The Experimental Study Of Rho Kinase Inhibitor Y-27632 On The Mechanism Of Lowering The Intraocular Pressue

PURPOSE: To investigate the roles of glucocorticoid (Dexamethasone, DEX) and Rho kinase inhibitor. Y-27632 the cytoskeleton on human trabecular meshwork (HTM) cells in vitro. To establish a kind model of corticosteroid -induced elevation of IOP in cats by topical adminstration of Dexamethasone drops and subconjunctival injections of triamcinoIone.To observe the role of Rho kinase inhibitor Y-27632 in the regulating intraocular pressure in the cats with corticosteroid-induced ocular hypertension. To investigate the pathogenesis of corticosteroid-induced glaucoma and the mechanism lowering the intraocular pressue of Rho kinase inhibitor Y-27632.METHODS:1. HTM cells (sixth passage) were cultured to a confluent stage with stableendothelium-like morphology and incubated with DEX (200mM) for 2 weeks. Then the cells were treated with 10μM of Y-27632 for 3, 24hrs respectively. Then we observed the changs on cell morphology and actin and vinculin.2. Adult domestic cats were trained to accept ocular drug administration andtonometry under topical anesthesia. To establish intraocular pressure (IOP) baseline IOP values, tonometry was performed at the same time of day for 3 consecutive days, with TONO-PEN XL tonometer. Beginning on day 4, thirty-two cats received either 0.5% dexamethasone (right eye) or vehicle (left eye) administered topically three times a day for approximately 56 days, and subconjunctival injections of 3mg triamcinolone in right eyes under topical anesthesia weekly. IOP was monitored once a week after the corticosteroid administration. Then, the tissue of trabecular meshwork (TM) was collected and was examined by electric microscopy.3. The cats with corticosteroid-induced ocular hypertension were used in this study. Rho kinase-specific inhibitor, Y-27632, was topically administered to one eye of the cats with corticosteroid-induced ocular hypertension.Y-27632 (30mM or 10mM) or phosphate buffered saline (PBS) was administered to the central cornea as three 5-μl drops at intervals of 30 seconds. TONO-PEN XL tonometer was used to monitor the IOP. It was measured before the administration of Y-27632 and at 0.5, 1, 3, 6, 9, and 24 hours after administration.RESULTS:1. Compared with DEX-free cultured HTM cells, there was an increased amount of the actin cytoskeleton, even formed cross-linked actin networks, and vinculin-positive focal adhesions increased in DEX-treated cells. Y-27632 treated HTM cells appeared to be retracted and round up. The actin cytoskeleton was disrupted or reduced, vinculin-positive focal adhesions were reduced in number or disappeared in Y-27632 treated cells. These alterations were found to be reversible after drug was withdrawn. 2. The baseline IOP of the rest 32 cats was 18.64±2.42 mmHg. Introcular pressure began to increase after 2 weeks of treatment in 89% of the cat eyes receiving corticosteroid, and reached a peak 4 weeks later. The peak IOP differences between the corticosteroid-treated eye and the fellow control eye reached up to 7.38mmHg (P<0.05). The changes of the trabecular cells ultrastucture, in the corticosteroid-induced hypertension cat eyes, including dysmorphic change of nucleus, enlargement of the endoplasmic and Golgi apparatus, and deposition of TM extracellular matrix.3. In cat eyes with corticosteroid-induced ocular hypertension, topical administration of Y-27632 resulted in a significant decrease in IOP. Compared with PBS-treated control eyes, the IOP in Y-27632-treated eyes was significantly lowered 0.5 hours after topical administration of Y-27632 eye drops (P < 0.05). The average maximum decreases was seen after 60 minutes administration of Y-27632 .The IOP reduction was observed between 0.5 and 3 hours (P < 0.05) with 30 mM of Y-27632 and the average maximum decreases was 6.13mmHg. With the 10mM concentration eye drops, the reduction were seen between 0.5 and 1 hour (P < 0.05) and the average maximum decreases was 5.12mmHg.CONCLUSIONS:1. Corticosteriod caused an obvious change in the organization of actin cytoskeleton and vinculin-positive focal adhesions in the cultured HTM cells.we infer that the cocorticoid-mediated changes in the HTM cytoskeleton maybe the pathogenesis of corticosteroid-induced glaucoma. Because Y-27632 can inhibit the DEX-induced changes in the HTM cells. It indicate that Rho kinase inhibitor Y-27632 can decrease the cocorticoid-induced intraocular pressure.2. The domestic cat eyes exhibit a robust steroid-induced ocular hypertensive response, with 89% occurrence in this trial. The high prevalence of corticosteroid-induced elevation of IOP in the cat eyes will permit studies on the mechanism of steroid-induced glaucoma and the pressure-lowering effect of drugs.3. Administration of Y-27632 caused a reduction in IOP in cat eyes with corticosteroid-induced ocular hypertension in a dose-dependent manner. The Rho kinase-specific inhibitor Y-27632 is a promising treatment for glaucoma therapy in the next generation and IOP-lowering effects of Y-27632 may be related to the altered cytoskeleton behavior of TM cells.