Purchase this article with an account.
MinHee K Ko, Eun Kyoung Kim, James C H Tan; Glaucoma drug studies in live mice: effect of latrunculin-B on outflow facility and actomyosin contractility. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5674.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To determine the effect of latrunculin-B (Lat-B), an F-actin modulating agent, on outflow facility and aqueous drainage tissue actomyosin contractility in live mice.
Drug delivery of Lat-B at 2.5, 5, and 10µM into the anterior chamber of live C57BL/6 mice was performed at a fixed perfusion rate of 0.63µl/min (total 5µl) and then drug was incubated for 1 hour. Independent mice received ethanol 0.02% (vehicle) or phosphate buffered solution (PBS) in the same manner. Live mouse outflow facility was then measured by feedback-controlled 2-level constant pressure perfusion over 2 hours. Eyes were post-fixed with 4% paraformaldehyde, cryosectioned then immunostained with fluorescence-conjugated phalloidin labeling of filamentous actin (F-actin). F-actin labeling intensity in the tissues was analyzed quantitatively in confocal microscopy images. Tissue structure was examined by H&E staining.
Twenty live mice underwent perfusion. Mean outflow facility determined by 2-level constant pressure perfusion was 0.0027±0.00036 (mean ± SEM)µL/min/mmHg in PBS controls and 0.0023±0.0005µL/min/mmHg in vehicle controls. No time-dependent facility increase (washout) was seen in PBS (p=0.76) or vehicle (p=0.29) control groups during perfusions lasting 2 hours. Following Lat-B, outflow facility increased time-dependently over 2 hours for delivered doses of 2.5µM (0.0082±0.0066µl/min/mmHg at 2 hours; mean=0.0057µl/min/mmHg; p=0.29); 5µM (0.043±0.0094µl/min/mmHg at 2 hours; mean=0.015µl/min/mmHg; p=0.039); and 10µM (0.088±0.0029µl/min/mmHg at 2 hours; mean=0.049µl/min/mmHg; p=0.001). This represented a dose-dependent increase in mean outflow facility of 3.6, 19, 38 fold for Lat-B doses of 2.5, 5 and 10µM respectively compared with vehicle controls. F-actin labeling intensity was decreased by 46±5.6% in ciliary muscle and 37±8.3% in trabecular meshwork (both p<0.005) relative to controls.
Lack of washout indicates the strength of the perfusion approach and mouse model for in vivo drug screening and probing of aqueous outflow dynamics. Lat-B delivered by perfusion into the anterior chamber dose- and time-dependently increased aqueous outflow facility in live mice relative to controls. This was associated with significantly decreased actin polymerization in both trabecular meshwork and ciliary muscle that was verifiable by quantitative analysis.
This PDF is available to Subscribers Only