Cell Adhesiveness and the Response of Chick Embryonic Retinal Neurons to Photoelectric Dye–Coupled Polyethylene Film as a Prototype of Retinal Prostheses

Abstract

Abstract: : Purpose: Photoelectric dyes absorb light and convert photon energy to electric potentials. We previously reported a simple in vitro system for screening photoelectric dyes towards their use for retinal prostheses (Acta Medica Okayama 2003;57:257–260). Furthermore, one of these dyes coupled with polyethylene film surface as a prototype of retinal prostheses could induce intracellular calcium elevation in chick embryonic retinal tissues (Artificial Organs 2005 in press). In this study, we assessed cell adhesiveness and the response of chick embryonic retinal neurons to our retinal prostheses. Methods: Carboxyl moieties were first introduced to polyethylene film surface by treatment with fuming nitric acid. A dye, 2–[2–[4–(dibutylamino)phenyl]ethenyl]–3–carboxymethylbenzothiazolium bromide (NK–5962, Hayashibara Biochemical Laboratories), was coupled with the carboxyl moiety–bearing polyethylene film by amide linkage. For surface modification, type 1 collagen was then reacted with the unused carboxyl moieties of the dye–coupled film surface. Retinal neurons were isolated from the eyes of chick embryos at the 10–day embryonic stage at which no retinal photoreceptor cells yet developed. Cell suspension was delivered to wells of a 24–well multidish with the dye–coupled films attached to the bottom. On day 7 of culture, the number of retinal neurons at five areas on the surface of the dye–coupled films were counted and summed. The response of retinal neurons to the dye–coupled film was observed by calcium imaging: retinal cells in culture loaded with Fluo–4 were attached to the dye–coupled film and placed on the stage of a fluorescent inverted microscope with a CCD camera to monitor intracellular calcium levels. Results: The number of retinal neurons on the dye–coupled polyethylene film coated with collagen was significantly larger than the number of neurons on the dye–coupled film with no surface modification. Intracellular calcium elevation was observed in retinal neurons attached to the dye–coupled film, in contrast to neurons which had no contact with the film. Conclusions: The photoelectric dye, NK–5962, coupled with polyethylene film surface, absorbed light under an inverted microscope and stimulated chick retinal neurons. Surface modification of the dye–coupled film with collagen improved the compatibility between retinal prostheses and retinal neurons.

Keywords: retinal culture • retina: neurochemistry • imaging/image analysis: non-clinical