Discovery of Cross-linked Actin Networks (CLANs) in Outflow Tissues from Dexamethasone-treated Human Eyes

Abstract

Abstract: : Purpose: There is a re-organization of the actin cytoskeleton to form cross-linked actin networks (CLANs) in cultured glaucomatous trabecular meshwork (TM) cells and in dexamethasone (DEX)-treated TM cell cultures. Our goal was to determine whether these CLAN-like structures also occur in situ in aqueous outflow tissues. Methods: Anterior segments of normal human eyes were perfusion cultured with or without DEX (10^-7M) for 5-10 days prior to fixation at constant pressure. Some tissue samples were dissected by opening Schlemm’s canal, and triple staining with rhodamine-phalloidin, Sytox, and either anti-laminin or anti-CD-31 to label the f-actin, nuclei, basement membrane and endothelia respectively. The inner wall of Schlemm’s canal, JCT, and corneoscleral TM were examined using confocal laser microscopy. Additionally, TM tissue was carefully dissected and sections were stained with fluorescently labeled phalloidin prior to en-face optical sectioning from the uveal meshwork (UM) through to the corneoscleral meshwork (CSM) using a confocal laser microscope. Results: In control eyes, inner wall cells contained marginal actin bands, while JCT, CSM and UM cells contained lightly stained, apparently randomly distributed actin arrays. In DEX-treated eyes, the actin distribution in some samples was indistinguishable from that in controls. However, in other samples the actin distribution appeared much more disordered than in control eyes, and included actin-tangles and clear examples of CLANs. CLANs seemed to be more numerous in UM and CSM cells when compared to JCT or inner wall cells. "Normal" and "tangled" actin were frequently found in adjacent quadrants from the same DEX-treated eyes. Conclusion: The geodesic dome-like CLAN structures previously reported in cultured TM cells also are found in whole TM tissue from DEX-treated eyes. DEX treatment appears to affect F-actin structure in outflow tissues by an unknown mechanism. Although still unclear, it is possible that these changes in actin structure may have an effect on aqueous outflow resistance, possibly by making TM cells less deformable.

Keywords: cytoskeleton • trabecular meshwork • microscopy: confocal/tunneling