Abstract
Purpose: :
The trabecular meshwork’s (TM) actin cytoskeleton plays an important role in intraocular pressure (IOP) regulation and is a treatment target for glaucoma. We have sought to: 1) characterize 3-dimensional (3D) features of filamentous actin (F-actin) within the human TM by 2-photon excitation fluorescence (TPEF) optical sectioning; and 2) analyze actin disruption in situ.
Methods: :
We have used a novel 3D tissue model derived from human corneoscleral donor explants (n=31) containing the intact TM and Schlemm’s Canal (SC) for in situ TPEF imaging (Leica SP5). Tissue viability was assayed by Calcein AM/propidium iodide (PI) double labeling followed by TPEF. Viable explants were incubated with latrunculin-A (LAT-A, 1μM), lysophosphatidic acid (LPA, 20μM), or vehicle control for 24 h. Tissue was then fixed, permeabilized, and incubated with Alexa 546-conjugated phalloidin (F-actin) and Hoechst (nuclei). TM beams were autofluorescent (AF) under TPEF; visible without labeling. Image analysis and 3D reconstruction was performed (Leica LAS and Imaris).
Results: :
The TM’s fine AF structure varied from uveal to corneoscleral to juxtacanalicular (JCT) regions. Beyond the JCT, SC was evident as a signal void in image slices. Within the TM, F-actin was seen adjacent to nuclei as a mixture of punctate, cytosolic distribution and more dense and continuous cortical distribution at cell borders, allowing cell-cell associations to be seen. In the uveal TM, F-actin was wrapped around the beams as a fine lacy network, coinciding with cellular attachments. F-actin intermittently bridged gaps between beams. In the corneoscleral TM, F-actin formed an interconnected network across adjacent cells sometimes spanning tissue pores. In the JCT, F-actin formed a continuous network across cells. Arrays of fine AF fibers were seen amongst cells here. At SC endothelium, F-actin distinctly outlined cell-cell borders giving a confluent monolayer appearance in the near absence of AF fibers. Following incubation with LAT-A, cortical F-actin was lost; staining was exclusively punctate and diffuse. Following LPA incubation, F-actin was almost exclusively cortical and outlined cell borders. Calcein AM staining revealed intact cells and was prevalent throughout all layers prior to and following actin manipulation. Double labeling with PI suggested viability greater than 75%. 3D reconstruction provided striking perspectives of the human TM.
Conclusions: :
We have applied advanced imaging approaches to a novel in situ model of the human TM to visualize and characterize F-actin in 3D within the tissue. We could also analyze in situ actin dynamics in response to actin-modulating probes. Our findings are pertinent to deciphering the biology and pharmacology of the TM.
Keywords: trabecular meshwork • outflow: trabecular meshwork • microscopy: confocal/tunneling