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I. Fernandez-Bueno, J. C. Pastor, M. J. Gayoso, I. Alcalde, M. T. Garcia; Müller Cells Modifications After Interact With Macrophage-Like Cells in an Organotypic Culture of Porcine Neuroretina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4886. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To analyze the in vitro response of Müller cells to the addition of a mononuclear blood fraction (MNF, monocytes and lymphocytes), as a source of macrophages, in an organotypic culture model of porcine neuroretina.
Neuroretina explants were cultured with and without (control group) porcine MNF and collected at days 3, 6 and 9. Specimens were processed for epoxy-resin embedding and cryosectioning. Light and immunofluorescence microscopy were performed, using toluidine blue staining and antibodies against glial fibrillary acidic protein (GFAP), as a reactive gliosis marker, and cellular retinaldehide-binding protein (CRALBP), as a specifically marker of Müller cells. Cellular nuclei were identified by 4’,6-diamino-2-phenilindole dihydrochloride (DAPI) fluorescent staining.
Explants co-cultured with MNF revealed an increased cellular disorganization, and large multinuclear tissue invasion of the subretinal space at culture day 9, compared to control group. Furthermore, immunohistochemical labelling revealed increased GFAP+ reactivity and significant changes in the Müller cells at all culture periods. More Müller cells were GFAP immunoreactive than in control neuroretinas, and their cytoplasm was wider. In addition, at day 9, co-cultured explants showed multiple intermediate filaments GFAP+ (IF), corresponding to Müller cell extensions, that invaded the subretinal space and were distributed in several fibrous layers. Numerous IF also occupied the nerve fibre layer and invaded the ganglion cell layer during the culture, corresponding to astrocytic extensions, as GFAP and CRALBP co-labelling revealed.
Addition of MNF seems to stimulate the modifications of the Müller cells, producing a wider intraretinal reactive gliosis and the tissue proliferation at the subretinal space (outer layers of the retina). These findings emphasize the role of macrophage-like cells in the production of retinal structural changes observed after retinal detachment.
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