Our hypothesis is that the cells of the aqueous outflow pathway can modulate aqueous humor outflow resistance through changes in cell shape, contraction and attachment; that the trabecular cell cytoskeleton, which is involved in these cellular events, is a useful target for aqueous outflow drug therapy and that abnormalities in certain cytoskeletal functions might underlie some forms of glaucoma. We wish to investigate the relationship between cytoskeletal induced changes in trabecular cell shape and attachments and changes in tissue conductivity (outflow facility) by correlating cellular assays (cell contraction assay (silicon rubber deformation); cell retraction assay (using high resolution DIC microscopy and video time lapse); cytoskeletal response to stretch (immunofluorescence after passive stretch); cytoplasmic calcium release upon pharmacological stimulation (using fura 2); and monolayer permeability assays) with intact tissue assays. We will study bovine, porcine, and human trabecular cells (including human glaucoma trabecular cells), putative Schlemm's canal cells (outer wall), and calf pulmonary artery endothelial cells (as a possible model for Schlemm's canal cells) in the cellular assays after pharmacological and/or mechanical manipulation, and seek correlations with bovine and human short- term whole eye and long-term anterior segment organ culture outflow studies. Quantitative anterior chamber perfusion and subsequent morphological evaluation in living monkey experiments are planned for final confirmation of relevance to living tissue function. We wish to investigate the role of microtubules in such trabecular cell functions by utilizing recognized tubulin techniques such as cellular injection methods, immunofluorescence labeling of stabilized versus dynamic microtubules, kinesin assays, and polymerization/depolymerization methods. We will evaluate in outflow pathway cells a tensegrity model of tubulin acting in concert with actin for derived effects on tissue conductivity. Using ethacrynic acid as a lead probe, we also wish to determine whether other tubulin active drugs can increase outflow facility.
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