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Kathryn E Bollinger, Barbara Mysona, jing zhao, Cecelia Hernandez, Graydon Gonsalvez; Comparison of TGF-β stimulated optic nerve head astrocytes in 2D versus 3D cultures. Invest. Ophthalmol. Vis. Sci. 2018;59(9):309.
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© ARVO (1962-2015); The Authors (2016-present)
Our understanding of mechanisms that underlie optic nerve head astrocyte (ONHA) responses to glaucomatous injury is limited. ONHAs are the main glial cell supporting retinal ganglion cell (RGC) axons at the optic nerve head (ONH), a region vulnerable to glaucoma-induced damage. ONHAs undergo changes in morphology, secretory and migration patterns that likely play an important role in the initiation of axonal damage. TGF-β is a cytokine elevated in aqueous humor of many glaucoma patients. TGF-β acts on and is secreted by ONHAs but whether TGF-β mediated changes in ONHAs are beneficial or detrimental to RGC axons is unknown. Better in vitro models are needed to elucidate mechanisms of TGF-β stimulated ONHA responses. In particular, 3D ONHA culture may be a superior model for study of ONHA responses than traditional 2D culture.
Primary, rat ONHAs were isolated from 3-5 day Sprague-Dawley rats as previously published by our group. Cells were used between passage 2 and 4. In 2D experiments, rat ONHAs were grown to confluence, switched to serum free media overnight, injured by scratch wound, then treated with varying levels of FBS (0-1%) and TGF-β1 (0.1 ng/ml –10 ng/ml) for 24 hours. In 3D cultures rat ONHAs were seeded in a Matrigel Matrix (BD Biosciences) at a concentration of 2 x 106 cells per ml and treated with TGF-β1 (10 ng/ml). The ONHA response to TGF-β1 was evaluated by migration assay, immunocytochemistry for actin stress fibers and GFAP, and Western blot.
In 2D culture, treatment with 1% FBS and 10 ng/ml TGF-β1 was accompanied by significant increases in ONHA migration, actin stress fibers and GFAP. BDNF, an important neurotrophin for RGC health, was also significantly elevated in TGF-β stimulated ONHAs. 2D results were from three independent experiments utilizing ONHAs derived from separate isolations, P < 0.05. While ONHAs in 2D culture had a broad flat morphology, ONHAs in 3D culture exhibited an angular phenotype and thin processes similar to in vivo. 3D rat ONHAs formed clusters in response to TGF-β1 but whether this was indicative of increased migration, proliferation, or astrocyte-mediated extracellular matrix change remains to be determined.
In both 2D and 3D cultures, TGF-β1 stimulated changes in rat ONHAs. The angular 3D ONHA morphology suggested that 3D cultures are a promising alternative to conventional 2D approaches to study TGF-β mediated responses.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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