Elevated stiffness of cultured glaucomatous Schlemm’s canal (SC) cells correlates with impaired pore formation

Abstract

 

Purpose

 

SC cells form pores in response to a transcellular pressure drop, likely facilitating aqueous outflow into SC. A characteristic feature of glaucoma is reduced SC cell pore density. We hypothesize that impaired pore formation in glaucoma is due to elevated SC cell stiffness.

 

Methods

 

SC cells were isolated from 7 normal and 6 glaucomatous human donor eyes as previously described (Stamer, 1998); 6 strains were used for cell monolayer perfusion to measure pores, and 11 were used for atomic force microscopy (AFM) to measure stiffness. For monolayer perfusion, SC cells were seeded at confluence onto track-etch filter membranes and cultured for 2 days. Monolayers were perfused in the basal-to-apical direction at 6 mmHg for 30 minutes, followed by fixation at the same pressure. Pores were imaged at 12 randomly selected regions (~5500 µm² each) per cell layer using scanning electron microscopy, and quantified for total pore area and density. AFM measurements were performed on subconfluent SC cells with pyramidal tips or spherical (4.5 or 10 μm) tips, and Young’s modulus (E) was determined using a modified Hertz model. Finite element modeling (FEM) was used to assess the effect of the cell cortex on AFM measurements.

 

Results

 

Glaucomatous SC cells were stiffer and formed fewer pores. In comparison to normal SC cell strains, glaucomatous SC cell strains had decreased total pore area (86.9 ± 35.8 vs. 599 ± 128 µm²/mm²; mean ± standard error; p=0.071) and decreased pore density (68.8 ± 14.8 vs 224 ± 53.9 pores/mm²; p<0.03). Glaucomatous cells were stiffer (larger E) than normal cells when measured using spherical AFM tips (1.33 ± 0.13 vs 0.90 ± 0.11 kPa; p<0.02), but not pyramidal tips (7.69 ± 1.46 vs. 7.99 ± 0.96 kPa). FEM revealed that stiffness measured by pyramidal tips was more strongly influenced by the cortex, while stiffness measured by spherical tips better reflected the internal cytoskeleton. Comparing cell strains examined by both AFM and perfusions, pore density decreased as SC cell stiffness increased, as measured with spherical tips (see Figure).

 

Conclusions

 

Glaucomatous SC cells have impaired pore-forming ability in vitro that correlates with increased cell stiffness. This elevated stiffness appears to reside in the subcortical cytoskeleton as opposed to the cell cortex. Targeting SC cell stiffness may provide a therapeutic approach to lower IOP for glaucoma therapy.

 

 

Keywords: 633 outflow: trabecular meshwork • 493 cytoskeleton