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G. A. Pinzon-Duarte, G. Daly, D. D. Hunter, W. J. Brunken; Defective Formation of the Inner Limiting Membrane in the ß23 Laminin Null Retina Alters Number and Spatial Organization of Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5686.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we have shown that the deletion of both ß2 and γ3 chains of laminin leads to the disruption of the inner limiting membrane (ILM) leading to retinal dysplasia. Because retinal ganglion cell (RGC) development and survival are associated with the integrity of this membrane, we examine how this disruption affects their density and organization.
Retinas from wild type (WT) and double null mice were collected on postnatal day 15, and were examined by conventional histology, immunohistochemistry and electron microscopy. DAPI was used to label non-specifically cell nuclei; the following markers were used: Brn-3a and DiI for RGCs; glutamine synthetase and vimentin for Müller cells; perlecan for the basement membrane.
In whole mounted WT retinas, the ILM was continuous and the Müller cell endfoot ended in a regular array encasing the cells in the RGC layer. Moreover, Brn-3a and DiI labeled cells appeared even spaced throughout the RGC layer forming a regular array. In stark contrast, the ILM of the ß2γ3 mutant retina was discontinuous and Müller cell endfeet were in disarray, bearing no relation to the cells in the RGC layer and loosing contact with the disrupted ILM and failing to surround RGCs. DAPI and cell specific markers demonstrated that RGCs distribution was disrupted as well. Specifically, the cell density varied regionally- i.e., regions of normally distributed cells alternated with regions of clustered cells or regions nearly devoid of cells. DAPI and Brn-3a negative regions were spatially restricted to those regions in which the ILM was disrupted, i.e. perlecan negative regions. Light and electronic microscopic studies of radial sections confirmed the disruption of the ILM and the alterations in the spatial array of RGCs; studies are underway to determine if RGC fates (subtype phenotypes) are disrupted in these animals.
Our hypothesis is that the absence of ß2γ3 laminin causes disruption in the ILM, which results in an abnormal organization, distribution and spatial relationships among the cells of the RGC layer. These studies may have implications in the surgical stripping of the ILM used in some treatment of human disease.
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