Tissue-based multiphoton analysis of actomyosin and structural responses in human trabecular meshwork

The contractile trabecular meshwork (TM) modulates aqueous humor outflow resistance and intraocular pressure. The primary goal was to visualize and quantify human TM contractile state by analyzing actin polymerization (F-actin) by 2-photon excitation fluorescence imaging (TPEF) in situ. A secondary goal was to ascertain if structural extracellular matrix (ECM) configuration changed with contractility. Viable ex vivo human TM was incubated with latrunculin-A (Lat-A) or vehicle prior to Alexa-568-phalloidin labeling and TPEF. Quantitative image analysis was applied to 2-dimensional (2D) optical sections and 3D image reconstructions. After Lat-A exposure, (a) the F-actin network reorganized as aggregates; (b) F-actin-associated fluorescence intensity was reduced by 48.6% (mean; p = 0.007; n = 8); (c) F-actin 3D distribution was reduced by 68.9% (p = 0.040); (d) ECM pore cross-sectional area and volume were larger by 36% (p = 0.032) and 65% (p = 0.059) respectively and pores appeared more interconnected; (e) expression of type I collagen and elastin, key TM structural ECM proteins, were unaltered (p = 0.54); and (f) tissue viability was unchanged (p = 0.39) relative to vehicle controls. Thus Lat-A-induced reduction of actomyosin contractility was associated with TM porous expansion without evidence of reduced structural ECM protein expression or cellular viability. These important subcellular-level dynamics could be visualized and quantified within human tissue by TPEF.