PURPOSE. Limited integration is consistently observed between subretinal transplants and host retinas. In the current study, an in vitro model system for studying connections forming between two abutting retinas was developed. METHODS. Neuroretinas were dissected from normal wild-type (WT) mice and green fluorescent protein (GFP) transgenic mice (obtained at postnatal days [P]0, P5, or P60), as well as from adult rd mice. Pieces from two different retinas (WT-WT, GFP-WT, GFP-rd) were placed side-by-side (contacting each other at the margins) or overlapping each other in organ cultures for 7 or 12 days. The abutting retinal pieces derived from animals of the same age (P5-P5; P60-P60) or of different ages (P0-P60; P5-P60). Retinal cells and fibers were visualized in wholemount preparations and in cross sections by immunocytochemistry using antibodies against neurofilament (NFϩ), neuronal nitric oxide synthase (NOSϩ), and protein kinase C (PKCϩ) and by GFP fluorescence (GFPϩ). RESULTS. In side-by-side pairs (WT-WT, GFP-WT), numerous horizontal cell fibers (NFϩ) and amacrine cell fibers (NOSϩ) crossed the interface between the two pieces, forming continuous plexiform layers. In overlapping pairs, NFϩ, NOSϩ, and PKCϩ fibers displayed parallel plexiform layers, and no crossover of fibers was observed in any of the pair combinations examined (WT-WT, GFP-WT, GFP-rd). Some integration was seen only in small areas where the structure of both retinal pieces was disrupted at the interface. CONCLUSIONS. The results demonstrate the ability of neurites to extend between abutting retinas and to make appropriate target choices when they are placed side-by-side. However, this ability is limited when they overlap each other, similar to that observed in subretinal transplantation. (Invest Ophthalmol Vis Sci. 2003;44:4936 -4946 ). Prompted by the problem of poor graft-host integration, we developed a modified culture system in which the outgrowth of fibers between two retinal pieces could be analyzed. The system consists of two abutting retinal pieces, placed overlapping each other, which is analogous to the in vivo situation of subretinal transplantation, or side by side. Using specific neuronal markers, we examined in wholemount preparations and in transverse sections, whether neuronal fibers can extend from one retinal piece into the abutting piece. Pairs were formed using retinal pieces derived from 5-day-old (P5) mice and cultured for 7 days, thus encompassing a time window (P5-P12) during which, in normal mouse development, substantial outgrowth of retinal cell processes occurs within the synaptic layers, and synaptic maturation is initiated. 11,12 MATERIALS AND METHODS Animals and Tissue Culture Preparation The experiments were conducted with the approval of the local animal experimentation and ethics committee. Animals were handled according to the guidelines on care and use of experimental animals set forth by the Government Committee on Animal Experimentation at the University of Lund and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The organ culture condition has been described in detail. 13 Retinas were dissected from normal mice (C57BL/6), from GFP mice (harboring a transgene consisting of enhanced GFP [EGFP] cDNA under the control of a chicken -actin promoter and a cytomegalovirus enhancer), After the superior and nasal cornea were marked, the eyes were enucleated under sterile conditions and transferred to a dish containing serum-free medium (R16; Invitrogen-Gibco, Gaithersburg, MD). 15 Retinas were dissected from the retinal pigment epithelium (RPE) and from hyaloid vessels. Each retina was cut under fresh medium into four pieces along the superior-inferior and the nasal-temporal axes (Fig
