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Ivan Fernandez-Bueno, Maria Teresa Garcia-Gutierrez, GirishKumar Srivastava, Manuel J Gayoso, Jose-Carlos Pastor; TNFα spontaneously secreted in an organotypic culture of porcine neuroretina is blocked by adalimumab. Invest. Ophthalmol. Vis. Sci. 2013;54(15):721.
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© ARVO (1962-2015); The Authors (2016-present)
Previous experiments (Fernandez-Bueno et al. ARVO 2011 Abstract 1853) revealed that reactive gliosis modifications enhanced by exogenous TNFα in an organotypic culture of porcine neuroretina can be reduce by adalimumab addition. The purpose of the present study is to determine wether TNFα is spontaneously secreted by retinal cells, and if concomitant adalimumab diminishes spontaneous glial cells modifications.
Porcine retinal explants from 10 eyeballs were cultured in Transwell® dishes (Corning Inc.). Cultures with 10 µg/ml adalimumab (Humira®, Abbott Laboratories Ltd.) added at day 0 and controls without the TNF-blocker were maintained 9 days (n=20 on each). Freshly detached retinas were processed in parallel. TNFα concentration was determined in control culture supernatants collected at medium exchange, on days 1, 3, 5, 7, and 9 of culture (n=5 each), by quantitative enzyme-linked immunosorbent assays specific for porcine TNFα (Quantiquine®; R&D Systems). Cryostat sections were doubly immunostained for glial fibrillary acidic protein (GFAP, 1:500 rabbit polyclonal; DakoCytomation Inc.), a marker for reactive gliosis, and retinaldehyde-binding protein (CRALBP, 1:1000 mouse monoclonal [B2]; Abcam plc.), a marker for Müller cells. Sections were also labeled with the isolectin IB4 (1:50, from Griffonia simplicifolia; Molecular Probes®), a label for microglia/macrophages.
The supernatants of cultured neuroretinas contained low, but detectable, levels of TNFα at day 1. Although at 3, 5, 7 and 9 days TNFα concentrations were below the detectable level. GFAP upregulation and CRALBP reduction were noted during culture, compared to fresh neuroretinal samples. Adalimumab inhibited spontaneous increases in GFAP and maintained CRALBP immunoreactivity expression. No appreciable changes in IB4 labelling were observed.
In cultured porcine neuroretina TNFα was secreted by retinal cells at day 1. This factor could be implicated in the further spontaneous reactive gliosis and retinal disorganization observed. These gliotic modifications were reduced by adalimumab addition. This finding, which involves the inhibition of TNFα spontaneously produced by retinal cells, emphasises the molecular role of this cytokine in development of retinal reactive gliosis and the importance of its early blockade for prevention of retinal changes after retinal detachment.
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