Purchase this article with an account.
Jeremy M. Sivak, Adrian Nahirny, Xiaoxin Guo; Regulation of Chronic Oxidative Stress Responses in Adult Retinal Astrocytes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2012.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Astrocytes are the most prominent glial cell type found in the nerve fiber layer (NFL) and optic nerve head (ONH). During ‘normal’ tissue function they perform an essential role maintaining retinal ganglion cell (RGC) homeostasis. Following retinal insult, they rapidly activate, undergoing a variety of cellular and biochemical changes, including; secretion of cytokines and growth factors, and producing antioxidants around the sensitive RGCs and NFL axons. The molecular mechanisms regulating this response are still unclear, but oxidative stress has been established as an important factor contributing to progression of a variety of diseases affecting the inner retina, including glaucoma and diabetic retinopathy. In order to investigate the influence of oxidative stress on NFL astrocytes directly, we have developed a new model exposing primary adult rat NFL astrocytes to chronic production of reactive oxygen species (ROS).
Primary NFL astrocytes from three to four week old Wistar rats were isolated and cultured using a protocol adapted from methods developed for neonatal brains. The resulting cells were characterized through morphology and immunofluorescence staining by a panel of marker proteins. Astrocyte cultures were then subjected to ROS exposure by acute and chronic methods, using H2O2, or the redox cycling chemical paraquot, respectively. ROS generation, cell death, and expression and concentration of markers for glial activation, and oxidative stress were subsequently investigated.
This culture system produced relatively pure populations of NFL astrocytes with little or no contamination from other retinal cell types. Increasing levels of ROS and cell death showed a clear correlation, with chronic exposure producing more severe effects over time. Molecular and biochemical analyses revealed concomitant changes in glial reactivity, and a panel of oxidative and metabolic stress response factors, including increases in GFAP and HSP70, and a surprising decrease in glutamate synthetase (GS).
This in vitro system demonstrates the damaging effect chronic ROS exposure can have on this important mechanism regulating RGC homeostasis. We propose this new model for further study of the factors regulating chronic oxidative stress responses in adult NFL astrocytes.
This PDF is available to Subscribers Only