Abstract
Purpose :
Micron-sized pores provide a pathway for aqueous humour outflow across the inner wall of Schlemm’s canal (SC). Cultured SC cells form pores when exposed to 10% or 20% stretch (Braakman, Exp Eye Res, 2014), (in vivo range: 3-36% (Overby, Mechanobiology Handbook, 2011), and pore formation is impaired in glaucomatous SC cells (Overby, PNAS, 2014). However, the dependence of pore formation on other stretch levels is unexamined. We hypothesise that glaucomatous SC cells form fewer transcellular “I” pores than non-glaucomatous regardless of stretch level.
Methods :
Human SC cells were isolated and characterized from one non-glaucomatous (SC75) and one glaucomatous donor (SC64G) (Perkumas and Stamer, Exp Eye Res, 2012). SC cells were seeded at low confluency onto elastomeric membranes coated with biotinylated gelatin. Cells were incubated with green avidin for 2 mins to label pores present at 0% stretch. Equibiaxial stretch was then applied at 5%, 12%, 18%, 22% or 34%. After 2 mins, samples were incubated with red avidin for 1.5 mins to label pores formed during stretch. Between 338 and 923 cells were examined per stretch level by a masked observer, and the number of I-pores per cell was compared using the E-test for Poisson statistics.
Results :
At 0% and 5% strain, pore counts were similar between SC75 and SC64G (Figure). However, for strain levels ≥12%, pore counts were significantly reduced in the glaucomatous relative to non-glaucomatous cells (p<0.001). At ~20% strain, pore counts increased sharply in both groups, but the peak pore count was 4-fold larger in SC75 relative to SC64G (p<0.001). Cell size was similar between groups (p=0.4).
Conclusions :
Stretch-induced pore formation is impaired in glaucomatous SC cells across multiple stretch levels. As controlled stretch eliminates the effect of cell stiffness, impaired pore formation in glaucoma cannot be attributed to elevated SC cell stiffness alone (Overby, PNAS, 2014). Above a critical strain of ~20%, SC cells exhibit a step-increase in pore formation. A better understanding of pore formation would provide key insights into the pathogenesis of outflow dysfunction in glaucoma.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.