Abstract
Purpose :
Ex-vivo human anterior segment perfusion cultured system is widely used to study the effects of glaucoma factors on intraocular pressure (IOP) and trabecular meshwork (TM) pathology. Due to considerable high cost and failure rates with perfused human donor eyes, we explored whether rejected corneas containing intact TM ring can be cultured for several days and whether cultured TM tissue can be used to study the effects of glaucoma factors on TM pathology.
Methods :
Human rejected corneas (anterior segments) with intact TM ring were obtained from the Lions Eye Institute (Tampa, Florida) in accordance with Declaration of Helsinki guidelines. The anterior segments were dissected into 4 equal quadrants and each quadrant was cultured in a 24 well plate using DMEM medium supplemented with 10% FBS and 1% Pen-strep. The anterior segment quadrants were either treated with 100 nM dexamethasone (Dex), 5 ng/ml recombinant TGFβ2 (in 0.5% FBS containing media) or transduced with Ad5 expressing WT and mutant myocilin for 7 days with appropriate controls. Conditioned medium was collected and TM tissue was examined by Western blot analysis, immunostaining, H &E staining.
Results :
H&E staining and TUNEL assay demonstrates well preserved structure and integrity of TM tissues in 7-days cultured human anterior segments. Treatment of anterior segment with Dex or TGFβ2 resulted in glaucomatous TM changes including increased extracellular matrix (ECM) proteins and increased endoplasmic reticulum stress compared to control TM. Western blot analysis of conditioned medium showed an increase of myocilin, fibronectin and collagen IV levels in Dex or TGFβ2 treated samples. Ad5 treatment of anterior segment tissue resulted in expression of WT and mutant myocilin in TM tissues. Western blot analysis on conditioned medium revealed decreased secretion of mutant myocilin in Ad5-mutant-myocilin transduced segments compared to Ad5-wildtype-myocilin.
Conclusions :
Our findings suggest that ex-vivo culture human anterior segment model is cost-effective and can be used as a pre-screening tool to study the effects of glaucoma factors and anti-glaucoma therapeutics on the TM. However, the unfeasibility of this model system in IOP studies is a limitation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.