dehydrogenase release and bis-benzimide staining. While inhibitors directed to classical (alpha, beta, and gamma) or novel PKCs (delta or epsilon) had no effect, the PKCzeta inhibitor completely prevented the NMDA-induced necrotic neuronal death. Confocal microscopy confirmed that NMDA induced PKCzeta translocation, which was blocked by the PKCzeta inhibitor. The NMDA-induced changes in intracellular free Ca2+ were not affected by the peptides. In situ hybridization experiments demonstrated that PKCzeta mRNA is induced in the cortex after focal brain ischemia. Altogether, the results indicate that PKCzeta activation is a downstream signal in NMDA-induced death of cortical neurons.

    View details for DOI 10.1046/j.1471-4159.2003.01846.x

    View details for Web of Science ID 000183900300018

    View details for PubMedID 12871585

  • Copper-zinc superoxide dismutase affects Akt activation after transient focal cerebral ischemia in mice STROKE Noshita, N., Sugawara, T., Lewen, A., Hayashi, T., Chan, P. H. 2003; 34 (6): 1513-1518

    Abstract

    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.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).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.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.

    View details for DOI 10.1161/01.STR.0000072986.46924.F4

    View details for Web of Science ID 000183348300034

    View details for PubMedID 12738898

  • Effects of cold injury-induced trauma in manganese superoxide dismutase-deficient mice JOURNAL OF NEUROTRAUMA Grzeschik, S. M., Maier, C. M., Chan, P. H. 2003; 20 (6): 571-581

    Abstract

    Manganese superoxide dismutase (Mn-SOD, SOD2) is an inducible antioxidant localized to the mitochondria, which have been shown to be both the sites of superoxide anion (O(2)*-)) production and the target of free radical attacks. Knock-out mice with targeted disruption of Sod2 (SOD2-KO) are more susceptible to ischemic damage than their wild-type (WT) counterparts, showing increased loss of mitochondrial cytochrome c after trauma, but less apoptotic cell death in the first 24 h following controlled cortical injury. In this study, we sought to investigate whether oxidative stress plays a significant role in the development of secondary brain damage following cold injury-induced brain trauma (CIBT), a model of vasogenic edema. We first measured the levels of O(2)(*-) production 2 h after CIBT by means of in situ hydroethidine oxidation. We then examined lesion size, brain swelling, apoptosis by morphology and TUNEL-staining, neutrophil infiltration, and hemorrhage rates in both SOD2-KO and WT mice at 1, 3, and 7 days post-CIBT. We found no significant differences between SOD2-KO and WT littermates in any of the paradigms or endpoints studied. There was, however, a significant increase in hemorrhagic transformations in all animals that paralleled a robust inflammatory response at 3 days post insult compared with the 24-h endpoint. In the CIBT model used in this study, a 50% reduction in SOD2 activity did not appear to alter the injury response, suggesting that accumulation of free radicals does not play a significant role in secondary brain damage as previously thought with this particular model.

    View details for Web of Science ID 000183926900005

    View details for PubMedID 12906741

  • Cyclosporin A prevents calpain activation despite increased intracellular calcium concentrations, as well as translocation of apoptosis-inducing factor, cytochrome c and caspase-3 activation in neurons exposed to transient hypoglycemia JOURNAL OF NEUROCHEMISTRY Ferrand-Drake, M., Zhu, C. L., Gido, G., Hansen, A. J., Karlsson, J. O., Bahr, B. A., Zamzami, N., Kroemer, G., Chan, P. H., Wieloch, T., Blomgren, K. 2003; 85 (6): 1431-1442

    Abstract

    Blockade of mitochondrial permeability transition protects against hypoglycemic brain damage. To study the mechanisms downstream from mitochondria that may cause neuronal death, we investigated the effects of cyclosporin A on subcellular localization of apoptosis-inducing factor and cytochrome c, activation of the cysteine proteases calpain and caspase-3, as well as its effect on brain extracellular calcium concentrations. Redistribution of cytochrome c occurred at 30 min of iso-electricity, whereas translocation of apoptosis-inducing factor to nuclei occurred at 30 min of recovery following 30 min of iso-electricity. Active caspase-3 and calpain-induced fodrin breakdown products were barely detectable in the dentate gyrus and CA1 region of the hippocampus of rat brain exposed to 30 or 60 min of insulin-induced hypoglycemia. However, 30 min or 3 h after recovery of blood glucose levels, fodrin breakdown products and active caspase-3 markedly increased, concomitant with a twofold increase in caspase-3-like enzymatic activity. When rats were treated with neuroprotective doses of cyclosporin A, but not with FK 506, the redistribution of apoptosis-inducing factor and cytochrome c was reduced and fodrin breakdown products and active caspase-3 immuno-reactivity was diminished whereas the extracellular calcium concentration was unaffected. We conclude that hypoglycemia leads to mitochondrial permeability transition which, upon recovery of energy metabolism, mediates the activation of caspase-3 and calpains, promoting cell death.

    View details for DOI 10.1046/j.1471-4159.2003.01794.x

    View details for Web of Science ID 000183308300009

    View details for PubMedID 12787063

  • Role of superoxide in poly(ADP-ribose) polymerase upregulation after transient cerebral ischemia MOLECULAR BRAIN RESEARCH Narasimhan, P., Fujimura, M., Noshita, N., Chan, P. H. 2003; 113 (1-2): 28-36

    Abstract

    Oxidative stress plays a pivotal role in ischemic-reperfusion cell injury. Oxygen-derived free radicals trigger DNA strand damage, which is responsible for the activation of poly(ADP-ribose) polymerase (PARP). Recent studies have shown that peroxynitrite is the primary mediator of DNA damage and, hence, PARP activation after ischemia. PARP activation depletes NAD and ATP pools, ultimately resulting in necrotic cell death by loss of energy stores. Our study shows that PARP is upregulated as early as 15 min after 1 h of transient focal cerebral ischemia and remains for 8 h. We also examined the role of superoxide in PARP induction using copper/zinc-superoxide dismutase transgenic mice. Immunohistochemical and Western blotting data showed that there was no increased induction in PARP expression in these mice, suggesting that one of the mechanisms by which ischemic injury is attenuated in these mice might be by the inhibition of PARP induction. Furthermore, double staining of ischemic tissue with a PARP antibody and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) indicated that most cells that are positive for TUNEL do not stain for the PARP antibody, confirming recent reports that PARP activation is involved in necrotic cell death rather than apoptosis during ischemic-reperfusion injury.

    View details for DOI 10.1016/S0169-328X(03)00062-7

    View details for Web of Science ID 000183245100003

    View details for PubMedID 12750003

  • Aspirin inhibits p44/42 mitogen-activated protein kinase and is protective against hypoxia/reoxygenation neuronal damage STROKE Vartiainen, N., Goldsteins, G., Keksa-Goldsteine, V., Chan, P. H., Koistinaho, J. 2003; 34 (3): 752-757

    Abstract

    Acetylsalicylic acid (ASA) is preventive against stroke and protects against focal brain ischemia in rats. We studied the mechanisms of the manner in which ASA provides neuroprotection against hypoxia/reoxygenation (H/R) injury.Spinal cord cultures exposed to 20 hours of hypoxia followed by reoxygenation were treated with a vehicle, ASA or inhibitors of inducible nitric oxide synthase (iNOS), mitogen-activated protein kinases p38 MAPK and ERK1/2, or an N-methyl-d-aspartic acid (NMDA) receptor antagonist. Cell viability was assessed by LDH release measurement and cell counts. Prostaglandin production was measured by enzyme immunoassay, MAPK signaling by immunoblotting, and DNA binding of nuclear factor-kappaB (NF-kappaB) and activating protein-1 (AP-1) by electrophoretic mobility shift assay.One to 3 mmol/L ASA inhibited H/R-induced neuronal death when present during H/R but not when administered only for the reoxygenation period. Prostaglandin E2 production was very low and was not altered by ASA. The AP-1 and NF-kappaB DNA binding activities increased after H/R. ASA increased the H/R-induced AP-1 binding but had no effect on NF-kappaB binding. H/R induced a sustained ERK1/2 activation followed by neuronal death, whereas no changes in p38 or c-Jun N-terminal kinase were detected. ASA strongly inhibited this ERK1/2 activation. PD98059, an ERK1/2 inhibitor, was also neuroprotective, prevented H/R-induced ERK1/2 activation, and had no effect on NF-kappaB binding activity. Inhibition of NMDA receptors, iNOS, or p38 MAPK did not provide neuroprotection.Inhibition of the sustained activation of ERK1/2 may partially contribute to neuroprotection achieved by ASA against H/R injury.

    View details for DOI 10.1161/01.STR.0000057813.31798.1F

    View details for Web of Science ID 000181466700038

    View details for PubMedID 12624303

  • Overexpression of copper/zinc superoxide dismutase in transgenic mice protects against neuronal cell death after transient focal ischemia by blocking activation of the bad cell death signaling pathway JOURNAL OF NEUROSCIENCE Saito, A., Hayashi, T., Okuno, S., Ferrand-Drake, M., Chan, P. H. 2003; 23 (5): 1710-1718

    Abstract

    The Bad signaling pathway contributes to the regulation of apoptosis after a variety of cell death stimuli, and Bad plays a key role in determining cell death or survival. We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduces apoptotic cell death after transient focal cerebral ischemia (tFCI). However, both the role of the Bad pathway after tFCI and the role of oxygen free radicals in the regulation of apoptosis remain unknown. To clarify these issues, we used an in vivo tFCI model of SOD1 transgenic mice and wild-type mice. Moreover, to examine the role of protein kinase A (PKA) in the Bad pathway after tFCI, we administered the PKA inhibitor, H89, into the mouse brain after tFCI. Immunohistochemistry and Western blot analysis showed that dephosphorylation and translocation of Bad were detected early after tFCI and that they were promoted by H89 treatment but prevented by SOD1. Coimmunoprecipitation revealed that the dimerization of Bad progressed with 14-3-3 (Bad/14-3-3) and with Bcl-x(L) (Bad/Bcl-x(L)) after tFCI. Moreover, Bad/14-3-3 was prevented by H89 treatment but promoted by SOD1. Bad/Bcl-x(L) was prevented by SOD1 but promoted by H89 treatment. A cell death assay revealed that apoptotic-related DNA fragmentation was aggravated by H89 treatment but reduced by SOD1. These results suggest that the Bad pathway mediated by PKA is involved in apoptotic cell death after tFCI and that overexpression of SOD1 may attenuate this apoptotic cell death.

    View details for Web of Science ID 000181450600020

    View details for PubMedID 12629175

  • Temporal profile of angiogenesis and expression of related genes in the brain after ischemia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Hayashi, T., Noshita, N., Sugawara, T., Chan, P. H. 2003; 23 (2): 166-180

    Abstract

    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.

    View details for DOI 10.1097/00004647-200302000-00004

    View details for Web of Science ID 000180893800004

    View details for PubMedID 12571448

  • Protective effects of superoxide dismutase against ischemia-reperfusion injury: Development and application of a transgenic animal model PLASTIC AND RECONSTRUCTIVE SURGERY Klein, M. B., Chan, P. H., Chang, J. 2003; 111 (1): 251-255

    Abstract

    Reperfusion of ischemic tissues can be associated with structural and functional injury, which is referred to as ischemia-reperfusion injury. Superoxide dismutase is an endogenous free radical scavenger that converts toxic oxygen derived free radicals to hydrogen peroxide. With the development of gene cloning technology, the potential of manipulating cells to overexpress endogenous proteins has been realized. Transgenic mice capable of overexpressing superoxide dismutase, and knockout mice in which the gene responsible for its production has been deleted, were used as a model to examine the protective effects of superoxide dismutase against ischemia-reperfusion injury. Epigastric island flaps were elevated in wild-type (control), transgenic superoxide dismutase 1, and knockout superoxide dismutase 1 mice and subjected to ischemic intervals of 0, 3, 6, 9, or 12 hours. Five animals were studied at each time point in each study group. Flap viability was assessed on postoperative day 7. Baseline wild-type flap survival was 100 percent after 3 hours of ischemia and subsequent reperfusion; survival decreased to 21 percent after 9 hours of ischemia. Transgenic mice had significantly higher flap survival than wild-type animals after 6 hours of ischemia and subsequent reperfusion (97.0 versus 85.2 percent) and after 9 hours of ischemia (82 versus 21 percent, p < 0.01). In knockout mice, there was complete flap necrosis after as little as 3 hours of ischemia. This study confirms the protective effects of superoxide dismutase against ischemia-reperfusion injury. In addition, its deficiency results in a dramatic susceptibility to ischemic injury.

    View details for DOI 10.1097/01.PRS.0000034938.58120.12

    View details for Web of Science ID 000180191700042

    View details for PubMedID 12496586

  • Overexpression of SOD1 protects vulnerable motor neurons after spinal cord injury by attenuating mitochondrial cytochrome c release. FASEB journal Sugawara, T., Lewén, A., Gasche, Y., Yu, F., Chan, P. H. 2002; 16 (14): 1997-1999

    Abstract

    Defective Cu,Zn-superoxide dismutase (SOD1) is responsible for some types of amyotrophic lateral sclerosis, and ventral horn motor neurons (VMN) have been shown to die through a mitochondria-dependent apoptotic pathway after chronic exposure to high levels of reactive oxygen species (ROS). VMN are also selectively vulnerable to mild spinal cord injury (SCI); however, the involvement of SOD1, ROS, and apoptosis in their death has not been clarified. Mild compression SCI was induced in SOD1-overexpressing transgenic rats and wild-type littermates. Superoxide production, mitochondrial release of cytochrome c, and activation of caspase-9 were examined, and apoptotic DNA injury was also characterized. In the wild-type animals, increased superoxide production, mitochondrial release of cytochrome c, and cleaved caspase-9 were observed exclusively in VMN after SCI. Subsequently, a majority of VMN (75%) selectively underwent delayed apoptotic cell death. Transgenic animals showed less superoxide production, mitochondrial cytochrome c release, and caspase-9 activation, resulting in death of only 45% of the VMN. These results suggest that the ROS-initiated mitochondrial signaling pathway possibly plays a pivotal role in apoptotic VMN death after SCI and that increased levels of SOD1 in VMN reduce oxidative stress, thereby attenuating the activation of the pathway and delayed cell death.

    View details for PubMedID 12368231

  • Overexpression of SOD1 protects vulnerable motor neurons after spinal cord injury by attenuating mitochondrial cytochrome c release FASEB JOURNAL Sugawara, T., Lewen, A., Gasche, Y., Yu, F. S., Chan, P. H. 2002; 16 (12): 1997-?
  • Effects of mild hypothermia on superoxide anion production, superoxide dismutase expression, and activity following transient focal cerebral ischemia NEUROBIOLOGY OF DISEASE Maier, C. M., Sun, G. H., Cheng, D. Y., Yenari, M. A., Chan, P. H., Steinberg, G. K. 2002; 11 (1): 28-42

    Abstract

    Following a transient ischemic insult there is a marked increase in free radical (FR) production within the first 10-15 min of reperfusion and again at the peak of the inflammatory process. Hypothermia decreases lipid peroxidation following global ischemia, raising the possibility that it may act by reducing FR production early on and by maintaining or increasing endogenous antioxidant systems. By means of FR fluorescence, Western blot, immunohistochemistry, and enzymatic assay, we studied the effects of mild hypothermia on superoxide (O(-*)(2)) anion production, superoxide dismutase SOD expression, and activity following focal cerebral ischemia in rats. Mild hypothermia significantly reduced O(-*)(2) generation in the ischemic penumbra and corresponding contralateral region, but did not alter the bilateral SOD expression. SOD enzymatic activity in the ischemic core was slightly reduced in hypothermia-treated animals compared with normothermic controls. Our results suggest that the neuroprotective effect of mild hypothermia may be due, in part, to a reduction in neuronal and endothelial O(-*)(2) production during early reperfusion.

    View details for DOI 10.1006/nbdi.2002.0513

    View details for Web of Science ID 000179314100003

    View details for PubMedID 12460544

  • Copper/zinc superoxide dismutase attenuates neuronal cell death by preventing extracellular signal-regulated kinase activation after transient focal cerebral ischemia in mice JOURNAL OF NEUROSCIENCE Noshita, N., Sugawara, T., Hayashi, T., Lewen, A., Omar, G., Chan, P. H. 2002; 22 (18): 7923-7930

    Abstract

    Recent studies have revealed that activation of extracellular signal-regulated kinase (ERK) may contribute to apoptosis, a cell death process involved in oxidative stress. We examined phosphorylation of ERK1/2 and oxidative stress after transient focal cerebral ischemia (FCI) using transgenic (Tg) mice that overexpress copper/zinc superoxide dismutase (SOD1). The mice were subjected to 60 min of middle cerebral artery (MCA) occlusion by intraluminal suture blockade followed by 1, 4, and 24 hr of reperfusion. Immunohistochemistry and Western blot analysis showed that phospho-ERK1 was markedly increased in the cortex within the MCA territory at 1 hr of reperfusion (p < 0.01), followed by a decrease at 24 hr in wild-type mice. Double staining with phospho-ERK1/2 and neuron-specific nuclear protein showed that phospho-ERK1/2 was primarily expressed in neurons. In SOD1 Tg mice, phospho-ERK1/2 was prominently reduced compared with nonischemic controls, shown by immunohistochemistry. Western blot analysis confirmed a significant decrease in phospho-ERK1/2 1 hr after FCI in the ischemic cortex (p < 0.005). Apoptotic-related DNA fragmentation was reduced in the ischemic cortex of SOD1 Tg mice compared with wild-type mice using a cell death assay. These results suggest that phosphorylation of ERK1/2 may be involved in apoptosis or cell death after transient FCI and that SOD1 may attenuate apoptotic cell death mediated by the mitogen-activated protein kinase/ERK pathway.

    View details for Web of Science ID 000177916000012

    View details for PubMedID 12223545

  • Role of superoxide dismutases in oxidative damage and neurodegenerative disorders NEUROSCIENTIST Maier, C. M., Chan, P. H. 2002; 8 (4): 323-334

    Abstract

    In recent years, oxidative stress has been implicated in a variety of degenerative processes, diseases, and syndromes. Some of these include atherosclerosis, myocardial infarction, stroke, and ischemia/reperfusion injury; chronic and acute inflammatory conditions such as wound healing; central nervous system disorders such as forms of familial amyotrophic lateral sclerosis (ALS) and glutathione peroxidase-linked adolescent seizures; Parkinson's disease and Alzheimer's dementia; and a variety of other age-related disorders. Among the various biochemical events associated with these conditions, emerging evidence suggests the formation of superoxide anion and expression/activity of its endogenous scavenger, superoxide dismutase (SOD), as a common denominator. This review summarizes the function of SOD under normal physiological conditions as well as its role in the cellular and molecular mechanisms underlying oxidative tissue damage and neurological abnormalities. Experimental evidence from laboratory animals that either overexpress (transgenics) or are deficient (knockouts) in antioxidant enzyme/protein levels and the genetic SOD mutations observed in some familial cases of ALS are also discussed.

    View details for Web of Science ID 000177126500015

    View details for PubMedID 12194501

  • Involvement of superoxide in excitotoxicity and DNA fragmentation in striatal vulnerability in mice after treatment with the mitochondrial toxin, 3-nitropropionic acid JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Kim, G. W., Chan, P. H. 2002; 22 (7): 798-809

    Abstract

    Oxidative stress and excitotoxicity have been implicated in selective striatal vulnerability caused by the mitochondrial toxin, 3-nitropropionic acid (3-NP), which may simulate Huntington's disease in animals and humans. The detailed mechanism of the role of superoxide in striatal vulnerability induced by 3-NP is still unknown. The authors investigated oxidative cellular injury and DNA fragmentation after systemic 3-NP injection in wild-type (Wt) mice and mutant mice with a deficiency in manganese superoxide dismutase (MnSOD; Sod2 -/+). Furthermore, they investigated the effects of decortication after 3-NP treatment in Sod2 -/+ mice, and copper/zinc SOD (CuZnSOD) treatment in recently developed Sod2 -/+ mice that overexpress CuZnSOD (SOD1 +/- / Sod2 -/+ mice). Oxidized hydroethidine, 8-hydroxyguanosine immunoreactivity, and nitrotyrosine immunoreactivity were increased in the Sod2 -/+ mice compared with the Wt mice after 3-NP treatment (P < 0.001). Decortication completely abolished oxidative striatal damage after 3-NP treatment in the Sod2 -/+ mice. Increased CuZnSOD attenuated DNA fragmentation and striatal lesion volume after 3-NP treatment in the Sod2 -/+ mice (P < 0.001). These data suggest that production of superoxide may be a critical step to excitotoxicity and subsequent DNA fragmentation in selective striatal vulnerability after 3-NP treatment.

    View details for Web of Science ID 000176689700005

    View details for PubMedID 12142565

  • Carbohydrate source influences gelatinase production by mouse astrocytes in vitro GLIA Massengale, J. L., Gasche, Y., Chan, P. H. 2002; 38 (3): 240-245

    Abstract

    Molecular mediators of ischemic brain injury include intercellular adhesion molecule-1 (ICAM-1) and matrix metalloproteinase-9 (MMP-9), involved in the alteration of blood-brain barrier permeability and induced in astroglial cultures by tumor necrosis factor-alpha (TNF-alpha). Hyperglycemia is known to aggravate in vivo ischemic brain damage, while treatment with sorbitol shows benefit in reducing vasogenic brain edema. This study investigated whether a culture medium carbohydrate source could alter the astrocyte production of MMP-9 and ICAM-1 in vitro. The growth of astrocytes in 12.5 mM glucose, 25 mM glucose, or 25 mM sorbitol for 14 days did not alter cellular release of lactate dehydrogenase, uptake of Trypan blue, or surface expression of glial fibrillary acidic protein (GFAP). ICAM-1 levels were similar in astrocytes grown in glucose or sorbitol both under basal conditions and after TNF-alpha stimulation for 48 h. In contrast, levels of proMMP-9 released from astrocytes cultured for 14 days in 25 mM sorbitol reached only 55-28% of those obtained from cultures in 25 mM glucose after stimulation with 1,000 U/ml (P = 0.05) or 5,000 U/ml (P < 0.025) TNF-alpha, respectively. Limiting the duration of pre-stimulation sorbitol exposure to 48 h resulted in lower proMMP-9 levels than in glucose cultures after 5,000, but not 1,000, U/ml TNF-alpha, and differences were not significant when sorbitol exposure was further reduced to 24 h. Incubation in mixed glucose/sorbitol media did not affect the release of proMMP-9. These findings suggest that MMP-9 production may be increased in astrocytes as a consequence of glucose metabolism, which can be avoided by growth in sorbitol alone.

    View details for DOI 10.1002/glia.10070

    View details for Web of Science ID 000175523600006

    View details for PubMedID 11968061

  • Akt phosphorylation and neuronal survival after traumatic brain injury in mice NEUROBIOLOGY OF DISEASE Noshita, N., Lewen, A., Sugawara, T., Chan, P. H. 2002; 9 (3): 294-304

    Abstract

    The serine-threonine kinase, Akt, is involved in the survival signaling pathways in many cell systems. The present study examined phosphorylation of Akt at serine-473 and DNA fragmentation after traumatic brain injury (TBI) in mice. Immunohistochemistry showed phospho-Akt was decreased in the injured cortex 1 h after TBI, whereas it was temporally increased at 4 h in the perifocal damaged cortex. In the CA1 region of the hippocampus, phospho-Akt was increased after TBI. Western blot analysis showed that Akt was significantly decreased as early as 1 h after trauma; however, the phosphorylation was accelerated at 4 h. Double staining with phospho-Akt and phospho-BAD or phospho-GSK-3beta revealed the colocalization of phospho-Akt and downstream elements. Double staining with phospho-Akt and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling showed different cellular distributions after TBI. The present study implicates Akt phosphorylation in the signaling pathways that are involved in cell survival after TBI.

    View details for DOI 10.1006/nbdi.2002.0482

    View details for Web of Science ID 000175193500003

    View details for PubMedID 11950275

  • Manganese superoxide dismutase deficiency exacerbates cerebral infarction after focal cerebral ischemia/reperfusion in mice - Implications for the production and role of superoxide radicals STROKE Kim, G. W., Kondo, T., Noshita, N., Chan, P. H. 2002; 33 (3): 809-815

    Abstract

    Superoxide anion radicals (O2*-) are implicated in ischemia/reperfusion injury, although a direct relationship has not been elucidated. Recently, a specific method of hydroethidine (HEt) oxidation by O2*- was developed to detect O2*- production in a variety of experimental brain injury models. To clarify the role of O2*- in the mechanism of ischemia/reperfusion, we investigated O2*- production after ischemia/reperfusion and ischemia/reperfusion injury in mutant mice deficient in mitochondrial manganese superoxide dismutase (MnSOD) and in wild-type littermates.Ischemia/reperfusion was performed for 60 minutes using intraluminal suture blockade of the middle cerebral artery in the mutant or wild-type mice. We evaluated fluorescent kinetics of HEt or ethidium, the oxidized form of HEt, in brains after an intravenous injection of HEt, followed by measurement of cellular O2*- production using specific HEt oxidation by O2*- before and after ischemia/reperfusion. Furthermore, we compared O2*- production and subsequent infarct volume in the mice using triphenyltetrazolium chloride after ischemia/reperfusion.HEt oxidation to ethidium is primarily a result of mitochondrially produced O2*- under physiological conditions. Cerebral ischemia/reperfusion produced O2*- prominently in neurons shortly after reperfusion, followed by a delayed increase in endothelial cells. A deficiency in MnSOD in mutant mice increased mitochondrial O2*- production and exacerbated cerebral infarction, worsening neurological deficits after ischemia/reperfusion.These results suggest that mitochondrial O2*- production may be a critical step underlying the mechanism of ischemia/reperfusion injury and that MnSOD may protect against ongoing oxidative cell death after ischemia/reperfusion.

    View details for Web of Science ID 000174200600029

    View details for PubMedID 11872908

  • Effects of global ischemia duration on neuronal, astroglial, oligodendroglial, and microglial reactions in the vulnerable hippocampal CA1 subregion in rats JOURNAL OF NEUROTRAUMA Sugawara, T., Lewen, A., Noshita, N., Gasche, Y., Chan, P. H. 2002; 19 (1): 85-98

    Abstract

    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.

    View details for Web of Science ID 000173611300008

    View details for PubMedID 11852981

  • Overexpression of copper/zinc superoxide dismutase in transgenic rats protects vulnerable neurons against ischemic damage by blocking the mitochondrial pathway of caspase activation JOURNAL OF NEUROSCIENCE Sugawara, T., Noshita, N., Lewen, A., Gasche, Y., Ferrand-Duke, M., Fujimura, M., Morita-Fujimura, Y., Chan, P. H. 2002; 22 (1): 209-217

    Abstract

    Mitochondria are known to be involved in the early stage of apoptosis by releasing cytochrome c, caspase-9, and the second mitochondria-derived activator of caspases (Smac). We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduced superoxide production and ameliorated neuronal injury in the hippocampal CA1 subregion after global ischemia. However, the role of oxygen free radicals produced after ischemia/reperfusion in the mitochondrial signaling pathway has not been clarified. Five minutes of global ischemia was induced in male SOD1-transgenic (Tg) and wild-type (Wt) littermate rats. Cytosolic expression of cytochrome c and Smac and activation of caspases were evaluated by immunohistochemistry, Western blot, and caspase activity assay. Apoptotic cell death was characterized by DNA nick end and single-stranded DNA labeling. In the Wt animals, early superoxide production, mitochondrial release of cytochrome c, Smac, and cleaved caspase-9 were observed after ischemia. Active caspase-3 was subsequently increased, and 85% of the hippocampal CA1 neurons showed apoptotic DNA damage 3 d after ischemia. Tg animals showed less superoxide production and cytochrome c and Smac release. Subsequent active caspase-3 expression was not evident, and only 45% of the neurons showed apoptotic DNA damage. A caspase-3 inhibitor (N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone) reduced cell death only in Wt animals. These results suggest that overexpression of SOD1 reduced oxidative stress, thereby attenuating the mitochondrial release of cytochrome c and Smac, resulting in less caspase activation and apoptotic cell death. Oxygen free radicals may play a pivotal role in the mitochondrial signaling pathway of apoptotic cell death in hippocampal CA1 neurons after global ischemia.

    View details for Web of Science ID 000172905700029

    View details for PubMedID 11756504

  • Mild hypothermia attenuates cytochrome C release but does not alter Bcl-2 expression or caspase activation after experimental stroke JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Yenari, M. A., Iwayama, S., Cheng, D. Y., Sun, G. H., Fujimura, M., Morita-Fujimura, Y., Chan, P. H., Steinberg, G. K. 2002; 22 (1): 29-38

    Abstract

    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.

    View details for Web of Science ID 000172930900004

    View details for PubMedID 11807391

  • Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Noshita, N., Lewen, A., Sugawara, T., Chan, P. H. 2001; 21 (12): 1442-1450

    Abstract

    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.

    View details for Web of Science ID 000172574400009

    View details for PubMedID 11740206

  • Matrix metalloproteinase inhibition prevents oxidative stress-associated blood-brain barrier disruption after transient focal cerebral ischemia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Gasche, Y., Copin, J. C., Sugawara, T., Fujimura, M., Chan, P. H. 2001; 21 (12): 1393-1400

    Abstract

    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.

    View details for Web of Science ID 000172574400003

    View details for PubMedID 11740200

  • Delayed treatment with polynitroxyl albumin reduces infarct size after stroke in rats NEUROREPORT Sugawara, T., Yu, F. S., Ma, L., Hsia, C. J., Chan, P. H. 2001; 12 (16): 3609-3612

    Abstract

    Nitroxides are antioxidants that are known to protect cells from oxidative damage. Polynitroxyl albumin (PNA) is a compound of human serum albumin covalently labeled with nitroxides that exhibits a prolonged half-life and an enhanced antioxidant activity. Adult male Sprague-Dawley rats were subjected to 90 min intraluminal middle cerebral artery occlusion and the drug was administered intravenously immediately or 2 h after reperfusion. The effects of the drug were evaluated 24 h after reperfusion. Infarct volume was significantly reduced in immediate (79% reduction) and delayed (53% reduction) PNA-treated groups. The efficacy of a single, delayed i.v. injection of PNA suggests that PNA has great promise in the treatment of acute human stroke.

    View details for Web of Science ID 000172175000046

    View details for PubMedID 11733721

  • Superoxide during reperfusion contributes to caspase-8 expression and apoptosis after transient focal stroke STROKE Morita-Fujimura, Y., Fujimura, M., Yoshimoto, T., Chan, P. H. 2001; 32 (10): 2356-2361

    Abstract

    Reactive oxygen species produced during reperfusion may play a detrimental role in focal cerebral ischemia (FCI). We examined the protein expression of caspase-8, which plays a major role in both Fas-dependent and cytochrome c-dependent apoptotic pathways after FCI with or without reperfusion. Caspase-8 expression after transient FCI was compared between wild-type and transgenic mice that overexpress the cytosolic antioxidant copper/zinc superoxide dismutase (SOD1).Adult male CD-1 mice were subjected to 1 hour of FCI and reperfusion or to permanent FCI by intraluminal blockade of the middle cerebral artery. DNA fragmentation was evaluated by genomic DNA gel electrophoresis. Caspase-8 expression was analyzed by Western blot.Caspase-8 was significantly induced 4 hours after transient FCI and remained at an increased level until 24 hours, whereas it was not modified after permanent FCI. Genomic DNA gel electrophoresis showed DNA laddering in a pattern similar to that seen in apoptosis, with a small amount of background smear 24 hours after transient FCI, whereas 25 hours of permanent FCI resulted in less DNA laddering with a strong background smear. Caspase-8 induction was significantly reduced in SOD1 transgenic mice compared with wild-type mice 4 hours after transient FCI.The results suggest that increased reactive oxygen species production during reperfusion may contribute to the induction of caspase-8, thereby exacerbating apoptosis after FCI.

    View details for Web of Science ID 000171488800030

    View details for PubMedID 11588326

  • Neuronal expression of the DNA repair protein Ku 70 after ischemic preconditioning corresponds to tolerance to global cerebral ischemia STROKE Sugawara, T., Noshita, N., Lewen, A., Kim, G. W., Chan, P. H. 2001; 32 (10): 2388-2393

    Abstract

    Oxidative stress after ischemia/reperfusion has been shown to induce DNA damage and subsequent DNA repair activity. Ku 70/86, multifunctional DNA repair proteins, bind to broken DNA ends and trigger a DNA repair pathway. We investigated the involvement of these proteins in the development of neuronal tolerance to global cerebral ischemia after ischemic preconditioning.Adult male Sprague-Dawley rats were subjected to either 5 minutes of lethal global ischemia with or without 3 minutes of sublethal ischemic preconditioning or 3 minutes of ischemia only. Neuronal injury was histologically assessed, and DNA damage was visualized by in situ labeling of DNA fragmentation and DNA gel electrophoresis. Ku expression was also examined by immunohistochemistry and Western blot analysis.Hippocampal CA1 neurons underwent DNA-fragmented cell death 3 days after 5 minutes of ischemia. However, these neurons showed a strong tolerance to 5 minutes of ischemia 1 to 3 days after ischemic preconditioning. Immunohistochemistry showed virtually no constitutive expression of Ku proteins in CA1 neurons; however, ischemic preconditioning induced neuronal Ku 70 expression 1 to 3 days later. Western blot confirmed an increase in Ku 70 in this region at the same time.The temporal and spatial expression of Ku 70 corresponded to tolerance of the hippocampal CA1 neurons to subsequent ischemia, suggesting the involvement of Ku proteins in the development of neuronal tolerance after ischemic preconditioning.

    View details for Web of Science ID 000171488800035

    View details for PubMedID 11588331

  • Oxidative stress-dependent release of mitochondrial cytochrome c after traumatic brain injury JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Lewen, A., Fujimura, M., Sugawara, T., Matz, P., Copin, J. C., Chan, P. H. 2001; 21 (8): 914-920

    Abstract

    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.

    View details for Web of Science ID 000170228000003

    View details for PubMedID 11487726

  • Neuronal, but not microglial, accumulation of extravasated serum proteins after intracerebral hemolysate exposure is accompanied by cytochrome c release and DNA fragmentation JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Matz, P. G., Lewen, A., Chan, P. H. 2001; 21 (8): 921-928

    Abstract

    Vasogenic edema after oxidative injury has been accompanied by intracellular accumulation of serum proteins and nuclear damage. This study sought to determine whether serum protein accumulation, along with other markers of brain injury, was present after exposure to intracerebral hemolysate, an oxidant model of intracerebral hemorrhage (ICH). Saline (n = 24) or hemolysate (n = 30) was injected into the caudate-putamen of adult Sprague-Dawley rats. Compared with saline, hemolysate deposition was associated with intracellular accumulation of serum proteins as evidenced by Evans blue uptake in neurons and microglia at 4 and 24 hours. Intracellular Evans blue colocalized with DNA fragmentation detected by nick end-labeling and whose presence was confirmed by gel electrophoresis. Immunoblots of cytosolic fractions confirmed cytochrome c release. Immunostaining established colocalization of cytosolic cytochrome c and intracellular Evans blue at 4 hours. At 24 hours, cytosolic cytochrome c was evident in astrocytes surrounding Evans blue-positive cells. Immunoblot analysis and immunostaining revealed HSP70 induction at 24 hours in regions adjacent to intracellular serum accumulation. Neuronal accumulation of extravasated serum proteins in this model of ICH was associated with cytochrome c release, DNA fragmentation, and cell death. Stress protein induction in adjacent regions suggested that vasogenic edema might have exacerbated cellular dysfunction and cell death after ICH.

    View details for Web of Science ID 000170228000004

    View details for PubMedID 11487727

  • Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism JOURNAL OF NEUROCHEMISTRY Chen, Y. M., Vartiainen, N. E., Ying, W. H., Chan, P. H., Koistinaho, J., Swanson, R. A. 2001; 77 (6): 1601-1610

    Abstract

    Nitric oxide (NO) contributes to neuronal death in cerebral ischemia and other conditions. Astrocytes are anatomically well positioned to shield neurons from NO because astrocyte processes surround most neurons. In this study, the capacity of astrocytes to limit NO neurotoxicity was examined using a cortical co-culture system. Astrocyte-coated dialysis membranes were placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium. The utility of this system was tested by comparing neuronal death produced by glutamate, which is rapidly cleared by astrocytes, and N-methyl-D-aspartate (NMDA), which is not. The presence of an astrocyte layer increased the LD(50) for glutamate by approximately four-fold, but had no effect on NMDA toxicity. Astrocyte effects on neuronal death produced by the NO donors S-nitroso-N-acetyl penicillamine and spermine NONOate were examined by placing these compounds into the medium of co-cultures containing either a control astrocyte layer or an astrocyte layer depleted of glutathione by prior exposure to buthionine sulfoximine. Neurons in culture with the glutathione-depleted astrocytes exhibited a two-fold increase in cell death over a range of NO donor concentrations. These findings suggest that astrocytes protect neurons from NO toxicity by a glutathione-dependent mechanism.

    View details for Web of Science ID 000169392400017

    View details for PubMedID 11413243

  • Early decrease in DNA repair proteins, Ku70 and Ku86, and subsequent DNA fragmentation after transient focal cerebral ischemia in mice STROKE Kim, G. W., Noshita, N., Sugawara, T., Chan, P. H. 2001; 32 (6): 1401-1407

    Abstract

    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.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.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.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.

    View details for Web of Science ID 000169184600033

    View details for PubMedID 11387505

  • Oxidative cellular damage and the reduction of APE/Ref-1 expression after experimental traumatic brain injury NEUROBIOLOGY OF DISEASE Lewen, A., Sugawara, T., Gasche, Y., Fujimura, M., Chan, P. H. 2001; 8 (3): 380-390

    Abstract

    The DNA repair enzyme, apurinic/apyrimidinic endonuclease (or redox effector factor-1, APE/Ref-1), is involved in base excision repair of apurinic/apyrimidinic sites after oxidative DNA damage. We investigated the expression of APE/Ref-1 and its relationship to oxidative stress after severe traumatic brain injury produced by controlled cortical impact in normal mice, and in mice over- or underexpressing copper-zinc superoxide dismutase (SOD1TG and SOD1KO, respectively). Oxygen free radical-mediated cellular injury was visualized with 8-hydroxyguanine immunoreactivity as a marker for DNA oxidation, and in situ hydroethidine oxidation as a marker for superoxide production. After trauma there was a reduced expression of APE/Ref-1 in the ipsilateral cortex and hippocampus that correlated with the gene dosage levels of cytosolic superoxide dismutase. The decrease in APE/Ref-1 expression preceded DNA fragmentation. There was also a close correlation between APE/Ref-1 protein levels 4 h after trauma and the volume of the lesion 1 week after injury. Our data have demonstrated that reduction of APE/Ref-1 protein levels correlates closely with the level of oxidative stress after traumatic brain injury. We suggest that APE/Ref-1 immunoreactivity is a sensitive marker for oxidative cellular injury.

    View details for Web of Science ID 000169459000002

    View details for PubMedID 11447995

  • Manganese superoxide dismutase affects cytochrome c release and caspase-9 activation after transient focal cerebral ischemia in mice JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Noshita, N., Sugawara, T., Fujimura, M., Morita-Fujimura, Y., Chan, P. H. 2001; 21 (5): 557-567

    Abstract

    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.

    View details for Web of Science ID 000168487500010

    View details for PubMedID 11333366

  • Overexpression of copper-zinc superoxide dismutase attenuates acute activation of activator protein-1 after transient focal cerebral ischemia in mice STROKE Huang, C. Y., Fujimura, M., Chang, Y. Y., Chan, P. H. 2001; 32 (3): 741-747

    Abstract

    Reactive oxygen species (ROS) have been implicated in reperfusion injury after focal cerebral ischemia (FCI). ROS are known to regulate the activity of transcription factors such as activator protein-1 (AP-1), which is a dimer consisting of members of the Jun and Fos families. We investigated the role of ROS in AP-1 activity after FCI using transgenic mice that overexpressed copper-zinc superoxide dismutase (SOD1) and that had reduced infarction volume after FCI.The SOD1 transgenic mice and their wild-type littermates were subjected to middle cerebral artery occlusion and reperfusion by intraluminal suture blockade. After 60 minutes of middle cerebral artery occlusion, mice were allowed to recover for 1, 2, and 4 hours before euthanasia. Protein expression of c-Jun and c-Fos was examined by immunohistochemistry and Western blotting. AP-1 DNA-protein binding activity was assessed by electrophoretic mobility shift assays.In wild-type mice, immunohistochemistry demonstrated acute c-Jun and c-Fos activation in ischemic cortex and its outer boundary. Expression of both was reduced in SOD1 transgenic mice. Western blotting confirmed that SOD1 overexpression was associated with reduced c-Jun and c-Fos protein levels in ischemic brain. Electrophoretic mobility shift assays revealed that the ischemia-enhanced DNA binding activity observed in wild-type mice was reduced in SOD1 transgenic mice. Supershift assays indicated that c-Jun participated in the bound AP-1 complex.SOD1 overexpression prevents early activation of AP-1 after transient FCI in mice. This may block the expression of downstream target genes that are injurious, thereby reducing the infarction volume after transient FCI in mice.

    View details for Web of Science ID 000167354500025

    View details for PubMedID 11239196

  • Increased cytochrome c-mediated DNA fragmentation and cell death in manganese-superoxide dismutas-deficient mice after exposure to subarachnoid hemolysate STROKE Matz, P. G., Fujimura, M., Lewen, A., Morita-Fujimura, Y., Chan, P. H. 2001; 32 (2): 506-515

    Abstract

    We sought to investigate the mechanisms for oxidative injury caused by subarachnoid hemolysate, a pro-oxidant.Injection of 50 microL of subarachnoid hemolysate or saline was performed in CD1 mice (n=75), mutant mice deficient in Mn-superoxide dismutase (Sod2+/-; n=23), and their wild-type littermates (n=23). Subcellular location of cytochrome c was studied by immunocytochemistry, immunofluorescence, and immunoblotting of cellular fractions. DNA fragmentation was assessed though DNA laddering and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL). Cell death was examined through basic histology.Cytochrome c immunoreactivity was present in the cytosol of neurons at 2 hours after hemolysate injection and increased by 4 hours compared with saline-injected animals (P:<0.02). Cytosolic cytochrome c was more abundant in Sod2+/- mutants. DNA fragmentation was evident at 24 hours, but not 4 hours, after hemolysate injection as determined by DNA laddering and TUNEL staining (P:<0.02). DNA fragmentation colocalized to cells with cytosolic cytochrome c and iron. In Sod2+/- mutants, the extent of fragmentation was increased as determined by TUNEL staining (52% increase; P:<0.02) and DNA laddering (optical density=0.819 versus 0.391; P:<0.01). Cell death was evident on basic histology as early as 4 hours after hemolysate injection. No cell death was evident in controls. In Sod2+/- mutants, cell death was increased by 51% compared with wild-type littermates (P:<0.05).These results demonstrate that subarachnoid blood products are associated with the presence of cytochrome c in the cytosol and subsequent cell death in neurons. It appears that Mn-superoxide dismutase plays a role in preventing cell death after exposure to subarachnoid blood products.

    View details for Web of Science ID 000166851100022

    View details for PubMedID 11157190

  • SOD1 down-regulates NF-kappa B and c-mye expression in mice after transient focal cerebral ischemia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Huang, C. Y., Fujimura, M., Noshita, N., Chang, Y. Y., Chan, P. H. 2001; 21 (2): 163-173

    Abstract

    Reactive oxygen species (ROS) are implicated in reperfusion injury after focal cerebral ischemia (FCI). Reactive oxygen species regulate activity of transcription factors like NF-kappaB. The authors investigated the role of ROS in NF-kappaB 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-kappaB p65 immunoreactivity. Colocalization of NF-kappaB and the neuronal marker, microtubule-associated proteins (MAPs), showed that NF-kappaB was up-regulated in neurons after FCI. Electrophoretic mobility shift assays showed that SODI overexpression reduced ischemia-induced NF-kappaB DNA binding activity. Supershift assays showed that DNA-protein complexes contained p65 and p50 subunits. Immunoreactivity of c-myc, an NF-kappaB downstream gene, was increased in the ischemic cortex and colocalized with NF-kappaB. Western blotting showed that SOD1 overexpression reduced NF-kappaB 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-kappaB 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.

    View details for Web of Science ID 000166839400008

    View details for PubMedID 11176282

  • Prolonged hypoxia during cell development protects mature manganese superoxide dismutase-deficient astrocytes from damage by oxidative stress FASEB JOURNAL Copin, J. C., Gasche, Y., Li, Y. B., Chan, P. H. 2001; 15 (2): 525-534

    Abstract

    Mouse astrocytes deficient in the mitochondrial form of superoxide dismutase do not grow in culture under 20% atmospheric O2 levels. By flow cytometry, immunocytochemistry, and enzymatic analysis we have shown that the oxygen block of cell division is due to a decrease in the number of cells entering the S phase of the cell cycle and is concomitant with higher DNA oxidation and impairment of mitochondrial functions. Seeding the cells under 5% O2 until the cultures become confluent can circumvent this problem. An initial hypoxic environment increases the resistance of manganese superoxide dismutase-deficient astrocytes to superoxide radicals artificially produced by paraquat treatment, preserves respiratory activity, and allows normoxic division during a subsequent passage. DNA oxidation is then not higher than in wild-type control cells. However, the adaptation of the cells is not due to compensation by other enzymes of the antioxidant defense system and is specific to cells totally lacking manganese superoxide dismutase. Alteration of the phenotype by prior hypoxia exposure in the SOD2-deficient mutant provide a unique model to study adaptative mechanisms of cellular resistance to oxygen toxicity.

    View details for Web of Science ID 000166872900034

    View details for PubMedID 11156968

  • Oxidative stress and neuronal DNA fragmentation mediate age-dependent vulnerability to the mitochondrial toxin, 3-nitropropionic acid, in the mouse striatum NEUROBIOLOGY OF DISEASE Kim, G. W., Chan, P. H. 2001; 8 (1): 114-126

    Abstract

    Oxidative stress is involved in the neuropathology of several neurodegenerative diseases and stroke, all of which are related to excitotoxicity. Age-dependent vulnerability is characteristic of these conditions. It is not clear whether apoptosis-related neuronal death is involved in age-dependent vulnerability to excitotoxicity. We evaluated whether apoptosis-related neuronal death after treatment with 3-nitropropionic acid (3-NP) is age-dependent in the mouse striatum. We have demonstrated that oxidative stress occurs early after 3-NP treatment and even more so in aged mice. DNA fragmentation with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling staining and gel electrophoresis occurred in an age-dependent fashion. Expression of the DNA repair enzyme, apurinic/apyrimidinic endonuclease, was more attenuated in old mice. Therefore, these results suggest that oxidative stress induces age-dependent neuronal apoptosis in the mouse striatum after 3-NP treatment, which in turn produces an age-dependent vulnerability to 3-NP.

    View details for Web of Science ID 000167069500011

    View details for PubMedID 11162245

  • Reduction of copper,zinc-superoxide dismutase in knockout mice does not affect edema or infarction volumes and the early release of mitochondrial cytochrome c after permanent focal cerebral ischemia BRAIN RESEARCH Fujimura, M., Morita-Fujimura, Y., Copin, J. C., Yoshimoto, T., Chan, P. H. 2001; 889 (1-2): 208-213

    Abstract

    Copper,zinc-superoxide dismutase (SOD1) was shown to be highly protective against ischemia/reperfusion injury in the brain. We have recently reported that SOD1 prevents the release of mitochondrial cytochrome c and subsequent apoptosis after ischemia/reperfusion in mice. To investigate its dose dependent effect on permanent focal cerebral ischemia, we examined neurological deficit scores, infarction volume, and the amount of hemisphere enlargement after 24 h of focal cerebral ischemia in both knockout mutants of SOD1 (Sod1 -/+ and Sod1 -/-) and wild-type littermates. We also examined the release of cytochrome c and subsequent DNA fragmentation after ischemia. There were no differences in the neurological deficit scores, infarction volumes and edema formation. There was also no difference of the amount cytosolic cytochrome c at 2 h and of the amount of DNA fragmentation at 24 h after focal cerebral ischemia. The results indicate that the SOD1 enzyme does not appear to affect cerebral infarction, cerebral edema nor the mitochondrial signaling pathway for apoptosis following permanent focal cerebral ischemia where there is no reperfusion injury.

    View details for Web of Science ID 000166818800024

    View details for PubMedID 11166705

  • Piroxicam and NS-398 rescue neurones from hypoxia/reoxygenation damage by a mechanism independent of cyclo-oxygenase inhibition JOURNAL OF NEUROCHEMISTRY Vartiainen, N., Huang, C. Y., Salminen, A., Goldsteins, G., Chan, P. H., Koistinaho, J. 2001; 76 (2): 480-489

    Abstract

    We studied whether NS-398, a selective cyclo-oxygenase-2 (COX-2) enzyme inhibitor, and piroxicam, an inhibitor of COX-2 and the constitutively expressed COX-1, protect neurones against hypoxia/reoxygenation injury. Rat spinal cord cultures were exposed to hypoxia for 20 h followed by reoxygenation. Hypoxia/reoxygenation increased lactate dehydrogenase (LDH) release, which was inhibited by piroxicam (180-270 microM) and NS-398 (30 microM). Cell counts confirmed the neuroprotection. Western blotting revealed no COX-1 or COX-2 proteins even after hypoxia/reoxygenation. Production of prostaglandin E2 (PGE2), a marker of COX activity, was barely measurable and piroxicam and NS-398 had no effect on the negligible PGE2 production. Hypoxia/reoxygenation increased nuclear factor-kappa B (NF-kappaB) binding activity, which was inhibited by piroxicam but not by NS-398. AP-1 binding activity after hypoxia/reoxygenation was inhibited by piroxicam but strongly enhanced by NS-398. However, both COX inhibitors induced activation of extracellular signal-regulated kinase (ERK) in neurones and phosphorylation of heavy molecular weight neurofilaments, cytoskeletal substrates of ERK. It is concluded that piroxicam and NS-398 protect neurones against hypoxia/reperfusion. The protection is independent of COX activity and not solely explained by modulation of NF-kappaB and AP-1 binding activity. Instead, piroxicam and NS-398-induced phosphorylation through ERK pathway may contribute to the increased neuronal survival.

    View details for Web of Science ID 000166458200017

    View details for PubMedID 11208911

  • The cytosolic antioxidant, copper/zinc superoxide dismutase, attenuates blood-brain barrier disruption and oxidative cellular injury after photothrombotic cortical ischemia in mice NEUROSCIENCE Kim, G. W., Lewen, A., Copin, J. C., Watson, B. D., Chan, P. H. 2001; 105 (4): 1007-1018

    Abstract

    Oxidative stress has been associated with the development of blood-brain barrier disruption and cellular injury after ischemia. The cytosolic antioxidant, copper/zinc superoxide dismutase, has been shown to protect against blood-brain barrier disruption and infarction after cerebral ischemia-reperfusion. However, it is not clear whether copper/zinc superoxide dismutase can protect against evolving ischemic lesions after thromboembolic cortical ischemia. In this study, the photothrombotic ischemia model, which is physiologically similar to thromboembolic stroke, was used to develop cortical ischemia. Blood-brain barrier disruption and oxidative cellular damage were investigated in transgenic mice that overexpress copper/zinc superoxide dismutase and in littermate wild-type mice after photothrombotic ischemia, which was induced by both injection of erythrosin B (30 mg/kg) and irradiation using a helium neon laser for 3 min. Free radical production, particularly superoxide, was increased in the lesioned cortex as early as 4 h after ischemia using hydroethidine in situ detection. The transgenic mice showed a prominent decrease in oxidative stress compared with the wild-type mice. Blood-brain barrier disruption, evidenced by quantitation of Evans Blue leakage, occurred 1 h after ischemia and gradually increased up to 24 h. Compared with the wild-type mice, the transgenic mice showed less blood-brain barrier disruption, a decrease in oxidative DNA damage using 8-hydroxyguanosine immunohistochemistry, a subsequent decrease in DNA fragmentation using the in situ nick-end labeling technique, and decreased infarct volume after ischemia. From these results we suggest that superoxide anion radical is an important factor in blood-brain barrier disruption and oxidative cellular injury, and that copper/zinc superoxide dismutase could protect against the evolving infarction after thromboembolic cortical ischemia.

    View details for Web of Science ID 000171009800021

    View details for PubMedID 11530238

  • Reactive oxygen radicals in signaling and damage in the ischemic brain JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Chan, P. H. 2001; 21 (1): 2-14

    Abstract

    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.

    View details for Web of Science ID 000166016600002

    View details for PubMedID 11149664

  • Neuronal death is an active, caspase-dependent process after moderate but not severe DNA damage JOURNAL OF NEUROCHEMISTRY Gobbel, G. T., Chan, P. H. 2001; 76 (2): 520-531

    Abstract

    Mild insults to neurons caused by ischemia or glutamate induce apoptosis, whereas severe insults induce non apoptotic death, such as necrosis. The molecular targets that are damaged by these insults and ultimately induce cell death are not fully established. To determine if DNA damage can induce apoptotic or non apoptotic death depending on the severity, neurons were treated with up to 128 Gy of ionizing radiation. Such treatment induced a dose-related increase in DNA single-strand breaks but no immediate membrane disruption or lipid peroxidation. Following moderate doses of < or = 32 Gy, neuronal death had many characteristics of apoptosis including nuclear fragmentation and DNA laddering. Nuclear fragmentation and membrane breakdown after moderate DNA damage could be blocked by inhibition of active protein synthesis with cycloheximide and by inhibition of caspases. In contrast, cell death after doses of > 32 Gy was not blocked by cycloheximide or caspase inhibitors, and membrane breakdown occurred relatively early in the cell death process. These data suggest that cell death after high dose irradiation and severe DNA damage can occur by non apoptotic mechanisms and that blocking apoptotic pathways may not prevent death after severe damage.

    View details for Web of Science ID 000166458200021

    View details for PubMedID 11208915

  • Involvement of oxidative stress and caspase-3 in cortical infarction after photothrombotic ischemia in mice JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Kim, G. W., Sugawara, T., Chan, P. H. 2000; 20 (12): 1690-1701

    Abstract

    Apoptosis-related cell death is linked to oxidative stress and caspases in experimental cerebral ischemia. However, the role of oxidative stress in caspase activation and subsequent apoptotic cell death after cerebral ischemia is unknown. The authors evaluated the role of oxidative stress in ischemic cerebral infarction after photothrombosis and the relation between oxidative stress and caspase-related cell death 6 and 24 hours after ischemia with and without U-74389G, a potent free radical scavenger (10 mg/kg, 30 minutes before and after ischemia induction). Reactive oxygen species, detected by hydroethidine oxidation, and cytosolic cytochrome c were detected in early ischemic lesions. Western blot analysis showed the cleaved form and the increased level of the proform of caspase-3 in the ischemic lesion 24 hours after ischemia. Decreased caspase-3 immunoreactivity was detected in the antioxidant-treated group after ischemia. Decreased DNA fragmentation and laddering were detected and the lesion was smaller in the treated group after ischemia compared with the untreated group. Oxidative stress and cytochrome c release occur in the ischemic lesion after photothrombotic ischemia. The free radical scavenger attenuated caspase-3 up-regulation, DNA fragmentation, and the final lesion. The authors concluded that oxidative stress may mediate caspase-related apoptotic cell death and subsequent cortical infarction after photothrombotic ischemia.

    View details for Web of Science ID 000165696700008

    View details for PubMedID 11129785

  • Free radical pathways in CNS injury JOURNAL OF NEUROTRAUMA Lewen, A., Matz, P., Chan, P. H. 2000; 17 (10): 871-890

    Abstract

    Free radicals are highly reactive molecules implicated in the pathology of traumatic brain injury and cerebral ischemia, through a mechanism known as oxidative stress. After brain injury, reactive oxygen and reactive nitrogen species may be generated through several different cellular pathways, including calcium activation of phospholipases, nitric oxide synthase, xanthine oxidase, the Fenton and Haber-Weiss reactions, by inflammatory cells. If cellular defense systems are weakened, increased production of free radicals will lead to oxidation of lipids, proteins, and nucleic acids, which may alter cellular function in a critical way. The study of each of these pathways may be complex and laborious since free radicals are extremely short-lived. Recently, genetic manipulation of wild-type animals has yielded species that over- or under-express genes such as, copper-zinc superoxide dismutase, manganese superoxide dismutase, nitric oxide synthase, and the Bcl-2 protein. The introduction of the species has improved the understanding of oxidative stress. We conclude here that substantial experimental data links oxidative stress with other pathogenic mechanisms such as excitotoxicity, calcium overload, mitochondrial cytochrome c release, caspase activation, and apoptosis in central nervous system (CNS) trauma and ischemia, and that utilization of genetically manipulated animals offers a unique possibility to elucidate the role of free radicals in CNS injury in a molecular fashion.

    View details for Web of Science ID 000089894300007

    View details for PubMedID 11063054

  • Cell death after exposure to subarachnoid hemolysate correlates inversely with expression of CuZn-Superoxide dismutase STROKE Matz, P. G., Copin, J. C., Chan, P. H. 2000; 31 (10): 2450-2458

    Abstract

    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.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.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.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.

    View details for Web of Science ID 000089655900029

    View details for PubMedID 11022079

  • Effect of hypotension severity on hippocampal CA1 neurons in a rat global ischemia model BRAIN RESEARCH Sugawara, T., Kawase, M., Lewen, A., Noshita, N., Gasche, Y., Fujimura, M., Chan, P. H. 2000; 877 (2): 281-287

    Abstract

    Neuronal death in the hippocampal CA1 subregion has been shown to occur in a delayed manner after transient global ischemia. The 2-vessel occlusion model is one of the most frequently used global ischemia paradigms in rodents. Although researchers often fail to induce bilateral delayed CA1 neuronal death, the importance of hypotension severity has not been fully discussed. We induced 10 min of global ischemia with 2-vessel occlusion and various severities of hypotension in rats, and the subsequent neuronal damage and neurogenesis in the hippocampal CA1 pyramidal cell layer were immunohistochemically studied. Neuronal apoptosis after global ischemia was also characterized by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL). The mean arterial blood pressure of 31-35 mmHg was the most appropriate range of hypotension in this model because of low mortality and consistent bilateral CA1 injury. Most of the neurons in the CA1 pyramidal cell layer lost neuron specific nuclear protein and became TUNEL-positive 3 days after ischemia. There was no evidence of apoptosis or neurogenesis at 7-28 days. There were ischemia-tolerant neurons in the CA1 pyramidal cell layer that survived delayed neurodegeneration, however, further studies are necessary to characterize the property of these neurons.

    View details for Web of Science ID 000089388500019

    View details for PubMedID 10986342

  • Spreading depression-induced expression of c-fos and cyclooxygenase-2 in transgenic mice that overexpress human copper/zinc-superoxide dismutase JOURNAL OF NEUROTRAUMA Yrjanheikki, J., Koistinaho, J., Copin, J. C., de Crespigny, A., Moseley, M. E., Chan, P. H. 2000; 17 (8): 713-718

    Abstract

    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.

    View details for Web of Science ID 000088951600007

    View details for PubMedID 10972246

  • Reduction of the DNA base excision repair protein, XRCC1, may contribute to DNA fragmentation after cold injury-induced brain trauma in mice BRAIN RESEARCH Fujimura, M., Morita-Fujimura, Y., Noshita, N., Yoshimoto, T., Chan, P. H. 2000; 869 (1-2): 105-111

    Abstract

    The X-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in the DNA base excision repair pathway by interacting with DNA ligase III and DNA polymerase beta. The present study examined the protein expression of XRCC1 and DNA fragmentation before and after cold injury-induced brain trauma (CIBT) in mice, in which apoptosis is assumed to participate. Immunohistochemistry showed the nuclear expression of XRCC1 in the entire region of the control brains. Fifteen minutes after CIBT, nuclear immunoreactivity was predominantly decreased in the inner boundary of the lesion, followed by a significant reduction of XRCC1 in the entire lesion 4 h after CIBT. A characteristic 70-kDa band was detected in the non-traumatic area, and was markedly decreased after CIBT as shown by Western blot analysis. DNA fragmentation was also observed after CIBT, and double staining with XRCC1 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed a spatial relationship between XRCC1 loss and DNA fragmentation 24 h after CIBT. These data indicate that early decrease of XRCC1 and failure of the DNA repair mechanism may contribute to DNA-damaged neuronal cell death after CIBT.

    View details for Web of Science ID 000087990300013

    View details for PubMedID 10865064

  • Overexpression of copper/zinc superoxide dismutase does not prevent neonatal lethality in mutant mice that lack manganese superoxide dismutase FREE RADICAL BIOLOGY AND MEDICINE Copin, J. C., Gasche, Y., Chan, P. H. 2000; 28 (10): 1571-1576

    Abstract

    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.

    View details for Web of Science ID 000088732700017

    View details for PubMedID 10927183

  • Spreading depression-induced cyclooxygenase-2 expression in the cortex NEUROCHEMICAL RESEARCH Koistinaho, J., Chan, P. H. 2000; 25 (5): 645-651

    Abstract

    Spreading depression (SD) is a wave of sustained depolarization challenging the energy metabolism of the cells without causing irreversible damage. However, brain injury, especially focal ischemic stroke, triggers SD-like waves, which in the vicinity of the original damage site contribute to enlargement of the dying brain tissue. Brain injury induces expression of several genes, which are thought to play a role in neuronal death, and therefore represent potential targets for therapy. One such gene is cyclooxygenase-2 (COX-2), an inducible prostaglandin and superoxide producing enzyme. Here we review our recent studies on the regulation of COX-2 in SD.

    View details for Web of Science ID 000087857300014

    View details for PubMedID 10905626

  • The cytosolic antioxidant copper/zinc-superoxide dismutase prevents the early release of mitochondrial cytochrome c in ischemic brain after transient focal cerebral ischemia in mice JOURNAL OF NEUROSCIENCE Fujimura, M., Morita-Fujimura, Y., Noshita, N., Sugawara, T., Kawase, M., Chan, P. H. 2000; 20 (8): 2817-2824

    Abstract

    Release of mitochondrial cytochrome c into the cytosol is a critical step in apoptosis. We have reported that early release of cytochrome c in vivo occurs after permanent focal cerebral ischemia (FCI) and is mediated by the mitochondrial antioxidant manganese superoxide dismutase (SOD). However, the role of reactive oxygen species produced after ischemia-reperfusion in the mitochondrial apoptosis process is still unknown, although overexpression of copper/zinc-SOD (SOD1), a cytosolic isoenzyme, protects against ischemia-reperfusion. We now hypothesize that the overexpression of SOD1 also prevents apoptosis after FCI. To address this issue, we examined the subcellular distribution of the cytochrome c protein in both wild-type mice and in SOD1 transgenic (Tg) mice after transient FCI. Cytosolic cytochrome c was detected as early as 2 hr after reperfusion, and correspondingly, mitochondrial cytochrome c was significantly reduced after FCI. Cytosolic cytochrome c was significantly lower in the SOD1 Tg mice compared with wild types 2 (p < 0.0001) and 4 (p < 0.05) hr after FCI. Apaf-1, which interacts with cytochrome c and activates caspases, was constitutively expressed in both groups of animals, with no alteration after FCI. Double staining with cytochrome c immunohistochemistry and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed a spatial relationship between cytosolic cytochrome c expression and DNA fragmentation. A significant amount of DNA laddering was detected 24 hr after ischemia and was reduced in SOD1 Tg mice. These data suggest that SOD1 blocks cytosolic release of cytochrome c and could thereby reduce apoptosis after transient FCI.

    View details for Web of Science ID 000086412100011

    View details for PubMedID 10751433

  • Subarachnoid hemolysate produces DNA fragmentation in a pattern similar to apoptosis in mouse brain BRAIN RESEARCH Matz, P. G., Fujimura, M., Chan, P. H. 2000; 858 (2): 312-319

    Abstract

    Stroke and traumatic brain/spinal cord injuries are often associated with hemorrhage. Despite the relative frequency of hemorrhage in the central nervous system (CNS), little is known about what role blood and hemoglobin (Hb) play in mediating cellular injury. Since Hb and hemolysate have been associated with generation of oxidative stress and cell injury, we examined whether apoptosis was present after cortical exposure to subarachnoid hemolysate. Subarachnoid hemorrhage (SAH) was induced in CD-1 mice (n=25) by injection of 50 microl of autologous hemolysate over the right parietal cortex. Saline-injected mice (n=13) were used as controls. Subjects were sacrificed at 24 h. Transcardiac perfusion fixation was performed on a subgroup of hemolysate- (n=15) and saline-injected (n=9) animals. Sections were stained for DNA fragmentation using the terminal deoxyuridine nick end-labeling (TUNEL) method and also immunostained for the hemeoxygenase-1 (HO-1) protein to assess blood distribution. In the remaining animals (n=6 SAH, n=4 saline), DNA was extracted and precipitated from 40 mg of tissue and subjected to electrophoresis on a 1.5% agarose gel. DNA fragmentation was evident on TUNEL staining in 10/15 subjects injected with hemolysate as compared to 0/9 subjects injected with saline (p<0.01, Fisher exact test). TUNEL-positive cells were most abundant closest to the site of cortical SAH, as evidenced by HO-1 immunoreactivity. TUNEL-positive cells were also seen remotely in the hippocampus and basal forebrain. The presence of apoptosis was suggested by DNA laddering on electrophoresis in the hemolysate-injected subjects (4/6 animals). No laddering was evident in saline-injected subjects (n=4). These results provide evidence that the presence of subarachnoid blood products is associated with DNA fragmentation and apoptotic cell death.

    View details for Web of Science ID 000085814800009

    View details for PubMedID 10708682

  • Aquaporin-4 deletion in mice reduces brain edema after acute water intoxication and ischemic stroke NATURE MEDICINE Manley, G. T., Fujimura, M., Ma, T. H., Noshita, N., Filiz, F., Bollen, A. W., Chan, P., Verkman, A. S. 2000; 6 (2): 159-163

    Abstract

    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.

    View details for Web of Science ID 000085016900035

    View details for PubMedID 10655103

  • Excitotoxicity is required for induction of oxidative stress and apoptosis in mouse striatum by the mitochondrial toxin, 3-nitropropionic acid JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Kim, G. W., Copin, J. C., Kawase, M., Chen, S. F., Sato, S., Gobbel, G. T., Chan, P. H. 2000; 20 (1): 119-129

    Abstract

    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.

    View details for Web of Science ID 000085169600016

    View details for PubMedID 10616800

  • Overexpression of copper and zinc superoxide dismutase in transgenic mice prevents the induction and activation of matrix metalloproteinases after cold injury-induced brain trauma JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Morita-Fujimura, Y., Fujimura, M., Gasche, Y., Copin, J. C., Chan, P. H. 2000; 20 (1): 130-138

    Abstract

    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.

    View details for Web of Science ID 000085169600017

    View details for PubMedID 10616801

  • Inhibition of inflammation and neuroprotection by tetracyclines PHARMACOLOGY OF CEREBRAL ISCHEMIA 2000 Yrjanheikki, J., Tikka, T., Goldsteins, G., Koistinaho, M., KEINANEN, R., Chan, P. H., Koistinaho, J. 2000: 267-274
  • Neuroprotective effects of an antioxidant in cortical cerebral ischemia: prevention of early reduction of the apurinic/apyrimidinic endonuclease DNA repair enzyme NEUROSCIENCE LETTERS Chang, Y. Y., Fujimura, M., Morita-Fujimura, Y., Kim, G. W., Huang, C. Y., Wu, H. S., Kawase, M., Copin, J. C., Chan, P. H. 1999; 277 (1): 61-64

    Abstract

    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.

    View details for Web of Science ID 000084345000016

    View details for PubMedID 10643898

  • Mitochondrial release of cytochrome c corresponds to the selective vulnerability of hippocampal CA1 neurons in rats after transient global cerebral ischemia JOURNAL OF NEUROSCIENCE Sugawara, T., Fujimura, M., Morita-Fujimura, Y., Kawase, M., Chan, P. H. 1999; 19 (22)

    Abstract

    Release of cytochrome c from mitochondria to the cytosol is a critical step in apoptotic cell death after focal cerebral ischemia. The relationship among cytochrome c release, selective vulnerability, and delayed death of hippocampal CA1 neurons after transient global ischemia was examined. Global ischemia was induced by 10 min of bilateral common carotid artery occlusion and hypotension in rats. Cytosolic expression of cytochrome c was evaluated by immunohistochemistry and Western blotting. Apoptosis after global ischemia was also characterized by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) staining and DNA gel electrophoresis. Immunohistochemistry showed cytosolic cytochrome c-positive cells exclusively in the CA1 subregion of the hippocampus as early as 2 hr after ischemia. Double fluorescent immunostaining confirmed that CA1 neurons and a small number of astrocytes expressed cytochrome c. Western blot analysis revealed a band (15 kDa) of cytochrome c in the cytosolic fraction and a corresponding decrease in the mitochondrial fraction. A significant number of TUNEL-positive cells appeared only in the CA1 pyramidal cell layer of the hippocampus, and DNA gel electrophoresis showed a significant amount of DNA fragmentation 3-5 d after ischemia. Our data provide the first evidence that cytochrome c was released to the cytosol from mitochondria in CA1 neurons after global ischemia and that the release preceded DNA fragmentation. These findings suggest cytochrome c involvement in the delayed death of hippocampal CA1 neurons in rats after transient global ischemia.

    View details for Web of Science ID 000083607700002

    View details for PubMedID 10559429

  • A tetracycline derivative, minocycline, reduces inflammation and protects against focal cerebral ischemia with a wide therapeutic window PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yrjanheikki, J., Tikka, T., KEINANEN, R., Goldsteins, G., Chan, P. H., Koistinaho, J. 1999; 96 (23): 13496-13500

    Abstract

    The only treatment of patients with acute ischemic stroke is thrombolytic therapy, which benefits only a fraction of stroke patients. Both human and experimental studies indicate that ischemic stroke involves secondary inflammation that significantly contributes to the outcome after ischemic insult. Minocycline is a semisynthetic second-generation tetracycline that exerts antiinflammatory effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against focal brain ischemia with a wide therapeutic window. Using a rat model of transient middle cerebral artery occlusion, we show that daily treatment with minocycline reduces cortical infarction volume by 76 +/- 22% when the treatment is started 12 h before ischemia and by 63 +/- 35% when started even 4 h after the onset of ischemia. The treatment inhibits morphological activation of microglia in the area adjacent to the infarction, inhibits induction of IL-1beta-converting enzyme, and reduces cyclooxygenase-2 expression and prostaglandin E(2) production. Minocycline had no effect on astrogliosis or spreading depression, a wave of ionic transients thought to contribute to enlargement of cortical infarction. Treatment with minocycline may act directly on brain cells, because cultured primary neurons were also salvaged from glutamate toxicity. Minocycline may represent a prototype of an antiinflammatory compound that provides protection against ischemic stroke and has a clinically relevant therapeutic window.

    View details for Web of Science ID 000083649400094

    View details for PubMedID 10557349

  • Early decrease of XRCC1, a DNA base excision repair protein, may contribute to DNA fragmentation after transient focal cerebral ischemia in mice STROKE Fujimura, M., Morita-Fujimura, Y., Sugawara, T., Chan, P. H. 1999; 30 (11): 2456-2462

    Abstract

    DNA damage and the DNA repair mechanism are known to be involved in ischemia/reperfusion injury in the brain. The x-ray repair cross-complementing group 1 (XRCC1) protein plays a central role in the DNA base excision repair pathway by interacting with DNA ligase III and DNA polymerase beta. The present study examined the protein expression of XRCC1 and DNA fragmentation before and after transient focal cerebral ischemia (FCI).Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal blockade of the middle cerebral artery. XRCC1 protein expression was analyzed by immunohistochemistry and Western blot analysis. DNA damage was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL). The spatial relationship between XRCC1 expression and DNA damage was examined by double staining with XRCC1 and TUNEL after FCI.Immunohistochemistry showed the nuclear expression of XRCC1 in all regions of the control brains and that it was predominant in the hippocampus. The XRCC1 level was markedly reduced in the caudate putamen at 10 minutes, further decreased in the entire middle cerebral artery territory at 1 hour, and remained reduced until 4 and 24 hours after FCI. Western blot analysis of the normal control brain showed a characteristic band of 70 kDa, which decreased after FCI. A significant amount of DNA fragmentation was detected by DNA gel electrophoresis 24 hours but not 4 hours after FCI. Double staining showed that the neurons that lost XRCC1 immunoreactivity became TUNEL positive.These results suggest that the early decrease of XRCC1 and the failure of the DNA repair mechanism may contribute, at least in part, to DNA fragmentation after FCI.

    View details for Web of Science ID 000083434100039

    View details for PubMedID 10548684

  • Copper-zinc superoxide dismutase prevents the early decrease of apurinic/apyrimidinic endonuclease and subsequent DNA fragmentation after transient focal cerebral ischemia in mice STROKE Fujimura, M., Morita-Fujimura, Y., Narasimhan, P., Copin, J. C., Kawase, M., Chan, P. H. 1999; 30 (11): 2408-2415

    Abstract

    DNA damage and its repair mechanism are thought to be involved in ischemia/reperfusion injury in the brain. We have previously shown that apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, rapidly decreased after transient focal cerebral ischemia (FCI) before the peak of DNA fragmentation. To further investigate the role of reactive oxygen species in APE/Ref-1 expression in vivo, we examined the expression of APE/Ref-1 and DNA damage after FCI in wild-type and transgenic mice overexpressing copper-zinc superoxide dismutase.Transgenic mice overexpressing copper-zinc superoxide dismutase and wild-type littermates were subjected to 60 minutes of transient FCI by intraluminal blockade of the middle cerebral artery. APE/Ref-1 protein expression was analyzed by immunohistochemistry and Western blot analysis. DNA damage was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL).A similar level of APE/Ref-1 was detected in the control brains from both groups. APE/Ref-1 was significantly reduced 1 hour after transient FCI in both groups, whereas the transgenic mice had less reduction than that seen in wild-type mice 1 and 4 hours after FCI. DNA laddering was detected 24 hours after FCI and was decreased in transgenic mice. Double staining with APE/Ref-1 and TUNEL showed that the neurons that lost APE/Ref-1 immunoreactivity became TUNEL positive.These results suggest that reactive oxygen species contribute to the early decrease of APE/Ref-1 and thereby exacerbate DNA fragmentation after transient FCI in mice.

    View details for Web of Science ID 000083434100027

    View details for PubMedID 10548678

  • Early appearance of activated matrix metalloproteinase-9 and blood-brain barrier disruption in mice after focal cerebral ischemia and reperfusion BRAIN RESEARCH Fujimura, M., Gasche, Y., Morita-Fujimura, Y., Massengale, J., Kawase, M., Chan, P. H. 1999; 842 (1): 92-100

    Abstract

    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.

    View details for Web of Science ID 000082809300011

    View details for PubMedID 10526099

  • Early appearance of activated matrix metalloproteinase-9 after focal cerebral ischemia in mice: A possible role in blood-brain barrier dysfunction JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Gasche, Y., Fujimura, M., Morita-Fujimura, Y., Copin, J. C., Kawase, M., Massengale, J., Chan, P. H. 1999; 19 (9): 1020-1028

    Abstract

    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.

    View details for Web of Science ID 000084883700010

    View details for PubMedID 10478654

  • Exacerbation of delayed cell injury after transient global ischemia in mutant mice with CuZn superoxide dismutase deficiency STROKE Kawase, M., Murakami, K., Fujimura, M., Morita-Fujimura, Y., Gasche, Y., Kondo, T., Scott, R. W., Chan, P. H. 1999; 30 (9): 1962-1968

    Abstract

    We have demonstrated that copper-zinc superoxide dismutase (CuZn-SOD), a cytosolic isoenzyme of SODs, has a protective role in the pathogenesis of superoxide radical-mediated brain injury. Using mice bearing a disruption of the CuZn-SOD gene (Sod1), the present study was designed to clarify the role of superoxide anion in the pathogenesis of selective vulnerability after transient global ischemia.Sod1 knockout homozygous mutant mice (Sod1 -/-) with a complete absence of endogenous CuZn-SOD activity, heterozygous mutant mice (Sod1 +/-) with a 50% decrease in the activity, and littermate wild-type mice (male, 35 to 45 g) were subjected to global ischemia. Since the plasticity of the posterior communicating artery (PcomA) has been reported to influence the outcome of hippocampal injury, we assessed the relation between the plasticity of PcomAs and the decrease of regional cerebral blood flow in global ischemia.The fluorescence intensity of hydroethidine oxidation, a measurement of ethidium fluorescence for superoxide radicals, was increased in mutant mice 1 day after both 5 and 10 minutes of global ischemia, compared with wild-type mice. Hippocampal injury in the PcomA hypoplastic brains showed significant exacerbation in mutant mice compared with wild-type littermates 3 days after 5 minutes of global ischemia, although a marked difference was not observed at 1 day.These data suggest that superoxide radicals play an important role in the pathogenesis of delayed injury in the vulnerable hippocampal CA1 subregion after transient global ischemia.

    View details for Web of Science ID 000082278400043

    View details for PubMedID 10471451

  • Expression of cyclooxygenase-2 mRNA after global ischemia is regulated by AMPA receptors and glucocorticoids STROKE Koistinaho, J., Koponen, S., Chan, P. H. 1999; 30 (9): 1900-1905

    Abstract

    Cyclooxygenase-2 (COX-2) is implicated in ischemic neuronal death. In focal ischemia, its mRNA induction is mediated through N-methyl-D-aspartic acid (NMDA) receptors and phospholipase A(2). Because mechanisms of neuronal death involving COX-2 in global ischemia are unclear, we studied the time course and regulation of COX-2 expression in rat brain global ischemia.Global ischemia was induced by a 4-vessel occlusion method. COX-2 mRNA levels were demonstrated with in situ hybridization and COX-2 protein with immunocytochemistry. Several animals were pretreated with MK-801, an NMDA receptor antagonist; 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX), an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist; and dexamethasone.In the cortex, the CA3 hippocampal region and dentate gyrus expression of COX-2 mRNA peaked at 4 to 8 hours, while in the CA1 region COX-2 mRNA levels were high at 4 to 24 hours. COX-2 protein was induced in the corresponding regions at 12 to 24 hours, but in the CA1 neurons the protein was still seen at 3 days. COX-2 mRNA induction in the cortex was inhibited by NBQX and dexamethasone and in CA1 neurons was inhibited by NBQX. MK-801 did not suppress COX-2 induction.COX-2 is differentially induced in the cortex and hippocampal structures after global ischemia. The prolonged COX-2 expression in the vulnerable CA1 neurons is regulated by AMPA receptors, suggesting that COX-2 expression is likely to be associated with AMPA receptor-mediated neuronal death in global ischemia. Glucocorticoids may not be efficiently used to inhibit ischemia-induced COX-2 expression in the hippocampus.

    View details for Web of Science ID 000082278400027

    View details for PubMedID 10471443

  • Release of mitochondrial cytochrome c and DNA fragmentation after cold injury-induced brain trauma in mice: possible role in neuronal apoptosis NEUROSCIENCE LETTERS Morita-Fujimura, Y., Fujimura, M., Kawase, M., Chen, S. F., Chan, P. H. 1999; 267 (3): 201-205

    Abstract

    Recent studies have shown that release of mitochondrial cytochrome c is a critical step in the apoptosis process. In this study, we examined the subcellular distribution of the cytochrome c protein after cold injury (CI), in which apoptosis is assumed to participate. Western blotting and immunohistochemistry showed cytosolic cytochrome c as early as 1 h after CI, and correspondingly, there was a reduction in mitochondrial cytochrome c after injury. Neuronal distribution of cytosolic cytochrome c was shown by double staining with a neuronal nuclear marker by immunohistochemistry. A significant amount of DNA laddering was detected 4 h after CI, and increased in a time-dependent manner. These data suggest that early cytochrome c release from mitochondria may contribute to apoptosis induction after traumatic brain injury.

    View details for Web of Science ID 000080653600014

    View details for PubMedID 10381011

  • Inhibition of interleukin-1 beta converting enzyme family proteases (caspases) reduces cold injury-induced brain trauma and DNA fragmentation in mice JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Morita-Fujimura, Y., Fujimura, M., Kawase, M., Murakami, K., Kim, G. W., Chan, P. H. 1999; 19 (6): 634-642

    Abstract

    The authors examined the effect of z-VAD.FMK, an inhibitor that blocks caspase family proteases, on cold injury-induced brain trauma, in which apoptosis as well as necrosis is assumed to play a role. A vehicle alone or with z-VAD.FMK was administered into the cerebral ventricles of mice 15 minutes before and 24 and 48 hours after cold injury. At 24 hours after cold injury, infarction volumes in the z-VAD.FMK-treated animals were significantly smaller than infarction volumes in the vehicle-treated animals, and were further decreased at 72 hours (0.92 +/- 1.80 mm3, z-VAD.FMK-treated animals; 7.46 +/- 3.53 mm3, vehicle-treated animals; mean +/- SD, n = 7 to 8). The amount of DNA fragmentation was significantly decreased in the z-VAD.FMK-treated animals compared with the vehicle-treated animals, as shown by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling staining and DNA gel electrophoresis. By Western blot analysis, both the proform and activated form of interleukin-1beta converting enzyme (caspase 1) were detected in the control brain, and the activated form showed moderate reduction after cold injury-induced brain trauma. These results indicate that caspase inhibitors could reduce cold injury-induced brain trauma by preventing neuronal cell death by DNA damage. The caspase family proteases appear to contribute to the mechanisms of cell death in cold injury-induced brain trauma and to provide therapeutic targets for traumatic brain injury.

    View details for Web of Science ID 000084948700006

    View details for PubMedID 10366193

  • Manganese superoxide dismutase mediates the early release of mitochondrial cytochrome C and subsequent DNA fragmentation after permanent focal cerebral ischemia in mice JOURNAL OF NEUROSCIENCE Fujimura, M., Morita-Fujimura, Y., Kawase, M., Copin, J. C., Calagui, B., Epstein, C. J., Chan, P. H. 1999; 19 (9): 3414-3422

    Abstract

    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 vivo occurred 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 c in 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.

    View details for Web of Science ID 000079883700015

    View details for PubMedID 10212301

  • Early decrease of apurinic/apyrimidinic endonuclease expression after transient focal cerebral ischemia in mice JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Fujimura, M., Morita-Fujimura, Y., Kawase, M., Chan, P. H. 1999; 19 (5): 495-501

    Abstract

    The authors examined the protein expression of apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, before and after transient focal ischemia in mice. Immunohistochemistry showed the nuclear expression of APE/Ref-1 in the entire region of the control brains. Nuclear immunoreactivity was decreased as early as 5 minutes after 60 minutes of ischemia in the ischemic core, which was followed by a significant reduction of APE/Ref-1-positive cells in the entire middle cerebral artery territory. Western blot analysis of the sample from the nonischemic brain showed a characteristic 37-kDa band, which was reduced after ischemia. A significant amount of DNA fragmentation was observed at 24 hours, but not at 4 hours, after ischemia. The authors' data provide the first evidence that APE/Ref-1 rapidly decreases after transient focal ischemia, and that this reduction precedes the peak of DNA fragmentation in the brain regions that are destined to show necrosis and apoptosis. Although further examination is necessary to elucidate the direct relationship between the APE/Ref-1 decrease and ischemic necrosis and apoptosis, our results suggest the possibility that rapid decrease of APE/Ref-1 and the failure of the DNA repair mechanism may contribute to necrosis or apoptosis after transient focal ischemia.

    View details for Web of Science ID 000084948600003

    View details for PubMedID 10326716

  • Cold injury in mice: A model to study mechanisms of brain edema and neuronal apoptosis PROGRESS IN NEUROBIOLOGY Murakami, K., Kondo, T., Yang, G. Y., Chen, S. F., Morita-Fujimura, Y., Chan, P. H. 1999; 57 (3): 289-299

    Abstract

    Small rodents, mice in particular, have been widely used for genetic manipulation because of the extensive knowledge in development, embryology and other molecular aspects of this species. However, the use of mice for neurobiology research in the area of brain edema and neuronal injury has not been common. Here we summarize the studies of cold injury-induced brain edema and neuronal apoptosis using mice. Blood-brain barrier (BBB) permeability, demonstrated by extravasation of a serum albumin tracer, Evans Blue, was increased immediately after the injury and returned to the control level by 24 hr. Water content was maximized at 24 hr, whereas a secondary lesion gradually progressed up to 72 hr after cold injury. The mechanism of the development of the cold injury-induced edema and the secondary lesion, involving of oxygen radicals in particular, was determined using superoxide dismutase (SOD)-1 transgenic (Tg) mice with overexpressed copper, zinc-SOD. All of the parameters, BBB permeability, water content and secondary lesion, were attenuated in the Tg mice as compared to littermate non-Tg mice. This clearly demonstrates that oxygen radicals, superoxide anion in particular, mediate cold injury. We also studied whether apoptosis contributes to brain injury following cold injury. Staining with terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed the apoptotic cells widespread throughout the entire lesion while still remaining in the margin. DNA laddering was exhibited by gel electrophoresis. These studies indicate that oxidative mediates the development of cold injury-induced edema and the secondary injury, and induces apoptotic cell death. We believe that cold injury in mice provides a simple animal model to study the pathogenesis of brain edema and apoptosis in genetically altered animals.

    View details for Web of Science ID 000077641000001

    View details for PubMedID 10096842

  • Reduction of apurinic/apyrimnidinic endonuclease expression after transient global cerebral ischemia in rats - Implication of the failure of DNA repair in neuronal apoptosis STROKE Kawase, M., Fujimura, M., Morita-Fujimura, Y., Chan, P. H. 1999; 30 (2): 441-448

    Abstract

    To clarify the relationship between apurinic/apyrimidinic endonuclease (APE/Ref-1), a multifunctional protein in the DNA base excision repair pathway, and delayed neuronal cell death associated with apoptosis, we examined the expression of APE/Ref-1 before and after transient global ischemia in rats.Global ischemia was induced by bilateral common carotid artery occlusion and hypotension. Expression of the APE/Ref-1 protein was evaluated by Western blot and immunohistochemical analyses. Apoptosis after global ischemia was observed by DNA electrophoresis and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining.Immunohistochemistry showed the nuclear expression of APE/Ref-1 in the control brains. Nuclear immunoreactivity of APE/Ref-1 was significantly decreased 2 days after 10 minutes of ischemia in the hippocampal CA1 subregion. Western blot analysis of a sample from the normal brains showed a characteristic 37-kDa band, which was reduced in the hippocampal CA1 subregion after ischemia. A significant amount of DNA fragmentation was observed at 3 days but not at 1 day after ischemia. Double staining with APE/Ref-1 and TUNEL clearly showed that the neurons that lost APE/Ref-1 immunoreactivity became TUNEL positive.Our data provide evidence that APE/Ref-1 decreased in hippocampal CA1 neurons after transient global ischemia and that this reduction precedes DNA fragmentation, which is destined to cause apoptosis. Our results suggest the possibility that a decrease of APE/Ref-1 activity and the failure of DNA repair may underlie the mechanism of apoptosis after transient focal ischemia.

    View details for Web of Science ID 000078381600030

    View details for PubMedID 9933285

  • Early decrease in apurinic/apyrimidinic endonuclease is followed by DNA fragmentation after cold injury-induced brain trauma in mice NEUROSCIENCE Morita-Fujimura, Y., Fujimura, M., Kawase, M., Chan, P. H. 1999; 93 (4): 1465-1473

    Abstract

    Apurinic/apyrimidinic endonuclease, a multifunctional protein in the DNA base excision repair pathway, plays a central role in repairing DNA damage caused by reactive oxygen species. We examined protein expression of apurinic/apyrimidinic endonuclease before and after cold injury-induced brain trauma in mice, where we have previously shown reactive oxygen species to participate. Immunohistochemistry showed the nuclear expression of apurinic/apyrimidinic endonuclease in the entire region of control brains. One hour after cold injury-induced brain trauma, nuclear immunoreactivity was predominantly decreased in the inner boundary of the lesion, whereas there was a slight increase in the outer boundary area. Four hours after cold injury-induced brain trauma, nuclear immunoreactivity was almost absent in the entire lesion, and remained so until 24 h. At this time, a marked increase in apurinic/apyrimidinic endonuclease immunoreactivity was seen in the outer boundary zone. Western blot analysis of the sample from the non-ischemic area showed a characteristic 37,000 mol. wt band, which decreased markedly 24 h after cold injury-induced brain trauma. A time-dependent increase in DNA fragmentation was also observed after cold injury-induced brain trauma. Our data provide the first evidence that apurinic/apyrimidinic endonuclease decreased rapidly in the lesion after cold injury-induced brain trauma, whereas it was significantly increased at the outer boundary zone. Although further examination is necessary to elucidate the direct relationship between apurinic/apyrimidinic endonuclease alteration and the pathogenesis of cold injury-induced brain trauma, our results suggest the possibility that an early decrease in apurinic/apyrimidinic endonuclease and failure of the DNA repair mechanism may contribute to DNA-damaged neuronal cell death after cold injury-induced brain trauma.

    View details for Web of Science ID 000082436000028

    View details for PubMedID 10501471

  • Glutamatergic receptors regulate expression, phosphorylation and accumulation of neurofilaments in spinal cord neurons NEUROSCIENCE Vartiainen, N., Tikka, T., KEINANEN, R., Chan, P. H., Koistinaho, J. 1999; 93 (3): 1123-1133

    Abstract

    Glutamatergic regulation of neurofilament expression, phosphorylation and accumulation in cultured spinal cord neurons was studied. At seven days in culture, 0.15% of the neurons were immunoreactive for non-phosphorylated neurofilaments, but essentially no cells immunoreactive for phosphorylated neurofilaments were seen. The number and size of the immunoreactive cells in culture corresponded well to those of rat and human spinal cord neurons in vivo. In spinal cord cultures, sublethal, long-lasting stimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate or metabotrophic receptors, but not N-methyl-D-aspartate receptors, dose-dependently increased the number of non-phosphorylated neurofilament-immunoreactive cells, which was blocked by nifedipine, an antagonist of voltage-sensitive Ca2+ channels. Stimulation of kainate or all non-N-methyl-D-aspartate receptors decreased the expression of medium-molecular-weight neurofilament messenger RNA. Blockade of AMPA/kainate receptors, but not of N-methyl-D-aspartate receptors, increased the amount of phosphorylated neurofilament protein and the number of phosphorylated neurofilament-immunoreactive cell bodies. The phosphorylated neurofilament-immunoreactive cell population was different from the non-phosphorylated neurofilament-immunoreactive neurons, which lost their axonal non-phosphorylated neurofilament immunoreactivity but showed intense cytoplasmic labeling in response to the blockade of AMPA/ kainate receptors. Immunoreactivity for phosphoserine did not change upon glutamate receptor stimulation and blockade. The results show that activation of AMPA/kainate receptors decreases the expression of neurofilament messenger RNA and neurofilament phosphorylation in spinal cord neurons by a mechanism involving active voltage-sensitive Ca2+ channels. Blockade of these receptors seems to disturb axonal neurofilament transport. Because AMPA/kainate receptors mediate chronic glutamatergic death of spinal motor neurons and these receptors have been suggested to be involved in the pathogenesis of amyotrophic lateral sclerosis, the observed alteration in neurofilament phosphorylation and distribution may contribute to the pathogenesis of chronic motor neuron diseases.

    View details for Web of Science ID 000082018700029

    View details for PubMedID 10473276

  • Spreading depression-induced gene expression is regulated by plasma glucose STROKE Koistinaho, J., Pasonen, S., Yrjanheikki, J., Chan, P. H. 1999; 30 (1): 114-119

    Abstract

    Plasma glucose and spreading depression (SD) are both determinants of brain ischemia. The purpose of this study was to examine whether plasma glucose affects SD-induced gene expression in the cortex.SD was induced by topical application of KCl. Hyperglycemia and hypoglycemia were induced by intraperitoneal injection of glucose and insulin, respectively. The expression of c-fos, cyclooxygenase-2 (COX-2), protein kinase C-delta (PKCdelta), and heme oxygenase-1 (HO-1) was determined by in situ hybridization.SD alone induced expression of c-fos (by 340%), COX-2 (210%), HO-1 (470%), and PKCdelta (410%). Hypoglycemia (2.4+/-0.9 mmol/L) alone did not induce gene expression, and hyperglycemia (22.1+/-3.7 mmol/L) alone induced only c-fos by 42%. When hypoglycemia was induced 30 minutes before SD, c-fos induction was enhanced by 145%, but the induction of HO-1 and PKCdelta was reduced to 43% and 64%, respectively. When hyperglycemia was induced 30 minutes before SD, c-fos induction was enhanced by 388% and COX-2 expression by 53%, whereas the induction of PKCdelta and HO-1 was reduced to 54% and 51%, respectively. The frequency, amplitude, and duration of direct current potentials were unaltered in hyperglycemic SD animals, whereas in hypoglycemic animals the duration was increased by 47%.While SD induces expression of several genes, the availability of glucose regulates the extent of the gene induction. The effect of glucose is different on early-response genes (c-fos and COX-2) compared with late-response genes. Plasma glucose may contribute to neuronal damage partially by regulating gene expression.

    View details for Web of Science ID 000077934200020

    View details for PubMedID 9880398

  • Spreading depression induces expression of calcium-independent protein kinase C subspecies in ischaemia-sensitive cortical, layers: Regulation by N-methyl-D-aspartate receptors and glucocorticoids NEUROSCIENCE Koponen, S., KEINANEN, R., Roivainen, R., HIRVONEN, T., Narhi, M., Chan, P. H., Koistinaho, J. 1999; 93 (3): 985-993

    Abstract

    Spreading depression is a wave of sustained depolarization challenging the energy metabolism of the cells without causing irreversible damage. In the ischaemic brain, sreading depression-like depolarization contributes to the evolution of ischaemia to infarction. The depolarization is propagated by activation of N-methyl-D-aspartate receptors, but changes in signal transduction downstream of the receptors are not known. Because protein phosphorylation is a general mechanism whereby most cellular processes are regulated, and inhibition of N-methyl-D-aspartate receptors or protein kinase C is neuroprotective, the expression of protein kinase C subspecies in spreading depression was examined. Cortical treatment with KCl induced an upregulation of protein kinase Cdelta and zeta messenger RNA at 4 and 8 h, whereas protein kinase Calpha, beta, gamma and epsilon did not show significant changes. The gene induction was the strongest in layers 2 and 3, and was followed by an increased number of protein kinase Cdelta-immunoreactive neurons. Protein kinase Cdelta and zeta inductions were inhibited by pretreatment with an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate, which also blocked spreading depression propagation, and with dexamethasone, which acted without blocking the propagation. Quinacrine, a phospholipase A2 inhibitor, reduced only protein kinase C5 induction. In addition, N(G)(-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, did not influence protein kinase Cdelta or zeta induction, whereas 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist, and the cyclo-oxygenase inhibitors indomethacin and diclophenac tended to increase gene expression. The data show that cortical spreading depression induces Ca2(+)-independent protein kinase C subspecies delta and zeta, but not Ca(2+)-dependent subspecies, through activation of N-methyl-D-aspartate receptors and phospholipase A2. Even though the signal pathway is similar to the induction described previously in ischaemia for genes implicated in delayed neuronal death, the gene inductions observed here are not necessarily pathogenetic, but may represent a general reaction to metabolic stress.

    View details for Web of Science ID 000082018700016

    View details for PubMedID 10473263

  • Reduced mitochondrial manganese-superoxide dismutase activity exacerbates glutamate toxicity in cultured mouse cortical neurons BRAIN RESEARCH Li, Y. B., Copin, J. C., Reola, L. F., Calagui, B., Gobbel, G. T., Chen, S. F., Sato, S., Epstein, C. J., Chan, P. H. 1998; 814 (1-2): 164-170

    Abstract

    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.

    View details for Web of Science ID 000077849300017

    View details for PubMedID 9838093

  • Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Fujimura, M., Morita-Fujimura, Y., Murakami, K., Kawase, M., Chan, P. H. 1998; 18 (11): 1239-1247

    Abstract

    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.

    View details for Web of Science ID 000076764600010

    View details for PubMedID 9809513

  • Overexpression of SOD1 in transgenic rats protects vulnerable neurons against ischemic damage after global cerebral ischemia and reperfusion JOURNAL OF NEUROSCIENCE Chan, P. H., Kawase, M., Murakami, K., Chen, S. F., Li, Y. B., Calagui, B., Reola, L., Carlson, E., Epstein, C. J. 1998; 18 (20): 8292-8299

    Abstract

    Transient global cerebral ischemia resulting from cardiac arrest is known to cause selective death in vulnerable neurons, including hippocampal CA1 pyramidal neurons. It is postulated that oxygen radicals, superoxide in particular, are involved in cell death processes. To test this hypothesis, we first used in situ imaging of superoxide radical distribution by hydroethidine oxidation in vulnerable neurons. We then generated SOD1 transgenic (Tg) rats with a five-fold increase in copper zinc superoxide dismutase activity. The Tg rats and their non-Tg wild-type littermates were subjected to 10 min of global ischemia followed by 1 and 3 d of reperfusion. Neuronal damage, as assessed by cresyl violet staining and DNA fragmentation analysis, was significantly reduced in the hippocampal CA1 region, cortex, striatum, and thalamus in SOD1 Tg rats at 3 d, as compared with the non-Tg littermates. There were no changes in the hippocampal CA3 subregion and dentate gyrus, resistant areas in both SOD1 Tg and non-Tg rats. Quantitative analysis of the damaged CA1 subregion showed marked neuroprotection against transient global cerebral ischemia in SOD1 Tg rats. These results suggest that superoxide radicals play a role in the delayed ischemic death of hippocampal CA1 neurons. Our data also indicate that SOD1 Tg rats are useful tools for studying the role of oxygen radicals in the pathogenesis of neuronal death after transient global cerebral ischemia.

    View details for Web of Science ID 000076317600017

    View details for PubMedID 9763473

  • Trolox and 6,7-dinitroquinoxaline-2,3-dione prevent necrosis but not apoptosis in cultured neurons subjected to oxygen deprivation BRAIN RESEARCH Copin, J. C., Li, Y. B., Reola, L., Chan, P. H. 1998; 784 (1-2): 25-36

    Abstract

    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.

    View details for Web of Science ID 000072586800004

    View details for PubMedID 9518539

  • Attenuation of acute and chronic damage following traumatic brain injury in copper, zinc-superoxide dismutase transgenic mice JOURNAL OF NEUROSURGERY Mikawa, S., Kinouchi, H., Kamii, H., Gobbel, G. T., Chen, S. F., Carlson, E., Epstein, C. J., Chan, P. H. 1996; 85 (5): 885-891

    Abstract

    To elucidate the role of oxygen-derived free radicals and superoxide dismutase in traumatic brain injury (TBI), blood-brain barrier (BBB) permeability, brain edema, behavioral function, and necrotic cavity volume (CV) were evaluated after TBI using nontransgenic (nTg) mice and heterozygous and homozygous transgenic (Tg) mice with a 1.5- (Tg 1.5x), 3.1-(Tg3.1x) and five- (Tg5x) fold increase in human copper, zinc-superoxide dismutase (CuZn-SOD) activity. Traumatic brain injury was produced by the weight-drop method. Evans blue dye leakage 4 hours after injury was attenuated in a CuZn-SOD dose-dependent manner with decreases of 18.6%, 40.9%, and 48.8%, in the Tg1.5x, Tg3.1x, and Tg5x groups, respectively. The water content 6 hours after injury in the Tg3.1x (79.64%) and Tg5x (79.45%) groups was significantly lower than in nTg mice (81.37%). There was an initial decrease in body weight and in motor performance, as measured by beam walk and beam balance tasks undertaken 1 day after TBI. However, the average reduction in beam balance and beam walk performance deficits and changes in body weight postinjury were significantly ameliorated in Tg mice. The CV was significantly smaller in Tg mice than in nTg mice (p < 0.01). These results indicate that superoxide radicals play a deleterious role following TBI. Furthermore, Tg mice provide a useful model for demonstrating the beneficial role of an antioxidant enzyme in TBI without the confounding effect of pharmacokinetics, toxicity, and BBB permeability associated with exogenous agents.

    View details for Web of Science ID A1996VP48900020

    View details for PubMedID 8893728

  • Effects of nitric oxide synthase inhibition on brain infarction in SOD-1-transgenic mice following transient focal cerebral ischemia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Kamii, H., Mikawa, S., Murakami, K., Kinouchi, H., Yoshimoto, T., Reola, L., Carlson, E., Epstein, C. J., Chan, P. H. 1996; 16 (6): 1153-1157

    Abstract

    To investigate the role of superoxide in the toxicity of nitric oxide (NO), we examined the effect of nitric oxide synthase (NOS) inhibition on brain infarction in transgenic mice overexpressing CuZn-superoxide dismutase (SOD-1). Male SOD-transgenic mice and non-transgenic littermates (30-35 g) were subjected to 60 min of middle cerebral artery occlusion followed by 24 h of reperfusion. Either NG-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg), a mixed neuronal and endothelial NOS inhibitor, or 7-nitroindazole (7-NI; 25 mg/kg), a selective neuronal NOS inhibitor, was administered intraperitoneally 5 min after the onset of ischemia. At 24 h of reperfusion, the mice were decapitated and the infarct volume was evaluated in each group. In the nontransgenic mice, L-NAME significantly increased the infarct volume as compared with the vehicle, while 7-NI significantly decreased it. In the SOD-transgenic mice, L-NAME-treated animals showed a significantly larger infarct volume than vehicle-treated ones, whereas there were no significant differences between 7-NI- and vehicle-treated mice. Our findings suggest that selective inhibition of neuronal NOS ameliorates ischemic brain injury and that both neuronal and endothelial NOS inhibition may result in the deterioration of ischemic injury due to vasoconstriction of the brain. Since L-NAME increased infarct volume even in SOD-transgenic mice, the protective effect of SOD could result from the vasodilation by increased endothelial NO as well as the reduction of neuronal injury due to less production of peroxynitrite compared to wild-type mice. Moreover, the neurotoxic role of NO might not be dependent on NO itself, but the reaction with superoxide to form peroxynitrite, because of no additive effects of SOD and a neuronal NOS inhibitor.

    View details for Web of Science ID A1996VP54000009

    View details for PubMedID 8898687

  • 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 JOURNAL OF NEUROTRAUMA Copin, J. C., Reola, L. F., CHAN, T. Y., Li, Y. B., Epstein, C. J., Chan, P. H. 1996; 13 (5): 233-244

    Abstract

    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.

    View details for Web of Science ID A1996UT29500001

    View details for PubMedID 8797173

  • Neuroprotective role of CuZn-superoxide dismutase in ischemic brain damage CELLULAR AND MOLECULAR MECHANISMS OF ISCHEMIC BRAIN DAMAGE Chan, P. H., Epstein, C. J., Kinouchi, H., Kamii, H., Chen, S. F., Carlson, E., Gafni, J., Yang, G. Y., Reola, L. 1996; 71: 271-280

    View details for Web of Science ID A1996BG71K00020

    View details for PubMedID 8790805

  • EXPRESSION OF C-FOS AND HSP70 MESSENGER-RNA AFTER TRAUMATIC BRAIN INJURY IN TRANSGENIC MICE OVEREXPRESSING CUZN-SUPEROXIDE DISMUTASE MOLECULAR BRAIN RESEARCH Mikawa, S., Sharp, F. R., Kamii, H., Kinouchi, H., Epstein, C. J., Chan, P. H. 1995; 33 (2): 288-294

    Abstract

    The aim of this study was to determine the role of oxidative stress on c-fos and hsp70 gene expression in transgenic (Tg) mice overexpressing CuZn-superoxide dismutase (SOD-1) following traumatic brain injury (TBI). hsp70 mRNA, as investigated using in situ hybridization, was induced around the lesion at 4 and 24 h, but not at 1 and 48 h, in both Tg and non-transgenic (nTg) mice littermates. The degree of hsp70 induction was somewhat greater in nTg than Tg mice at 4 and 24 h after TBI. c-fos mRNA was induced throughout cortex, hippocampus, caudate putamen and the ventricular wall in Tg and nTg mice. TBI induced c-fos bilaterally in the cortex in both animals. There was a time-dependent difference in cortical c-fos expression between nTg and Tg mice. The induction of c-fos mRNA in the striatum was greater in nTg at 24 h and decreased in both animals by 48 h. Edema of the injured cortex was significantly attenuated in Tg mice at all time points (1-48 h). These data show that the degree of hsp70 induction and the degree, extent, and duration of c-fos induction produced by TBI are affected by levels of superoxide dismutase activity. It is proposed that superoxide radicals affect spreading depression and brain edema produced by TBI and that this effect may either directly or indirectly modulate the expression of the c-fos and hsp70 genes after TBI.

    View details for Web of Science ID A1995TA39300013

    View details for PubMedID 8750888

  • MK-801 INHIBITS THE INDUCTION OF IMMEDIATE-EARLY GENES IN CEREBRAL-CORTEX, THALAMUS, AND HIPPOCAMPUS, BUT NOT IN SUBSTANTIA-NIGRA FOLLOWING MIDDLE CEREBRAL-ARTERY OCCLUSION NEUROSCIENCE LETTERS Kinouchi, H., Sharp, F. R., Chan, P. H., Mikawa, S., Kamii, H., Arai, S., Yoshimoto, T. 1994; 179 (1-2): 111-114

    Abstract

    Middle cerebral artery (MCA) occlusion in rats induced c-fos and junB mRNA 4h later in all ipsilateral cortex outside the MCA distribution and in many subcortical structures: medial striatum; most of thalamus including medial and lateral geniculate nuclei: substantia nigra; and hippocampus. The N-methyl-D-aspartate (NMDA) antagonist, MK-801 (4 mg/kg, i.p.) inhibited c-fos and junB mRNA induction in the cortex, striatum, thalamus, and hippocampus but not in the substantia nigra. These data show that c-fos and junB mRNA induction in cortex, striatum, thalamus, hippocampus involves the activation of NMDA receptors whereas different receptors must be implicated in the induction in substantia nigra.

    View details for Web of Science ID A1994PL51100028

    View details for PubMedID 7845604

  • INDUCTION OF C-FOS, JUNB, C-JUN, AND HSP70 MESSENGER-RNA IN CORTEX, THALAMUS, BASAL GANGLIA, AND HIPPOCAMPUS FOLLOWING MIDDLE CEREBRAL-ARTERY OCCLUSION JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Kinouchi, H., Sharp, F. R., Chan, P. H., Koistinaho, J., Sagar, S. M., Yoshimoto, T. 1994; 14 (5): 808-817

    Abstract

    Middle cerebral artery (MCA) occlusion in halothane-anesthetized rats induced c-fos, junB, and c-jun immediate early gene mRNAs and hsp70 heat shock gene mRNA in brain. In situ hybridization studies showed that c-fos and junB were induced throughout all of the cortex at 1 and 4 h following MCA occlusion. hsp70 was induced in the core and margins of the MCA ischemia. By 24 h, there was little expression of c-fos, junB, c-jun, and hsp70 in the core of the MCA infarct; there was modest induction of hsp70 at the margins of the infarct; and there was diffuse induction of c-fos, junB, and c-jun in all of the cortex outside the infarct. MCA occlusion also induced these genes in subcortical structures. c-fos, junB, and hsp70 were induced in ipsilateral medial striatum, most of thalamus including medial and lateral geniculate nuclei, substantia nigra, and hippocampus. Most of these structures, except for the striatum, are not supplied by the MCA. These data show that changes in gene expression can occur in regions remote from an infarction.

    View details for Web of Science ID A1994PC77000012

    View details for PubMedID 8063876

  • PROLONGED EXPRESSION OF HSP70 MESSENGER-RNA FOLLOWING TRANSIENT FOCAL CEREBRAL-ISCHEMIA IN TRANSGENIC MICE OVEREXPRESSING CUZN-SUPEROXIDE DISMUTASE JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Kamii, H., Kinouchi, H., Sharp, F. R., Koistinaho, J., Epstein, C. J., Chan, P. H. 1994; 14 (3): 478-486

    Abstract

    The distribution of heat shock protein hsp70 mRNA after 10 min of middle cerebral artery (MCA) occlusion was investigated through in situ hybridization in transgenic (Tg) mice overexpressing CuZn-superoxide dismutase (CuZn-SOD) and in control nontransgenic (nTg) littermates. In the ischemic cortex of nTg mice, hsp70 mRNA was detected 1 h after reperfusion and was observed for up to 6 h. In Tg mice, however, it was still detectable within the cortex even at 24 h. In the caudate putamen, hsp70 mRNA appeared at 1 h and was present for up to 6 h in both nTg and Tg mice. Although hsp70 mRNA was detected in the thalamus only at 1 h in nTg mice, it was observed for up to 6 h in Tg mice. Similarly, hsp70 mRNA was detected in the hippocampus of nTg mice only at 1 h, whereas it was detected in Tg mice at 1 h and continued up to 24 h, with high intensity in the CA1 subfield. Despite the significant amounts of hsp70 mRNA in both Tg and nTg mice following ischemia, there was no observable neuronal necrosis (as assessed using hematoxylin and eosin staining) for up to 7 days. Cortical cerebral blood flow (CBF), measured by laser-Doppler flowmetry, did not differ between nTg and Tg mice during ischemia and reperfusion, despite exhibiting hyperemia following hypoperfusion. These results suggest that oxidative stress affects the expression of hsp70 following temporary focal ischemia. An alteration in oxidation stress, which resulted from reduced levels of superoxide radicals in the presence of the CuZn-SOD transgenes, may permit the prolonged expression of hsp70.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1994NG20900013

    View details for PubMedID 8163590

  • INDUCTION OF NGFI-A MESSENGER-RNA FOLLOWING MIDDLE CEREBRAL-ARTERY OCCLUSION IN RATS - IN-SITU HYBRIDIZATION STUDY NEUROSCIENCE LETTERS Kinouchi, H., Sharp, F. R., Chan, P. H., Koistinaho, J., Sagar, S. M., Yoshimoto, T. 1994; 171 (1-2): 163-166

    Abstract

    Middle cerebral artery (MCA) occlusion in halothane-anesthetized rats induced the zinc finger gene, NGFI-A, in brain. In situ hybridization studies showed that NGFI-A was induced throughout all of the cortex following MCA occlusion. By 24 h after MCA occlusion there was little expression of NGFI-A mRNA in the core of the MCA infarct, but the mRNA was still induced in all of cortex outside the infarct. MCA occlusion also induced this gene in subcortical structures: ipsilateral medial striatum; most of thalamus including medial and lateral geniculate nuclei; substantia nigra; and hippocampus at 4 h of MCA occlusion which generally disappeared by 24 h of MCA occlusion. Most of these structures, except for the striatum, are not supplied by the MCA. These data show that changes in brain gene expression can occur in many regions remote from an infarction.

    View details for Web of Science ID A1994NH34500042

    View details for PubMedID 8084481

  • BRAIN INFARCTION IS NOT REDUCED IN SOD-1 TRANSGENIC MICE AFTER A PERMANENT FOCAL CEREBRAL-ISCHEMIA NEUROREPORT Chan, P. H., Kamii, H., Yang, G. Y., Gafni, J., Epstein, C. J., Carlson, E., Reola, L. 1993; 5 (3): 293-296

    Abstract

    Using a mouse model with intraluminal blockade of the middle cerebral artery (MCA) which produced both cortical and striatal infarction, the effect that superoxide radicals have on cerebral infarction, local cerebral blood flow, and neurological deficits after 24 h of permanent focal cerebral ischemia in transgenic mice (Tg) overexpressing human CuZn-superoxide dismutase (SOD-1) was examined. There were no difference between SOD-1 Tg mice and non-Tg littermates observed in the infarct areas of brain slices, the infarct volume, the local cerebral blood flow, or the neurological deficits. These data suggest that pre-existing high levels of antioxidant enzyme failed to provide neuronal protection against permanent focal cerebral ischemia.

    View details for Web of Science ID A1993MP00200028

    View details for PubMedID 8298091

  • INDUCTION OF 70-KDA HEAT-SHOCK PROTEIN AND HSP70 MESSENGER-RNA FOLLOWING TRANSIENT FOCAL CEREBRAL-ISCHEMIA IN THE RAT JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Kinouchi, H., Sharp, F. R., Hill, M. P., Koistinaho, J., Sagar, S. M., Chan, P. H. 1993; 13 (1): 105-115

    Abstract

    Induction of the 70-kDa heat shock protein (HSP70) was demonstrated immunocytochemically in adult rats 4 h to 7 days following temporary middle cerebral artery (MCA) occlusions lasting 30, 60, or 90 min. Maximal HSP70 induction occurred approximately 24 h following ischemia. Thirty minutes of ischemia induced HSP70 in neurons throughout the cortex in the MCA distribution, whereas 90 min of ischemia induced HSP70 in neurons in the penumbra. HSP70 protein was induced in endothelial cells in infarcted neocortex following 60-90 min of MCA occlusion, and HSP70 was induced in endothelial cells in infarcted regions of lateral striatum following 30-90 min of MCA occlusion. hsp70 mRNA was induced in the MCA distribution in cortex and to a lesser extent in striatum at 2 h to 3 days following 60 min of ischemia. It is proposed that brief ischemia induces hsp70 mRNA and HSP70 protein in the cells most vulnerable to ischemia--the neurons. HSP70 protein is not induced in most neurons and glia following 60-90 min of ischemia in areas destined to infarct, whereas it is induced in vascular endothelial cells.

    View details for Web of Science ID A1993KE73000011

    View details for PubMedID 8416999

Conference Proceedings


  • The role of mitochondria and oxidative stress in neuronal death Chan, P. H., Fujimura, M., Lewen, A., Noshita, N., Sugawara, T., Morita-Fujimura, Y. WILEY-BLACKWELL. 2001: 203-203

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