Abstract
Purpose :
Pores in Schlemm’s canal (SC) endothelial cells are thought to be an important modulator of IOP and are triggered by mechanical stretch. We aimed to develop an in vitro assay to rapidly assess intracellular pore (I-pore) formation in SC cells exposed to local cellular stretch.
Methods :
Glass substrates were coated with biotinylated gelatin cross-linked with transglutaminase. Carboxyl ferromagnetic particles (dia. ∼5 µm, Spherotech) were added on the substrate and then primary glaucomatous human SC cells were seeded (gSC57, passage 5). Pores were detected using fluorescently labeled streptavidin tracers (Braakman+, IOVS, 2014). After 5-7 h of incubation, a first tracer was added to the media for 5 min. The first tracer was then washed away and a second tracer was added for 20 min, during which time particles were exposed to a magnetic force, thereby creating local cellular stretch in the basal to apical direction. Cells were fixed, imaged, and analyzed by counting punctate tracer signals that colocalized with particles under cells.
Results :
The tracers reached and bound to the substrate at locations not covered by the cells, including at I-pore sites (Figure). The I-pores were categorized as Type I (first tracer, i.e. present without magnetic force/stretching) and Type II (second tracer, i.e. present with local cell stretching). Type I and II pores were associated with 9.1 ± 3.2% (mean ± std dev) and 19.0 ± 5.1% of particles under cells, respectively (n = 6 wells, 200 analyzed cells/well, 73 ± 24 particles/well). There were more Type II pores than Type I (2-tailed t-test, p = 0.006).
Conclusions :
Our approach allows assessment of I-pores in SC cells. Some pores form spontaneously (without magnets, Type I) through mechanisms that are unclear; however, the greater number of Type II pores supports the association between pore formation and local mechanical stretch. Morphological studies of I-pores formed in this assay are ongoing.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.