title: ROS detoxification and proinflammatory cytokines are linked by p38 MAPK signaling in a model of mature astrocyte activation.
abstract: Astrocytes are the most abundant glial cell in the retinal nerve fiber layer (NFL) and optic nerve head (ONH), and perform essential roles in maintaining retinal ganglion cell (RGC) detoxification and homeostasis. Mature astrocytes are relatively quiescent, but rapidly undergo a phenotypic switch in response to insult, characterized by upregulation of intermediate filament proteins, loss of glutamate buffering, secretion of pro-inflammatory cytokines, and increased antioxidant production. These changes result in both positive and negative influences on RGCs. However, the mechanism regulating these responses is still unclear, and pharmacologic strategies to modulate select aspects of this switch have not been thoroughly explored. Here we describe a system for rapid culture of mature astrocytes from the adult rat retina that remain relatively quiescent, but respond robustly when challenged with oxidative damage, a key pathogenic stress associated with inner retinal injury. When primary astrocytes were exposed to reactive oxygen species (ROS) we consistently observed characteristic changes in activation markers, along with increased expression of detoxifying genes, and secretion of proinflammatory cytokines. This in vitro model was then used for a pilot chemical screen to target specific aspects of this switch. Increased activity of p38α and β Mitogen Activated Protein Kinases (MAPKs) were identified as a necessary signal regulating expression of MnSOD, and heme oxygenase 1 (HO-1), with consequent changes in ROS-mediated injury. Additionally, multiplex cytokine profiling detected p38 MAPK-dependent secretion of IL-6, MCP-1, and MIP-2α, which are proinflammatory signals recently implicated in damage to the inner retina. These data provide a mechanism to link increased oxidative stress to proinflammatory signaling by astrocytes, and establish this assay as a useful model to further dissect factors regulating the reactive switch.
authors.affiliation: Department of Vision Sciences, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada.
authors.forename: Adrian
authors.initials: A
authors.lastname: Nahirnyj
authors.affiliation: Department of Vision Sciences, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada.
authors.forename: Izhar
authors.initials: I
authors.lastname: Livne-Bar
authors.affiliation: Department of Vision Sciences, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada.
authors.forename: Xiaoxin
authors.initials: X
authors.lastname: Guo
authors.affiliation: Department of Vision Sciences, Toronto Western Research Institute, University Health Network, Toronto, Ontario, Canada ; Department of Ophthalmology and Vision Science, University of Toronto, Toronto, Ontario, Canada ; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Sivak
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018932:Chemokine CCL2 / Q000096:biosynthesis / Q000378:metabolism; D054426:Chemokine CXCL2 / Q000096:biosynthesis / Q000378:metabolism; D005786:Gene Expression Regulation; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015850:Interleukin-6 / Q000096:biosynthesis / Q000378:metabolism; D048290:Mitogen-Activated Protein Kinase 11 / Q000235:genetics / Q000378:metabolism*; D048308:Mitogen-Activated Protein Kinase 14 / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D010269:Paraquat / Q000494:pharmacology; D061251:Primary Cell Culture; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism
chemical_list: C491766:Ccl2 protein, rat; D018932:Chemokine CCL2; D054426:Chemokine CXCL2; D015850:Interleukin-6; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D006419:Heme Oxygenase (Decyclizing); C491523:Hmox1 protein, rat; D013482:Superoxide Dismutase; D048290:Mitogen-Activated Protein Kinase 11; D048308:Mitogen-Activated Protein Kinase 14; D010269:Paraquat