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ABSTRACT: Reactive oxygen species (ROS) produced by brain-infiltrating macrophages and neutrophils, as well as resident microglia, are pivotal to pathogen clearance during viral brain infection. However, unchecked free radical generation is also responsible for damage to and cytotoxicity of critical host tissue bystander to primary infection. These unwanted effects of excessive ROS are combated by local cellular production of antioxidant enzymes, including heme oxygenase-1 (HO-1) and glutathione peroxidase 1 (Gpx1). In this study, we showed that experimental murine herpes encephalitis triggered robust ROS production, as well as an opposing upregulation of the antioxidants HO-1 and Gpx1. This antioxidant response was insufficient to prevent tissue damage, neurotoxicity, and mortality associated with viral brain infection. Previous studies corroborate our data supporting astrocytes as the major antioxidant producer in brain cell cultures exposed to HSV-1 stimulated microglia. We hypothesized that stimulating opposing antioxidative responses in astrocytes, as well as neurons, would mitigate the effects of ROS-mediated neurotoxicity both in vitro and during viral brain infection in vivo. Here, we demonstrate that the addition of sulforaphane, a potent stimulator of antioxidant responses, enhanced HO-1 and Gpx1 expression in astrocytes through the activation of nuclear factor-E2-related factor 2 (Nrf2). Additionally, sulforaphane treatment was found to be effective in reducing neurotoxicity associated with HSV-stimulated microglial ROS production. Finally, intraperitoneal injections of sulforaphane into mice during active HSV infection reduced neuroinflammation via a decrease in brain-infiltrating leukocytes, macrophage- and neutrophil-produced ROS, and MHCII-positive, activated microglia. These data support a key role for astrocyte-produced antioxidants in modulating oxidative stress and neuronal damage in response to viral infection.
PLoS ONE 01/2012; 7(4):e36216. · 4.09 Impact Factor
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ABSTRACT: Production of reactive oxygen species (ROS) and proinflammatory cytokines by microglial cells in response to viral brain infection contributes to both pathogen clearance and neuronal damage. In the present study, we examined the effect of herpes simplex virus (HSV)-1-induced, NADPH oxidase-derived ROS in activating mitogen-activated protein kinases (MAPKs) as well as driving cytokine and chemokine expression in primary murine microglia.
Oxidation of 2', 7'-dichlorodihydrofluorescin diacetate (H2DCFDA) was used to measure production of intracellular ROS in microglial cell cultures following viral infection. Virus-induced cytokine and chemokine mRNA and protein levels were assessed using real-time RT-PCR and ELISA, respectively. Virus-induced phosphorylation of microglial p38 and p44/42 (ERK1/2) MAPKs was visualized using Western Blot, and levels of phospho-p38 were quantified using Fast Activated Cell-based ELISA (FACE assay). Diphenyleneiodonium (DPI) and apocynin (APO), inhibitors of NADPH oxidases, were used to investigate the role of virus-induced ROS in MAPK activation and cytokine, as well as chemokine, production.
Levels of intracellular ROS were found to be highly elevated in primary murine microglial cells following infection with HSV and the majority of this virus-induced ROS was blocked following DPI and APO treatment. Correspondingly, inhibition of NADPH oxidase also decreased virus-induced proinflammatory cytokine and chemokine production. In addition, microglial p38 and p44/42 MAPKs were found to be phosphorylated in response to viral infection and this activation was also blocked by inhibitors of NADPH oxidase. Finally, inhibition of either of these ROS-induced signaling pathways suppressed cytokine (TNF-α and IL-1β) production, while chemokine (CCL2 and CXCL10) induction pathways were sensitive to inhibition of p38, but not ERK1/2 MAPK.
Data presented herein demonstrate that HSV infection induces proinflammatory responses in microglia through NADPH oxidase-dependent ROS and the activation of MAPKs.
Journal of Neuroinflammation 09/2011; 8:123. · 3.83 Impact Factor
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ABSTRACT: The rat auditory cortex is organized as a tonotopic map of sound frequency. This map is broadly tuned at birth and is refined during the first 3 weeks postnatal. The structural correlates underlying tonotopic map maturation and reorganization during development are poorly understood. We employed fluorescent dye ballistic labeling ("DiOlistics") alone, or in conjunction with immunohistochemistry, to quantify synaptogenesis in the auditory cortex of normal hearing rats. We show that the developmental appearance of dendritic protrusions, which include both immature filopodia and mature spines, on layers 2/3, 4, and 5 pyramidal and layer 4 spiny nonpyramidal neurons occurs in three phases: slow addition of dendritic protrusions from postnatal day 4 (P4) to P9, rapid addition of dendritic protrusions from P9 to P19, and a final phase where mature protrusion density is achieved (>P21). Next, we combined DiOlistics with immunohistochemical labeling of bassoon, a presynaptic scaffolding protein, as a novel method to categorize dendritic protrusions as either filopodia or mature spines in cortex fixed in vivo. Using this method we observed an increase in the spine-to-filopodium ratio from P9-P16, indicating a period of rapid spine maturation. Previous studies report mature spines as being shorter in length compared to filopodia. We similarly observed a reduction in protrusion length between P9 and P16, corroborating our immunohistochemical spine maturation data. These studies show that dendritic protrusion formation and spine maturation occur rapidly at a time previously shown to correspond to auditory cortical tonotopic map refinement (P11-P14), providing a structural correlate of physiological maturation.
The Journal of Comparative Neurology 07/2011; 519(16):3327-45. · 3.81 Impact Factor
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ABSTRACT: Congenital cytomegalovirus (CMV) infection is the leading cause of sensorineural hearing loss (SNHL) in children. During murine (M)CMV-induced encephalitis, the immune response is important for both the control of viral dissemination and the clearance of virus from the brain. While the importance of CMV-induced SNHL has been described, the mechanisms surrounding its pathogenesis and the role of inflammatory responses remain unclear. This study presents a neonatal mouse model of profound SNHL in which MCMV preferentially infected both cochlear perilymphatic epithelial cells and spiral ganglion neurons. Interestingly, MCMV infection induced cochlear hair cell death by 21 days post-infection, despite a clear lack of direct infection of hair cells and the complete clearance of the virus from the cochlea by 14 dpi. Flow cytometric, immunohistochemical, and quantitative PCR analysis of MCMV-infected cochlea revealed a robust and chronic inflammatory response, including a prolonged increase in reactive oxygen species production by infiltrating macrophages. These data support a pivotal role for inflammation during MCMV-induced SNHL.
Journal of NeuroVirology 03/2011; 17(3):201-11. · 2.31 Impact Factor
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ABSTRACT: Using a murine model of herpes simplex virus (HSV)-1 encephalitis, our laboratory has determined that induction of proinflammatory mediators in response to viral infection is largely mediated through a Toll-like receptor-2 (TLR2)-dependent mechanism. Published studies have shown that, like other inflammatory mediators, reactive oxygen species (ROS) are generated during viral brain infection. It is increasingly clear that ROS are responsible for facilitating secondary tissue damage during central nervous system infection and may contribute to neurotoxicity associated with herpes encephalitis.
Purified microglial cell and mixed neural cell cultures were prepared from C57B/6 and TLR2-/- mice. Intracellular ROS production in cultured murine microglia was measured via 2', 7'-Dichlorofluorescin diacetate (DCFH-DA) oxidation. An assay for 8-isoprostane, a marker of lipid peroxidation, was utilized to measure free radical-associated cellular damage. Mixed neural cultures obtained from beta-actin promoter-luciferase transgenic mice were used to detect neurotoxicity induced by HSV-infected microglia.
Stimulation with HSV-1 elevated intracellular ROS in wild-type microglial cell cultures, while TLR2-/- microglia displayed delayed and attenuated ROS production following viral infection. HSV-infected TLR2-/- microglia produced less neuronal oxidative damage to mixed neural cell cultures in comparison to HSV-infected wild-type microglia. Further, HSV-infected TLR2-/- microglia were found to be less cytotoxic to cultured neurons compared to HSV-infected wild-type microglia. These effects were associated with decreased activation of p38 MAPK and p42/p44 ERK in TLR2-/- mice.
These studies demonstrate the importance of microglial cell TLR2 in inducing oxidative stress and neuronal damage in response to viral infection.
Journal of Neuroinflammation 01/2010; 7:35. · 3.83 Impact Factor
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ABSTRACT: Protoplasmic astrocytes in mammalian CNS tissues in vivo have a highly complex 3D morphology, but in dissociated cell cultures they often assume a flattened, fibroblast-like morphology bearing only a few, simple processes. By fluorescent labeling and confocal reconstruction we show that many astrocytes in organotypic hippocampal slice cultures exhibit a more native complex cytoarchitecture. Although astrocytes at the surface of slice cultures show a reactive form with several thick glial fibrillary acidic protein (GFAP)-positive processes, astrocytes situated in deeper portions of tissue slices retain a highly complex 3D morphology with many fine spine- or veil-like protrusions. Dozens of astrocytes can be labeled in single slice cultures by gene gun-mediated ballistic delivery of gold or tungsten particles carrying cDNAs (Biolistics), lipophilic dyes (DiOlistics), or fluorescent intracellular calcium indicators (Calistics). Expression of a membrane-targeted form of eGFP (Lck-GFP) is superior to soluble eGFP for resolving fine astrocytic processes. Time-lapse confocal imaging of Lck-GFP transfected astrocytes or "calistically" labeled astrocytes show structural remodeling and calcium transients, respectively. This approach provides an in vitro system for investigating the functional architecture, development and dynamic remodeling of astrocytes and their relationships to neurons and glia in live mammalian brain tissues.
Journal of Neuroscience Methods 02/2005; 141(1):41-53. · 1.98 Impact Factor
