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Abstract #14040 Published in IGR 8-3
Biochemical activity of reactive oxygen species scavengers do not predict retinal ganglion cell survival
Schlieve CR; Lieven CJ; Levin LAInvestigative Ophthalmology and Visual Science 2006; 47: 3878-3886
PURPOSE: Retinal ganglion cells (RGCs) die as a result of axonal injury in a variety of optic neuropathies, including glaucoma. Reactive oxygen species (ROS) act as intracellular signaling molecules and initiate apoptosis in nerve growth factor-deprived sympathetic neurons and axotomized RGCs. Determination of the role of specific ROS relies on the use of small molecule or protein scavengers with various degrees of specificity. The pro- or anti-cell-death effect of several ROS generating and scavenging systems in cultured RGCs was correlated with their activity in cell-free assays. METHODS: Neonatal rat retinas were dissociated and incubated with ROS-generating systems for hydroxyl radical, superoxide anion (O2- ), and H2 O2 . Scavengers tested were catalase, polyethylene glycol-superoxide dismutase (PEG-SOD), manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), deferoxamine, and U-74389G. Viability of retrogradely labeled RGCs was determined with calcein-AM 24 hours after plating. O2- and H2 O2 scavenging in cell-free assays was measured with dihydroethidium and Amplex Red (Invitrogen, Carlsbad, CA), respectively. RESULTS: Systematic differences were found between ROS scavenging in cell-free assays and the ability of scavengers to protect RGCs in cell culture. Furthermore, many ROS scavengers lost specificity and protected against various ROS, whereas others failed to protect against their unique ROS target. These activities stray from commonly recognized specificities of individual ROS scavengers or generating systems and are important in understanding ROS biology. In addition, antioxidant defense mechanisms used by RGCs and other retinal cells interfere with responses expected from ROS scavengers in well-defined systems. Last, H2 O2 induced intramitochondrial O2- , whereas paraquat produced O2- outside of the mitochondria, and these areas of generation can mislead interpretations of ROS scavenger activity and effectiveness. CONCLUSIONS: There is discordance between ROS effects in cultured RGCs and cell-free assays, with several mechanisms accounting for this divergence. To identify the roles of ROS signaling in cell death accurately, several approaches should be used. These include using a panel of ROS scavengers and generators, testing the panel in primary neuronal cultures, and quantifying ROS with cell-free assays.
Dr. C.R. Schlieve, Department of Ophthalmology and Visual Sciences, University of Wisconsin Medical School, Madison, WI, USA
Classification:
2.13 Retina and retinal nerve fibre layer (Part of: 2 Anatomical structures in glaucoma)
3.7 Biochemistry (Part of: 3 Laboratory methods)
3.8 Pharmacology (Part of: 3 Laboratory methods)
3.9 Pathophysiology (Part of: 3 Laboratory methods)