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James C. Tan, Jose M. Gonzalez, Jr., Sarah F. Hamm-Alvarez, Martin Heur; In Situ 3D Modeling of the Human Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3245.
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© ARVO (1962-2015); The Authors (2016-present)
To describe a model for studying biological interactions within the 3-dimensional (3D) human trabecular meshwork (TM) by 2-photon excitation fluorescence (TPEF) optical sectioning and deep tissue imaging.
Viable ex vivo post-transplant human corneoscleral tissue containing the TM was imaged by TPEF (Leica SP5), aided by multiple approaches: autofluorescence (AF), second harmonic generation (SHG), intravital dye live cell labeling (Celltracker Red, R18, Hoechst, Propidium Iodide (PI), Calcein-AM), direct fluorescent dyes (eosin for elastin; phalloidin-rhodamine for filamentous actin (F-actin), baculovirus actin-RFP transduction (Cellular Lights, Invitrogen), indirect immunofluorescence (anti-type IV collagen, laminin, heparan sulfate, myocilin, alpha-smooth muscle actin (ASMA) primary antibodies), and 3D reconstruction (Imaris). Agents that induce TM protein expression (dexamethasone (Dex; 250nM), TGF-beta1 (50nM)) and affect actin (lysophosphatidic acid (LPA; 20uM), latrunculin-A (LAT-A; 1uM)) served as probes.
AF images revealed features of the uveal, corneoscleral and juxtacanalicular TM in great detail. AF corresponded with elastin (eosin) and collagen (SHG), key structural extracellular matrix (ECM) proteins of TM beam cores. Basement membrane-associated proteins such as type IV collagen, laminin and heparan sulfate surrounded the autofluorescent cores. Hoechst nuclear and celltracker cytosolic staining revealed live cellularity with respect to the 3D TM structure. 3D reconstructions allowed us to quantify and confirm cell viability by PI/calcein-AM double labeling. Tissue incubation with Dex induced myocilin that localized in basement membranes. TGF-beta1 induced ASMA that localized intracellularly. F-actin had a cortical distribution in cells, laced the 3D ECM, and was accentuated by LPA and diminished by LAT-A. Actin-RFP transduction provided an alternative method to visualize live-cell actin.
We have developed and characterized a unique in situ model in which cells, ECM and protein expression can simultaneously be analyzed within the 3D architecture of the TM. Live cells and cellular interactions such as actin cytoskeleton dynamics can be observed without conventional embedding and sectioning. The model provides an accessible and novel platform for TM biological and drug discovery.
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