Abstract
Abstract: :
Purpose: Reactive oxygen species (ROS) are candidates for intracellular signaling molecules transducing the effects of axotomy in retinal ganglion cells (RGCs). However, only a subset of ROS scavengers can delay RGC death in axotomized RGCs. To explore how ROS scavengers interact with specific ROS, we combined ROS generating systems and scavengers and assessed RGC survival. Methods: Postnatal 2–4 Long Evans rat RGCs were retrogradely labeled with DAPI. At postnatal days 11–13, retinas were dissociated and cultured in Neurobasal/B27 with ROS–generating systems and scavengers. ROS generating systems tested were: hydroxyl radical (2 µM CuSO4, 2 µM phenanthroline, and 100 µM ascorbic acid; CPA), O2– (30 µM paraquat), nitric oxide (250 µM SNAP), and H2O2 (30 µM). Scavengers tested were catalase (0.5–500 U/ml), PEG–SOD (3–300 U/ml), MnTMPyP (5–100 µM), Trolox (1–100 µM), deferoxamine (2–200 µM), and U–74389G (3–100 µM). RGC viability was determined with calcein–AM 24 hours after plating. Effect of scavengers on RGC survival was assessed by calculating the fraction rescued compared to survival with ROS alone. Each experiment was performed at least twice. Results: Cultures exposed to H2O2 were partially rescued with MnTMPyP (5–10 µM), deferoxamine (100 µM), Trolox (100 µM), catalase (5–500U/ml), or PEG–SOD (30–100 U/ml), and fully rescued with MnTMPyP (50–100 µM), deferoxamine (200 µM), or PEG–SOD (300 U/ml). Cultures exposed to paraquat were partially rescued with deferoxamine (10–200 µM) or PEG–SOD (30–100 U/ml), and fully rescued with Trolox (10–100 µM), catalase (0.5–500 U/ml), or PEG–SOD (300 U/ml). Cultures exposed to SNAP were partially rescued with MnTMPyP (50 µM), Trolox (100 µM), deferoxamine (2–200 µM), or catalase (0.5 U/ml), and fully rescued with MnTMPyP (100 µM) or catalase (5–500 U/ml). Cultures exposed to CPA were partially rescued with MnTMPyP (50 µM), Trolox (100 µM), deferoxamine (100–200 µM), or catalase (50–500 U/ml), and fully rescued with Trolox (100 µM). Conclusions: The effects of ROS scavengers on axotomized RGCs are not comparable to what would be expected from their chemical properties. Extrapolation of cell–free models to culture and in vivo studies should be done cautiously.
Keywords: apoptosis/cell death