Laminar Reorganisation in Retinal Reaggregates From the Neonatal Gerbil Starts at the Level of the IPL

Abstract

Abstract: : Purpose:The capacity of tissue regeneration decreases with increasing complexity of organisms. Retinal reaggregates from birds can reorganise into laminated spheres including all retinal layers. To analyse the reorganisation ability of mammalian retinal cells, two reaggregation systems of the gerbil retina (Meriones unguiculatus) were established to analyse cellular processes of rodent retinal histogenesis. Methods:3D–spheres derived from dissociated retinal cells of neonatal (P1) gerbils were grown in control reaggregation medium or in medium conditioned by RPE–monolayers. Reaggregates were collected after 10–15 days in rotation culture, then the spheres were fixed and cryosectioned. Differentiation and reorganisation were documented immunohistochemically with antibodies against specific cell–markers. Proliferation was detected by BrdU uptake, and TUNEL assay showed apoptosis during the culture period. Results:Distinctive histotypic reorganisation occurred in 3D–spheres of mammalian retinal cells, whereby most major retinal cell–types could be identified. Formation of an inner plexiform layer (IPL)–like matrix, and sorting out of amacrine and displaced amacrine cells in spheres grown in unconditioned medium was shown by the early differentiation marker calretinin. Moreover, signs of neuropil sublamination emerged. Advanced lamination occurred when spheres were grown in RPE–conditioned medium. Thereby, the period of proliferation was prolonged. Ganglion cells formed an outer ring, followed by a distinct IPL. Amacrine cells, Müller glia and horizontal cells were located inside in a correct but inverted manner. Noticeably, photoreceptors were absent in both types of spheres. Conclusions: Dissociated mammalian retinal cells are able to form histotypic laminated reaggregates. In contrast to spheres derived from birds or rats, this in vitro study shows that retinal histogenesis is possible in absence of photoreceptors, whereby self–organisation first takes place at the level of the IPL. Thereby, diffusible factors derived from the RPE influence proliferation, differentiation and lamination.

Keywords: retinal culture • regeneration • retinal development