title: Tissue-based multiphoton analysis of actomyosin and structural responses in human trabecular meshwork.
abstract: 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.
authors.affiliation: Doheny Eye Institute and Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Gonzalez
authors.affiliation: Doheny Eye Institute and Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA.
authors.forename: Minhee K
authors.initials: MK
authors.lastname: Ko
authors.affiliation: Doheny Eye Institute and Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Pouw
authors.affiliation: Doheny Eye Institute and Department of Ophthalmology, David Geffen School of Medicine at UCLA, Los Angeles, CA.
authors.forename: James C H
authors.initials: JC
authors.lastname: Tan
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000205:Actomyosin / Q000737:chemistry / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D021621:Imaging, Three-Dimensional; D036641:Microscopy, Fluorescence, Multiphoton; D055503:Protein Multimerization; D014129:Trabecular Meshwork / Q000378:metabolism*
chemical_list: D000199:Actins; D000205:Actomyosin