Experimental purpose and approaches: Cultured hfRPE cells obtained from three more donors were used for the assay. The cells were incubated with Tert-butyl-hydroperoxide (TBH) (Sigma) in serum free culture media containing MEM-alpha;modified medium (Sigma-Aldrich), glutamine-penicillin-streptomycin (Sigma-Aldrich) 1:100 mL/mL, and nonessential amino acid solution (Sigma-Aldrich) 1:100 mL/mL for various time points. Trans epithelial potential (TER) was measured prior to or immediately after the treatment of TBH. Live-dead assay was used to determine the level of cell death and fluorescent images were captured with a microscope equipped with high resolution AxioCam camera (Axioplan 2 with Axiovision 3.4 software AxioCam-MRm, Carl Zeiss Meditec, Inc., Dublin, CA). In case of cell death quantification, the whole filter from insert was imaged through MosaiX Axiovision module;the numbers of positive nuclei were counted via NIH Image J 13.6 equipped with ITCN (image based tool for counting nuclei). For chronic injury process, nonlethal concentrations of TBH will be applied to the culture for up to a week, the impact of TBH on TER will be assayed and mitochondrial potential will be monitored using the fluorescent dye-JC-1 (molecular probes, invitrogen). Changes of expression and distribution o f junction proteins will be examined by Western blot analysis and immunocytochemistry. Potential interaction of TBH and cytokines(other immunoregulators)in RPE injury will be studied as well. Results: TBH induced a dose dependent cell death and TER reduction in hfRPE. Lethal concentrations of TBH treatment produced a robust cell death when applied for more than 6 hours. TER reduction appears to be gradual events. The level of cell death and TER reduction appear to be associated. Conclusion: Although lethal concentrations of oxidative stimulant, such as TBH can elicit cell death in hfRPE, the acute and robust process doesn't possibly mimics the process as occurred in age-related -macular degeneration (AMD). Treating hfRPE with low levels of oxidant or/and inflammatory stimulations, two types of risk factors that are associated AMD, will employ an relevant injury process.
