Abstract
Glaucoma is the second leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG), the most common form of glaucoma, is often associated with elevation of intraocular pressure (IOP) due to the dysfunction of trabecular meshwork (TM) tissues. Currently, an ex vivo human anterior segment perfusion cultured system is widely used to study the effects of glaucoma factors and disease modifying drugs on physiological parameters like aqueous humor (AH) dynamics and IOP homeostasis. This system requires the use of freshly enucleated intact human eyes, which are sparsely available at very high cost. In this study, we explored the feasibility of using human donor corneoscleral segments for modeling morphological and biochemical changes associated with POAG. Among the number of corneas donated each year, many are deemed ineligible for transplantation due to stringent acceptance criteria. These ineligible corneoscleral segments were obtained from the Lions Eye Bank, Tampa, Florida. Each human donor anterior corneoscleral segment was dissected into four equal quadrants and cultured for 7 days by treating with the glaucoma factors dexamethasone (Dex) or recombinant transforming growth factor (TGF) β2 or transduced with lentiviral expression vectors containing wild type (WT) and mutant myocilin. Hematoxylin and Eosin (H&E) staining analysis revealed that the TM structural integrity is maintained after 7 days in culture. Increased TUNEL positive TM cells were observed in corneoscleral quadrants treated with glaucoma factors compared to their respective controls. However, these TUNEL positive cells were mainly confined to the scleral region adjacent to the TM. Treatment of corneoscleral quadrants with Dex or TGFβ2 resulted in glaucomatous changes at the TM, which included increased extracellular matrix (ECM) proteins and induction of endoplasmic reticulum (ER) stress. Western blot analysis of the conditioned medium showed an increase in ECM (fibronectin and collagen IV) levels in Dex- or TGFβ2-treated samples compared to control. Lentiviral transduction of quadrants resulted in expression of WT and mutant myocilin in TM tissues. Western blot analysis of conditioned medium revealed decreased secretion of mutant myocilin compared to WT myocilin. Moreover, increased ECM deposition and ER stress induction was observed in the TM of mutant myocilin transduced quadrants. Our findings suggest that the ex-vivo cultured human corneoscleral 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.
Highlights
Glaucoma is the second leading cause of irreversible blindness affecting nearly 70 million people worldwide [1]
Fixed tissues were utilized for H & E and immunohistochemical analysis while trabecular meshwork (TM) tissues and conditioned medium were analyzed by Western blot (Fig 1)
We cultured the corneoscleral segments with two different serum conditions; i) Standard serum media (10% FBS, suitable for Dex and myocilin treatments) and ii) Low serum media (0.5% FBS, suitable for TGFβ2 treatment)
Summary
Glaucoma is the second leading cause of irreversible blindness affecting nearly 70 million people worldwide [1]. The normal role for wild type (WT) myocilin is unknown, mutations in MYOC cause a deleterious gain-of-function leading to inhibition of its secretion [10], intracellular accumulation and protein stress within TM cells [11,12,13]. Another major contributor to glaucomatous TM damage is the increased expression of the pro-fibrotic cytokine, transforming growth factor (TGF) β2 in the aqueous humor and TM of POAG patients [14,15,16,17]. Both Dex and TGFβ2-induced OHT are associated with changes to the TM cytoskeleton [33, 36] and ECM deposition [28], which stiffen the TM [35, 37]
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