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J.-C. Pastor, I. Fernandez-Bueno, M. Garcia, M. Gayoso, A. Corell, R. Reinoso, A. Enriquez de Salamanca, C. Garcia; Neuroretinal Reactive Gliosis is Not Appreciable If Macrophage-Like Cells Do Not Produce TNF Alpha and/or IL-1b. Preliminary Results. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5758.
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To evaluate neuroretina degeneration as stimulating factor for the production of TNFα and/or IL-1b by macrophage-like cells, and to study the possible boosting role of these cytokines in neuroretinal reactive gliosis, in an organotypic culture of porcine neuroretina.
Porcine neuroretina explants were cultured in Transwell® dishes (Corning Inc, USA). Porcine CD14+ monocytes were added over the explant or in the culture medium at day 0. Explants without any kind of external cells addition were used as controls. CD14+ monocytes were obtained from porcine blood after percoll gradient and posterior magnetic selection (CD3-, Sigma-Aldrich Co., UK; CD8a- & CD45RA-, AbD Serotec, UK; and CD14+, Miltenyi Biotec MACS, Germany). Culture supernates were changed each couple days and collected for quantitative evaluation of porcine TNFα and IL-1b (Quantikine®, R&D Systems, UK). According to datasheets, minimun detectable doses of these kits were >3,7 pg/mL and >10 pg/mL respectively. Neuroretina samples were collected at days 3, 6 and 9 of culture. Specimens were embedded in OCT and 5 µm sections were immunostained with rabbit anti-cow GFAP (glial fibillary acidic protein, DakoCytomation Inc., USA) and mouse anti-human CRALBP (cellular retinaldehide-binding protein, Abcam plc., UK). Cellular nuclei were stained with DAPI dye (4’,6-diamino-2-phenilindole dihydrochloride, Molecular Probes, USA).
Neuroretina degeneration happened along the culture. No significant increase levels in TNFα or IL-1b were present in supernates during coculture. No differences in reactive gliosis changes, evaluated by stimation of Müller cells GFAP+, increased cells wideness and modifications in astrocytes distribution through the neuroretinas, were observed during co-culture, compared to controls.
No increase reactive gliosis appeared in the neuroretina after coculture with CD14+ monocytes that did not secrete significant TNFα and/or IL-1b levels in this model. Neuroretina degeneration does not seem to induce CD14+ monocytes to secrete considerable TNFα and/or IL-1b levels in this model.
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