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A. Uji, T. Matsuo, S. Ishimaru, A. Kajiura, T. Yamashita, K. Shimamura, H. Ohtsuki, Y. Dan–oh, S. Suga; Photoelectric dye–coupled polyethylene film as a prototype of retinal prostheses tested in vitro by chick embryonic retinal tissue . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4180.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We previously reported a simple in vitro system for screening photoelectric dyes using embryonic chick retinal cells in culture and calcium imaging, and showed the feasibility of these dyes as components for retinal prostheses (Acta Medica Okayama 2003;57:257–260). In this study, a photoelectric dye screened by this method was coupled with polyethylene film surface and tested in vitro using retinal tissues from chick embryos which had not yet developed photoreceptor cells. Methods: Carboxyl moieties were introduced to polyethylene film surface with fuming nitric acid at 80 C for 20 minutes. A photoelectric dye, NK–5962 (see the structure at http://hayashibara.co.jp), was coupled with the polyethylene film through amide linkage by ethylenediamine between carboxyl moieties of the film and the dye under catalytic presence of dicyclohexylcarbodiimide in a reaction solvent, chlorobenzene, at 35 C for 24 hours. The reaction was monitored by infrared absorption spectrum. The dye–coupled film was finally washed with chlorobenzene for 48 hours. The retinal tissues were isolated from 12–day chick embryos, placed in wells of a 24–well multidish containing 300 microliter HEPES buffer, and incubated with Fluo–4 acetoxymethylester at 37 C for 60 minutes. After washed with the buffer, the multidish with 300 microliter HEPES buffer in each well was placed on the stage of a fluorescent dissecting microscope attached to a CCD camera in order to monitor intracellular calcium levels of retinal tissues. The images were captured by a software (ViewFinder, Nikon, Tokyo, Japan). Results: A dye, NK–5962, was successfully coupled with polyethylene film surface by this method. Intracellular calcium elevation was observed in retinal tissues placed on the dye–coupled polyethylene film, in contrast to retinal tissues which had no contact with the film. When retinal tissues on the film were removed from the film and placed at the distance of the film, intracellular calcium elevation in the retinal tissues returned to a low level. Conclusions: The photoelectric dye, NK–5962, coupled with polyethylene film surface, absorbed light under a dissecting microscope and stimulated neurons in chick embryonic retinal tissues which had not yet developed photoreceptor cells. This in vitro study showed that the photoelectric dye, NK–5962–coupled polyethylene film could be used as a prototype of retinal prostheses.
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