Increased stiffness and flow resistance of the inner wall of Schlemm's canal in glaucomatous human eyes

Abstract

The cause of the elevated outflow resistance and consequent ocular hypertension characteristic of glaucoma is unknown. To investigate possible causes for this flow resistance, we used atomic force microscopy (AFM) with 10-µm spherical tips to probe the stiffness of the inner wall of Schlemm's canal as a function of distance from the tissue surface in normal and glaucomatous postmortem human eyes, and 1-µm spherical AFM tips to probe the region immediately below the tissue surface. To localize flow resistance, perfusion and imaging methods were used to characterize the pressure drop in the immediate vicinity of the inner wall using giant vacuoles that form in Schlemm's canal cells as micropressure sensors. Tissue stiffness increased with increasing AFM indentation depth. Tissues from glaucomatous eyes were stiffer compared with normal eyes, with greatly increased stiffness residing within ∼1 µm of the inner-wall surface. Giant vacuole size and density were similar in normal and glaucomatous eyes despite lower flow rate through the latter due to their higher flow resistance. This implied that the elevated flow resistance found in the glaucomatous eyes was localized to the same region as the increased tissue stiffness. Our findings implicate pathological changes to biophysical characteristics of Schlemm's canal endothelia and/or their immediate underlying extracellular matrix as cause for ocular hypertension in glaucoma.

Keywords: biophysics; extracellular matrix; primary open-angle glaucoma.

Fig. 1.

Aqueous humor outflow pathway is shown schematically (Left) (9). Light micrograph of this tissue (Middle). Transmission electron micrograph of the inner-wall region of Schlemm’s canal (SC) (Right). Aqueous humor flows (red arrows) from the anterior chamber (AC) to the trabecular meshwork (TM), into Schlemm’s canal and then enters collector channels (CC) spaced periodically around the eye. From here, the aqueous humor flows into the episcleral veins. CM, ciliary muscle; SS, scleral spur. The 2 colored lines (Right) are 1 µm (magenta) and 3 µm (orange) from Schlemm’s canal and represent approximate boundaries of the regions probed by the 1- and 10-µm AFM probes, respectively (Discussion). (Scale bars, 100 μm [Middle] and 2 µm [Right].)

Fig. 2.

Images of extracted tissue. (A) Scanning electron micrograph of extracted tissue showing the inner wall of Schlemm’s canal (arrows). (B) Higher magnification showing the inner-wall tissue. Bulges are nuclei of the Schlemm’s canal endothelial cells, and the arrow shows a tear in tissue likely made during tissue extraction. (C) Confocal image of the inner-wall endothelium demonstrates a similar cell pattern as the electron micrograph; nuclei (blue) and F-actin (red) show stretched cell layout and their peripheral cortices (arrows). (D) AFM probe indenting the tissue that is illuminated from below.

Fig. 3.

Modulus (E) as a function of depth of AFM indentation into the tissue using a 10-µm AFM tip showing 5 different patterns (A–E). Each plot represents a single set of AFM measurements at 1 location. For a homogeneous material, the modulus would be independent of indentation depth. The common pattern of increasing values of E with indentation seen in B–E reflects the tissue being stiffer as the AFM tip probes deeper into the tissue. E₁ (interpreted as SC stiffness) and E₂ (interpreted as substrate stiffness) are average moduli of the plateaus seen in the results, determined as described in the text. E_last is the modulus measured at the deepest indentation.

Fig. 4.

Results from AFM measurements. (A) Box and whisker plots of elastic moduli of normal and glaucomatous inner-wall tissue as measured with a 10-µm AFM tip. (B) Outflow resistance (inverse outflow facility) of human eyes as a function of E₁ (P < 0.02) as measured with a 10-µm AFM tip. (C) Optic nerve counts of human eyes as a function of E₁ (P < 0.03) as measured with a 10-µm AFM tip. (D and E) Typical data for modulus as a function of indentation for inner-wall tissue from 1 normal and 1 glaucomatous eye as measured with a 1- or 10-µm AFM tip. (D and E, Insets) Regions where indentations are <300 nm.

Fig. 5.

Results from measurements of vacuolar parameters. (A and B) Representative images of the trabecular meshwork and inner wall of Schlemm’s canal (SC) of a normal (77-y-old) and glaucomatous (63-y-old) eye. Images are composites of multiple images along the inner wall of SC. Vacuoles (arrowheads) are seen in both normal and diseased tissue. The canal in the normal eye is entirely open but partially collapsed in the glaucomatous eye. (C–E) Vacuole measurements in the SC comparing normal (filled) with glaucomatous (open) human eyes. (C) Density of giant vacuoles along the length of the SC (total SC) or along the open aspects (open SC). (D) Giant vacuole size. (E) Giant vacuole size distribution. None of the differences are statistically significant. Normal, filled; glaucomatous, empty; mean ± SE.