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.
Highlights
Astrocytes play a critical role in maintaining neuronal homeostasis in the central nervous system (CNS) through secretion of trophic factors, neurotransmitter recycling, nutrient and oxygen balancing, and free radical scavenging [1,2,3,4]
Cultures were consistently $98% positive for astrocyte proteins glial fibrillary acidic protein (GFAP), Vimentin, Glutamine Synthetase (GS), Pax-2, and S100A, as we have previously reported as markers of human optic nerve head (ONH) astrocytes [17] (Figure 1C)
A Model of Mature Astrocyte Reactivity Critically, these cells appear to maintain a quiescent phenotype for the first three weeks of culture, but during this period are capable of robust responses to titrated levels of oxidative stress, including characteristic changes in markers associated with activation, such as increases in GFAP, HSP70, and PGC-1a, and a dramatic loss of GS, as well as increased expression of reactive oxygen species (ROS) detoxifying genes, and cytokine secretion
Summary
Astrocytes play a critical role in maintaining neuronal homeostasis in the central nervous system (CNS) through secretion of trophic factors, neurotransmitter recycling, nutrient and oxygen balancing, and free radical scavenging [1,2,3,4]. In response to injury or stress, astrocytes undergo a phenotypic switch, characterized by; upregulation of intermediate filament proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, loss of glutamate buffering function, secretion of pro-inflammatory cytokines, and increased production of antioxidants [2,4,5] Both positive and negative influences of astrocyte re-activation have been implicated in a wide variety of neurodegenerative processes. Along with astrocytic radial Muller glia, they rapidly re-activate following oxidative stress [4,12] and have been proposed to both preserve inner retinal tissue homeostasis, and generate a detrimental para-inflammatory response [13,14,15,16] These effects are accomplished through increased antioxidant activity, and secretion of proinflammatory cytokines that activate resident microglia, increase vascular permeability, and induce direct damage or protection to retinal ganglion cells (RGCs) [4,14,17,18]. The molecular link between oxidative stress and proinflammatory signaling in these cells has not been established
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