title: Adalimumab (tumor necrosis factor-blocker) reduces the expression of glial fibrillary acidic protein immunoreactivity increased by exogenous tumor necrosis factor alpha in an organotypic culture of porcine neuroretina.
abstract: OBJECTIVE
  To determine if exogenous addition of tumor necrosis factor alpha (TNFα) exacerbates retinal reactive gliosis in an organotypic culture of porcine neuroretina and to evaluate if concomitant adalimumab, a TNF-blocker, diminishes it.
  
  
  METHODS
  Porcine retinal explants from 20 eyeballs were cultured. Cultures with 100 pg/ml TNFα, 10 µg/ml adalimumab, 100 pg/ml TNFα plus 10 µg/ml adalimumab, or controls without additives were maintained for 9 days. Freshly detached retinas were processed in parallel. TNFα levels in control culture supernatants were quantified with enzyme-linked immunosorbent assay. Cryostat sections were doubly immunostained for glial fibrillary acidic protein (GFAP), a marker for reactive gliosis, and cellular retinaldehyde-binding protein (CRALBP), a marker for Müller cells. Sections were also labeled with the isolectin IB4, a label for microglia/macrophages.
  
  
  RESULTS
  TNFα in control culture supernatants was detected only at day 1. Compared to the fresh neuroretinal samples, upregulation of GFAP and downregulation of CRALBP occurred during the 9 days of culture. Exogenous TNFα stimulated glial cells to upregulate GFAP and downregulate CRALBP immunoreactivity. TNFα-treated cultures also initiated the growth of gliotic membranes and underwent retinal disorganization. Adalimumab inhibited the spontaneous increases in GFAP and maintained CRALBP. In combination with TNFα, adalimumab reduced GFAP expression and conserved CRALBP, with only slight retinal disorganization. No appreciable changes in IB4 labeling were observed under the different culture conditions.
  
  
  CONCLUSIONS
  In cultured porcine neuroretina, spontaneous reactive gliosis and retinal disorganization were exacerbated by exogenous TNFα. Adalimumab reduced spontaneous changes and those induced by TNFα. Therefore, inhibiting TNFα may represent a novel approach to controlling retinal fibrosis observed in some human diseases.
authors.affiliation: Instituto Universitario de Oftalmobiología Aplicada (IOBA), University of Valladolid, Valladolid, Spain. ifernandezb@ioba.med.uva.es
authors.forename: I
authors.initials: I
authors.lastname: Fernandez-Bueno
authors.forename: M T
authors.initials: MT
authors.lastname: Garcia-Gutierrez
authors.forename: G K
authors.initials: GK
authors.lastname: Srivastava
authors.forename: M J
authors.initials: MJ
authors.lastname: Gayoso
authors.forename: J M
authors.initials: JM
authors.lastname: Gonzalo-Orden
authors.forename: J C
authors.initials: JC
authors.lastname: Pastor
mesh_terms: D000068879:Adalimumab; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism*; D006801:Humans; D037121:Plant Lectins / Q000378:metabolism; D012160:Retina / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013194:Staining and Labeling; D034421:Sus scrofa; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000494:pharmacology*
chemical_list: C020932:11-cis-retinal-binding protein; D061067:Antibodies, Monoclonal, Humanized; D002352:Carrier Proteins; D005904:Glial Fibrillary Acidic Protein; C045475:Griffonia simplicifolia lectins; D037121:Plant Lectins; D014409:Tumor Necrosis Factor-alpha; D000068879:Adalimumab