Alterations in stiffness of the trabecular meshwork (TM) may play an important role in primary open-angle glaucoma (POAG), the second leading cause of blindness. Specifically, certain data suggest an association between elevated intraocular pressure (IOP) and increased TM stiffness; however, the underlying link between TM stiffness and IOP remains unclear and requires further study. We here first review the literature on TM stiffness measurements, encompassing various species and based on a number of measurement techniques, including direct approaches such as atomic force microscopy (AFM) and uniaxial tension tests, and indirect methods based on a beam deflection model. We also briefly review the effects of several factors that affect TM stiffness, including lysophospholipids, rho-kinase inhibitors, cytoskeletal disrupting agents, dexamethasone (DEX), transforming growth factor-β (TGF-β), nitric oxide (NO) and cellular senescence. We then describe a method we have developed for determining TM stiffness measurement in mice using a cryosection/AFM-based approach, and present preliminary data on TM stiffness in C57BL/6J and CBA/J mouse strains. Finally, we investigate the relationship between TM stiffness and outflow facility between these two strains. The method we have developed shows promise for further direct measurements of mouse TM stiffness, which may be of value in understanding mechanistic relations between outflow facility and TM biomechanical properties.
Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.
Elevated intraocular pressure (IOP) is the main risk factor for glaucoma. Exogenous nitric oxide (NO) decreases IOP by increasing outflow facility, but whether endogenous NO production contributes to the physiological regulation of outflow facility is unclear. Outflow facility was measured by pressure-controlled perfusion in ex vivo eyes from C57BL/6 wild-type (WT) or transgenic mice expressing human endothelial NO synthase (eNOS) fused to green fluorescent protein (GFP) superimposed on the endogenously expressed murine eNOS (eNOS-GFPtg). In WT mice, exogenous NO delivered by 100 μM S-nitroso-N-acetylpenicillamine (SNAP) increased outflow facility by 62 ± 28% (SD) relative to control eyes perfused with the inactive SNAP analog N-acetyl-d-penicillamine (NAP; n = 5, P = 0.016). In contrast, in eyes from eNOS-GFPtg mice, SNAP had no effect on outflow facility relative to NAP (-9 ± 4%, P = 0.40). In WT mice, the nonselective NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 10 μM) decreased outflow facility by 36 ± 13% (n = 5 each, P = 0.012), but 100 μM l-NAME had no detectable effect on outflow facility (-16 ± 5%, P = 0.22). An eNOS-selective inhibitor (cavtratin, 50 μM) decreased outflow facility by 19 ± 12% in WT (P = 0.011) and 39 ± 25% in eNOS-GFPtg (P = 0.014) mice. In the conventional outflow pathway of eNOS-GFPtg mice, eNOS-GFP expression was localized to endothelial cells lining Schlemm's canal and the downstream vessels, with no apparent expression in the trabecular meshwork. These results suggest that endogenous NO production by eNOS within endothelial cells of Schlemm's canal or downstream vessels contributes to the physiological regulation of aqueous humor outflow facility in mice, representing a viable strategy to more successfully lower IOP in glaucoma.
Aqueous humor drains from the eye through Schlemm's canal, a small endothelial-lined collecting duct. Schlemm's canal endothelial cells may be important in controlling the pressure within the eye (and hence are of interest in glaucoma), and are subject to an unusual combination of shear stress and a basal-to-apical pressure gradient. We sought to characterize this biomechanical environment and determine its effects on F-actin architecture in situ. A theoretical model of flow in Schlemm's canal was used to estimate shear stresses applied to endothelial cells by flowing aqueous humor. Alignment of Schlemm's canal endothelial cells in human eyes was quantified by scanning electron microscopy. F-actin architecture was visualized by fluorescent labeling and compared for closely adjacent cells exposed to different biomechanical environments. We found that, despite the relatively low flow rate of aqueous humor, shear stresses experienced by Schlemm's canal endothelial cells could reach those in the arterial system. Schlemm's canal endothelial cells showed a statistically significant preferential alignment, consistent with a shear-mediated effect. Schlemm's canal endothelial cells subjected to a basal-to-apical pressure gradient due to transendothelial flow showed a prominent marginal band of F-actin with relatively few cytoplasmic filaments. Adjacent cells not subject to this gradient showed little marginal F-actin, with a denser cytoplasmic random network. We conclude that Schlemm's canal endothelial cells experience physiologically significant levels of shear stress, promoting cell alignment. We speculate that this may help control the calibre of Schlemm's canal. F-actin distribution depends critically on the presence or absence of transendothelial flow and its associated pressure gradient. In the case of this pressure gradient, mechanical reinforcement around the cell periphery by F-actin seems to be critical.
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