Bio

Academic Appointments


Honors & Awards


  • NIH Clinical Investigator Award, NIH (1990-1995)
  • Young Investigator Award, Foundation for Anesthesia Education and Research (1991-1992)
  • Ellen Weaver Award, Association for Women in Science, Northern California Chapters (1997)
  • Frontiers in Anesthesia Research Award, International Anesthesia Research Society (1998-2003)
  • AHA/Bugher Award, American Heart Association (2000-2004)
  • Ellis Cohen Achievement Award, Stanford Department of Anesthesia (2009)

Professional Education


  • Ph.D., Stanford University, Structural Biology
  • M.D., Stanford University, Medicine

Research & Scholarship

Current Research and Scholarly Interests


Brain injury from stroke, head trauma, and chronic neurologic degenerative diseases, is a major cause of morbidity and mortality. We are particularly interested in the cellular consequences of brain injury. To study this problem we work with primary cultures of neurons and astrocytes from mice in addition to employing rodent models of stroke. Current work focuses on: 1) the role of astrocytes in brain injury; 2) the interaction of neurons and glia during injury; 3) the role of specific microRNAs in ischemia 4)protection using heat shock proteins and other protective proteins 4) changes in mitochondrial function and signaling in injury and ways to protect mitochondria; 5) the interaction of oxidative stress and inflammation in stroke; 6) computational modeling of cell death.

We use gene transfer techniques to express genes and miRNAs of interest in brain cells and intact brain and analyze ways in which these can provide protection. We use fluorescent probes for pH, intracellular calcium, ROS, mitochondrial membrane potential, as well as morphologically evaluate outcome, and quantitate injury. We also use transgenic mice to analyze the effects of overexpression or loss of expression of specific genes on outcome from stroke. Mitochondria are central to both energy metabolism and the regulation of inflammation and cell death. We study changes in mitochondria with stress. We are also interested in the interaction of oxidative stress and inflammation in stroke.

A new area for us is computational modeling of cell death and the effects of heat shock proteins. We are working on an ODE control model.

Teaching

2013-14 Courses


Postdoctoral Advisees


Graduate and Fellowship Programs


Publications

Journal Articles


  • Genomic Analysis of Reactive Astrogliosis JOURNAL OF NEUROSCIENCE Zamanian, J. L., Xu, L., Foo, L. C., Nouri, N., Zhou, L., Giffard, R. G., Barres, B. A. 2012; 32 (18): 6391-6410

    Abstract

    Reactive astrogliosis is characterized by a profound change in astrocyte phenotype in response to all CNS injuries and diseases. To better understand the reactive astrocyte state, we used Affymetrix GeneChip arrays to profile gene expression in populations of reactive astrocytes isolated at various time points after induction using two mouse injury models, ischemic stroke and neuroinflammation. We find reactive gliosis consists of a rapid, but quickly attenuated, induction of gene expression after insult and identify induced Lcn2 and Serpina3n as strong markers of reactive astrocytes. Strikingly, reactive astrocyte phenotype strongly depended on the type of inducing injury. Although there is a core set of genes that is upregulated in reactive astrocytes from both injury models, at least 50% of the altered gene expression is specific to a given injury type. Reactive astrocytes in ischemia exhibited a molecular phenotype that suggests that they may be beneficial or protective, whereas reactive astrocytes induced by LPS exhibited a phenotype that suggests that they may be detrimental. These findings demonstrate that, despite well established commonalities, astrocyte reactive gliosis is a highly heterogeneous state in which astrocyte activities are altered to respond to the specific injury. This raises the question of how many subtypes of reactive astrocytes exist. Our findings provide transcriptome databases for two subtypes of reactive astrocytes that will be highly useful in generating new and testable hypotheses of their function, as well as for providing new markers to detect different types of reactive astrocytes in human neurological diseases.

    View details for DOI 10.1523/JNEUROSCI.6221-11.2012

    View details for Web of Science ID 000303598900032

    View details for PubMedID 22553043

  • Neuroprotection from Stroke in the Absence of MHCI or PirB NEURON Adelson, J. D., Barreto, G. E., Xu, L., Kim, T., Brott, B. K., Ouyang, Y., Naserke, T., Djurisic, M., Xiong, X., Shatz, C. J., Giffard, R. G. 2012; 73 (6): 1100-1107

    Abstract

    Recovery from stroke engages mechanisms of neural plasticity. Here we examine a role for MHC class I (MHCI) H2-Kb and H2-Db, as well as PirB receptor. These molecules restrict synaptic plasticity and motor learning in the healthy brain. Stroke elevates neuronal expression not only of H2-Kb and H2-Db, but also of PirB and downstream signaling. KbDb knockout (KO) or PirB KO mice have smaller infarcts and enhanced motor recovery. KO hippocampal organotypic slices, which lack an intact peripheral immune response, have less cell death after in vitro ischemia. In PirB KO mice, corticospinal projections from the motor cortex are enhanced, and the reactive astrocytic response is dampened after MCAO. Thus, molecules that function in the immune system act not only to limit synaptic plasticity in healthy neurons, but also to exacerbate brain injury after ischemia. These results suggest therapies for stroke by targeting MHCI and PirB.

    View details for DOI 10.1016/j.neuron.2012.01.020

    View details for Web of Science ID 000301998700008

    View details for PubMedID 22445338

  • miR-181 regulates GRP78 and influences outcome from cerebral ischemia in vitro and in vivo NEUROBIOLOGY OF DISEASE Ouyang, Y., Lu, Y., Yue, S., Xu, L., Xiong, X., White, R. E., Sun, X., Giffard, R. G. 2012; 45 (1): 555-563

    Abstract

    MicroRNAs (miRNA) are short (~22nt) single stranded RNAs that downregulate gene expression. Although recent studies indicate extensive miRNA changes in response to ischemic brain injury, there is currently little information on the roles of specific miRNAs in this setting. Heat shock proteins (HSP) of the HSP70 family have been extensively studied for their multiple roles in cellular protection, but there is little information on their regulation by miRNAs. We used bioinformatics to identify miR-181 as a possible regulator of several HSP70 family members. We validated GRP78/BIP as a target by dual luciferase assay. In response to stroke in the mouse we find that miR-181 increases in the core, where cells die, but decreases in the penumbra, where cells survive. Increased levels of miR-181a are associated with decreased GRP78 protein levels, but increased levels of mRNA, implicating translational arrest. We manipulated levels of miR-181a using plasmid overexpression of pri-miR-181ab or mimic to increase, and antagomir or inhibitor to reduce levels. Increased miR-181a exacerbated injury both in vitro and in the mouse stroke model. Conversely, reduced levels were associated with reduced injury and increased GRP78 protein levels. Studies in C6 cells show that if GRP78 levels are maintained miR-181a no longer exerts a toxic effect. These data demonstrate that miR-181 levels change in response to stroke and inversely correlate with levels of GRP78. Importantly, reducing or blocking miR-181a protects the brain from stroke.

    View details for DOI 10.1016/j.nbd.2011.09.012

    View details for Web of Science ID 000297883500061

    View details for PubMedID 21983159

  • Mitochondrial Protection Attenuates Inflammation-Induced Impairment of Neurogenesis In Vitro and In Vivo JOURNAL OF NEUROSCIENCE Voloboueva, L. A., Lee, S. W., Emery, J. F., Palmer, T. D., Giffard, R. G. 2010; 30 (37): 12242-12251

    Abstract

    The impairment of hippocampal neurogenesis has been linked to the pathogenesis of neurological disorders from chronic neurodegenerative disease to the progressive cognitive impairment of children who receive brain irradiation. Numerous studies provide evidence that inflammation downregulates neurogenesis, with multiple factors contributing to this impairment. Although mitochondria are one of the primary targets of inflammatory injury, the role of mitochondrial function in the modulation of neurogenesis remains relatively unstudied. In this study, we used neurosphere-derived cells to show that immature doublecortin (Dcx)-positive neurons are uniquely sensitive to mitochondrial inhibition, demonstrating rapid loss of mitochondrial potential and cell viability compared with glial cells and more mature neurons. Mitochondrial inhibition for 24 h produced no significant changes in astrocyte or oligodendrocyte viability, but reduced viability of mature neurons by 30%, and reduced survival of Dcx(+) cells by 60%. We demonstrate that protection of mitochondrial function with mitochondrial metabolites or the mitochondrial chaperone mtHsp75/mortalin partially reverses the inflammation-associated impairment of neurogenesis in vitro and in irradiated mice in vivo. Our findings highlight mitochondrial mechanisms involved in neurogenesis and indicate mitochondria as a potential target for protective strategies to prevent the impairment of neurogenesis by inflammation.

    View details for DOI 10.1523/JNEUROSCI.1752-10.2010

    View details for Web of Science ID 000281798700003

    View details for PubMedID 20844120

  • Astrocyte Targeted Overexpression of Hsp72 or SOD2 Reduces Neuronal Vulnerability to Forebrain Ischemia GLIA Xu, L., Emery, J. F., Ouyang, Y., Voloboueva, L. A., Giffard, R. G. 2010; 58 (9): 1042-1049

    Abstract

    Brief forebrain ischemia is a model of the delayed hippocampal neuronal loss seen in patients following cardiac arrest and resuscitation. Previous studies demonstrated that selective dysfunction of hippocampal CA1 subregion astrocytes occurs hours to days before delayed neuronal death. In this study we tested the strategy of directing protection to astrocytes to protect neighboring neurons from forebrain ischemia. Two well-studied protective proteins, heat shock protein 72 (Hsp72) or superoxide dismutase 2 (SOD2), were genetically targeted for expression in astrocytes using the astrocyte-specific human glial fibrillary acidic protein (GFAP) promoter. The expression constructs were injected stereotacticly immediately above the hippocampal CA1 region on one side of the rat brain two days prior to forebrain ischemia. Cell type specific expression was confirmed by double label immunohistochemistry. When the expression constructs were injected two days before transient forebrain ischemia, the loss of CA1 hippocampal neurons observed seven days later was significantly reduced on the injected side compared with controls. This neuroprotection was associated with significantly better preservation of astrocyte glutamate transporter-1 immunoreactivity at 5-h reperfusion and reduced oxidative stress. Improving the resistance of astrocytes to ischemic stress by targeting either the cytosolic or mitochondrial compartment was thus associated with preservation of CA1 neurons following forebrain ischemia. Targeting astrocytes is a promising strategy for neuronal preservation following cardiac arrest and resuscitation.

    View details for DOI 10.1002/glia.20985

    View details for Web of Science ID 000278198400003

    View details for PubMedID 20235222

  • Overexpression of Heat Shock Protein 72 Attenuates NF-?B Activation Using a Combination of Regulatory Mechanisms in Microglia. PLoS computational biology Sheppard, P. W., Sun, X., Khammash, M., Giffard, R. G. 2014; 10 (2)

    Abstract

    Overexpression of the inducible heat shock protein 70, Hsp72, has broadly cytoprotective effects and improves outcome following stroke. A full understanding of how Hsp72 protects cells against injury is elusive, though several distinct mechanisms are implicated. One mechanism is its anti-inflammatory effects. We study the effects of Hsp72 overexpression on activation of the transcription factor NF-κB in microglia combining experimentation and mathematical modeling, using TNFα to stimulate a microglial cell line stably overexpressing Hsp72. We find that Hsp72 overexpression reduces the amount of NF-κB DNA binding activity, activity of the upstream kinase IKK, and amount of IκBα inhibitor phosphorylated following TNFα application. Simulations evaluating several proposed mechanisms suggest that inhibition of IKK activation is an essential component of its regulatory activities. Unexpectedly we find that Hsp72 overexpression reduces the initial amount of the RelA/p65 NF-κB subunit in cells, contributing to the attenuated response. Neither mechanism in isolation, however, is sufficient to attenuate the response, providing evidence that Hsp72 relies upon multiple mechanisms to attenuate NF-κB activation. An additional observation from our study is that the induced expression of IκBα is altered significantly in Hsp72 expressing cells. While the mechanism responsible for this observation is not known, it points to yet another means by which Hsp72 may alter the NF-κB response. This study illustrates the multi-faceted nature of Hsp72 regulation of NF-κB activation in microglia and offers further clues to a novel mechanism by which Hsp72 may protect cells against injury.

    View details for DOI 10.1371/journal.pcbi.1003471

    View details for PubMedID 24516376

  • Inhibition of microRNA-181 reduces forebrain ischemia-induced neuronal loss JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Moon, J., Xu, L., Giffard, R. G. 2013; 33 (12): 1976-1982

    Abstract

    MicroRNA (miRNA), miR-181a, is enriched in the brain, and inhibition of miR-181a reduced astrocyte death in vitro and infarct volume after stroke in vivo. This study investigated the role of miR-181a in neuronal injury in vitro and hippocampal neuronal loss in vivo after forebrain ischemia. miR-181a levels were altered by transfection with mimic or antagomir. N2a cells subjected to serum deprivation and oxidative stress showed less cell death when miR-181a was reduced and increased death when miR-181a increased; protection was associated with increased Bcl-2 protein. In contrast, transfected primary neurons did not show altered levels of cell death when miR-181a levels changed. Naive male rats and rats stereotactically infused with miR-181a antagomir or control were subjected to forebrain ischemia and cornus ammonis (CA)1 neuronal survival and protein levels were assessed. Forebrain ischemia increased miR-181a expression and decreased Bcl-2 protein in the hippocampal CA1 region. miR-181a antagomir reduced miR-181a levels, reduced CA1 neuronal loss, increased Bcl-2 protein, and significantly prevented the decrease of glutamate transporter 1. Thus, miR-181a antagomir reduced evidence of astrocyte dysfunction and increased CA1 neuronal survival. miR-181a inhibition is thus a potential target in the setting of forebrain or global cerebral ischemia as well as focal ischemia.

    View details for DOI 10.1038/jcbfm.2013.157

    View details for Web of Science ID 000328002200019

    View details for PubMedID 24002437

  • MicroRNAs Regulate the Chaperone Network in Cerebral Ischemia TRANSLATIONAL STROKE RESEARCH Ouyang, Y., Giffard, R. G. 2013; 4 (6): 693-703

    Abstract

    The highly evolutionarily conserved 70 kDa heat shock protein (HSP70) family was first understood for its role in protein folding and response to stress. Subsequently, additional functions have been identified for it in regulation of organelle interaction, of the inflammatory response, and of cell death and survival. Overexpression of HSP70 family members is associated with increased resistance to and improved recovery from cerebral ischemia. MicroRNAs (miRNAs) are important posttranscriptional regulators that interact with multiple target messenger RNAs (mRNA) coordinately regulating target genes, including chaperones. The members of the HSP70 family are now appreciated to work together as networks to facilitate organelle communication and regulate inflammatory signaling and cell survival after cerebral ischemia. This review will focus on the new concept of the role of the chaperone network in the organelle network and its novel regulation by miRNA.

    View details for DOI 10.1007/s12975-013-0280-3

    View details for Web of Science ID 000327465200012

    View details for PubMedID 24323423

  • Astrocyte-enriched miR-29a targets PUMA and reduces neuronal vulnerability to forebrain ischemia. Glia Ouyang, Y., Xu, L., Lu, Y., Sun, X., Yue, S., Xiong, X., Giffard, R. G. 2013; 61 (11): 1784-1794

    Abstract

    Following transient forebrain ischemia, astrocytes play a key role in determining whether or not neurons in the hippocampal CA1 sector go on to die in a delayed fashion. MicroRNAs (miRNAs) are a novel class of RNAs that control gene expression at the post-transcriptional level and the miR-29 family is highly expressed in astrocytes. In this study we assessed levels of miR-29 in hippocampus following forebrain ischemia and found that after transient forebrain ischemia and short periods of reperfusion, miR-29a significantly increased in the resistant dentate gyrus, but decreased in the vulnerable CA1 region of the hippocampus. We demonstrate that miR-29a targets BH3-only proapoptotic BCL2 family member PUMA by luciferase reporter assay and by Western blot. Comparing primary neuron and astrocyte cultures, and postnatal brain, we verified the strongly astrocytic expression of miR-29a. We further found that miR-29a mimic protects and miR-29a inhibitor aggravates cell injury and mitochondrial function after ischemia-like stresses in vitro. Lastly, by overexpressing and reducing miR-29a we demonstrate the protective effect of miR-29a on CA1 delayed neuronal death after forebrain ischemia. Our data suggest that by targeting a pro-apoptotic BCL2 family member, increasing levels of miR-29a might emerge as a strategy for protection against ischemia-reperfusion injury. GLIA 2013;61:1784-1794.

    View details for DOI 10.1002/glia.22556

    View details for PubMedID 24038396

  • Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar BRITISH JOURNAL OF ANAESTHESIA Jevtovic-Todorovic, V., Absalom, A. R., Blomgren, K., Brambrink, A., Crosby, G., Culley, D. J., Fiskum, G., Giffard, R. G., Herold, K. F., Loepke, A. W., Ma, D., Orser, B. A., Planel, E., Slikker, W., Soriano, S. G., Stratmann, G., Vutskits, L., Xie, Z., Hemmings, H. C. 2013; 111 (2): 143-151

    Abstract

    Although previously considered entirely reversible, general anaesthesia is now being viewed as a potentially significant risk to cognitive performance at both extremes of age. A large body of preclinical as well as some retrospective clinical evidence suggest that exposure to general anaesthesia could be detrimental to cognitive development in young subjects, and might also contribute to accelerated cognitive decline in the elderly. A group of experts in anaesthetic neuropharmacology and neurotoxicity convened in Salzburg, Austria for the BJA Salzburg Seminar on Anaesthetic Neurotoxicity and Neuroplasticity. This focused workshop was sponsored by the British Journal of Anaesthesia to review and critically assess currently available evidence from animal and human studies, and to consider the direction of future research. It was concluded that mounting evidence from preclinical studies reveals general anaesthetics to be powerful modulators of neuronal development and function, which could contribute to detrimental behavioural outcomes. However, definitive clinical data remain elusive. Since general anaesthesia often cannot be avoided regardless of patient age, it is important to understand the complex mechanisms and effects involved in anaesthesia-induced neurotoxicity, and to develop strategies for avoiding or limiting potential brain injury through evidence-based approaches.

    View details for DOI 10.1093/bja/aet177

    View details for Web of Science ID 000322337900005

    View details for PubMedID 23722106

  • Selective activation of protein kinase C? in mitochondria is neuroprotective in vitro and reduces focal ischemic brain injury in mice. Journal of neuroscience research Sun, X., Budas, G. R., Xu, L., Barreto, G. E., Mochly-Rosen, D., Giffard, R. G. 2013; 91 (6): 799-807

    Abstract

    Activation of protein kinase C? (PKC?) confers protection against neuronal ischemia/reperfusion. Activation of PKC? leads to its translocation to multiple intracellular sites, so a mitochondria-selective PKC? activator was used to test the importance of mitochondrial activation to the neuroprotective effect of PKC?. PKC? can regulate key cytoprotective mitochondrial functions, including electron transport chain activity, reactive oxygen species (ROS) generation, mitochondrial permeability transition, and detoxification of reactive aldehydes. We tested the ability of mitochondria-selective activation of PKC? to protect primary brain cell cultures or mice subjected to ischemic stroke. Pretreatment with either general PKC? activator peptide, TAT-??RACK, or mitochondrial-selective PKC? activator, TAT-??HSP90, reduced cell death induced by simulated ischemia/reperfusion in neurons, astrocytes, and mixed neuronal cultures. The protective effects of both TAT-??RACK and TAT-??HSP90 were blocked by the PKC? antagonist ?V1-2 , indicating that protection requires PKC? interaction with its anchoring protein, TAT-?RACK. Further supporting a mitochondrial mechanism for PKC?, neuroprotection by TAT-??HSP90 was associated with a marked delay in mitochondrial membrane depolarization and significantly attenuated ROS generation during ischemia. Importantly, TAT-??HSP90 reduced infarct size and reduced neurological deficit in C57/BL6 mice subjected to middle cerebral artery occlusion and 24 hr of reperfusion. Thus selective activation of mitochondrial PKC? preserves mitochondrial function in vitro and improves outcome in vivo, suggesting potential therapeutic value clinically when brain ischemia is anticipated, including neurosurgery and cardiac surgery.

    View details for DOI 10.1002/jnr.23186

    View details for PubMedID 23426889

  • Inflammatory response of microglial BV-2 cells includes a glycolytic shift and is modulated by mitochondrial glucose-regulated protein 75/mortalin FEBS LETTERS Voloboueva, L. A., Emery, J. F., Sun, X., Giffard, R. G. 2013; 587 (6): 756-762

    Abstract

    Recent studies suggest a link between mitochondria and proinflammatory cytokine generation. We previously demonstrated that overexpression of mitochondrial chaperone glucose-regulated protein75 (Grp75/mortalin) protects mitochondria. In this study we investigated the modulation of the lipopolisaccharide (LPS)-induced inflammatory response of microglial BV-2 cells by Grp75. We demonstrate that LPS-induced activation promotes significant metabolic changes suppressing mitochondrial function and increasing glycolysis. Overexpression of Grp75 attenuates the LPS-induced oxidative and metabolic responses, and suppresses proinflammatory activation, which depends on both NF-?B activation and lactate. Thus overexpression of Grp75 provides a novel strategy to modulate proinflammatory cytokine production of relevance to inflammation-associated pathologies.

    View details for DOI 10.1016/j.febslet.2013.01.067

    View details for Web of Science ID 000315947400034

    View details for PubMedID 23395614

  • Mitigation of Murine Focal Cerebral Ischemia by the Hypocretin/Orexin System is Associated With Reduced Inflammation STROKE Xiong, X., White, R. E., Xu, L., Yang, L., Sun, X., Zou, B., Pascual, C., Sakurai, T., Giffard, R. G., Xie, X. (. 2013; 44 (3): 764-770

    Abstract

    Brain ischemia causes immediate and delayed cell death that is exacerbated by inflammation. Recent studies show that hypocretin-1/orexin-A (Hcrt-1) reduces ischemic brain injury, and Hcrt-positive neurons modulate infection-induced inflammation. Here, we tested the hypothesis that Hcrt plays a protective role against ischemia by modulating inflammation.Orexin/ataxin-3 (AT) mice, a transgenic strain in which Hcrt-producing neurons degenerate in early adulthood, and wild-type mice were subjected to transient middle cerebral artery occlusion (MCAO). Infarct volume, neurological score, and spontaneous home cage activity were assessed. Inflammation was measured using immunohistochemistry, ELISA, and assessment of cytokine mRNA levels.Infarct volumes 24 and 48 hours after MCAO were significantly larger, neurological score was worse, and spontaneous activity decreased in AT compared with wild-type mice. Macrophage/microglial infiltration and myeloperoxidase-positive cells were higher in AT compared with wild-type mice. Pre-MCAO intracerebroventricular injection of Hcrt-1 significantly reduced infarct volume and macrophage/microglial infiltration in both genotypes and improved neurological score in AT mice. Post-MCAO treatment decreased infarct size in both wild-type and AT mice, but had no effect on neurological score in either genotype. Microglia express the Hcrt-1 receptor after MCAO. Tumor necrosis factor-? production by lipopolysaccharide-stimulated microglial BV2 cells was significantly reduced by Hcrt-1 pretreatment. Sham AT mice exhibit increased brain tumor necrosis factor-? and interleukin-6 mRNA, suggesting chronic inflammation.Loss of Hcrt neurons in AT mice resulted in worsened stroke outcomes, which were reversed by administration of exogenous Hcrt-1. The mechanism underlying Hcrt-mediated neuroprotection includes attenuation of inflammatory responses after ischemic insult.

    View details for DOI 10.1161/STROKEAHA.112.681700

    View details for Web of Science ID 000315447400037

    View details for PubMedID 23349191

  • microRNAs: Innovative Targets for Cerebral Ischemia and Stroke CURRENT DRUG TARGETS Ouyang, Y., Stary, C. M., Yang, G., Giffard, R. 2013; 14 (1): 90-101

    Abstract

    Stroke is one of the leading causes of death and disability worldwide. Because stroke is a multifactorial disease with a short therapeutic window many clinical stroke trials have failed and the only currently approved therapy is thrombolysis. MicroRNAs (miRNA) are endogenously expressed noncoding short single-stranded RNAs that play a role in the regulation of gene expression at the post-transcriptional level, via degradation or translational inhibition of their target mRNAs. The study of miRNAs is rapidly growing and recent studies have revealed a significant role of miRNAs in ischemic disease. miRNAs are especially important candidates for stroke therapeutics because of their ability to simultaneously regulate many target genes and since to date targeting single genes for therapeutic intervention has not yet succeeded in the clinic. Although there are already quite a few review articles about miRNA in ischemic heart disease, much less is currently known about miRNAs in cerebral ischemia. This review summarizes current knowledge about miRNAs and cerebral ischemia, focusing on the role of miRNAs in ischemia, both changes in expression and identification of potential targets, as well as the potential of miRNAs as biomarkers and therapeutic targets in cerebral ischemia.

    View details for Web of Science ID 000316871200010

    View details for PubMedID 23170800

  • Effects of heat shock protein 72 (Hsp72) on evolution of astrocyte activation following stroke in the mouse EXPERIMENTAL NEUROLOGY Barreto, G. E., White, R. E., Xu, L., Palm, C. J., Giffard, R. G. 2012; 238 (2): 284-296

    Abstract

    Astrocyte activation is a hallmark of the response to brain ischemia consisting of changes in gene expression and morphology. Heat shock protein 72 (Hsp72) protects from cerebral ischemia, and although several protective mechanisms have been investigated, effects on astrocyte activation have not been studied. To identify potential mechanisms of protection, microarray analysis was used to assess gene expression in the ischemic hemispheres of wild-type (WT) and Hsp72-overexpressing (Hsp72Tg) mice 24 h after middle cerebral artery occlusion or sham surgery. After stroke both genotypes exhibited changes in genes related to apoptosis, inflammation, and stress, with more downregulated genes in Hsp72Tg and more inflammation-related genes increased in WT mice. Genes indicative of astrocyte activation were also upregulated in both genotypes. To measure the extent and time course of astrocyte activation after stroke, detailed histological and morphological analyses were performed in the cortical penumbra. We observed a marked and persistent increase in glial fibrillary acidic protein (GFAP) and a transient increase in vimentin. No change in overall astrocyte number was observed based on glutamine synthetase immunoreactivity. Hsp72Tg and WT mice were compared for density of astrocytes expressing activation markers and astrocytic morphology. In animals with comparable infarct size, overexpression of Hsp72 reduced the density of GFAP- and vimentin-expressing cells, and decreased astrocyte morphological complexity 72 h following stroke. However, by 30 days astrocyte activation was similar between genotypes. These data indicate that early modulation of astrocyte activation provides an additional novel mechanism associated with Hsp72 overexpression in the setting of ischemia.

    View details for DOI 10.1016/j.expneurol.2012.08.015

    View details for Web of Science ID 000311763100025

    View details for PubMedID 22940431

  • MicroRNA-320 induces neurite outgrowth by targeting ARPP-1 NEUROREPORT White, R. E., Giffard, R. G. 2012; 23 (10): 590-595

    Abstract

    MicroRNAs are important in the development, functioning, and pathophysiology of the central nervous system. Here, we show that increasing the levels of microRNA-320 (miR-320) for 3 days markedly increases neurite length, and at 4 days, reduces the total cell number in Neuro-2A cells. In-silico analysis of possible miR-320 targets identified cAMP-regulated phosphoprotein-19 kDa (ARPP-19) and semaphorin 3a as potential targets that could be involved. ARPP-19 was validated by showing reduced mRNA and protein levels when miR-320 was overexpressed, whereas miR-320 had no effect on semaphorin 3a expression. ARPP-19 is known to inhibit protein phosphatase-2A activity, which inhibits mitosis and induces neurite outgrowth, making this the likely mechanism. Thus, increased levels of miR-320 lead to decreased levels of ARPP-19, increased neurite length, and fewer total cells. These data suggest that miR-320 could play a role in neuronal development and might be a target to enhance neuronal regeneration following injury.

    View details for DOI 10.1097/WNR.0b013e3283540394

    View details for Web of Science ID 000305501000003

    View details for PubMedID 22617447

  • Stroke-induced activation of the alpha 7 nicotinic receptor increases Pseudomonas aeruginosa lung injury FASEB JOURNAL Lafargue, M., Xu, L., Carles, M., Serve, E., Anjum, N., Iles, K. E., Xiong, X., Giffard, R., Pittet, J. 2012; 26 (7): 2919-2929

    Abstract

    Infectious complications, predominantly pneumonia, are the most common cause of death in the postacute phase of stroke, although the mechanisms underlying the corresponding immunosuppression are not fully understood. We tested the hypothesis that activation of the ?7 nicotinic acetylcholine receptor (?7nAChR) pathway is important in the stroke-induced increase in lung injury caused by Pseudomonas aeruginosa pneumonia in mice. Prior stroke increased lung vascular permeability caused by P. aeruginosa pneumonia and was associated with decreased lung neutrophil recruitment and bacterial clearance in mice. Pharmacologic inhibition (methyllycaconitine IC(50): 0.2-0.6 nM) or genetic deletion of the ?7nAChR significantly (P<0.05) attenuates the effect of prior stroke on lung injury and mortality caused by P. aeruginosa pneumonia in mice. Finally, pretreatment with PNU-282987, a pharmacologic activator of the ?7nAChR (EC(50): 0.2 ?M), significantly (P<0.05) increased lung injury caused by P. aeruginosa pneumonia, significantly (P<0.05) decreased the release of KC, a major neutrophil chemokine, and significantly (P<0.05) decreased intracellular bacterial killing by a mouse alveolar macrophage cell line and primary mouse neutrophils. In summary, the ?7 nicotinic cholinergic pathway plays an important role in mediating the systemic immunosuppression observed after stroke and directly contributes to more severe lung damage induced by P. aeruginosa.

    View details for DOI 10.1096/fj.11-197384

    View details for Web of Science ID 000305912500020

    View details for PubMedID 22490926

  • miR-181 targets multiple Bcl-2 family members and influences apoptosis and mitochondrial function in astrocytes MITOCHONDRION Ouyang, Y., Lu, Y., Yue, S., Giffard, R. G. 2012; 12 (2): 213-219

    Abstract

    Mitochondria are central to the execution of apoptosis, and the Bcl-2 protein family of pro- and anti-apoptotic proteins interacts with mitochondria to regulate apoptosis. Using bioinformatics we predicted that miR-181, a microRNA expressed in brain, could target the 3'UTRs of Bcl-2 family members Bcl-2-L11/Bim, Mcl-1, and Bcl-2. Using the luciferase reporter assay we confirmed these targets. We used mimic and inhibitor to alter miR-181a levels in primary astrocyte cultures and found miR-181a reduction was associated with increased Bcl-2 and Mcl-1 protein levels. Decreased miR-181a levels reduced glucose deprivation induced apoptosis, mitochondrial dysfunction, and loss of mitochondrial membrane potential in astrocytes.

    View details for DOI 10.1016/j.mito.2011.09.001

    View details for Web of Science ID 000301906600005

    View details for PubMedID 21958558

  • ER-Mitochondria Crosstalk during Cerebral Ischemia: Molecular Chaperones and ER-Mitochondrial Calcium Transfer. International journal of cell biology Ouyang, Y., Giffard, R. G. 2012; 2012: 493934-?

    Abstract

    It is commonly believed that sustained elevations in the mitochondrial matrix Ca(2+) concentration are a major feature of the intracellular cascade of lethal events during cerebral ischemia. The physical association between the endoplasmic reticulum (ER) and mitochondria, known as the mitochondria-associated ER membrane (MAM), enables highly efficient transmission of Ca(2+) from the ER to mitochondria under both physiological and pathological conditions. Molecular chaperones are well known for their protective effects during cerebral ischemia. It has been demonstrated recently that many molecular chaperones coexist with MAM and regulate the MAM and thus Ca(2+) concentration inside mitochondria. Here, we review recent research on cerebral ischemia and MAM, with a focus on molecular chaperones and ER-mitochondrial calcium transfer.

    View details for DOI 10.1155/2012/493934

    View details for PubMedID 22577383

  • Mice lacking the beta 2 adrenergic receptor have a unique genetic profile before and after focal brain ischaemia ASN NEURO White, R. E., Palm, C., Xu, L., Ling, E., Ginsburg, M., Daigle, B. J., Han, R., Patterson, A., Altman, R. B., Giffard, R. G. 2012; 4 (5): 343-356

    View details for DOI 10.1042/AN20110020

    View details for Web of Science ID 000308887200005

  • Inflammation, Mitochondria, and the Inhibition of Adult Neurogenesis JOURNAL OF NEUROSCIENCE RESEARCH Voloboueva, L. A., Giffard, R. G. 2011; 89 (12): 1989-1996

    Abstract

    The process of neurogenesis continues throughout life, with thousands of new neurons generated every day in the mammalian brain. Impairment of hippocampal neurogenesis has been suggested to be involved in neurodegenerative conditions, including the cognitive decline associated with aging, Alzheimer's disease, Parkinson's disease, and ionizing radiation. These neurodegenerative conditions are all characterized by proinflammatory changes and increased numbers of activated microglia. Activated microglia produce a variety of proinflammatory factors, including interleukin-6, tumor necrosis factor-?, reactive oxygen species, and nitric oxide, all of which are antineurogenic. These same factors have also been shown to suppress mitochondrial function, but the role of mitochondria in neurogenesis remains barely investigated. This brief review summarizes the findings of several studies that support a role for mitochondrial impairment as part of the mechanism of the reduction of neurogenesis associated with inflammation.

    View details for DOI 10.1002/jnr.22768

    View details for Web of Science ID 000296420400012

    View details for PubMedID 21910136

  • Astrocyte Proliferation Following Stroke in the Mouse Depends on Distance from the Infarct PLOS ONE Barreto, G. E., Sun, X., Xu, L., Giffard, R. G. 2011; 6 (11)

    Abstract

    Reactive gliosis is a hallmark of brain pathology and the injury response, yet the extent to which astrocytes proliferate, and whether this is central to astrogliosis is still controversial. We determined the fraction of mature astrocytes that proliferate in a mouse stroke model using unbiased stereology as a function of distance from the infarct edge. Cumulatively 11.1±1.2% of Aldh1l1(+) astrocytes within 400 µm in the cortical penumbra incorporate BrdU in the first week following stroke, while the overall number of astrocytes does not change. The number of astrocytes proliferating fell sharply with distance with more than half of all proliferating astrocytes found within 100 µm of the edge of the infarct. Despite extensive cell proliferation primarily of microglia and neutrophils/monocytes in the week following stroke, few mature astrocytes re-enter cell cycle, and these are concentrated close to the infarct boundary.

    View details for DOI 10.1371/journal.pone.0027881

    View details for Web of Science ID 000297789900028

    View details for PubMedID 22132159

  • Significance of Marrow-Derived Nicotinamide Adenine Dinucleotide Phosphate Oxidase in Experimental Ischemic Stroke ANNALS OF NEUROLOGY Tang, X. N., Zheng, Z., Giffard, R. G., Yenari, M. A. 2011; 70 (4): 606-615

    Abstract

    Reperfusion after stroke leads to infiltration of inflammatory cells into the ischemic brain. Nicotinamide adenine dinucleotide phosphate oxidase (NOX2) is a major enzyme system that generates superoxide in immune cells. We studied the effect of NOX2 derived from the immune cells in the brain and in blood cells in experimental stroke.To establish whether NOX2 plays a role in brain ischemia, strokes were created in mice, then mice were treated with the NOX2 inhibitor apocynin or vehicle and compared to mice deficient in NOX2's gp91 subunit and their wild-type littermates. To determine whether NOX2 in circulating cells versus brain resident cells contribute to ischemic injury, bone marrow chimeras were generated by transplanting bone marrow from wild-type or NOX2-deficient mice into NOX2 or wild-type hosts, respectively.Apocynin and NOX2 deletion both significantly reduced infarct size, blood-brain barrier disruption, and hemorrhagic transformation of the infarcts, compared to untreated wild-type controls. This was associated with decreased matrix metalloproteinase 9 expression and reduced loss of tight junction proteins. NOX2-deficient mice receiving wild-type marrow had better outcomes compared to the wild-type mice receiving wild-type marrow. Interestingly, wild-type mice receiving NOX2-deficient marrow had even smaller infarct sizes and less hemorrhage than NOX2-deficient mice receiving wild-type marrow.This indicates that NOX2, whether present in circulating cells or brain resident cells, contributes to ischemic brain injury and hemorrhage. However, NOX2 from the circulating cells contributed more to the exacerbation of stroke than that from brain resident cells. These data suggest the importance of targeting the peripheral immune system for treatment of stroke.

    View details for DOI 10.1002/ana.22476

    View details for Web of Science ID 000296396700014

    View details for PubMedID 22028221

  • Quantitative characterization and analysis of the dynamic NF-kappa B response in microglia BMC BIOINFORMATICS Sheppard, P. W., Sun, X., Emery, J. F., Giffard, R. G., Khammash, M. 2011; 12

    Abstract

    Activation of the NF-κB transcription factor and its associated gene expression in microglia is a key component in the response to brain injury. Its activation is dynamic and is part of a network of biochemical species with multiple feedback regulatory mechanisms. Mathematical modeling, which has been instrumental for understanding the NF-κB response in other cell types, offers a valuable tool to investigate the regulation of NF-κB activation in microglia at a systems level.We quantify the dynamic response of NF-κB activation and activation of the upstream kinase IKK using ELISA measurements of a microglial cell line following treatment with the pro-inflammatory cytokine TNFα. A new mathematical model is developed based on these data sets using a modular procedure that exploits the feedback structure of the network. We show that the new model requires previously unmodeled dynamics involved in the stimulus-induced degradation of the inhibitor IκBα in order to properly describe microglial NF-κB activation in a statistically consistent manner. This suggests a more prominent role for the ubiquitin-proteasome system in regulating the activation of NF-κB to inflammatory stimuli. We also find that the introduction of nonlinearities in the kinetics of IKK activation and inactivation is essential for proper characterization of transient IKK activity and corresponds to known biological mechanisms. Numerical analyses of the model highlight key regulators of the microglial NF-κB response, as well as those governing IKK activation. Results illustrate the dynamic regulatory mechanisms and the robust yet fragile nature of the negative feedback regulated network.We have developed a new mathematical model that incorporates previously unmodeled dynamics to characterize the dynamic response of the NF-κB signaling network in microglia. This model is the first of its kind for microglia and provides a tool for the quantitative, systems level study the dynamic cellular response to inflammatory stimuli.

    View details for DOI 10.1186/1471-2105-12-276

    View details for Web of Science ID 000294044200001

    View details for PubMedID 21729324

  • Increased Brain Injury and Worsened Neurological Outcome in Interleukin-4 Knockout Mice After Transient Focal Cerebral Ischemia STROKE Xiong, X., Barreto, G. E., Xu, L., Ouyang, Y. B., Xie, X., Giffard, R. G. 2011; 42 (7): 2026-2032

    Abstract

    Stroke causes brain injury with activation of an inflammatory response that can contribute to injury. We tested the hypothesis that the anti-inflammatory cytokine interleukin-4 (IL-4) reduces injury after stroke using IL-4 knockout (KO) adult male mice.IL-4 KO and wild-type mice were subjected to transient middle cerebral artery occlusion. Outcome was assessed by triphenyltetrazolium chloride staining for infarct volume, neuroscore and spontaneous activity for behavioral outcome, and immunostaining and stereological counting for cellular response.Infarction volume at 24 hours was significantly larger in IL-4 KO mice, neurological score was significantly worse, and spontaneous activity was reduced compared with wild-type mice. Increased macrophage/microglial infiltration, increased numbers of myeloperoxidase-positive cells, and increased Th1/Th2 ratio were observed in the infarct core in IL-4 KO mice. Reduced astrocyte activation was observed in the cortical penumbra in IL-4 KO mice. Recombinant IL-4 administered intracerebroventricularly before middle cerebral artery occlusion significantly reduced infarct volume, improved neurological score, reduced macrophages/microglia, and lowered the Th1/Th2 ratio in IL-4 KO mice, but not in wild-type.Loss of IL-4 signaling in KO mice was associated with worse outcome, and this was reversed by giving exogenous IL-4. Worsened outcome was associated with increased inflammation in the core, which was reversed in IL-4 KO but not significantly changed in wild-type mice by exogenous IL-4. This is consistent with IL-4 signaling leading to reduced inflammation in the core and a possible beneficial role for activated astrocytes in the penumbra.

    View details for DOI 10.1161/STROKEAHA.110.593772

    View details for Web of Science ID 000292090900051

    View details for PubMedID 21597016

  • Astrocytes: targets for neuroprotection in stroke. Central nervous system agents in medicinal chemistry Barreto, G., White, R. E., Ouyang, Y., Xu, L., Giffard, R. G. 2011; 11 (2): 164-173

    Abstract

    In the past two decades, over 1000 clinical trials have failed to demonstrate a benefit in treating stroke, with the exception of thrombolytics. Although many targets have been pursued, including antioxidants, calcium channel blockers, glutamate receptor blockers, and neurotrophic factors, often the focus has been on neuronal mechanisms of injury. Broader attention to loss and dysfunction of non-neuronal cell types is now required to increase the chance of success. Of the several glial cell types, this review will focus on astrocytes. Astrocytes are the most abundant cell type in the higher mammalian nervous system, and they play key roles in normal CNS physiology and in central nervous system injury and pathology. In the setting of ischemia astrocytes perform multiple functions, some beneficial and some potentially detrimental, making them excellent candidates as therapeutic targets to improve outcome following stroke and in other central nervous system injuries. The older neurocentric view of the central nervous system has changed radically with the growing understanding of the many essential functions of astrocytes. These include K+ buffering, glutamate clearance, brain antioxidant defense, close metabolic coupling with neurons, and modulation of neuronal excitability. In this review, we will focus on those functions of astrocytes that can both protect and endanger neurons, and discuss how manipulating these functions provides a novel and important strategy to enhance neuronal survival and improve outcome following cerebral ischemia.

    View details for PubMedID 21521168

  • Heat Shock Protein 72 Overexpression Prevents Early Postoperative Memory Decline after Orthopedic Surgery under General Anesthesia in Mice ANESTHESIOLOGY Vizcaychipi, M. P., Xu, L., Barreto, G. E., Ma, D., Maze, M., Giffard, R. G. 2011; 114 (4): 891-900

    Abstract

    Problems with learning and memory are common after surgery in the elderly and are associated with high morbidity. Heat shock protein 72 (Hsp72) confers neuroprotection against acute neurologic injury. We hypothesized that overexpression of Hsp72 would prevent the development of postoperative memory loss.C57BL/6 wild-type and Hsp72 overexpressing transgenic mice were randomly allocated to the following: control, isoflurane anesthesia alone, or tibial fracture during isoflurane anesthesia. Animals were trained 24 h before surgery using a fear conditioning protocol and assessed in their training environment and in a novel context on posttreatment days 1, 3, and 7. Microglial activation was assessed by immunostaining.Adult male C57BL/6 wild-type mice exhibited reduced memory evidenced by a decreased percentage freezing time on days 1 and 3 after anesthesia alone (58.8 ± 5, 46.5 ± 5 mean ± SEM) and after surgery (53.4 ± 6, 44.1 ± 7), compared with controls (78.8 ± 5, 63.4 ± 6; P < 0.05 and P < 0.001, respectively). Hsp72 mice showed no difference by treatment on any day. Similarly, nonhippocampal-dependent memory was significantly impaired on days 1 and 3 after surgery and day 3 after anesthesia. The genotype effect was significant on days 1 and 7. CD68-immunopositive activated microglia in the hippocampus varied modestly with subregion and time; on day 7, there was a significant treatment effect with no genotype effect, with more activated microglia after surgery in all regions.Hsp72 overexpression is associated with prevention of postoperative hippocampal-dependent and -independent memory deficit induced by anesthesia and/or surgery. Memory deficit is not correlated with numbers of activated hippocampal microglia.

    View details for Web of Science ID 000288694400020

    View details for PubMedID 21317632

  • Endotoxin-activated microglia injure brain derived endothelial cells via NF-kappa B, JAK-STAT and JNK stress kinase pathways JOURNAL OF INFLAMMATION-LONDON Kacimi, R., Giffard, R. G., Yenari, M. A. 2011; 8

    Abstract

    We previously showed that microglia damage blood brain barrier (BBB) components following ischemic brain insults, but the underlying mechanism(s) is/are not well known. Recent work has established the contribution of toll-like receptor 4 (TLR4) activation to several brain pathologies including ischemia, neurodegeneration and sepsis. The present study established the requirement of microglia for lipopolysaccharide (LPS) mediated endothelial cell death, and explored pathways involved in this toxicity. LPS is a classic TLR4 agonist, and is used here to model aspects of brain conditions where TLR4 stimulation occurs.In monocultures, LPS induced death in microglia, but not brain derived endothelial cells (EC). However, LPS increased EC death when cocultured with microglia. LPS led to nitric oxide (NO) and inducible NO synthase (iNOS) induction in microglia, but not in EC. Inhibiting microglial activation by blocking iNOS and other generators of NO or blocking reactive oxygen species (ROS) also prevented injury in these cocultures. To assess the signaling pathway(s) involved, inhibitors of several downstream TLR-4 activated pathways were studied. Inhibitors of NF-?B, JAK-STAT and JNK/SAPK decreased microglial activation and prevented cell death, although the effect of blocking JNK/SAPK was rather modest. Inhibitors of PI3K, ERK, and p38 MAPK had no effect.We show that LPS-activated microglia promote BBB disruption through injury to endothelial cells, and the specific blockade of JAK-STAT, NF-?B may prove to be especially useful anti-inflammatory strategies to confer cerebrovascular protection.

    View details for DOI 10.1186/1476-9255-8-7

    View details for Web of Science ID 000288615000001

    View details for PubMedID 21385378

  • Overexpressing GRP78 influences Ca2+ handling and function of mitochondria in astrocytes after ischemia-like stress MITOCHONDRION Ouyang, Y., Xu, L., Emery, J. F., Lee, A. S., Giffard, R. G. 2011; 11 (2): 279-286

    Abstract

    Ca(2+) transfer from endoplasmic reticulum (ER) to mitochondria at contact sites between the organelles can induce mitochondrial dysfunction and programmed cell death after stress. The ER-localized chaperone glucose-regulated protein 78kDa (GRP78/BiP) protects neurons against excitotoxicity and apoptosis. Here we show that overexpressing GRP78 protects astrocytes against ischemic injury, reduces net flux of Ca(2+) from ER to mitochondria, increases Ca(2+) uptake capacity in isolated mitochondria, reduces free radical production, and preserves respiratory activity and mitochondrial membrane potential after stress. We conclude that GRP78 influences ER-mitochondrial Ca(2+) crosstalk to maintain mitochondrial function and protect astrocytes from ischemic injury.

    View details for DOI 10.1016/j.mito.2010.10.007

    View details for Web of Science ID 000287112100006

    View details for PubMedID 21047562

  • Heat shock protein 72 (Hsp72) improves long term recovery after focal cerebral ischemia in mice NEUROSCIENCE LETTERS Xu, L., Xiong, X., Ouyang, Y., Barreto, G., Giffard, R. 2011; 488 (3): 279-282

    Abstract

    Many brain protective strategies have been tested over short survival intervals, but few have been examined for long term benefit. The inducible member of the Heat shock protein 70 (Hsp70) family, Heat shock protein 72 (Hsp72), has been widely found to reduce ischemic injury. Here we assessed outcome in Hsp72 transgenic overexpressing mice and wild type littermates for one month following transient focal ischemia. Hsp72 reduced infarct area lost and improved behavioral outcome on rotarod and foot fault at one month. Thus protection by Hsp72 overexpression is long lasting, and includes improved recovery of motor function over one month.

    View details for DOI 10.1016/j.neulet.2010.11.047

    View details for Web of Science ID 000286793000013

    View details for PubMedID 21108992

  • Direct protection of cultured neurons from ischemia-like injury by minocycline. Anatomy & cell biology Huang, W. C., Qiao, Y., Xu, L., Kacimi, R., Sun, X., Giffard, R. G., Yenari, M. A. 2010; 43 (4): 325-331

    Abstract

    Minocycline, a tetracycline antibiotic, is now known to protect cells via an anti-inflammatory mechanism. We further explored this effect using an in vitro model of ischemia-like injury to neurons. Coculturing neurons with microglia, the brain's resident immune cell, modestly increased cell death due to oxygen and glucose deprivation (OGD), compared to neurons alone. Treatment of cocultures with minocycline decreased cell death to a level significantly lower than that of neurons alone. Treatment of cocultures with minocycline or inhibitors of various immune mediators, also led to decreased cell death. Importantly, treatment of neuron cultures without added microglia with these same inhibitors of tissue plasminogen activator, matrix metalloproteinases, TNF-alpha and inducible nitric oxide synthase as well as minocycline also led to decreased cell death. Thus, anti-inflammatory treatments appear to be directly protective of neurons from in vitro ischemia.

    View details for DOI 10.5115/acb.2010.43.4.325

    View details for PubMedID 21267407

  • Age-related Defects in Sensorimotor Activity, Spatial Learning, and Memory in C57BL/6 Mice JOURNAL OF NEUROSURGICAL ANESTHESIOLOGY Barreto, G., Huang, T., Giffard, R. G. 2010; 22 (3): 214-219

    Abstract

    Impaired locomotor activity and spatial memory are common features in the natural aging process, and aging is an important risk factor for neurodegenerative disease and postoperative cognitive dysfunction. To characterize age-related changes in psychomotor performance, we assessed sensorimotor activity, spatial learning, and memory in C57BL/6 mice using the Rotarod, foot fault, and Barnes Maze tests. Old mice exhibit significant deficits in locomotor activity and spatial memory relative to young mice, but improve with training. These tests will be useful to assess outcome in neurodegenerative disease and postoperative cognitive dysfunction models carried out in aged mice.

    View details for DOI 10.1097/ANA.0b013e3181d56c98

    View details for Web of Science ID 000278709200006

    View details for PubMedID 20479674

  • Inflammation and NF kappa B activation is decreased by hypothermia following global cerebral ischemia NEUROBIOLOGY OF DISEASE Webster, C. M., Kelly, S., Koike, M. A., Chock, V. Y., Giffard, R. G., Yenari, M. A. 2009; 33 (2): 301-312

    Abstract

    We previously showed that hypothermia attenuates inflammation in focal cerebral ischemia (FCI) by suppressing activating kinases of nuclear factor-kappa B (NFkappaB). Here we characterize the inflammatory response in global cerebral ischemia (GCI), and the influence of mild hypothermia. Rodents were subjected to GCI by bilateral carotid artery occlusion. The inflammatory response was accompanied by microglial activation, but not neutrophil infiltration, or blood brain barrier disruption. Mild hypothermia reduced CA1 damage, decreased microglial activation and decreased nuclear NFkappaB translocation and activation. Similar anti-inflammatory effects of hypothermia were observed in a model of pure brain inflammation that does not cause brain cell death. Primary microglial cultures subjected to oxygen glucose deprivation (OGD) or stimulated with LPS under hypothermic conditions also experienced less activation and less NFkappaB translocation. However, NFkappaB regulatory proteins were not affected by hypothermia. The inflammatory response following GCI and hypothermia's anti-inflammatory mechanism is different from that observed in FCI.

    View details for DOI 10.1016/j.nbd.2008.11.001

    View details for Web of Science ID 000263120500018

    View details for PubMedID 19063968

  • Overexpression of mitochondrial Hsp70/Hsp75 in rat brain protects mitochondria, reduces oxidative stress, and protects from focal ischemia JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Xu, L., Voloboueva, L. A., Ouyang, Y., Emery, J. F., Giffard, R. G. 2009; 29 (2): 365-374

    Abstract

    Mitochondria are known to be central to the cell's response to ischemia, because of their role in energy generation, in free radical generation, and in the regulation of apoptosis. Heat shock protein 75 (Hsp75/Grp75/mortalin/TRAP1) is a member of the HSP70 chaperone family, which is targeted to mitochondria. Overexpression of Hsp75 was achieved in rat brain by DNA transfection, and expression was observed in both astrocytes and neurons. Rats were subjected to 100 mins middle cerebral artery occlusion followed by assessment of infarct volume, neurological score, mitochondrial function, and levels of oxidative stress at 24 h reperfusion. Overexpression of Hsp75 reduced infarct area from 44.6%+/-21.1% to 25.7%+/-12.1% and improved neurological outcome significantly. This was associated with improved mitochondrial function as shown by protection of complex IV activity, marked reduction of free radical generation detected by hydroethidine fluorescence, reduction of lipid peroxidation detected by 4-hydroxy-2-nonenol immunoreactivity, and increased preservation of ATP levels. This suggests that targeting mitochondria for protection may be a useful strategy to reduce ischemic brain injury.

    View details for DOI 10.1038/jcbfm.2008.125

    View details for Web of Science ID 000263119900016

    View details for PubMedID 18985056

  • Postischemic Brain Injury Is Attenuated in Mice Lacking the beta(2)-Adrenergic Receptor ANESTHESIA AND ANALGESIA Han, R., Ouyang, Y., Xu, L., Agrawal, R., Patterson, A. J., Giffard, R. G. 2009; 108 (1): 280-287

    Abstract

    Several beta-adrenergic receptor (betaAR) antagonists have been shown to have neuroprotective effects against cerebral ischemia. However, clenbuterol, a beta(2)AR agonist, was shown to have neuroprotective activity by increasing nerve growth factor expression. We used beta(2)AR knockout mice and a beta(2) selective antagonist to test the effect of loss of beta(2)ARs on outcome from transient focal cerebral ischemia.Ischemia was induced by the intraluminal suture method, for 60 min of middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion. Neurological score was determined at 24 h reperfusion and infarct size was determined by cresyl violet or 2,3,5-triphenyltetrazolium chloride staining. beta(2)AR knockout mice and wild-type congenic FVB/N controls were studied, as well as 2 groups of wild type mice given either ICI 118,551 (0.2 mg/kg) or 0.9% saline intraperitoneally 30 min before MCAO (n = 10 per group). Changes in expression of heat shock protein (Hsp)72 after ischemia were examined by immunohistochemistry and western blots.Compared with wild type littermates, infarct volume was decreased by 22.3% in beta(2)AR knockout mice (39.7 +/- 10.7 mm(3) vs 51.0 +/- 11.4 mm(3), n = 10/group, P = 0.034) after 60 min of MCAO followed by 24 h reperfusion. Pretreatment with a beta(2)AR selective antagonist, ICI 118,551, also decreased infarct size significantly, by 25.1%, compared with the saline control (32.8 +/- 11.9 mm(3) vs 43.8 +/- 10.3 mm(3), n = 10/group, P = 0.041). Neurological scores were also significantly improved in mice lacking the beta(2)AR or pretreated with ICI 118,551. After cerebral ischemia, total levels of Hsp72 and the number of Hsp72 immunopositive cells were greater in mice lacking beta(2) AR.Brain injury is reduced and neurological outcome improved after MCAO in mice lacking the beta(2)AR, or in wild type mice pretreated with a selective beta(2)AR antagonist. This is consistent with a shift away from prosurvival signaling to prodeath signaling in the presence of beta(2)AR activation in cerebral ischemia. Protection is associated with higher levels of Hsp72, a known antideath protein. The effect of beta(2)AR signaling in the setting of cerebral ischemia is complex and warrants further study.

    View details for DOI 10.1213/ane.0b013e318187ba6b

    View details for Web of Science ID 000261963000043

    View details for PubMedID 19095863

  • Regulation of apoptotic and inflammatory cell signaling in cerebral ischemia - The complex roles of heat shock protein 70 ANESTHESIOLOGY Giffard, R. G., Han, R., Emery, J. F., Duan, M., Pittet, J. F. 2008; 109 (2): 339-348

    Abstract

    Although heat shock proteins have been studied for decades, new intracellular and extracellular functions in a variety of diseases continue to be discovered. Heat shock proteins function within networks of interacting proteins; they can alter cellular physiology rapidly in response to stress without requiring new protein synthesis. This review focuses on the heat shock protein 70 family and considers especially the functions of the inducible member, heat shock protein 72, in the setting of cerebral ischemia. In general, inhibiting apoptotic signaling at multiple points and up-regulating survival signaling, heat shock protein 70 has a net prosurvival effect. Heat shock protein 70 has both antiinflammatory and proinflammatory effects depending on the cell type, context, and intracellular or extracellular location. Intracellular effects are often antiinflammatory with inhibition of nuclear factor-kappaB signaling. Extracellular effects can lead to inflammatory cytokine production or induction of regulatory immune cells and reduced inflammation.

    View details for Web of Science ID 000257939600021

    View details for PubMedID 18648242

  • Overexpression of mitochondrial Hsp70/Hsp75 protects astrocytes against ischemic injury in vitro JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Voloboueva, L. A., Duan, M., Ouyang, Y., Emery, J. F., Stoy, C., Giffard, R. G. 2008; 28 (5): 1009-1016

    Abstract

    Mitochondrial heat shock protein 70 (mtHsp70/Hsp75/Grp75/mortalin/TRAP-1/PBP74) is an essential mitochondrial chaperone and a member of the heat shock protein 70 (HSP70) family. Although many studies have shown the protective properties of overexpression of the cytosolic inducible member of the HSP70 family, Hsp72, few studies have investigated the protective potential of Hsp75 against ischemic injury. Mitochondria are one of the primary targets of ischemic injury in astrocytes. In this study, we analyzed the effects of Hsp75 overexpression on cellular levels of reactive oxygen species (ROS), mitochondrial membrane potential, ATP levels, and viability during the ischemia-like conditions of oxygen-glucose deprivation (OGD) or glucose deprivation (GD) in primary astrocytic cultures. We show that Hsp75 overexpression decreases ROS production and preserves mitochondrial membrane potential during GD, and preserves ATP levels and cell viability during OGD. These findings indicate that Hsp75 can provide protection against ischemia-like in vitro injury and suggest that it should be further studied as a potential candidate for protection against ischemic injury.

    View details for DOI 10.1038/sj.jcbfm.9600600

    View details for Web of Science ID 000255261300015

    View details for PubMedID 18091755

  • Inhibition of mitochondrial function in astrocytes: implications for neuroprotection JOURNAL OF NEUROCHEMISTRY Voloboueva, L. A., Suh, S. W., Swanson, R. A., Giffard, R. G. 2007; 102 (4): 1383-1394

    Abstract

    Much evidence suggests that astrocytes protect neurons against ischemic injury. Although astrocytes are more resistant to some insults than neurons, few studies offer insight into the real time changes of astrocytic protective functions with stress. Mitochondria are one of the primary targets of ischemic injury in astrocytes. We investigated the time course of changes in astrocytic ATP levels, plasma membrane potential, and glutamate uptake, a key protective function, induced by mitochondrial inhibition. Our results show that significant functional change precedes reduction in astrocytic viability with mitochondrial inhibition. Using the mitochondrial inhibitor fluorocitrate (FC, 0.25 mmol/L) that is preferentially taken by astrocytes we found that inhibition of astrocyte mitochondria increased vulnerability of co-cultured neurons to glutamate toxicity. In our studies, the rates of FC-induced astrocytic mitochondrial depolarization were accelerated in mixed astrocyte/neuron cultures. We hypothesized that the more rapid mitochondrial depolarization was promoted by an additional energetic demand imposed be the co-cultured neurons. To test this hypothesis, we exposed pure astrocytic cultures to 0.01-1 mmol/L aspartate as a metabolic load. Aspartate application accelerated the rates of FC-induced mitochondrial depolarization, and, at 1 mmol/L, induced astrocytic death, suggesting that strong energetic demands during ischemia can compromise astrocytic function and viability.

    View details for DOI 10.1111/j.1471-4159.2007.04634.x

    View details for Web of Science ID 000248884100034

    View details for PubMedID 17488276

  • NOx and ADMA changes with focal ischemia, amelioration with the chaperonin GroEL NEUROSCIENCE LETTERS Xu, L., Wang, B., Kaur, K., Kho, M. F., Cooke, J. P., Giffard, R. G. 2007; 418 (2): 201-204

    Abstract

    Both nitric oxide and asymmetric dimethylarginine (ADMA) play a critical role in the regulation of cerebral blood flow, though their neuroprotective and cytotoxic effects are still under investigation. In this study, we found that nitrate/nitrite (NOx) levels in plasma, ischemic brain tissue, and cerebrospinal fluid (CSF) increased significantly 24h after 2h transient middle cerebral artery occlusion (MCAO) in rats. ADMA levels were unchanged in plasma, but decreased significantly in CSF 24h following MCAO. The CSF ADMA/NOx ratio decreased markedly following ischemia. Rats protected by expression of the chaperonin GroEL or its folding deficient mutant D87K had lower plasma NOx levels at 24h reperfusion. ADMA, NO, and their ratio in CSF merit further study as biomarkers for ischemic brain injury.

    View details for DOI 10.1016/j.neulet.2007.03.021

    View details for Web of Science ID 000246871600017

    View details for PubMedID 17398004

  • Selective dysfunction of hippocampal CA1 astrocytes contributes to delayed neuronal damage after transient forebrain ischemia JOURNAL OF NEUROSCIENCE Ouyang, Y., Voloboueva, L. A., Xu, L., Giffard, R. G. 2007; 27 (16): 4253-4260

    Abstract

    Transient global ischemia, as with cardiac arrest, causes loss of CA1 hippocampal neurons 2-4 d later, whereas nearby dentate gyrus (DG) neurons are relatively resistant. Whether differential astrocyte vulnerability in ischemic injury contributes to CA1 neuronal death is uncertain. Here, we find that CA1 astrocytes are more sensitive to ischemia than DG astrocytes. In rats subjected to transient forebrain ischemia, CA1 astrocytes lose glutamate transport activity and immunoreactivity for GFAP, S100beta, and glutamate transporter GLT-1 within a few hours of reperfusion, but without astrocyte cell death. Oxidative stress may contribute to the observed selective CA1 changes, because CA1 astrocytes show early increases in mitochondrial free radicals and reduced mitochondrial membrane potential. Similar changes were not observed in DG astrocytes. Upregulation of GLT-1 expression in astrocytes with ceftriaxone protected CA1 neurons from forebrain ischemia. We suggest that greater oxidative stress and loss of GLT-1 function selectively in CA1 astrocytes is central to the well known delayed death of CA1 neurons.

    View details for DOI 10.1523/JNEUROSCI.0211-07.2007

    View details for Web of Science ID 000245810200004

    View details for PubMedID 17442809

  • Regulation of the rat brain Na+-driven Cl-/HCO3- exchanger involves protein kinase A and a multiprotein signaling complex FEBS LETTERS Lee, Y., Ouyang, Y., Giffard, R. G. 2006; 580 (20): 4865-4871

    Abstract

    The Na(+)-driven Cl(-)/HCO(3)(-) exchanger (NCBE) plays an important role in the regulation of intracellular pH (pH(i)). We previously identified two variants of NCBE from rat brain of which the variant with a carboxyterminal PSD-95/Dlg/ZO-1 (PDZ) motif (rb2NCBE) colocalized with the actin cytoskeleton. Increased rb2NCBE activity by PKA inhibition and reduction by forskolin and cAMP agonist suggest PKA regulation of NCBE. Disruption of actin filaments also decreased rb2NCBE activity. EBP50 and FLAG-rb2NCBE were reciprocally co-immunoprecipitated from rb2NCBE transfected cells. It is concluded that NCBE activity is inhibited by PKA and depends on the integrity of the actin cytoskeleton within a multiprotein complex at the plasma membrane.

    View details for DOI 10.1016/j.febslet.2006.07.075

    View details for Web of Science ID 000240371000017

    View details for PubMedID 16916513

  • 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

  • The carboxyl-terminal domain of inducible Hsp70 protects from ischemic injury in vivo and in vitro JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Sun, Y., Ouyang, Y., Xu, L., Chow, A. M., Anderson, R., Hecker, J. G., Giffard, R. G. 2006; 26 (7): 937-950

    Abstract

    Heat shock protein (Hsp)70 can suppress both necrosis and apoptosis induced by various injuries in vivo and in vitro. However, the relative importance of different functions and binding partners of Hsp70 in ischemic protection is unknown. To explore this question, we tested the ability of Hsp70-K71E, an adenosine triphosphate (ATP)ase-deficient point mutant, and Hsp70-381-640, a deletion mutant lacking the ATPase domain and encoding the carboxyl-terminal portion, to protect against ischemia-like injury in vivo and in vitro. Heat shock protein 70-wild type (-WT), -K71E, -381-640, and control vector plasmid LXSN were expressed in primary murine astrocyte cultures. Astrocytes overexpressing Hsp70-WT, -K71E, or -381-640 were all significantly protected from 4 h combined oxygen-glucose deprivation and 24 h reperfusion when assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or propidium iodide staining and cell counting (P < 0.05). Brains of rats were transfected with plasmids encoding Hsp70-WT, -K71E, -381-640, or LXSN 24 h before 2 h middle cerebral artery occlusion followed by 24 h reperfusion. Animals that overexpressed either of the mutant proteins or Hsp70-WT had significantly better neurological scores and smaller infarcts than control animals. Protection by both mutants was associated with reduced protein aggregation, as assessed by ubiquitin immunohistochemistry and reduced nuclear translocation of apoptosis-inducing factor. The results show that the carboxyl-terminal portion of Hsp70 is sufficient for neuroprotection. This indicates that neither the ability to fold denatured proteins nor interactions with cochaperones or other proteins that bind the amino-terminal half of Hsp70 are essential to ischemic protection.

    View details for DOI 10.1038/sj.jcbfm.9600246

    View details for Web of Science ID 000238589700009

    View details for PubMedID 16292251

  • Overexpression of inducible heat shock protein 70 and its mutants in astrocytes is associated with maintenance of mitochondrial physiology during glucose deprivation stress CELL STRESS & CHAPERONES Ouyang, Y., Xu, L., Sun, Y., Giffard, R. G. 2006; 11 (2): 180-186

    Abstract

    Wild-type inducible Hsp70 (WT) and 2 folding deficient mutants protect the brain against focal cerebral ischemia in vivo and brain cells from oxygen-glucose deprivation (OGD) in vitro, but the protective mechanisms remain unclear. Mitochondria are central to both normal physiological function and the regulation of cell death. We tested the effect of overexpressing Hsp70 and 2 mutants, Hsp70-K71 E, an adenosine triphosphatase (ATPase)-deficient point mutant, and Hsp70-381-640, a deletion mutant lacking the ATPase domain on mitochondrial physiology under glucose deprivation (GD) stress in primary cultured astrocytes. Mitochondrial membrane potential was assessed using a potentiometric fluorescent dye tetramethylrhodamine ethyl ester (TMRE). By 5 hours of GD, the mitochondria in the LXSN control transfected astrocytes had markedly reduced membrane potential. However, in the Hsp70-WT, -K71E, and -381-640 groups, there was no apparent change in TMRE signal during 5 hours of GD. Oxygen consumption was measured to assess oxidative respiration. Overexpression of Hsp70-K71 E and -381-640 prevented the decrease in state III respiration observed at 5 hours, and all 3 prevented the increase in state IV respiration found in LXSN controls after 5 hours of GD. Reactive oxygen species (ROS) production was assessed with hydroethidine. Hsp70 and its mutants all significantly reduced the increases in ROS accumulation during 5 hours of GD. The results demonstrate that the protective effect of the carboxyl-terminal half of Hsp70 and of the full-length molecule is associated with better maintained mitochondrial membrane potential, better maintained state IV respiration, and reduced ROS generation during GD.

    View details for Web of Science ID 000238467800011

    View details for PubMedID 16817324

  • Chaperonin GroEL and its mutant D87K protect from ischemia in vivo and in vitro NEUROBIOLOGY OF AGING Xu, L. J., Dayal, M., Ouyang, Y. B., Sun, Y. J., Yang, C. F., Frydman, J., Giffard, R. G. 2006; 27 (4): 562-569

    Abstract

    Protein aggregation and misfolding are central mechanisms of both acute and chronic neurodegeneration. Overexpression of chaperone Hsp70 protects from stroke in animal and cell culture models. Although it is accepted that chaperones protect cells, the mechanism of protection by chaperones in ischemic injury is poorly understood. In particular, the relative importance of preventing protein aggregation compared to facilitating correct protein folding during ischemia and recovery is not known. To test the importance of protein folding and minimize interaction with co-chaperones we studied the bacterial chaperonin GroEL (HSPD1) and a folding-deficient mutant D87K. Both molecules protected cells from ischemia-like injury, and reduced infarct volume and improved neurological outcome after middle cerebral artery occlusion in rats. Protection was associated with reduced protein aggregation, assessed by ubiquitin immunohistochemistry. Marked neuroprotection by the folding-deficient chaperonin demonstrates that inhibition of aggregation is sufficient to protect the brain from ischemia. This suggests that strategies to maintain protein solubility and inhibit aggregation in the face of acute insults such as stroke may be a useful protective strategy.

    View details for DOI 10.1016/j.neurobiolaging.2005.09.032

    View details for Web of Science ID 000236066200005

    View details for PubMedID 16257478

  • Microglia potentiate damage to blood-brain barrier constituents - Improvement by minocycline in vivo and in vitro STROKE Yenari, M. A., Xu, L. J., Tang, X. N., Qiao, Y. L., Giffard, R. G. 2006; 37 (4): 1087-1093

    Abstract

    Blood-brain barrier (BBB) disruption after stroke can worsen ischemic injury by increasing edema and causing hemorrhage. We determined the effect of microglia on the BBB and its primary constituents, endothelial cells (ECs) and astrocytes, after ischemia using in vivo and in vitro models.Primary astrocytes, ECs, or cocultures were prepared with or without added microglia. Primary ECs were more resistant to oxygen-glucose deprivation/reperfusion than astrocytes. ECs plus astrocytes showed intermediate vulnerability. Microglia added to cocultures nearly doubled cell death. This increase was prevented by minocycline and apocynin. In vivo, minocycline reduced infarct volume and neurological deficits and markedly reduced BBB disruption and hemorrhage in mice after experimental stroke.Inhibition of microglial activation may protect the brain after ischemic stroke by improving BBB viability and integrity. Microglial inhibitors may prove to be an important treatment adjunct to fibrinolysis.

    View details for DOI 10.1161/01.STR.0000206281.77178.ac

    View details for Web of Science ID 000236292100035

    View details for PubMedID 16497985

  • Antegrade cerebral perfusion reduces apoptotic neuronal injury in a neonatal piglet model of cardiopulmonary bypass JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY Chock, V. Y., Amir, G., DAVIS, C. R., Ramamoorthy, C., Riemer, R. K., Ray, D., Giffard, R. G., Reddy, V. M. 2006; 131 (3): 659-665

    Abstract

    Neonates with congenital heart disease might require surgical repair with deep hypothermic circulatory arrest, a technique associated with adverse neurodevelopmental outcomes. Antegrade cerebral perfusion is thought to minimize ischemic brain injury, although there are no supporting experimental data. We sought to evaluate and compare the extent of neurologic injury in a neonatal piglet model of deep hypothermic circulatory arrest and antegrade cerebral perfusion.Neonatal piglets undergoing cardiopulmonary bypass were randomized to deep hypothermic circulatory arrest or antegrade cerebral perfusion for 45 minutes. Animals were killed after 6 hours of recovery, and brain tissue was stained for evidence of cellular injury and for the apoptotic markers activated caspase 3 and cytochrome c translocation from mitochondria to cytosol.Piglets from the antegrade cerebral perfusion group exhibited less apoptotic or necrotic injury (4 +/- 3 vs 29 +/- 12 cells per field, P = .03). The piglets undergoing antegrade cerebral perfusion also had less evidence of apoptosis, with fewer cells staining for activated caspase 3 (57 +/- 8 vs 93 +/- 9 cells per field, P = .001) or showing cytochrome c translocation (6 +/- 2 vs 15 +/- 4 cells per field, P = .02).The use of antegrade cerebral perfusion in place of deep hypothermic circulatory arrest reduces evidence of apoptosis and histologic injury in neonatal piglets. Neonates with congenital heart disease might benefit from antegrade cerebral perfusion during complex cardiac surgery to improve their overall neurologic outcome.

    View details for DOI 10.1016/j.jtcvs.2005.09.005

    View details for Web of Science ID 000235940600024

    View details for PubMedID 16515920

  • Geldanamycin treatment reduces delayed CA1 damage in mouse hippocampal organotypic cultures subjected to oxygen glucose deprivation NEUROSCIENCE LETTERS Ouyang, Y. B., Xu, L. J., Giffard, R. G. 2005; 380 (3): 229-233

    Abstract

    Our prior work demonstrated that geldanamycin (GA) reduced injury due to oxygen-glucose deprivation (OGD) in primary astrocyte cultures. Using medium with an ionic composition similar to that observed during in vivo global ischemia, the selectivity and temporal profile of CA1 neuronal damage seen in vivo was mimicked with OGD in mouse hippocampal organotypic slice cultures. The present study tested the ability of GA to reduce delayed neuronal death in such cultures. Treating organotypic cultures with 100 nM GA for 24 h prior to OGD induced Hsp70 and significantly reduced CA1 neuronal damage. Staining with ubiquitin to identify protein aggregates revealed reduced redistribution of ubiquitin, consistent with reduced protein aggregation likely due at least in part to induction of Hsp70 by GA.

    View details for DOI 10.1016/j.neulet.2005.01.055

    View details for Web of Science ID 000229160000006

    View details for PubMedID 15862891

  • Ischemia-induced programmed cell death in astrocytes GLIA Giffard, R. G., Swanson, R. A. 2005; 50 (4): 299-306

    Abstract

    Astrocytes are essential for neuronal survival and function, neurogenesis, and neural repair. Although astrocytes are more resistant than neurons to most stress conditions in vitro, certain astrocyte subtypes, such as the glial fibrillary acidic protein (GFAP)-negative protoplasmic astrocytes that predominate in gray matter structures, may be equally or more sensitive than neurons to ischemia in vivo. Programmed cell death differs from passive, necrotic death in that cell constituents actively participate in cell demise. Like neurons, astrocytes undergo programmed cell death during normal development. Cell culture studies have shown that astrocytes can be induced to undergo apoptosis and other forms of programmed cell death by many factors relevant to ischemia, including acidosis, oxidative stress, substrate deprivation, and cytokines. Animal models of cerebral ischemia have confirmed nuclear condensation and upregulation of Bax and caspases in a subset of astrocytes exposed to ischemia, especially in immature brain. A causal role for these events in astrocyte death is supported by improved astrocyte survival after inhibition of caspase-dependent cell death pathways. Astrocyte survival is also improved by blocking the poly(ADP-ribose)-1 cell death pathway. Markers of programmed cell death are generally less evident and less widespread in astrocytes than in neighboring neurons. However, most studies to date have relied only on markers of classical apoptosis. In addition, these studies have relied almost exclusively on GFAP to identify astrocytes. Since most protoplasmic astrocytes are poorly immunoreactive for GFAP, the extent of ischemia-induced programmed cell death in this cell type remains uncertain.

    View details for DOI 10.1002/glia.20167

    View details for Web of Science ID 000228871600003

    View details for PubMedID 15846803

  • Overexpression of copper/zinc superoxide dismutase decreases ischemia-like astrocyte injury FREE RADICAL BIOLOGY AND MEDICINE Wang, H., Ma, J. H., Giffard, R. G. 2005; 38 (8): 1112-1118

    Abstract

    Overexpression of copper/zinc superoxide dismutase (SOD1) in transgenic mice protects from transient focal cerebral ischemia in adult animals, but increases oxidative injury in perinatal mice. The effect of SOD1 overexpression on astrocytes subjected to ischemia-like insults has not yet been determined. Overexpression of human SOD1 in astrocytes resulted in a 3-fold increase in SOD1 activity without coupled up-regulation of catalase or glutathione peroxidase activities. Cells subjected to oxygen-glucose deprivation (OGD) or glucose deprivation to mimic ischemic injury were protected by SOD1 overexpression. OGD injury was reduced 47.6+/-9.3%, assessed by release of lactate dehydrogenase. OGD also caused a significant increase in catalase activity which was moderated by SOD1 overexpression. The level of glutathione in astrocytes overexpressing SOD1 was maintained at higher levels following 5 h OGD compared to control cultures under the same conditions. Reduction of glutathione prior to OGD significantly increased cell death of SOD1-overexpressing astrocytes as well as controls, but SOD1 still provided significant protection, suggesting that both GSH-dependent scavenging and GSH-independent scavenging are relevant to SOD1 protection in astrocytes.

    View details for DOI 10.1016/j.freeradbiomed.2005.01.010

    View details for Web of Science ID 000228057700014

    View details for PubMedID 15780769

  • Development of neonatal murine microglia in vitro: Changes in response to lipopolysaccharide and ischemia-like injury PEDIATRIC RESEARCH Chock, V. Y., Giffard, R. G. 2005; 57 (4): 475-480

    Abstract

    Hypoxic/ischemic brain injury in the neonate can activate an inflammatory cascade, which potentiates cellular injury. The role of microglia in this inflammatory response has not been studied extensively. We used an in vitro model of murine microglia to investigate changes in microglial cytokine release and injury during early development. Isolated microglia were subjected to lipopolysaccharide (LPS) activation or injury by glucose deprivation (GD), serum deprivation (SD), or combined oxygen-glucose deprivation (OGD) for varying durations. The extent and the type of cell death were determined by trypan blue, terminal deoxynucleotidyl end-nick labeling, and annexin staining. Early-culture microglia (2-3 d in purified culture) showed significantly more apoptotic cell death after SD, GD, and OGD compared with microglia maintained in culture for 14-17 d. Measurements of tumor necrosis factor-alpha (TNF-alpha) and IL-1beta in culture media demonstrated that OGD induced greater release of both TNF-alpha and IL-1beta than LPS activation, with early-culture microglia producing more TNF-alpha compared with late-culture microglia. Microglia that are cultured for a short time are more sensitive to ischemia-like injury in vitro than those that are cultured for longer durations and may contribute to worsening brain injury by increased release of inflammatory cytokines. Inhibition of microglial activation and decreasing proinflammatory cytokine release may be targets for reduction of neonatal hypoxic/ischemic brain injury.

    View details for DOI 10.1203/01.PDR.0000155758.79523.44

    View details for Web of Science ID 000227746600003

    View details for PubMedID 15718374

  • Mild intraoperative hypothermia during surgery for intracranial aneurysm NEW ENGLAND JOURNAL OF MEDICINE Todd, M. M., Hindman, B. J., Clarke, W. R., Torner, J. C., Todd, M., Hindman, B., Clarke, W., Chaloner, K., Torner, J., Davis, P., Howard, M., Tranel, D., Anderson, S., Todd, M., Hindman, B., Weeks, J., Moss, L., Winn, J., Clarke, W., Chaloner, K., Wichman, M., Peters, R., Hansen, M., Anderson, D., Lang, J., Yoo, B., Adams, H., Clifton, G., Gelb, A., Loftus, C., Schubert, A., Warner, D., Young, W., Frankowski, R., Kieburtz, K., Prough, D., Sternau, L., Marler, J., Moy, C., Matta, B., Kirkpatrick, P., Chatfield, D., Skilbeck, C., Kirollos, R., Rasulo, F., English, K., Duffy, C., Pedersen, K., Scurrah, N., Burnstein, R., Prabhu, A., Salmond, C., Blackwell, A., Birrell, J., Jackson, S., Kassell, N., Pajewski, T., Fraley, H., Morris, A., Alden, T., Shaffrey, M., Bogdonoff, D., Durieux, M., Zuo, Z., Littlewood, K., Nemergut, E., Bedford, R., Stone, D., Balestrieri, P., Mason, J., Henry, G., Ting, P., Shafer, J., Blount, T., Kim, L., James, A., Farace, E., Clark, L., Irons, M., Sasaki, T., Webb, K., Short, T., Mee, E., Ormrod, J., Jane, J., Alden, T., Heppner, P., Olson, S., Ellegala, D., Lind, C., Sheehan, J., Woodfield, M., Law, A., Harrison, M., DAVIES, P., Campbell, D., Robertson, N., Fry, R., Sage, D., Laurent, S., Bradfield, C., Pedersen, K., Smith, K., Young, Y., Chambers, C., Hodkinson, B., Biddulph, J., Jensen, L., Ogden, J., Thayer, Z., Lee, F., Crump, S., Quaedackers, J., Wray, A., Roelfsema, V., Greif, R., Kleinpeter, G., Lothaller, C., Knosp, E., Pfisterer, W., Schatzer, R., Salem, C., Kutalek, W., Tuerkkan, E., Koller, L., Weber, T., Buchmann, A., Merhaut, C., Graf, M., Rapf, B., Lam, A., NEWELL, D., Tanzi, P., Lee, L., Domino, K., Vavilala, M., Bramhall, J., Souter, M., Britz, G., Winn, H., Bybee, H., Costello, T., Murphy, M., Harris, K., Thien, C., Nye, D., Han, T., McNeill, P., O'Brien, B., Cormack, J., Wyss, A., Grauer, R., Popovic, R., Jones, S., Deam, R., Heard, G., Watson, R., Evered, L., Bardenhagen, F., Meade, C., Haartsen, J., Kruger, J., Wilson, M., Maktabi, M., Traynelis, V., McAllister, A., Leonard, P., Hindman, B., Brian, J., Mensink, F., From, R., Papworth, D., Schmid, P., Dehring, D., Howard, M., Hitchon, P., VanGilder, J., Weeks, J., Moss, L., Manzel, K., Anderson, S., Tack, R., Taggard, D., Lennarson, P., Menhusen, M., Gelb, A., Lownie, S., Craen, R., Novick, T., Ferguson, G., Duggal, N., Findlay, J., Ng, W., Cowie, D., Badner, N., Herrick, I., Smith, H., Heard, G., Peterson, R., Howell, J., Lindsey, L., Carriere, L., von Lewinski, M., Schaefer, B., Bisnaire, D., Doyle-Pettypiece, P., McTaggart, M., Giannotta, S., Zelman, V., Thomson, E., Babayan, E., McCleary, C., Fishback, D., Samra, S., Thompson, B., Chandler, W., McGillicuddy, J., Tremper, K., Turner, C., Smythe, P., Dy, E., Pai, S., Portman, V., Palmisano, J., Auer, D., Quigley, M., Giordani, B., Freymuth, A., Scott, P., Silbergleit, R., Hickenbottom, S., Litt, L., Lawton, M., Hannegan, L., Gupta, D., Bickler, P., Dodson, B., Talke, P., Rampil, I., Chen, B., Wright, P., Mitchell, J., Ryan, S., Walker, J., Quinnine, N., Applebury, C., Myles, P., Rosenfeld, J., Hunt, J., Wallace, S., D'Urso, P., Thien, C., McMahon, J., Wadanamby, S., Siu, K., Malham, G., Laidlaw, J., Salerno, S., Alatakis, S., Madder, H., Cairo, S., Konstantatos, A., Smart, J., Lindholm, D., Bain, D., Machlin, H., Moloney, J., Buckland, M., Silvers, A., Downey, G., Molnar, A., Langley, M., McIlroy, D., Daly, D., Bennett, P., Forlano, L., Testa, R., Burnett, W., Johnson, F., Angliss, M., Fletcher, H., Manninen, P., Wallace, M., Lukitto, K., Tymianski, M., Porter, P., Gentili, F., El-Beheiry, H., Mosa, M., Mak, P., Balki, M., Shaikh, S., Sawyer, R., Quader, K., Chelliah, R., Berklayd, P., Merah, N., Ghazali, G., McAndrews, M., Ridgley, J., Odukoya, O., Yantha, S., Wilson, J., Petrozza, P., Miller, C., O'Brien, K., Tong, C., Olympio, M., Reynolds, J., Colonna, D., Glazier, S., Nobles, S., Hill, D., Hulbert, H., Jenkins, W., Lanier, W., Piepgras, D., Wilson, R., Meyer, F., Atkinson, J., Link, M., Weglinski, M., Berge, K., McGregor, D., Trenerry, M., Smith, G., Walkes, J., Felmlee-Devine, M., Van Aken, H., Greiner, C., Freise, H., Brors, H., Hahnenkamp, K., de Oliveira, N. M., Schul, C., Moskopp, D., Greiner, C., Woelfer, J., Hoenemann, C., Gramke, H., Bone, H., Gibmeier, I., Wirtz, S., Lohmann, H., Freyhoff, J., Bauer, B., Hogan, K., Dempsey, R., Rusy, D., Badie, B., Iskandar, B., Resnick, D., Deshmukh, P., Fitzpatrick, J., Sasse, F., Broderick, T., Willmann, K., Connery, L., Kish, J., Weasler, C., Page, N., Hermann, B., Jones, J., Dulli, D., Stanko, H., Geraghty, M., Elbe, R., Salevsky, F., Leblanc, R., Lapointe, N., MacGregor, H., Sinclair, D., Sirhan, D., Maleki, M., Abou-Madi, M., Chartrand, D., Angle, M., Milovan, D., Painchaud, Y., Mirski, M., Tamargo, R., Rice, S., Olivi, A., Kim, D., Rigamonti, D., Naff, N., Hemstreet, M., Berkow, L., Chery, P., Ulatowski, J., Moore, L., CUNNINGHAM, T., McBee, N., Hartman, T., Heidler, J., HILLIS, A., Tuffiash, E., Chase, C., Kane, A., Greene-Chandos, D., Torbey, M., Ziai, W., Lane, K., Bhardwaj, A., Subhas, N., Schubert, A., Mayberg, M., Beven, M., Rasmussen, P., Bhatia, S., Ebrahim, Z., Lotto, M., Vasarhelyi, F., Munis, J., GRAVES, K., Woletz, J., Chelune, G., Samples, S., Evans, J., Blair, D., Abou-Chebl, A., Shutway, F., Manke, D., Beven, C., Fogarty-Mack, P., Stieg, P., Eliazo, R., Li, P., Riina, H., Lien, C., Ravdin, L., Wang, J., Kuo, Y., Jaffe, R., Steinberg, G., Luu, D., Chang, S., Giffard, R., Lemmens, H., Morgan, R., Mathur, A., Angst, M., Meyer, A., Yi, H., Karzmark, P., Bell-Stephens, T., Marcellus, M., Sneyd, J., Pobereskin, L., Salsbury, S., Whitfield, P., Sawyer, R., Dashfield, A., Struthers, R., DAVIES, P., Rushton, A., Petty, V., Harding, S., Richardson, E., Yonas, H., Gyulai, F., Kirby, L., Kassam, A., Bircher, N., Meng, L., Krugh, J., Seever, G., Hendrickson, R., Gebel, J., Cowie, D., Fabinyi, G., Poustie, S., Davis, G., Drnda, A., Chandrasekara, D., Sturm, J., Phan, T., Shelton, A., Clausen, M., Micallef, S., Sills, A., Steinman, F., Sutton, P., Sanders, J., Van Alstine, D., Leggett, D., Cunningham, E., Hamm, W., Frankel, B., Sorenson, J., Atkins, L., Redmond, A., Dalrymple, S., Black, S., Fisher, W., Hall, C., Wilhite, D., Moore, T., Blanton, I. P., Sha, Z., Szmuk, P., Kim, D., Ashtari, A., Hagberg, C., Matuszczak, M., Shahen, A., Moise, O., Novy, D., Govindaraj, R., Jameson, L., Breeze, R., Awad, I., Mattison, R., ANDERSON, T., Salvia, L., Mosier, M., Loftus, C., Smith, J., Lilley, W., White, B., Lenaerts, M. 2005; 352 (2): 135-145

    Abstract

    Surgery for intracranial aneurysm often results in postoperative neurologic deficits. We conducted a randomized trial at 30 centers to determine whether intraoperative cooling during open craniotomy would improve the outcome among patients with acute aneurysmal subarachnoid hemorrhage.A total of 1001 patients with a preoperative World Federation of Neurological Surgeons score of I, II, or III ("good-grade patients"), who had had a subarachnoid hemorrhage no more than 14 days before planned surgical aneurysm clipping, were randomly assigned to intraoperative hypothermia (target temperature, 33 degrees C, with the use of surface cooling techniques) or normothermia (target temperature, 36.5 degrees C). Patients were followed closely postoperatively and examined approximately 90 days after surgery, at which time a Glasgow Outcome Score was assigned.There were no significant differences between the group assigned to intraoperative hypothermia and the group assigned to normothermia in the duration of stay in the intensive care unit, the total length of hospitalization, the rates of death at follow-up (6 percent in both groups), or the destination at discharge (home or another hospital, among surviving patients). At the final follow-up, 329 of 499 patients in the hypothermia group had a Glasgow Outcome Score of 1 (good outcome), as compared with 314 of 501 patients in the normothermia group (66 percent vs. 63 percent; odds ratio, 1.14; 95 percent confidence interval, 0.88 to 1.48; P=0.32). Postoperative bacteremia was more common in the hypothermia group than in the normothermia group (5 percent vs. 3 percent, P=0.05).Intraoperative hypothermia did not improve the neurologic outcome after craniotomy among good-grade patients with aneurysmal subarachnoid hemorrhage.

    View details for Web of Science ID 000226251700006

    View details for PubMedID 15647576

  • Antiapoptotic and anti-inflammatory mechanisms of heat-shock protein protection NEUROPROTECTIVE AGENTS Yenari, M. A., Liu, J. L., Zheng, Z., Vexler, Z. S., Lee, J. E., Giffard, R. G. 2005; 1053: 74-83

    Abstract

    We and others have previously shown that heat-shock proteins (HSPs) are involved in protecting the brain from a variety of insults including stroke, epilepsy, and other related insults. While the mechanism of this protection has largely been thought to be due to their chaperone functions (i.e., preventing abnormal protein folding or aggregation), recent work has shown that HSPs may also directly interfere with other cell death pathways such as apoptosis and inflammation. Using models of cerebral ischemic and ischemia-like injury, we overexpressed the 70-kDa heat-shock protein (HSP70) using gene transfer or by studying a transgenic mouse model. HSP70 protected neurons and astrocytes from experimental stroke and stroke-like insults. HSP70 transgenic mice also had better neurological scores following experimental stroke compared to their wild-type littermates. Overexpressing HSP70 was associated with less apoptotic cell death and increased expression of the antiapoptotic protein, Bcl-2. Furthermore, HSP70 suppressed microglial/monocyte activation following experimental stroke. HSP70 overexpression also led to the reduction of matrix metalloproteinases. We suggest that HSPs are capable of protecting brain cells from lethal insults through a variety of mechanisms and should be explored as a potential therapy against stroke and other neurodegenerative diseases.

    View details for DOI 10.1196/annals.1344.007

    View details for Web of Science ID 000235109800010

    View details for PubMedID 16179510

  • Susceptibility to apoptosis varies with time in culture for murine neurons and astrocytes: changes in gene expression and activity NEUROLOGICAL RESEARCH Xu, L. J., Chock, V. Y., Yang, E. Y., Giffard, R. G. 2004; 26 (6): 632-643

    Abstract

    Apoptotic pathways in the brain may differ depending on cell type and developmental stage. To understand these differences, we studied several apoptotic proteins in the murine cortex and primary cultures of neurons and astrocytes of various ages in culture. We then induced apoptosis in our cultures using serum deprivation (SD) and observed changes in these apoptotic proteins. When analyzed by nuclear morphology and TUNEL staining, early cultures showed greater apoptotic injury compared with late cultures, and neuronal cultures showed greater apoptosis than astrocyte cultures. The decrease in apoptosis with development correlated best with a down-regulation of procaspase-3 and bax and decreasing caspase activation. Early culture astrocytes had higher caspase-11 levels compared with neurons. Mitogen-activated protein (MAP) kinases were also differentially expressed with activation of extracellular signal-regulated kinase (ERK) and p38 higher in early culture astrocytes and stress-activated protein kinase/C-jun N-terminal kinase (SAPK/JNK) greater in early culture neurons. However, caspase inhibitors, but not MAP kinase inhibitors reduced cell death. Our findings demonstrate that apoptosis regulatory proteins display cell type and developmentally specific expression and activation.

    View details for DOI 10.1179/016164104225017587

    View details for Web of Science ID 000223832200005

    View details for PubMedID 15327753

  • Cellular neuroprotective mechanisms in cerebral ischemia: Bcl-2 family proteins and protection of mitochondrial function CELL CALCIUM Ouyang, Y. B., Giffard, R. G. 2004; 36 (3-4): 303-311

    Abstract

    Mitochondria are central to brain cell response to ischemia, with critical roles in generation of ATP, production of free radicals, and regulation of apoptotic cell death. Changes in the permeability of the outer mitochondrial membrane to regulators of apoptosis can control ischemic cell death and this permeability is directly controlled by the Bcl-2 family of proteins. The Bcl-2 family regulate apoptosis by several mechanisms including affecting the formation of apoptotic protein-conducting pores in the outer mitochondrial membrane. The anti-apoptotic protein Bcl-2 improves neuron survival following various insults, and is protective even when administered after stroke onset in a rat model of focal ischemia. Despite intense study, the precise molecular mechanisms underlying protection by the anti-apoptotic members of the Bcl-2 family are not completely understood. This review focuses on the mechanisms by which Bcl-2 family members control the permeability of the mitochondrial membrane and influence other aspects of mitochondrial function after brain ischemia, concluding with discussion of the potential use of Bcl-2 for the treatment of cerebral ischemia.

    View details for DOI 10.1016/j.ceca.2004.02.015

    View details for Web of Science ID 000223056500013

    View details for PubMedID 15261486

  • Agmatine reduces infarct area in a mouse model of transient focal cerebral ischemia and protects cultured neurons from ischemia-like injury EXPERIMENTAL NEUROLOGY Kim, J. H., Yenari, M. A., Giffard, R. G., Cho, S. W., Park, K. A., Lee, J. E. 2004; 189 (1): 122-130

    Abstract

    Agmatine is a primary amine formed by the decarboxylation of L-arginine synthesized in mammalian brain. In this study, we investigated the neuroprotective effect of agmatine on ischemic and ischemia-like insults. Primary cortical neuronal cultures were subjected to oxygen-glucose deprivation (OGD), a model of ischemia-like injury, and treated with agmatine before or at the start of OGD, or upon reperfusion. Neuronal death was reduced when agmatine was present during OGD, and this protection was associated with a reduction of nitric oxide (NO) and neuronal nitric oxide synthase (nNOS), but not inducible NOS (iNOS). Protection by agmatine was also studied at the in vivo level using a model of middle cerebral artery occlusion (MCAO) in mice. Mice were subjected to 2 h MCAO. Agmatine was administered either 30 min before ischemia, at the start of MCAO, at the start of reperfusion, or 2 or 5 h into reperfusion. Agmatine markedly reduced infarct area in all treatment groups except when treatment was delayed 5 h. The number of nNOS immunopositive cells was correlated with neuroprotection. Interestingly, immunoreactivity for iNOS was reduced only when agmatine was administered before and at the onset of MCAO. Our study suggests that agmatine may be a novel therapeutic strategy to reduce cerebral ischemic injury, and may act by inhibiting the detrimental effects of nNOS.

    View details for DOI 10.1016/j.expneurol.2004.05.029

    View details for Web of Science ID 000223253000014

    View details for PubMedID 15296842

  • Effect of overexpression of protective genes on mitochondrial function of stressed astrocytes JOURNAL OF BIOENERGETICS AND BIOMEMBRANES Giffard, R., Ouyang, Y. 2004; 36 (4): 313-315

    Abstract

    Antiapoptotic members of the Bcl-2 family have been shown to reduce ischemic brain injury in vivo and in vitro. Understanding early changes in respiration are important in understanding the cells response to stress and the mechanisms of protection afforded by overexpression of protective genes. This mini-review summarizes current knowledge regarding early responses of astrocytes to ischemia-like stress and the effects of overexpression of protective Bcl-2 family genes on astrocyte mitochondrial function. Overexpression of Bcl-x(L) improves mitochondrial respiratory function, normalizes mitochondrial membrane potential, and reduces production of free radicals early after the imposition of a stress in primary cultured murine astrocytes.

    View details for Web of Science ID 000223916300009

    View details for PubMedID 15377864

  • Chaperones, protein aggregation, and brain protection from hypoxic/ischemic injury JOURNAL OF EXPERIMENTAL BIOLOGY Giffard, R. G., Xu, L. J., Heng, Z., Carrico, W., Ouyang, Y. B., Qiao, Y. L., Sapolsky, R., Steinberg, G., Hu, B. R., Yenari, M. A. 2004; 207 (18): 3213-3220

    Abstract

    Chaperones, especially the stress inducible Hsp70, have been studied for their potential to protect the brain from ischemic injury. While they protect from both global and focal ischemia in vivo and cell culture models of ischemia/reperfusion injury in vitro, the mechanism of protection is not well understood. Protein aggregation is part of the etiology of chronic neurodegenerative diseases such as Huntington's and Alzheimer's, and recent data demonstrate protein aggregates in animal models of stroke. We now demonstrate that overexpression of Hsp70 in hippocampal CA1 neurons reduces evidence of protein aggregation under conditions where neuronal survival is increased. We have also demonstrated protection by the cochaperone Hdj-2 in vitro and demonstrated that this is associated with reduced protein aggregation identified by ubiquitin immunostaining. Hdj-2 can prevent protein aggregate formation by itself, but can only facilitate protein folding in conjunction with Hsp70. Pharmacological induction of Hsp70 was found to reduce both apoptotic and necrotic astrocyte death induced by glucose deprivation or oxygen glucose deprivation. Protection from ischemia and ischemia-like injury by chaperones thus involves at least anti-apoptotic, anti-necrotic and anti-protein aggregation mechanisms.

    View details for DOI 10.1242/jeb.01034

    View details for Web of Science ID 000224132000014

    View details for PubMedID 15299042

  • Cell-specific role for epsilon- and beta I-protein kinase C isozymes in protecting cortical neurons and astrocytes from ischemia-like injury NEUROPHARMACOLOGY Wang, J., Bright, R., Mochly-Rosen, D., Giffard, R. G. 2004; 47 (1): 136-145

    Abstract

    Activation of epsilon protein kinase C (epsilonPKC) has been shown to protect cardiac myocytes against ischemia and reperfusion injury. However, the role of PKC in ischemic brain injury is less well defined. Western blot analysis of murine neurons and astrocytes in primary culture demonstrated epsilon- and betaIPKC expression in both cell types. Activation of epsilonPKC increased in neuronal cultures in response to the ischemia-like insult of oxygen-glucose deprivation (OGD). Isozyme-specific peptide activators or inhibitors of PKC were applied at various times before, during and after the OGD period. Neuron-astrocyte mixed cultures pretreated with a selective epsilonPKC activator peptide showed a significant reduction in neuronal injury after OGD and reperfusion, compared to cultures pretreated with control peptide. The epsilonPKC activator peptide counteracted the increased damage induced by pretreatment with the epsilonPKC-selective inhibitor peptide in relatively pure neuronal cultures subjected to OGD. Neither epsilonPKC activator nor inhibitor peptides affected injury of neurons when applied after OGD onset. In contrast, the betaIPKC-selective inhibitor peptide increased injury in astrocyte cultures exposed to OGD at all application times tested. Our data demonstrate protection of neurons by selective activation of epsilonPKC but enhanced astrocyte cell death with selective inhibition of betaIPKC. Thus PKC isozymes exhibit cell type-specific effects on ischemia-like injury.

    View details for DOI 10.1016/j.neuropharm.2004.03.009

    View details for Web of Science ID 000222041600013

    View details for PubMedID 15165841

  • Changes in astrocyte mitochondrial function with stress: effects of Bcl-2 family proteins NEUROCHEMISTRY INTERNATIONAL Ouyang, Y. B., Giffard, R. G. 2004; 45 (2-3): 371-379

    Abstract

    Mitochondria are central to both apoptotic and necrotic cell death, as well as to normal physiological function. Astrocytes are crucial for neuronal metabolic, antioxidant, and trophic support, as well as normal synaptic function. In the setting of stress, such as during cerebral ischemia, astrocyte dysfunction may compromise the ability of neurons to survive. Despite their central importance, the response of astrocyte mitochondria to stress has not been extensively studied. Limited data already suggest clear differences in the response of neuronal and astrocytic mitochondria to oxygen-glucose deprivation (GD). Prominent mitochondrial alterations during stress that can contribute to cell death include changes in production of reactive oxygen species (ROS) and release of death regulatory and signaling molecules from the intermembrane space. In response to stress mitochondrial respiratory function and membrane potential also change, and these changes appear to depend on cell type. Bcl-2 family proteins are the best studied regulators of cell death, especially apoptosis, and mitochondria are a major site of action for these proteins. Although much data supports the role of Bcl-2 family proteins in the regulation of some of these mitochondrial alterations, this remains an area of active investigation. This mini-review summarizes current knowledge regarding mitochondrial control of cell survival and death in astrocytes and the effects of anti-apoptotic Bcl-2 proteins on astrocyte mitochondrial function.

    View details for DOI 10.1016/j.neuint.2003.07.006

    View details for Web of Science ID 000221783400016

    View details for PubMedID 15145551

  • The 70 kDa heat shock protein suppresses matrix metalloproteinases in astrocytes NEUROREPORT Lee, J. E., Kim, Y. J., Kim, Y. J., Lee, W. T., Yenari, M. A., Giffard, R. G. 2004; 15 (3): 499-502

    Abstract

    The 70 kDa heat shock protein (Hsp70) is synthesized in response to a variety of stresses, including ischemia, and is thought to act as a molecular chaperone to prevent protein denaturation and facilitate protein folding. Matrix metalloproteinases (MMPs), a family of serine proteases, are also upregulated by ischemia and are thought to promote cell death and tissue injury. We examined the influence of Hsp70 on expression and activity of MMPs. Astrocyte cultures were prepared from neonatal mice and transfected with retroviral vectors containing hsp70 or lacZ or mock infected, then exposed to oxygen-glucose deprivation followed by reperfusion. Zymograms and Western blots showed that Hsp70 over-expression suppressed MMP-2 and MMP-9. These findings suggest that Hsp70 may protect by regulating MMPs.

    View details for DOI 10.1097/01.wnr.0000111321.38420.95

    View details for Web of Science ID 000225140000023

    View details for PubMedID 15094511

  • Many mechanisms for Hsp70 protection from cerebral ischemia JOURNAL OF NEUROSURGICAL ANESTHESIOLOGY Giffard, R. G., Yenari, M. A. 2004; 16 (1): 53-61

    Abstract

    Overexpression of inducible Hsp70 has been shown to provide protection from cerebral ischemia both in animal models of stroke and in cell culture models. New work suggests that there are multiple routes of cell death, including apoptotic and necrotic cell death. Hsp70 is known to protect from both necrotic and apoptotic cell death. In addition to the well-studied role of Hsp70 as a molecular chaperone assisting in correct protein folding, several new mechanisms by which Hsp70 can prevent cell death have been described. Hsp70 is now known to regulate apoptotic cell death both directly by interfering with the function of several proteins that induce apoptotic cell death as well as indirectly by increasing levels of the anti-death protein bcl-2. Despite these new insights into the ways in which Hsp70 functions as an anti-death protein, further surprises are likely as we continue to gain insight into the functioning of this multifaceted protein.

    View details for Web of Science ID 000187659800010

    View details for PubMedID 14676570

  • Two variants of the rat brain sodium-driven chloride bicarbonate exchanger (NCBE): developmental expression and addition of a PDZ motif EUROPEAN JOURNAL OF NEUROSCIENCE Giffard, R. G., Lee, Y. S., Ouyang, Y. B., Murphy, S. L., Monyer, H. 2003; 18 (11): 2935-2945

    Abstract

    Regulation of pH in the central nervous system is critical to normal brain function and response to pathophysiological conditions. Here we identify two novel variants of the sodium-driven chloride bicarbonate exchanger (NCBE) from brain. The developmental expression pattern seen by in situ hybridization for the 90-bp containing insert (insert A) reveals strong expression in spinal cord and brain beginning in embryonic development. High-level expression is seen in cerebellar Purkinje cells and principal cells in hippocampus. The variant missing a 39-bp insert at the 3' end (insert B) encodes a protein in which the deduced carboxyterminal three amino acids are replaced with a unique 21 amino acid stretch terminating in a PDZ motif. rb2NCBE, the PDZ motif-encoding variant, is more highly expressed in astrocytes than is rb1NCBE. Both variants are expressed at similar levels in neurons. Expression varies with age and cell type. The FLAG epitope was fused in-frame at the amino terminus and each variant was expressed using a retroviral vector to study subcellular localization. Both variants were associated with the plasma membrane, but rb2NCBE colocalized with actin filaments to a greater extent, suggesting the PDZ form may interact with the cytoskeleton, whereas rb1NCBE was more often seen in intracellular vesicles. The PDZ motif-containing variant was much more active in pH regulation, with the expected ionic dependence on Na+, HCO3- and Cl- when expressed in 3T3 cells. These results are a first step towards understanding the regulation of expression and activity of this transporter in the brain.

    View details for DOI 10.1046/j.1460-9568.2003.03053.x

    View details for Web of Science ID 000187006400003

    View details for PubMedID 14656289

  • Overexpression of HDJ-2 protects Astrocytes from ischemia-like injury and reduces redistribution of ubiquitin staining in vitro JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Qiao, Y. L., Ouyang, Y. B., Giffard, R. G. 2003; 23 (10): 1113-1116

    Abstract

    HDJ-2, a member of the HSP40 family, functions as a cochaperone to promote protein folding both by binding to unfolded polypeptides and by regulating the activity of HSP70. This study tested whether HDJ-2 overexpression alone could provide significant protection from ischemia-like injury. Primary mouse astrocyte cultures were infected with an HDJ-2 encoding retroviral vector or control retrovirus lacking HDJ-2. Expression of HDJ-2 was confirmed by immunohistochemical staining and immunoblotting. Injury paradigms to mimic ischemia were glucose deprivation (GD) for 24 hours and oxygen-glucose deprivation (OGD) for 8 hours. Cell death was determined by trypan blue exclusion and cell counting. Overexpression of HDJ-2 alone significantly reduced astrocyte injury after both GD and OGD, independent of an elevation in HSP70. To further search for the mechanism of HDJ-2 protection, cultured astrocytes allowed to recover 16 hours after 8 hours GD were labeled with a monoclonal antiubiquitin antibody that recognizes both free ubiquitin and ubiquitinated proteins. The immunolabeling pattern changed from a relatively even distribution in both nuclei and cytoplasm in control cells to heterogeneous aggregates and marked reduction of nuclear staining in most of the cells after GD. When HDJ-2 was overexpressed, the number of cells with evidence of protein aggregation was significantly reduced. Thus, blocking protein aggregation may be an important mechanism by which HDJ-2 protects cells from damage.

    View details for DOI 10.1097/01.WCB.0000088765.02615.FE

    View details for Web of Science ID 000185755500001

    View details for PubMedID 14526221

  • Geldanamycin reduces necrotic and apoptotic injury due to oxygen-glucose deprivation in astrocytes NEUROLOGICAL RESEARCH Xu, L. J., Ouyang, Y. B., Giffard, R. G. 2003; 25 (7): 697-700

    Abstract

    Recent data show that geldanamycin can protect the brain against stroke in vivo, and this may be due to induction of heat shock proteins. Our previous results show that heat shock protein 70 expression by retroviral transfection protects astrocytes from necrotic injury by combined oxygen-glucose deprivation, an in vitro model of ischemia. This study tested the ability of geldanamycin to protect astrocytes from either necrotic or apoptotic injury induced by oxygen-glucose deprivation. Astrocytes were pre-treated with 0.1 microgram ml-1 geldanamycin in the medium 4, 8, or 16 h before as well as during oxygen-glucose deprivation. Increased protein levels of heat shock protein 70 were observed after 8 h pre-treatment with geldanamycin and increased further at 16 h pre-treatment, as detected by immunoblotting. Geldanamycin pre-treatment protected mature astrocytes from necrotic cell death and young astrocytes from apoptotic death. Geldanamycin protection of astrocytes against in vitro ischemia is likely due to upregulation of heat shock protein 70.

    View details for Web of Science ID 000186229300004

    View details for PubMedID 14579785

  • Perinatal brain injury: The role of development in vulnerability ANESTHESIOLOGY Giffard, R. G., Fiskum, G. 2003; 98 (5): 1039-1041

    View details for Web of Science ID 000182523200002

    View details for PubMedID 12717122

  • Bcl-x(L) maintains mitochondrial function in murine astrocytes deprived of glucose JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Ouyang, Y. B., Giffard, R. G. 2003; 23 (3): 275-279

    Abstract

    Bcl-XL is a protein that blocks both apoptotic and necrotic cell death. The authors have previously shown that it is effective in maintaining mitochondrial membrane potential during glucose deprivation in cultured astrocytes. To further investigate the mechanism involved, the authors studied mitochondrial function and cytochrome c release. Oxygen consumption was monitored to assess oxidative respiration. State III respiration decreased significantly as early as 3 h after removal of glucose. At this time mitochondria hyperpolarize but cytochrome c is not yet released. Damage to the electron transport chain is not responsible for this change because uncoupled respiration was unchanged at this time point. At 5 h of glucose deprivation, when mitochondrial depolarization was observed, state IV respiration increased significantly, cytochrome c began to be released, and mitochondrial morphology changed from elongated to punctate. When Bcl-XL was overexpressed normal state III respiration and mitochondrial morphology were maintained and cytochrome c release was inhibited in the face of glucose deprivation stress.

    View details for DOI 10.1097/01.WCB.0000055774.06337.F6

    View details for Web of Science ID 000181401200002

    View details for PubMedID 12621302

  • Gene therapy and hypothermia for stroke treatment NEUROPROTECTIVE AGENTS Yenari, M. A., Zhao, H., Giffard, R. G., Sobel, R. A., Sapolsky, R. M., Steinberg, G. K. 2003; 993: 54-68

    Abstract

    We have previously reported studies of gene therapy using a neurotropic herpes simplex viral (HSV) vector system containing bipromoter vectors to transfer various protective genes to neurons. Using this system in experimental models of stroke, cardiac arrest, and excitotoxicity, we found that it is possible to enhance neuron survival against such cerebral insults by overexpressing genes that target various facets of injury. Among the genes we studied, the anti-apoptotic protein BCL-2 improved neuron survival following various insults, and was protective even when administered after stroke onset. BCL-2 is thought to protect cells from apoptotic death by preventing cytochrome c release from the mitochondria and subsequent caspase activation. We and others have established that cooling the brain by a few degrees markedly reduces ischemic injury and improves neurologic deficits in models of cerebral ischemia and trauma. This hypothermic neuroprotection is also associated with BCL-2 upregulation in some instances. Furthermore, hypothermia suppresses many aspects of apoptotic death including cytochrome c release, caspase activation, and DNA fragmentation. Here we show that two different kinds of protective therapies, BCL-2 overexpression and hypothermia, both inhibit aspects of apoptotic cell death cascades, and that a combination treatment can prolong the temporal therapeutic window for gene therapy.

    View details for Web of Science ID 000184303000006

    View details for PubMedID 12853295

  • Caspase inhibitors reduce the apoptotic but not necrotic component of kainate injury in primary murine cortical neuronal cultures NEUROLOGICAL RESEARCH Glassford, A., Lee, J. E., Xu, L. J., Giffard, R. G. 2002; 24 (8): 796-800

    Abstract

    Excitotoxicity has been demonstrated to play a major role in ischemic neuronal injury. While the necrotic component of excitotoxicity has been well demonstrated, apoptosis has also been shown to play a role. We sought to quantitate and modulate the apoptotic component of kainate-induced injury. Experiments were performed in mouse primary cortical neuronal cultures after three or 10 days in vitro. Cell death was assessed by Hoechst/propidium iodide staining and cell counting. Apoptosis was further confirmed with inhibition by caspase inhibitors. Exposure of three-day old neurons to 100 microM kainate produced an injury in which 56% +/- 0.9% of cells showed apoptotic nuclei and 13.5% +/- 2.0% showed necrotic nuclei. After 10 days in vitro neurons were more easily injured by kainate, but the cell death had primarily necrotic characteristics. Inhibition of both caspases 1 and 3 significantly reduced the apoptotic injury of 3-day old neurons. Neither reduced the necrotic component. Inhibition of protein synthesis with cycloheximide was also effective in reducing the apoptotic injury without affecting the necrotic injury. Kainate injury causes both apoptosis and necrosis, with the injury depending on both the dose of kainate and the age of the culture. The apoptotic component can be selectively reduced by caspase inhibition or cycloheximide.

    View details for Web of Science ID 000179655900009

    View details for PubMedID 12500703

  • Neuroprotective effects of bcl-2 overexpression in hippocampal cultures: interactions with pathways of oxidative damage JOURNAL OF NEUROCHEMISTRY Howard, S., Bottino, C., Brooke, S., Cheng, E., Giffard, R. G., Sapolsky, R. 2002; 83 (4): 914-923

    Abstract

    Overexpression of bcl-2protects neurons from numerous necrotic insults, both in vitro and in vivo. While the bulk of such protection is thought to arise from Bcl-2 blocking cytochrome c release from mitochondria, thereby blocking apoptosis, the protein can target other steps in apoptosis, and can protect against necrotic cell death as well. There is evidence that these additional actions may be antioxidant in nature, in that Bcl-2 has been reported to protect against generators of reactive oxygen species (ROS), to increase antioxidant defenses and to decrease levels of ROS and of oxidative damage. Despite this, there are also reports arguing against either the occurrence, or the importance of these antioxidant actions. We have examined these issues in neuron-enriched primary hippocampal cultures, with overexpression of bcl-2 driven by a herpes simplex virus amplicon: (i) Bcl-2 protected strongly against glutamate, whose toxicity is at least partially ROS-dependent. Such protection involved reduction in mitochondrially derived superoxide. Despite that, Bcl-2 had no effect on levels of lipid peroxidation, which is thought to be the primary locus of glutamate-induced oxidative damage; (ii) Bcl-2 was also mildly protective against the pro-oxidant adriamycin. However, it did so without reducing levels of superoxide, hydrogen peroxide or lipid peroxidation; (iii) Bcl-2 protected against permanent anoxia, an insult likely to involve little to no ROS generation. These findings suggest that Bcl-2 can have antioxidant actions that may nonetheless not be central to its protective effects, can protect against an ROS generator without targeting steps specific to oxidative biochemistry, and can protect in the absence of ROS generation. Thus, the antioxidant actions of Bcl-2 are neither necessary nor sufficient to explain its protective actions against these insults in hippocampal neurons.

    View details for Web of Science ID 000179046200019

    View details for PubMedID 12421364

  • UV-vulnerability of human papilloma virus type-16 E7-expressing astrocytes is associated with mitochondrial membrane depolarization and caspase-3 activation MOLECULES AND CELLS Lee, W. T., Lee, S. H., Carriedo, S. G., Giffard, R. G., Yoon, Y. J., Kim, J. H., Park, K. A., Lee, J. E. 2002; 14 (2): 288-294

    Abstract

    The human papilloma virus-type 16 (HPV-16) E6 and E7 proteins interact with the p53 and pRb tumor suppressor proteins, respectively. The effect of E6 or E7 expression on UV irradiation (5 and 20 J/m2)-induced genotoxic injury of confluent primary murine astrocytes was determined. Retroviral vectors were used to overexpress E6 and E7. Astrocytes expressing E7 showed increased vulnerability to UV-induced apoptosis while E6 over expressing astrocytes were protected from the same insults. Cell death in the E7 overexpressing cells was apoptotic because it showed DNA ladders, activation of caspase-3, formation of apoptotic bodies and decreased DNA content to less than the G0 level. After UV-irradiation the level of E2F1 in E7-expressing astrocytes was higher than E6-, LXSN- or mock-infected cells, and caspase-3 was activated to a greater extent. E7-expressing astrocytes showed the highest levels of Bax under normal growth conditions. The mitochondrial membrane potential of E7-expressing astrocytes was depolarized by 90% after UV-irradiation while the depolarization in control cells was about 50%. E6 overexpression decreased while E7 overexpression increased UV-induced astrocyte apoptosis.

    View details for Web of Science ID 000178998400018

    View details for PubMedID 12442903

  • Advances in understanding protection from cerebral ischemia. Current opinion in anaesthesiology Giffard, R. G., Jaffe, R. A. 2002; 15 (5): 495-500

    Abstract

    Cerebral ischemia and protection is a large field, so for the purposes of this review, which focuses on results published in the last 9 months, we have chosen to discuss a few aspects of ischemia in which our understanding has advanced significantly in this period of time. Recent progress in the clinical use of hypothermia for neurological protection as well as laboratory progress on the role of stress proteins, estrogen and a few other potential adjuncts will be discussed.Two papers have now been published documenting improved neurological outcome in patients treated with hypothermia following cardiac arrest, both using randomized clinical trial designs. These reports and several laboratory studies identifying mechanisms of hypothermic brain protection are reviewed. In understanding the mechanisms underlying protection by estrogens, new results on both direct vascular effects and a demonstration that estrogens can reduce apoptosis are presented. The third area to be described is current progress in identifying mechanisms of stress protein protection from ischemia, in which new mechanisms have been identified with the demonstration of inhibition of several points in the cell death cascade. The remaining areas considered touch on the effects of approaches that reduce inflammation by blocking adhesion molecules, those that reduce free radical production and those that improve blood rheology.An important common theme in brain protection is reduction of cell death by blocking apoptosis or programmed cell death. While the use of hypothermia should now enter clinical practice, many areas of brain injury require further studies both to define injury mechanisms and to translate these understandings into clinically useful treatments to reduce ischemic brain injury.

    View details for PubMedID 17019244

  • Effect of Bcl-x(L) overexpression on reactive oxygen species, intracellular calcium, and mitochondrial membrane potential following injury in astrocytes FREE RADICAL BIOLOGY AND MEDICINE Ouyang, Y. B., Carriedo, S. G., Giffard, R. G. 2002; 33 (4): 544-551

    Abstract

    Many studies have demonstrated the protective effects of Bcl-x(L) against both apoptotic and necrotic cell death, but the mode of action of Bcl-x(L) remains unclear. This work analyzed effects of Bcl-x(L) overexpression on cellular levels of reactive oxygen species (ROS), intracellular calcium ([Ca(2+)](i)), and mitochondrial membrane potential (DeltaPsi(m)) in cultured mouse primary astrocytes after exposure to glucose deprivation (GD) or hydrogen peroxide (H(2)O(2)). Upon exposure to GD or H(2)O(2), uninfected and Lac-Z-expressing astrocytes showed an immediate, rapid increase in ROS accumulation that was slowed and or reduced by Bcl-x(L). Changes in DeltaPsi(m) in response to the two insults differed. H(2)O(2) induced a decrease in DeltaPsi(m) that was initially greater in Bcl-x(L) cells, but then held stable. DeltaPsi(m) in control and Lac-Z-expressing cells initially declined more slowly, but after about 20 min showed rapid deterioration. Five hours of GD caused mitochondrial membrane hyperpolarization followed by a decrease in DeltaPsi(m,) which was not observed with Bcl-x(L) overexpression. Bcl-x(L) failed to inhibit the calcium dysregulation seen in control cells exposed to 400 microM H(2)O(2), but still improved cell survival. There was no increase in [Ca(2+)](i) with 5 h of GD. These data thus dissociate the effect of Bcl-x(L) on calcium homeostasis from effects on ROS, DeltaPsi(m,) and for H(2)O(2) exposure, cell survival.

    View details for Web of Science ID 000177393800014

    View details for PubMedID 12160936

  • Gene transfer of HSP72 protects cornu ammonis 1 region of the hippocampus neurons from global ischemia: Influence of Bcl-2 ANNALS OF NEUROLOGY Kelly, S., Zhang, Z. J., Zhao, H., Xu, L. J., Giffard, R. G., Sapolsky, R. M., Yenari, M. A., Steinberg, G. K. 2002; 52 (2): 160-167

    Abstract

    We investigated whether HSV gene transfer of HSP72 in vivo and in vitro: (1) protected cornu ammonis 1 region of the hippocampus neurons from global cerebral ischemia; and (2) affected Bcl-2 expression. HSV vectors expressing HSP72 and beta-galactosidase (reporter) or beta-galactosidase only (control vector) were injected into cornu ammonis 1 region of the hippocampus 15 hours before induction of global cerebral ischemia (n = 10) and sham-operated rats (n = 8). HSP72 vector-treated rats displayed significantly more surviving transfected neurons (X-gal-positive, 31 +/- 8) compared with control vector-treated rats (10 +/- 4) after global cerebral ischemia. Sham-operated rats displayed similar numbers of X-gal-positive neurons (HSP72 vector 18 +/- 8 vs control vector 20 +/- 7). The percentage of beta-galactosidase and Bcl-2 coexpressing neurons in HSP72-treated rats after global cerebral ischemia (84 +/- 4%) was greater than that in control vector-treated rats (58 +/- 9%). The percentage of beta-galactosidase and Bcl-2 coexpressing neurons in sham-operated rats was similar in HSP72 (93 +/- 7%) and in control vector-treated rats (88 +/- 12%). HSP72 vector transfection led to 12 times as much Bcl-2 expression as the control vector in uninjured hippocampal neuronal cultures. In injured (oxygen-glucose deprivation) hippocampal neuron cultures, HSP72 vector transfection led to 2.8 times as much Bcl-2 expression as control vector. We show that HSP72 overexpression protects cornu ammonis 1 region of the hippocampus neurons from global cerebral ischemia, and that this protection may be mediated in part by increased Bcl-2 expression.

    View details for DOI 10.1002/ana.10264

    View details for Web of Science ID 000177140000005

    View details for PubMedID 12210785

  • Influence of mild hypothermia on inducible nitric oxide synthase expression and reactive nitrogen production in experimental stroke and inflammation JOURNAL OF NEUROSCIENCE Han, H. S., Qiao, Y. L., Karabiyikoglu, M., Giffard, R. G., Yenari, M. A. 2002; 22 (10): 3921-3928

    Abstract

    Mild hypothermia is neuroprotective, but the reasons are not well known. Inflammation contributes to ischemic damage; therefore, we examined whether the protection by hypothermia may be attributable to alterations in the inflammation. We examined whether hypothermia might alter the inflammatory cell-associated inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) and peroxynitrite generation in experimental stroke and inflammation. Rats underwent 2 hr of middle cerebral artery occlusion (MCAO). Brain inflammation was modeled by intravenous lipopolysaccharide (LPS) (2 mg/kg) injection. Temperature was maintained at 33 degrees C for 2 hr immediately after MCAO and LPS injection, delayed 2 hr after MCAO or maintained at 38 degrees C. Cultured microglia were activated with LPS and then incubated at 33 or 37 degrees C. Both intraischemic and delayed mild hypothermia attenuated infarct size by 40% (p < 0.05). Immunohistochemistry was performed to identify cell type, iNOS, and peroxynitrite. The majority of iNOS- and peroxynitrite-positive cells were activated microglia-macrophages, and mild hypothermia significantly decreased the numbers of immunoreactive cells at 72 hr by >50% (p < 0.05). After ischemia, mild hypothermia decreased NO production by 40%. Similarly, hypothermia attenuated NO and iNOS in LPS-injected rats, as well as in cultured microglia. Aminoguanidine, an iNOS inhibitor, also attenuated infarct size and NO in ischemic and inflammation models. We conclude that mild hypothermia significantly inhibits the inflammatory response by affecting microglial iNOS-NO generation. Therapies directed against microglia or their activation may be useful in treating stroke.

    View details for Web of Science ID 000175634900013

    View details for PubMedID 12019311

  • Mild hypothermia reduces apoptosis of mouse neurons in vitro early in the cascade JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Xu, L. J., Yenari, M. A., Steinberg, G. K., Giffard, R. G. 2002; 22 (1): 21-28

    Abstract

    Recent experimental work has shown that hypothermia with even small decreases in temperature is broadly neuroprotective, but the mechanism of this protection remains unclear. Although reduction of metabolism could explain protection by deep hypothermia, it does not explain the robust protection found with mild hypothermia. Several reports have suggested that ischemic apoptosis is reduced by hypothermia. The authors examined the effects of hypothermia on neuronal apoptosis using serum deprivation, a well-accepted model that induces neuronal apoptosis. Mild hypothermia (33 degrees C) significantly reduced the number of morphologically apoptotic neurons to less than half the number seen in normothermic culture temperatures (37 degrees C) after 48 hours. They examined the effect of hypothermia on several steps in the cascade. Caspase-3, -8, and -9 activity was significantly increased after 24 hours at 37 degrees C, and was significantly lower in cultures deprived of serum at 33 degrees C. Cytochrome c translocation was reduced by hypothermia. Western blot analysis failed to detect significant changes in Bax, bcl -2, or hsp -70 at early time points, whereas hypothermia significantly reduced cJun N-terminal kinase activation. The authors conclude that small decreases in temperature inhibit apoptosis very early, possibly at the level of the initiation of apoptosis, as suggested by reduced cJun N-terminal kinase activation and before the translocation of cytochrome c, with subsequent prevention of caspase activation.

    View details for Web of Science ID 000172930900003

    View details for PubMedID 11807390

  • Overexpression of HSP72 after induction of experimental stroke protects neurons from ischemic damage JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Hoehn, B., Ringer, T. M., Xu, L. J., Giffard, R. G., Sapolsky, R. M., Steinberg, G. K., Yenari, M. A. 2001; 21 (11): 1303-1309

    Abstract

    The 72-kD inducible heat shock protein (HSP72) can attenuate cerebral ischemic injury when overexpressed before ischemia onset. Whether HSP72 overexpression is protective when applied after ischemia onset is not known, but would have important clinical implications. Fifty-seven rats underwent middle cerebral artery occlusion for 1 hour. Defective herpes simplex viral (HSV) vectors expressing hsp72 with lacZ as a reporter were delivered 0.5, 2, and 5 hours after ischemia onset into each striatum. Control animals received an identical vector containing only lacZ. Striatal neuron survival at 2 days was improved by 23% and 15% when HSP72 vectors were delayed 0.5 and 2 hours after ischemic onset, respectively ( P < 0.05). However, when delayed by 5 hours, HSP72 overexpression was no longer protective. This is the first demonstration that HSP72 gene transfer even after ischemia onset is neuroprotective. Because expression from these HSV vectors begins 4 to 6 hours after injection, this suggests that the temporal therapeutic window for HSP72 is at least 6 hours after ischemia onset. Future strategies aimed at enhancing HSP72 expression after clinical stroke may be worth pursuing. The authors suggest that in the future HSP72 may be an effective treatment for stroke.

    View details for Web of Science ID 000172087000006

    View details for PubMedID 11702045

  • Human papilloma virus type 16 E7 genes protect astrocytes against apoptotic and necrotic death induced hy hydrogen peroxide YONSEI MEDICAL JOURNAL Lee, W. T., Lee, J. E., Lee, S. H., Jang, H. S., Giffard, R. G., Park, K. A. 2001; 42 (5): 471-479

    Abstract

    Hydrogen peroxide is considered to be a dose- and time-dependent mediator in apoptotic and necrotic death. In this study, we examined the signaling of the E6 and E7 proteins with respect to apoptosis or necrosis after H2O2 injury using an in vitro model with overexpressed E6 or E7 genes. For this purpose, the E6 and E7 gene expressing astrocytes were exposed to 10 micromole and 200 micromole H2O2 solutions. Twenty- four hours after treatment with the lower dosage(10 micromole H2O2), control, E6-expressing cells suffered about 45% injury and LXSN-expressing cells decreased by 67% as assessed by LDH release. However, E7-expressing cells showed less injury, resulting in 20-30% of LDH release. Astrocytes expressing E6, E7, LXSN and mock-infected cells showed a typical apoptotic death pattern on the DNA gel after treatment with a low-dose of H2O2 (10 micromole), however they died from necrotic death after a high-dose (200 micromole) H2O2. Overexpression of HPV-E7 genes protected the cells from apoptotic death after a low-dose of H2O2 and from necrotic death after a high-dose of H2O2, while the overexpression of E6 genes from the necrotic death. E7 expressing astrocytes showed higher catalase activity and the levels of E2F protein surged more than 100-folds compared with the control astrocytes. We believe that the activity of E7 protein to protect astrocytes from H2O2 injury was at least partly due to increased catalase, a scavenger protein.

    View details for Web of Science ID 000172040700001

    View details for PubMedID 11675674

  • Differential neuroprotection from human heat shock protein 70 overexpression in in vitro and in vivo models of ischemia and ischemia-like conditions EXPERIMENTAL NEUROLOGY Lee, J. E., Yenari, M. A., Sun, G. H., Xu, L. J., Emond, M. R., Cheng, D. Y., Steinberg, G. K., Giffard, R. G. 2001; 170 (1): 129-139

    Abstract

    We previously showed that overexpressing the 70-kDa inducible heat shock protein in primary astrocyte cultures and in a rodent stroke model using viral vectors resulted in protection from ischemia and ischemia-like injury. However, viral transfection could potentially provoke a stress response itself; therefore, we examined whether transgenic mice constitutively expressing human heat shock protein 70 were protected from ischemic insults. Astrocyte cultures from brains of heat shock protein 70 transgenic mice were resistant to hydrogen peroxide injury in a dose-dependent fashion, but were less resistant to hypoglycemia and oxygen-glucose deprivation. Because hydrogen peroxide exposure and glucose deprivation are partially dependent on glutathione levels, we determined whether heat shock protein 70 transgenic cultures had altered glutathione levels under normal growth conditions. However, there was no significant difference in glutathione levels between heat shock protein 70 transgenic and wildtype astrocytes. Hippocampal, but not cortical neuron cultures from these same transgenic mice were also protected against oxygen-glucose deprivation and glutamate toxicity. In an in vivo model of permanent focal cerebral ischemia, there was no significant difference in infarct size assessed 24 h postinsult. These results suggest that heat shock protein 70 protects against some but not all kinds of central nervous system injury. The protective effects may be related to the nature and severity of the insults, as well as subpopulations of brain cells and dose-dependent effects of HSP70 overexpression.

    View details for Web of Science ID 000169840400012

    View details for PubMedID 11421590

  • Differential sensitivity of murine astrocytes and neurons from different brain regions to injury EXPERIMENTAL NEUROLOGY Xu, L. J., Sapolsky, R. M., Giffard, R. G. 2001; 169 (2): 416-424

    Abstract

    Different brain regions show differential vulnerability to ischemia in vivo. Despite this, little work has been done to compare vulnerability of brain cells isolated from different brain regions to injury. Relatively pure neuronal and astrocyte cultures were isolated from mouse cortex, hippocampus, and striatum. Astrocyte vulnerability to 6 h oxygen-glucose deprivation was greatest in striatum (81.8 +/- 4.6% cell death), intermediate in hippocampus (59.8 +/- 4.8%), and least in cortex (37.0 +/- 3.5%). In contrast neurons deprived of oxygen and glucose for 3 h showed greater injury to cortical neurons (71.1 +/- 5.2%) compared to striatal (39.0 +/- 3.1%) or hippocampal (39.0 +/- 5.3%) neurons. Astrocyte injury from glucose deprivation or H(2)O(2) exposure was significantly greater in cells from cortex than from striatum or hippocampus. Neuronal injury resulting from serum deprivation was greater in cortical neurons than in those from striatum or hippocampus, while excitotoxic neuronal injury was equivalent between regions. Antioxidant status and apoptosis-regulatory genes were measured to assess possible underlying differences. Glutathione was higher in astrocytes and neurons isolated from striatum than in those from hippocampus. Superoxide dismutase activity was significantly higher in striatal astrocytes, while glutathione peroxidase activity and superoxide did not differ by brain region. Bcl-x(L) was significantly higher in striatal astrocytes than in astrocytes from other brain regions and higher in striatal and hippocampal neurons than in cortical neurons. Both neurons and astrocytes isolated from different brain regions demonstrate distinct patterns of vulnerability when placed in primary culture. Antioxidant state and levels of expression of bcl-x(L) can in part account for the differential injury observed. This suggests that different protective strategies may have different efficacies depending on brain region.

    View details for Web of Science ID 000169189800019

    View details for PubMedID 11358455

  • Ischemic vulnerability of primary murine microglial cultures NEUROSCIENCE LETTERS Yenari, M. A., Giffard, R. G. 2001; 298 (1): 5-8

    Abstract

    Microglia are known to secrete neurotoxic substances and have been implicated in potentiating injury in a variety of neuropathological settings including stroke. However, little is known about the susceptibility of microglia to ischemia. Here we characterize microglial vulnerabilities to ischemia-like insults. Microglia were remarkably resistant to hypoxia, but a majority of cells were killed after 30 h of aglycemia and 24 h continuous exposure to combined oxygen and glucose deprivation. Serum deprivation also resulted in significant cell death after 24 h. Interestingly, microglia activated by lipopolysaccharide were protected against death by serum deprivation, but not aglycemia. We conclude that microglia display susceptibility to ischemia-like insults that most resembles astrocytes, and that activation in some settings renders them capable of generating factors that enhance their own survival.

    View details for Web of Science ID 000166872500002

    View details for PubMedID 11154822

  • Principles of gene therapy: potential applications in the treatment of cerebral ischaemia BRITISH JOURNAL OF NEUROSURGERY Papadopoulos, M. C., Giffard, R. G., Bell, B. A. 2000; 14 (5): 407-414

    Abstract

    In this review we explore gene therapy as a possible treatment for conditions causing cerebral ischaemia and briefly consider other neurological pathologies such as brain tumours. DNA transfer may be achieved using retrovirus, herpes simplex virus, adenovirus, and adeno-associated virus vectors or liposomes. After cerebral ischaemia, these vectors are used to upregulate genes that increase survival and inhibit those that promote death in the injured cells. In contrast, in brain tumours gene therapy aims to kill the target cells. Examples from studies using cell culture, animal models and patients are presented. We conclude that manipulation of gene expression has potential for the treatment of cerebral ischaemia and brain tumours, although, at present, there are formidable technical obstacles to be overcome before clinical applications become a reality.

    View details for Web of Science ID 000165098800003

    View details for PubMedID 11198761

  • An introduction to the changes in gene expression that occur after cerebral ischaemia BRITISH JOURNAL OF NEUROSURGERY Papadopoulos, M. C., Giffard, R. G., Bell, B. A. 2000; 14 (4): 305-312

    Abstract

    Ischaemia is the final common pathway of brain cell death following a variety of insults. We consider the effect of cerebral ischaemia on gene expression, concentrating on immediate early genes, those encoding heat shock proteins, growth factors, cytokines, adhesion molecules, nitric oxide synthase and proteins involved in programmed cell death. We conclude that the changes in gene expression resulting from ischaemia are important determinants of neuronal and glial survival. In the future it may become possible to manipulate gene expression to limit the extent of damage arising from cerebral ischaemia.

    View details for Web of Science ID 000089235000002

    View details for PubMedID 11045194

  • The electrogenic sodium bicarbonate cotransporter: Developmental expression in rat brain and possible role in acid vulnerability JOURNAL OF NEUROSCIENCE Giffard, R. G., Papadopoulos, M. C., van Hooft, J. A., Xu, L. J., Giuffrida, R., Monyer, H. 2000; 20 (3): 1001-1008

    Abstract

    The electrogenic sodium bicarbonate cotransporter (NBC) is expressed in glial cells in the brain and plays an important role in the regulation of both intracellular and extracellular pH. Differential vulnerability to acidosis between neurons and glia has been noted and may contribute to infarction after cerebral ischemia. Ionic substitution studies and inhibition of injury by 4, 4'-di-isothiocyanostilbene-2,2'-disulfonic acid suggest that NBC is involved in astrocyte vulnerability to acidic injury. Recently two NBC cDNAs differing in 5'-untranslated and N-terminal coding sequence have been cloned from kidney and pancreas. We cloned one of these cDNAs from rat brain and demonstrate here that the clone is functional by expression in Xenopus oocytes. We determined the developmental and regional expression of NBC in the brain by in situ hybridization. Expression was observed in the spinal cord at embryonic day 17, whereas expression in brain was first seen at approximately postnatal day 0 (P0), increased at P15, and persisted in the adult brain. Expression was widespread throughout the cerebellum, cortex, olfactory bulb, and subcortical structures. Cellular resolution of the in situ hybridization signal and double labeling for glial fibrillary acidic protein were consistent with a glial localization for NBC. Expression of NBC in 3T3 cells that do not normally express this transporter rendered them vulnerable to acid injury. The expression profile suggests that this transporter is critical during the later stages of brain development and could be one of the factors contributing to the different patterns of injury seen in perinatal versus adult cerebral ischemia.

    View details for Web of Science ID 000084965300017

    View details for PubMedID 10648705

  • Cell death in the central nervous system: Therapeutic possibilities? REGIONAL ANESTHESIA AND PAIN MEDICINE Giffard, R. G., Morgan, R. L. 2000; 25 (1): 22-25

    View details for Web of Science ID 000084892000005

    View details for PubMedID 10660236

  • Overexpression of bcl-2, bcl-x(L) or hsp70 in murine cortical astrocytes reduces injury of co-cultured neurons NEUROSCIENCE LETTERS Xu, L. J., Lee, J. E., Giffard, R. G. 1999; 277 (3): 193-197

    Abstract

    Astrocytes perform many functions that protect neurons during stress, including transmitter uptake, metabolic support, and protection from oxidative stress. We asked whether astrocytes overexpressing either the anti-apoptotic genes bcl-2, or bcl-XL, or the inducible heat shock protein hsp70, could better protect neurons grown with them in co-culture than normal astrocytes or astrocytes expressing beta-galactosidase. Retroviral vectors were used to express these genes in primary astrocyte cultures. After antibiotic selection to eliminate untransformed astrocytes, neurons were plated on top of the astrocytes. Overexpression of any of the three genes in astrocytes reduced neuronal injury induced by combined oxygen-glucose deprivation, or glucose deprivation. Hsp70 overexpression reduced glutamate toxicity. As none of the genes studied is thought to be secreted, the likeliest explanation for the protection observed is improved astrocyte function.

    View details for Web of Science ID 000084405400014

    View details for PubMedID 10626846

  • The neuroprotective potential of heat shock protein 70 (HSP70) MOLECULAR MEDICINE TODAY Yenari, M. A., Giffard, R. G., Sapolsky, R. M., Steinberg, G. K. 1999; 5 (12): 525-531

    Abstract

    In response to many metabolic disturbances and injuries, including stroke, neurodegenerative disease, epilepsy and trauma, the cell mounts a stress response with induction of a variety of proteins, most notably the 70-kDa heat shock protein (HSP70). Whether stress proteins are neuroprotective has been hotly debated, as these proteins might be merely an epiphenomenon unrelated to cell survival. Only recently, with the availability of transgenic animals and gene transfer, has it become possible to overexpress the gene encoding HSP70 to test directly the hypothesis that stress proteins protect cells from injury. A few groups have now shown that overproduction of HSP70 leads to protection in several different models of nervous system injury. This review will cover these studies, along with the potential mechanisms by which HSP70 might mediate cellular protection.

    View details for Web of Science ID 000083729600007

    View details for PubMedID 10562718

  • More tips for users of the Bullard (TM) laryngoscope - In response ANESTHESIA AND ANALGESIA Habibi, A., Bushell, E., Jaffe, R. J., Giffard, R. G., Brock-Utne, J. G. 1999; 89 (1): 267-267
  • Two tips for users of Bullard (TM) intubating laryngoscope ANESTHESIA AND ANALGESIA Habibi, A., Bushell, E., Jaffe, R. A., Giffard, R. G., Brock-Utne, J. G. 1998; 87 (5): 1206-1208

    View details for Web of Science ID 000076692300044

    View details for PubMedID 9806711

  • The E6 and E7 genes of human papilloma virus-type 16 protect primary astrocyte cultures from injury BRAIN RESEARCH Lee, J. E., Kim, C. Y., Giaccia, A. J., Giffard, R. G. 1998; 795 (1-2): 10-16

    Abstract

    Many oncogenes are implicated in the regulation of apoptosis as well as in control of the cell cycle, and several have been shown to protect cells from injury. We tested whether the expression of human papillomavirus type 16 genes E6 and E7 could protect primary astrocytes from injury. Retroviral vectors were used to express E6, E7, or E6E7 in primary murine astrocyte cultures. Astrocytes expressing E6E7 suffered less than half the injury seen in controls after exposure to 400 microM H2O2. When we compared astrocytes expressing only E6 or E7, cells expressing E7 alone were protected to a greater extent and from more severe injury than those expressing E6. E6E7, E6 and E7 all provided protection from 30 h glucose deprivation, but again E7 provided the best protection, reducing injury to less than a third of that seen in controls. Expression of E7 alone decreases vulnerability to both hydrogen peroxide and glucose deprivation injury while E6 alone primarily decreases glucose deprivation injury. These results demonstrate that expression of human papillomavirus oncogenes can reduce the vulnerability of astrocytes to oxidative stress and nutrient deprivation.

    View details for Web of Science ID 000074806500002

    View details for PubMedID 9622584

  • Potentiation of murine astrocyte antioxidant defence by bcl-2: protection in part reflects elevated glutathione levels EUROPEAN JOURNAL OF NEUROSCIENCE Papadopoulos, M. C., Koumenis, I. L., Xu, L. J., Giffard, R. G. 1998; 10 (4): 1252-1260

    Abstract

    Overexpression of the proto-oncogene bcl-2 has been shown to protect a variety of cell types from oxidative and non-oxidative injury, blocking apoptotic and necrotic types of cell death. Retroviral vectors were used to stably overexpress bcl-2 in primary murine astrocyte cultures with more than 95% efficiency. Compared to beta-galactosidase-expressing and uninfected control cells, bcl-2 overexpressing astrocytes suffered < 40% injury after 24 h glucose deprivation, while controls were essentially completely injured. After exposure to 0.2 mM hydrogen peroxide, the bcl-2 overexpressing astrocytes suffered < 40% the injury seen in controls. In contrast, when the cultures were injured by combined oxygen-glucose deprivation, no difference in the extent or time course of injury was found between cells overexpressing bcl-2 and those expressing beta-galactosidase. To investigate one possible mechanism of bcl-2 protection, we measured the levels of glutathione and three antioxidant enzymes. Astrocytes overexpressing bcl-2 had elevated glutathione levels (130-200%), increased superoxide dismutase (170%) and glutathione peroxidase (140%) activities compared with beta-galactosidase-expressing controls. Bcl-2 overexpressing astrocytes suffered less lipid peroxidation after glucose deprivation, as assessed by cis-parinaric acid fluorescence, and demonstrated more rapid removal of hydrogen peroxide from the medium. When glutathione levels were decreased 80% by pretreatment with buthionine sulfoximine, the extent of protection from glucose deprivation of bcl-2 overexpressing cells was decreased by about half. Increased antioxidant defence contributes to protection from glucose deprivation in bcl-2 overexpressing astrocytes.

    View details for Web of Science ID 000072862100004

    View details for PubMedID 9749779

  • Acidosis reduces neuronal apoptosis NEUROREPORT Xu, L. J., Glassford, A. J., Giaccia, A. J., Giffard, R. G. 1998; 9 (5): 875-879

    Abstract

    Acidosis is a well established concomitant of tissue ischemia. Acidosis in the pH range 6.0-7.0 is seen in cerebral ischemia and within solid tumors. Extracellular acidosis of pH 6.0 and 6.4 provided essentially complete protection from 48 h serum deprivation induced apoptotic death of cultured primary murine neurons. We tested the effect of p53 using transformed mouse embryo fibroblasts of either p53+/+ or p53-/- genotype. Both were markedly protected from serum deprivation by acidity. Hypoxia induced fibroblast injury was also reduced at pH 6.8. Lower pH resulted in a shift from apoptotic to necrotic morphology after 42 h hypoxia. Acidosis reduces apoptosis of both normal and transformed cells, irrespective of p53 status.

    View details for Web of Science ID 000072808900021

    View details for PubMedID 9579683

  • Increasing vulnerability of astrocytes to oxidative injury with age despite constant antioxidant defenses NEUROSCIENCE Papadopoulos, M. C., Koumenis, I. L., Yuan, T. Y., Giffard, R. G. 1998; 82 (3): 915-925

    Abstract

    This paper investigates the vulnerability of astrocytes to oxidative injury as a function of age in culture in mice. Primary murine cortical astrocyte cultures of different ages were exposed to H2O2, combined oxygen-glucose deprivation or glucose deprivation. Astrocytes became more vulnerable to damage from each injury paradigm with age, showing transitions between 15 and 22 days. Both the antioxidant glutathione and superoxide dismutase activity increased after 30 days in culture, while catalase activity did not change up to 34 days. When the decrease in glutathione with injury was measured, young cells showed no change with H2O2 and decreases of < 20% after oxygen-glucose deprivation or glucose deprivation, while older cultures lost > 50% of their glutathione with the same insults. Since iron can be a catalyst for hydroxyl radical formation, we stained cultures and found both iron staining and ferritin immunoreactivity increased with age. Increased iron correlated with protection by deferoxamine against H2O2 injury. The three injury paradigms each had a unique pattern of protection by antioxidants. Dimethylthiourea, a hydrophilic antioxidant, protected from all three insults. Trolox, a lipophilic antioxidant, protected older astrocytes from oxygen-glucose deprivation and glucose deprivation. Deferoxamine provided near complete protection from H2O2, partial protection from oxygen-glucose deprivation and no protection from glucose deprivation. As evidence of increasing oxidative stress, lipid peroxidation resulting from oxygen-glucose deprivation increased with age, assessed with cis-parinaric acid. The increasing sensitivity of ageing astrocytes to oxidative injury occurs while antioxidant defenses are maintained. Increased sensitivity to H2O2 or oxygen-glucose deprivation correlates with iron accumulation.

    View details for Web of Science ID A1998YG39200023

    View details for PubMedID 9483545

  • Selection of human cervical epithelial cells that possess reduced apoptotic potential to low-oxygen conditions CANCER RESEARCH Kim, C. Y., Tsai, M. H., OSMANIAN, C., Graeber, T. G., Lee, J. E., Giffard, R. G., DiPaolo, J. A., Peehl, D. M., Giaccia, A. J. 1997; 57 (19): 4200-4204

    Abstract

    Since human papillomavirus (HPV) infection is strongly associated with cervical neoplasia and tumor hypoxia has prognostic significance in human cervical carcinomas, we examined the relationship between hypoxia and apoptosis in human cervical epithelial cells expressing high-risk HPV type 16 oncoproteins. In vitro, hypoxia stimulated both p53 induction and apoptosis in primary cervical epithelial cells infected with the HPV E6 and E7 genes but not in cervical fibroblasts infected with E6 and E7. Furthermore, cell lines derived from HPV-associated human cervical squamous cell carcinomas were substantially less sensitive to apoptosis induced by hypoxia, indicating that these cell lines have acquired additional genetic alterations that reduced their apoptotic sensitivity. Although the process of long-term cell culturing resulted in selection for subpopulations of HPV oncoprotein-expressing cervical epithelial cells with diminished apoptotic potential, the exposure of cells to hypoxia greatly accelerated the selection process. These results provide evidence for the role of hypoxia-mediated selection of cells with diminished apoptotic potential in the progression of human tumors and can in part explain why cervical tumors that possess low pO2 values are more aggressive.

    View details for Web of Science ID A1997XZ01100013

    View details for PubMedID 9331075

  • Increased production of extracellular glutamate by the mitochondrial glutaminase following neuronal death JOURNAL OF BIOLOGICAL CHEMISTRY Newcomb, R., Sun, X. Y., Taylor, L., Curthoys, N., Giffard, R. G. 1997; 272 (17): 11276-11282

    Abstract

    Elevated extracellular concentrations of the excitatory transmitter glutamate are an important cause of neuronal death in a variety of disorders of the nervous system. The concentrations and rates of clearance and production of extracellular glutamate were measured in the medium of primary cultures from mouse neocortex containing neurons, astrocytes, or both cell types. Measurements were performed in the presence and absence of 2 mM glutamine with or without neuronal injury caused by 5-h exposure to hypoxia or 500 microM N-methyl-D-aspartate or a freeze-thaw cycle. High rates of glutamate generation (0.5-0.8 microM/min in the 0.4-ml culture well) occurred if neurons were both damaged and exposed to glutamine. Intact neurons or glia exposed to glutamine generated only small amounts of glutamate (0.03 microM/min). Glutamate generation by damaged neurons was dependent on the presence of glutamine, activated by phosphate, and inhibited by 6-diazo-5-oxo-L-norleucine and p-chloromercuriphenylsulfonic acid (pCMPS), strongly implicating the mitochondrial glutaminase. Following 5-h exposure to 500 microM N-methyl-D-aspartate, the glutaminase was localized to fragments of damaged neurons and was accessible to inhibition by the membrane-impermeant pCMPS. The glutaminase activity from damaged neurons is sufficient to account for the neurotoxic concentrations of glutamate in hypoxic mixed neuronal-glial cultures exposed to 2 mM glutamine. Finally, pCMPS is neuroprotective and also prevents the increased rate of generation of glutamate observed in neuronal cultures after prolonged exposure to glutamine. The cumulative data indicate the following: 1) excitotoxic neuronal death activates the hydrolysis of extracellular glutamine by the mitochondrial glutaminase, and 2) the glutaminase in damaged neurons is sufficient to cause neuronal death in in vitro models of neuronal injury.

    View details for Web of Science ID A1997WW00900047

    View details for PubMedID 9111031

  • HSP70 protects murine astrocytes from glucose deprivation injury NEUROSCIENCE LETTERS Xu, L. J., Giffard, R. G. 1997; 224 (1): 9-12

    Abstract

    Expression of the 70 kDa heat shock protein (HSP70) induced by a first insult is associated with protection from a subsequent ischemic insult in brain. Expression of the human inducible HSP70 was previously shown to protect astrocytes in primary culture from combined oxygen-glucose deprivation. These studies have now been extended to demonstrate that HSP70 expression also protects from isolated glucose deprivation. Slight protection was seen against hydrogen peroxide (H2O2) exposure. Glutathione levels decrease less after glucose deprivation or H2O2 exposure (200 microM) in the cells overexpressing HSP70, compared to either beta-galactosidase expressing or uninfected controls (P < 0.01). These data suggest that the HSP70-expressing cells suffered less oxidative stress since their glutathione levels were better preserved.

    View details for Web of Science ID A1997WM86400003

    View details for PubMedID 9132695

  • Vulnerability to glucose deprivation injury correlates with glutathione levels in astrocytes BRAIN RESEARCH Papadopoulos, M. C., Koumenis, I. L., Dugan, L. L., Giffard, R. G. 1997; 748 (1-2): 151-156

    Abstract

    Astrocyte death from glucose deprivation appears to be mediated by free radicals. Reduced glutathione (GSH) was used as a measure of antioxidant defenses in primary cultures of cortical astrocytes. Glucose deprivation caused progressive, near complete loss of reduced glutathione (GSH). Astrocytes were protected by increasing endogenous GSH levels. Depletion of GSH to 21.4 +/- 3.3% of controls by the glutathione synthetase inhibitor buthionine sulfoximine resulted in more rapid injury by glucose deprivation, yet depletion of glutathione alone did not kill astrocytes. Both enhanced lipid peroxidation and membrane rigidification were caused by glucose deprivation, both indicators of oxidative damage. Membrane peroxidation was detected as a 24 +/- 2% decrease in cis-parinaric acid fluorescence, membrane rgidification as a 6.3 +/- 0.8% increase in fluorescence anisotropy using diphenylhexatriene. Glucose deprivation under normoxic conditions may occur clinically in patients such as diabetics. In addition, oxidative damage in the setting of energy depletion occurs with other insults, including ischemic brain injury. Glucose deprivation may thus be a clinically relevant model of hypoglycemic astrocyte injury, and may be useful to investigate the effects of glutathione and redox modulation on second messenger systems and gene regulation.

    View details for Web of Science ID A1997WL74900018

    View details for PubMedID 9067456

  • Mechanism of heat shock protein 72 induction in primary cultured astrocytes after oxygen-glucose deprivation NEUROLOGICAL RESEARCH Bergeron, M., Mivechi, N. F., Giaccia, A. J., Giffard, R. G. 1996; 18 (1): 64-72

    Abstract

    Induction of stress proteins is thought to be important in the protection of cells from a variety of environmental insults including heat, hypoxia and ischemia. The aim of this study was to compare the mechanism of induction of heat shock protein 72 (HSP72) in primary cultures of murine cortical astrocytes by heat and combined oxygen-glucose deprivation (OGD), a model of in vitro ischemia. 35S-methionine labeling and immunoblotting showed increased HSP72 synthesis and accumulation lasting for up to 24 h following heat or OGD. Heat induced a markedly greater amount of HSP72 mRNA and protein than did OGD. We then sought evidence of heat shock transcription factor-1 (HSF-1) activation. An increase in apparent molecular weight of nuclear HSF-1 after heat or OGD was observed, consistent with increased phosphorylation. To seek an explanation of the difference between heat and OGD as inducers of HSP72 we examined the binding activity of HSP72 + 73 to other proteins. More cellular protein was found to co-immunoprecipitate with HSP72 + 73, and more HSP72 + 73 was found in the pellet fraction after heat shock compared to OGD. These results suggest that HSP72 induction is regulated in astrocytes at least in part at the level of HSF activation, by both heat and OGD. Reduced availability of free HSP72 + 73 in heated cells could be responsible for the greater magnitude of HSP72 induction after heat compared to OGD.

    View details for Web of Science ID A1996TW49400013

    View details for PubMedID 8714540

  • Over-expression of HSP-7O protects astrocytes from combined oxygen-glucose deprivation NEUROREPORT Papadopoulos, M. C., Sun, X. Y., Cao, J. M., Mivechi, N. F., Giffard, R. G. 1996; 7 (2): 429-432

    Abstract

    Pretreatment by a sublethal insult is associated with induction of stress proteins and with protection from subsequent injury. Heat pretreatment protects the brain from subsequent ischemia, and is shown here to protect primary astrocyte cultures from subsequent oxygen-glucose deprivation. To determine whether the expression of a single stress protein, HSP-70, could account for much of this protection, we expressed HSP-70 or beta-galactosidase in astrocytes using retroviral vectors. Only 12% of astrocytes expressing HSP-70 died after 7 hours of oxygen-glucose deprivation compared to 65% of astrocytes expressing beta-galactosidase and 82% of normal astrocytes. Our data provide direct evidence that selective expression of HSP-70 enhances the survival of astrocytes challenged with heat or oxygen-glucose deprivation.

    View details for Web of Science ID A1996UC29900013

    View details for PubMedID 8730798

  • Ultrastructure of excitotoxic neuronal death in murine cortical culture BRAIN RESEARCH Regan, R. F., Panter, S. S., Witz, A., Tilly, J. L., Giffard, R. G. 1995; 705 (1-2): 188-198

    Abstract

    Ischemic and traumatic brain injury are likely to involve neuronal injury triggered by glutamate receptor overactivation. Although excitotoxic neuronal injury has been widely studied in the setting of primary culture, the extent to which these in vitro injury paradigms resemble in vivo ischemic injury morphologically has not previously been well studied. We studied glutamate receptor mediated neuronal death by transmission electron microscopy and light microscopy. Morphologic characteristics of neurons injured by 10 min exposure to 500 microM glutamate include rapid swelling of mitochondria and endoplasmic reticulum, and cytoplasmic and nuclear lucency. Both alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and kainic acid caused vacuolation, dilatation of the endoplasmic reticulum, cytoplasmic condensation and random condensation of chromatin with preserved mitochondria. None of these injuries was ameliorated by cycloheximide or actinomycin D; all were significantly lessened by aurintricarboxylic acid. Gel electrophoresis showed no increase in DNA fragmentation over control. The morphologic changes seen with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and kainate are distinct from the changes induced by glutamate. Excitotoxic injury in this system due to high concentrations of glutamate resembles necrosis while the other agonists produce a different form of cell death which is neither necrosis nor apoptosis.

    View details for Web of Science ID A1995TQ99800023

    View details for PubMedID 8821749

  • CORRELATION OF CGS-19755 NEUROPROTECTION AGAINST IN-VITRO EXCITOTOXICITY AND FOCAL CEREBRAL-ISCHEMIA JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM PEREZPINZON, M. A., Maier, C. M., Yoon, E. J., Sun, G. H., Giffard, R. G., Steinberg, G. K. 1995; 15 (5): 865-876

    Abstract

    The in vivo neuroprotective effect and brain levels of cis-4-phosphonomethyl-2-piperidine carboxylic acid (CGS 19755), a competitive N-methyl-D-aspartate (NMDA) antagonist, were compared with its in vitro neuroprotective effects. The dose-response for in vitro neuroprotection against both NMDA toxicity and combined oxygen-glucose deprivation (OGD) was determined in murine neocortical cultures. Primary cultures of neocortical cells from feta mice were injured by exposure to 500 microM NMDA for 10 min or to OGD for 45 min. The effect of CGS 19755 in both injury paradigms was assessed morphologically and quantitated by determination of lactate dehydrogenase release. Near complete neuroprotection was found at high doses of CGS 19755. The ED50 for protection against NMDA toxicity was 25.4 micro M, and against OGD the ED50 was 15.2 microM. For the in vivo paradigm rabbits underwent 2 h of left internal carotid, anterior cerebral, and middle cerebral artery occlusion followed by 4 h reperfusion; ischemic injury was assessed by magnetic resonance imaging and histopathology. The rabbits were treated with 40 mg/kg i.v. CGS 19755 or saline 10 min after arterial occlusion. CSF and brain levels of CGS 19755 were 12 microM and 5 microM, respectively, at 1 h, 6 microM and 5 microM at 2 h, and 13 microM and 7 microM at 4 h. These levels were neuroprotective in this model, reducing cortical ischemic edema by 48% and ischemic neuronal damage by 76%. These results suggest that a single i.v. dose penetrates the blood-brain barrier, attaining sustained neuroprotective levels that are in the range for in vitro neuroprotection.

    View details for Web of Science ID A1995RQ02000017

    View details for PubMedID 7673380

  • ACTIVATION OF CLASS-II OR CLASS-III METABOTROPIC GLUTAMATE RECEPTORS PROTECTS CULTURED CORTICAL-NEURONS AGAINST EXCITOTOXIC DEGENERATION EUROPEAN JOURNAL OF NEUROSCIENCE Bruno, V., Battaglia, G., Copani, A., Giffard, R. G., Raciti, G., Raffaele, R., Shinozaki, H., Nicoletti, F. 1995; 7 (9): 1906-1913

    Abstract

    Trans-1-aminocyclopentane-1,3-dicarboxylic acid, a mixed agonist of all metabotropic glutamate receptor (mGluR) subtypes, is known to produce either neurotoxic or neuroprotective effects. We have therefore hypothesized that individual mGluR subtypes differentially affect neurodegenerative processes. Selective agonists of subtypes which belong to mGluR class II or III, such as (2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)-glycine (DCG-IV) (specific for subtypes mGluR4, 6 or 7), were highly potent and efficacious in protecting cultured cortical neurons against toxicity induced by either a transient exposure to N-methyl-D-aspartate (NMDA) or a prolonged exposure to kainate. In contrast, agonists that preferentially activate class I mGluR subtypes (mGluR1 or 5), such as quisqualate or trans-azetidine-2,3-dicarboxylic acid, were inactive. DCG-IV was still neuroprotective when applied to cultures after the toxic pulse with NMDA. This delayed rescue effect was associated with a reduction in the release of endogenous glutamate, a process that contributes to the maturation of neuronal damage. We conclude that agonists of class II or III mGluRs are of potential interest in the experimental therapy of acute or chronic neurodegenerative disorders.

    View details for Web of Science ID A1995RV90200010

    View details for PubMedID 8528465

  • NEUROPROTECTION BY THE N-METHYL-D-ASPARTATE RECEPTOR ANTAGONIST CGP-40116 - IN-VIVO AND IN-VITRO STUDIES JOURNAL OF NEUROCHEMISTRY Maier, C. M., Sun, G. H., Kunis, D. M., Giffard, R. G., Steinberg, G. K. 1995; 65 (2): 652-659

    Abstract

    The goal of this study was to evaluate the effects of a novel competitive N-methyl-D-aspartate (NMDA) receptor antagonist, D-(E)-2-amino-4-methyl-5-phosphono-3-pentoic acid (CGP 40116), on neuronal damage in vivo and in vitro. We studied 20 rabbits that underwent a 2-h occlusion of the left internal carotid, anterior cerebral, and middle cerebral arteries followed by 4 h of reperfusion. Ten minutes after occlusion the animals were treated with either normal saline (n = 7) or CGP 40116 at two different doses (20 mg/kg, n = 6; 40 mg/kg, n = 7) administered over a 5-min period. Somatosensory evoked potentials were used to confirm adequate ischemia and neuronal injury was assessed by histopathology and magnetic resonance imaging. CGP 40116 decreased cortical ischemic neuronal damage by 74 and 77% (control, 37.8 +/- 13.1%; CGP 20 mg/kg, 9.9 +/- 3.6%; CGP 40 mg/kg, 8.7 +/- 3.7%; p < 0.01) and reduced cortical ischemic edema by 52 and 35% (control, 42.3 +/- 10.4%; CGP 20 mg/kg, 20.1 +/- 6.7%; CGP 40 mg/kg, 27.5 +/- 13.3%; p < 0.05) but did not protect against striatal injury. We performed a second study using primary cell cultures from mouse neocortex to determine the effects of CGP 40116 on neuronal death induced by a 10-min exposure to 500 microM NMDA or by 45 min of oxygen-glucose deprivation (OGD). Our results demonstrate that CGP 40116 was effective at attenuating neuronal death in a concentration-dependent manner (ED50 of 3.2 microM against NMDA toxicity and 23.1 microM against OGD) as measured by lactate dehydrogenase levels 24 h after the insult.(ABSTRACT TRUNCATED AT 250 WORDS)

    View details for Web of Science ID A1995RJ65700022

    View details for PubMedID 7616221

  • GLIA MODULATE THE RESPONSE OF MURINE CORTICAL-NEURONS TO EXCITOTOXICITY - GLIA EXACERBATE AMPA NEUROTOXICITY JOURNAL OF NEUROSCIENCE Dugan, L. L., BRUNO, V. M., Amagasu, S. M., Giffard, R. G. 1995; 15 (6): 4545-4555

    Abstract

    We have developed "pure" neuronal cultures (< 1% astrocytes) from mouse neocortex to study the effect of glial cells on the response of neurons to injury. Cortical neurons were found to require glial-conditioned medium to survive. Immature neurons, 2-4 d in vitro, deprived of glial-conditioned medium, underwent apoptosis over 48 hr, as suggested by condensed nuclear morphology, DNA fragmentation, and protection by inhibition of macromolecular synthesis. Apoptosis induced by trophic factor deprivation has been described for other neuronal populations, such as superior cervical ganglion and dorsal root ganglion cells. Cortical neurons in pure culture provide another neuronal population for the study of apoptosis induced by trophic factor deprivation. We then studied the interaction of neurons and glia under excitotoxic conditions. Experiments on mature cultures showed that pure neuronal cultures were at least 10-fold more sensitive to acute glutamate exposure than were neuronal-glial ("mixed") cocultures. The difference in sensitivity between pure neurons and mixed cultures was reduced when mixed cultures were treated with the glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC). In 24 hr exposure to N-methyl-D-aspartate (NMDA), or oxygen, glucose deprivation, pure neurons were more sensitive than mixed cultures; trans-PDC again increased the sensitivity of mixed cultures to nearly that of pure neuronal cultures. In contrast, mixed and pure neuronal cultures exposed to NMDA for 10 min, or to kainate for 24 hr, had similar injury dose-response curves, suggesting that glial glutamate uptake is a less important protective mechanism in these excitotoxic injuries. Surprisingly, pure neurons were less sensitive than mixed cultures to (RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) toxicity at concentrations up to 100 microM. This does not reflect astrocyte toxicity, as AMPA at concentrations to 1 mM did not injure astrocyte cultures. Glial cultures showed increased levels of glutamate in the extracellular medium in response to exposure to AMPA, but not NMDA or kainate. However, pure neuronal and mixed cultures exposed to the same concentration of AMPA did not have elevated levels of glutamate in the media. We found that glia were generally neuroprotective under excitotoxic conditions, likely through their ability to clear extracellular glutamate. However, the presence of glia exacerbated AMPA neurotoxicity.

    View details for Web of Science ID A1995RD29900034

    View details for PubMedID 7540679

  • NEUROPROTECTIVE EFFECT OF HYPOTHERMIA IN CORTICAL CULTURES EXPOSED TO OXYGEN-GLUCOSE DEPRIVATION OR EXCITATORY AMINO-ACIDS JOURNAL OF NEUROCHEMISTRY BRUNO, V. M., Goldberg, M. P., Dugan, L. L., Giffard, R. G., Choi, D. W. 1994; 63 (4): 1398-1406

    Abstract

    We examined the effect of moderate hypothermia (30 degrees C) on neuronal injury in murine cortical cell cultures. Lowering the temperature during and after a period of oxygen-glucose deprivation reduced both the release of glutamate to the bathing medium and accompanying neuronal degeneration. Hypothermia immediately after brief exposure to high concentrations of NMDA or glutamate also reduced the resulting neuronal degeneration. This protective effect was not eliminated when MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione were added immediately after washout of the exogenously added excitotoxin, suggesting that it was mediated by actions additional to reduction of endogenous late glutamate release. Hypothermia applied only during exposure to NMDA or glutamate, whether brief or prolonged, did not reduce subsequent cytosolic calcium accumulation or neuronal degeneration, suggesting that the postsynaptic induction of NMDA receptor-mediated excitotoxicity is not sensitive to temperature reduction. However, hypothermia during prolonged S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate exposure did reduce neuronal degeneration.

    View details for Web of Science ID A1994PH20200026

    View details for PubMedID 7523591

  • SECOBARBITAL ATTENUATES EXCITOTOXICITY BUT POTENTIATES OXYGEN-GLUCOSE DEPRIVATION NEURONAL INJURY IN CORTICAL CELL-CULTURE JOURNAL OF CEREBRAL BLOOD FLOW AND METABOLISM Giffard, R. G., Weiss, J. H., Swanson, R. A., Choi, D. W. 1993; 13 (5): 803-810

    Abstract

    We examined the effects of secobarbital and other sedative-hypnotic barbiturates on the neuronal death induced by exposure to excitatory amino acids or deprivation of oxygen or glucose in mouse cortical cell cultures. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionate, and kainate toxicities were attenuated in a concentration-dependent fashion by high concentrations of secobarbital or thiopental. Antagonism of NMDA toxicity was not overcome by increasing NMDA concentration and not mimicked by gamma-aminobutyrate. Despite these antiexcitotoxic actions, secobarbital exacerbated the neuronal death induced by deprivation of either glucose alone or oxygen and glucose together; death induced by oxygen deprivation alone was little affected. Thiopental and methohexital also increased oxygen-glucose deprivation injury. A possible explanation for this injury potentiation was provided by the observation that secobarbital enhanced the cellular ATP depletion induced by combined oxygen-glucose deprivation. Deleterious effects on ATP production may counterbalance the protective effects of barbiturates under some conditions.

    View details for Web of Science ID A1993LT80000009

    View details for PubMedID 8360287

  • NEUROPROTECTIVE EFFECTS OF GLUTAMATE ANTAGONISTS AND EXTRACELLULAR ACIDITY SCIENCE KAKU, D. A., Giffard, R. G., Choi, D. W. 1993; 260 (5113): 1516-1518

    Abstract

    Glutamate antagonists protect neurons from hypoxic injury both in vivo and in vitro, but in vitro studies have not been done under the acidic conditions typical of hypoxia-ischemia in vivo. Consistent with glutamate receptor antagonism, extracellular acidity reduced neuronal death in murine cortical cultures that were deprived of oxygen and glucose. Under these acid conditions, N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-kainate antagonists further reduced neuronal death, such that some neurons tolerated prolonged oxygen and glucose deprivation almost as well as did astrocytes. Neuroprotection induced by this combination exceeded that induced by glutamate antagonists alone, suggesting that extracellular acidity has beneficial effects beyond the attenuation of ionotropic glutamate receptor activation.

    View details for Web of Science ID A1993LF05800040

    View details for PubMedID 8389056

  • EXTRACELLULAR ALKALINITY EXACERBATES INJURY OF CULTURED CORTICAL-NEURONS STROKE Giffard, R. G., Weiss, J. H., Choi, D. W. 1992; 23 (12): 1817-1821

    Abstract

    We have previously shown that extracellular acidity protects cultured fetal murine neocortical neurons from glutamate toxicity and combined oxygen-glucose deprivation injury, an action at least in part mediated by reduction in N-methyl-D-aspartate receptor activation. We now investigate the effect of extracellular alkalinity on both glutamate neurotoxicity and injury due to combined oxygen-glucose deprivation.The effects of extracellular alkalinity during injury induced by exposure of murine neocortical cultures to glutamate (0.5 mM for 5 minutes) or oxygen-glucose deprivation are characterized morphologically and quantitated by efflux of lactate dehydrogenase from both neurons and glia to the bathing medium. Calcium accumulation is measured with calcium-45.Moderate extracellular alkalinity is well tolerated by cortical cells but significantly potentiates both glutamate neuronal toxicity and oxygen-glucose deprivation neuronal injury. In contrast, glial viability in the face of combined oxygen-glucose deprivation is little affected by extracellular alkalinity. Increased accumulation of calcium-45 during oxygen-glucose deprivation in alkalotic medium and blockade of this increase by MK-801 is demonstrated.These observations suggest that alkaline pH can exacerbate excitotoxic neuronal injury, most likely because of increased N-methyl-D-aspartate receptor activation. Metabolic alkalosis of any etiology may sensitize neurons to ischemic injury and potentiate reperfusion injury.

    View details for Web of Science ID A1992KA15100026

    View details for PubMedID 1360176

  • OXYGEN OR GLUCOSE DEPRIVATION-INDUCED NEURONAL INJURY IN CORTICAL CELL-CULTURES IS REDUCED BY TETANUS TOXIN NEURON Monyer, H., Giffard, R. G., Hartley, D. M., Dugan, L. L., Goldberg, M. P., Choi, D. W. 1992; 8 (5): 967-973

    Abstract

    We examined glutamate-mediated neurotoxicity in cortical cell cultures pretreated with 1-5 micrograms/ml tetanus toxin to attenuate the Ca(2+)-dependent release of neurotransmitters. Efficacy of the tetanus toxin pretreatment was suggested by blockade of electrical burst activity induced by Mg2+ removal and by reduction of glutamate efflux induced by high K+. Tetanus toxin reduced neuronal injury produced by brief exposure to elevated extracellular K+ or to glutamate, situations in which release of endogenous excitatory neurotransmitter is likely to play a role. Furthermore, although glutamate efflux evoked by anoxic conditions may occur largely via Ca(2+)-independent transport, tetanus toxin attenuated both glutamate efflux and neuronal injury following combined oxygen and glucose deprivation. With prolonged exposure periods, the neuroprotective efficacy of tetanus toxin was comparable to that of NMDA receptor antagonists. Presynaptic inhibition of Ca(2+)-dependent glutamate release may be a valuable approach to attenuating hypoxic-ischemic brain injury.

    View details for Web of Science ID A1992HV46700016

    View details for PubMedID 1350203

  • SELECTIVE VULNERABILITY OF CULTURED CORTICAL GLIA TO INJURY BY EXTRACELLULAR ACIDOSIS BRAIN RESEARCH Giffard, R. G., Monyer, H., Choi, D. W. 1990; 530 (1): 138-141

    Abstract

    Reduction of extracellular pH from 7.4 to 6.5 attenuated glutamate neurotoxicity in murine cortical neuronal and glial cultures, but if maintained for 24 h, resulted in morphological evidence of selective glial injury. Acid-induced gliotoxicity was examined quantitatively in cortical astrocyte cultures, using lactate dehydrogenase efflux as an index of cell damage. An exposure time of 9 h to pH 6.4 was sufficient to destroy about one third of the glia, whether or not 25 mM lactate was present. Furthermore, such acidosis increased the vulnerability of glia to injury by combined oxygen and glucose deprivation. These observations support the suggestion that the acidosis which accompanies ischemia in vivo may contribute to glial injury.

    View details for Web of Science ID A1990EF52800020

    View details for PubMedID 2176914

  • ACIDOSIS REDUCES NMDA RECEPTOR ACTIVATION, GLUTAMATE NEUROTOXICITY, AND OXYGEN-GLUCOSE DEPRIVATION NEURONAL INJURY IN CORTICAL CULTURES BRAIN RESEARCH Giffard, R. G., Monyer, H., Christine, C. W., Choi, D. W. 1990; 506 (2): 339-342

    Abstract

    The acidosis which accompanies cerebral ischemia in vivo has been thought to contribute to subsequent neuronal injury. However, recent electrophysiological recordings from hippocampal neurons suggest that H+ can attenuate N-methyl-D-aspartate (NMDA) receptor-mediated cation influx, likely a key event in the pathogenesis of ischemic neuronal injury. Here we report that moderate extracellular acidosis (pH 6.5) markedly reduced the inward whole cell current induced by NMDA on cultured cortical neurons; at pH 6.1, kainate-induced current was additionally reduced. Furthermore, such acidosis reduced the cortical neuronal injury caused by toxic glutamate exposure, as well as the neuronal degeneration and accumulation of 45Ca2+ induced by combined oxygen and glucose deprivation. These findings raise the possibility that moderate acidosis may decrease cortical neuronal vulnerability to ischemic damage.

    View details for Web of Science ID A1990CL25500028

    View details for PubMedID 1967968

  • Acute brain injury, NMDA receptors, and hydrogen ions: observations in cortical cell cultures. Advances in experimental medicine and biology Choi, D. W., Monyer, H., Giffard, R. G., Goldberg, M. P., Christine, C. W. 1990; 268: 501-504

    View details for PubMedID 2150160

  • Neuronal calcium channels. Journal of neurosurgical anesthesiology Giffard, R. G., Choi, D. W. 1989; 1 (4): 364-367

    View details for PubMedID 15815303

  • CA-2+-DEPENDENT BINDING OF SEVERIN TO ACTIN - A ONE-TO-ONE COMPLEX IS FORMED JOURNAL OF CELL BIOLOGY Giffard, R. G., Weeds, A. G., Spudich, J. A. 1984; 98 (5): 1796-1803

    Abstract

    Severin is a protein from Dictyostelium that severs actin filaments in a Ca2+-dependent manner and remains bound to the filament fragments (Brown, S. S., K. Yamamoto, and J. A. Spudich , 1982, J. Cell Biol., 93:205-210; Yamamoto, K., J. D. Pardee , J. Reidler , L. Stryer , and J. A. Spudich , 1982, J. Cell Biol. 95:711-719). Further characterization of the interaction of severin with actin suggests that it remains bound to the preferred assembly end of the fragmented actin filaments. Addition of severin in molar excess to actin causes total disassembly of the filaments and the formation of a high-affinity complex containing one severin and one actin. This severin -actin complex does not sever actin filaments. The binding of severin to actin, measured directly by fluorescence energy transfer, requires micromolar Ca2+, as does the severing and depolymerizing activity reported previously. Once bound to actin in the presence of greater than 1 microM Ca2+, severin is not released from the actin when the Ca2+ is lowered to less than 0.1 microM by addition of EGTA. Tropomyosin, DNase I, phalloidin, and cytochalasin B have no effect on the ability of severin to bind to or sever actin filaments. Subfragment 1 of myosin, however, significantly inhibits severin activity. Severin binds not only to actin filaments, but also directly to G-actin, as well as to other conformational species of actin.

    View details for Web of Science ID A1984SR27800021

    View details for PubMedID 6427234

  • CA-2+-SENSITIVE ISOLATION OF A CORTICAL ACTIN MATRIX FROM DICTYOSTELIUM AMEBAS JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY Giffard, R. G., Spudich, J. A., Spudich, A. 1983; 4 (1): 115-131

    Abstract

    A cortical actin matrix has been isolated from amoebae of Dictyostelium discoideum grown in liquid culture. The existence of this actin matrix in whole cells is indicated in electron micrographs as an area free of cytoplasmic organelles. The actin beneath the membrane is more clearly visible in sections of cells that are lysed gently with 0.5% Triton X-100 and fixed with 1% glutaraldehyde. Such Triton-lysed cells have fragments of plasma membrane associated with the cortical actin matrix. Isolation of the actin matrix, which sediments at 400 g, is inhibited by Ca2+. As much as 50% of the actin of the cell and about 12% of the total protein is found in the matrix isolated in lysis buffer containing no added Ca2+ and 2.5 mM EGTA, whereas less than 15% of the actin of the cell is recovered in a 400 g pellet when cells are lysed in buffer containing 2.5 mM Ca2+ and 2.5 mM EGTA. A 40 000 molecular weight protein that fragments F-actin in a Ca2+-dependent manner is not found in the isolated cortical actin matrix.

    View details for Web of Science ID A1983QD97100007

    View details for PubMedID 6404931

  • MOLECULAR ASPECTS OF CORTICAL ACTIN FILAMENT FORMATION UPON FERTILIZATION CELL DIFFERENTIATION Spudich, A., Giffard, R. G., Spudich, J. A. 1982; 11 (5-6): 281-284

Conference Proceedings


  • Overexpression of bcl-x(L) protects astrocyte from glucose deprivation and is associated with higher glutathione, ferritin, and iron levels Xu, L. J., Koumenis, I. L., Tilly, J. L., Giffard, R. G. LIPPINCOTT WILLIAMS & WILKINS. 1999: 1036-1046

    Abstract

    The possibility of altering outcome from ischemia-like injury by overexpressing the anti-cell death gene bcl-xL was studied. Cells are known to die by different pathways including apoptosis, or programmed cell death, and necrosis. The bcl-xL gene is a member of a family of apoptosis regulating genes and often displays the death-inhibiting properties of the prototype of this family, bcl-2. It is of special interest to study bcl-xL for possible brain protection, because, unlike bcl-2, it is important for normal brain development.Overexpression of bcl-xL was achieved in primary astrocyte cultures using a retroviral vector. Cultures of astrocytes overexpressing bcl-xL or a control gene were injured by hydrogen peroxide, glucose deprivation, or combined oxygen and glucose deprivation. Outcome was assessed morphologically and by release of lactate dehydrogenase. We assessed antioxidant effects by measuring glutathione using monochlorobimane, ferritin by immunoblotting, the level of iron spectrophotometrically, and superoxide using iodonitrotetrazolium violet and dihydroethidium.Protection by bcl-xL was found against glucose deprivation and hydrogen peroxide exposure but not combined oxygen and glucose deprivation. Higher levels of superoxide were found, without increased levels of lipid peroxidation. Overexpression of bcl-xL was associated with elevated glutathione levels, elevated ferritin levels, and increased amounts of iron. The increased glutathione contributed to the protection from glucose deprivation.Overexpression of bcl-xL protects astrocytes from oxidative injury with the same spectrum of protection seen previously for bcl-2. The increased antioxidant defense observed should be beneficial against both apoptotic and necrotic cell death. The effects on levels of ferritin and iron are novel and identify a new area of interest for this gene family. Whether this relates to the effects of these genes on mitochondrial function remains to be elucidated.

    View details for Web of Science ID 000082892200020

    View details for PubMedID 10519507

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