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A. Kardaszewska, Y. Zhang, T. van Veen, R. Caffé, M. Perez; Glial Barrier to Neuronal Integration in Abutting Retinas Culture System . Invest. Ophthalmol. Vis. Sci. 2003;44(13):498.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Poor integration is observed between subretinal neural grafts and the host retina. We established an in vitro model system that simulates subretinal transplantation for studying connectivity between two abutting retinas. Here, we further examined the factors influencing neuronal integration in this system. Methods: Retinas obtained from 5-days-old transgenic mice expressing green fluorescent protein (GFP) and from adult rd mice (rd) were cut into four pieces each. In the first group, two pieces from different retinas (GFP/rd) were placed flat, overlapping each other with the ganglion cell layer (GCL) of the GFP-mouse-derived piece facing the outer plexiform layer (OPL) of the rd-derived piece, forming laminar pairs. In the second group, the GFP-derived piece was fragmented and placed overlaying the OPL of the rd-derived piece, forming fragment-laminar pairs. The retinal pairs were cultured for seven days. Retinal cells and fibers were visualized by immunocytochemistry using neuronal and glial cell markers and by GFP fluorescence. Results:The structure of both retinal pieces in laminar pairs and of the rd-derived piece in fragment-laminar pairs appeared normal in the central regions, but often disrupted in the edges. GFP+ cells and fibers migrated and extended into the rd-derived tissue in both, laminar pairs and fragment-laminar pairs, some of which co-expressed nNOS (neuronal nitric oxide synthase), PKC (protein kinase C), calbindin, calretinin, or CRALBP (cellular retinaldehyde-binding protein). However, such integration was confined to the edges in laminar pairs, whereas it could be observed in several areas in fragment-laminar pairs. In the central region of laminar pairs, CRALBP+ and glial fibrillary acidic protein (GFAP)+ glial cells/processes of the two pieces intermingled, forming a boundary at the interface. A similar boundary was not seen in the edges of laminar pairs, and was thinner and discontinuous in fragment-laminar pairs. Conclusions: The results demonstrate that neuronal integration is limited at the inner and outer margins of the abutting retinal pieces. The accumulation of CRALBP and of GFAP in the areas where no integration occurred correlates with the presence of a glial structural/molecular barrier.
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