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MinHee K Ko, James C H Tan; In vivo RhoA activation increases trabecular meshwork contraction and decreases outflow facility. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3287.
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© ARVO (1962-2015); The Authors (2016-present)
Understanding mechanisms of intraocular pressure (IOP) regulation can help us better develop glaucoma therapies. We determined the effect of RhoA activation on TM contractility and outflow facility in live mice.
Lysophosphatidic acid (LPA), a RhoA activator, was perfused into the mouse anterior chamber (2 μM; total volume 5μl; fixed rate 0.63 μl/min). Separate mice received ethanol 0.02% (vehicle) as controls. Outflow facility was measured by 1-level constant pressure perfusion. Enucleated eyes were embedded in OCT compound, post-fixed with 4% paraformaldehyde, cryosectioned, and immunolabeled with fluorescence-conjugated phalloidin (for filamentous actin (F-actin)), alpha-smooth muscle actin, calponin, caldesmon, non-muscle myosin heavy chain IIb (MHC), and phospho-myosin light chain 2 (p-MLC2). Tissue fluorescence labeling intensity was analyzed in confocal microscopy images.
Mean outflow facility in LPA-treated mice was 0.0016 ± 0.002 (mean ± SEM) µL/min/mmHg compared with 0.0084 ± 0.003 µL/min/mmHg in vehicle controls (p<0.01). This represented a 5.2-fold decrease in mean outflow facility. F-actin and p-MLC2 labeling intensity in the TM increased in LPA-treated mice. Altered co-localization of caldesmon with MHC was seen. Levels of other contractile markers were unchanged. Evidence of increased LPA-induced ciliary muscle (CM) contraction was also seen.
In vivo RhoA activation by LPA caused increased drainage tissue contraction and decreased outflow facility. This supports the concept that aqueous drainage tissue contractility involving the TM and CM modulates outflow dynamics in vivo. The mouse is a versatile model in which to dissect out these contractile mechanisms pertinent to understanding IOP regulation and developing glaucoma therapy.
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