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P.A. Maher, A.M. Hanneken; Development and Characterization of an in vitro Model of Retinal Ganglion Cell Ischemia for the Identification of Neuroprotective Molecules . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4830.
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© ARVO (1962-2015); The Authors (2016-present)
Experimental evidence suggests that retinal ganglion cell death in glaucoma may result from a form of ischemia. The goal of these studies was to develop an in vitro ischemia model in order to identify molecules which could be neuroprotective for retinal ganglion cells and then use it to test the effects of potential neuroprotective molecules.
Ischemia was induced in the rat retinal ganglion cell line, RGC–5, using a brief treatment with iodoacetic acid (IAA). Removal of the IAA mimicked reperfusion. Cell death was monitored using the MTT assay. Levels of ATP and markers of oxidative stress (reactive oxygen species (ROS), glutathione (GSH)) were measured in control, IAA–treated and IAA–treated + reperfused cells. The ability of potential neuroprotective compounds such as flavonoids to prevent cell death in response to IAA/reperfusion as well as the changes in ATP levels and markers of oxidative stress was determined.
Brief treatment with IAA results in RGC–5 cell death within 24 hr. Similar to ischemia in vivo, IAA treatment causes a rapid loss of ATP to ∼50% of control levels which continue to decrease during the reperfusion period. In contrast, loss of GSH and increases in ROS are only seen during the period of reperfusion. Specific flavonoids when used at low micromolar concentrations were able to prevent the cell death caused by IAA treatment. Protection was seen both when the flavonoids were present during both the IAA treatment and the reperfusion or during the reperfusion period alone. Surprisingly, not only did many of these same flavonoids prevent the decrease in GSH levels but they also prevented the loss of ATP. In contrast, classical antioxidants such as vitamins C and E had little or no effect on the cell death seen in this in vitro ischemia model.
Using a combination of a retinal ganglion cell line and chemical ischemia, we have developed an in vitro ischemia model which has many of the features of ischemia in vivo. Preliminary results with this model indicate that specific flavonoids may be useful in preventing ischemia–induced retinal ganglion cell death in vivo.
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