Modulation of aqueous humor outflow facility by the Rho kinase-specific inhibitor Y-27632.

PURPOSE: The goal of this study was to investigate the role of Rho kinase in the modulation of aqueous humor outflow facility. Rho kinase, a critical downstream effector of Rho GTPase is recognized to control the formation of actin stress fibers, focal adhesions, and cellular contraction. METHODS: Expression of Rho GTPase, Rho kinase, and other downstream targets of Rho GTPase were determined in human trabecular meshwork (HTM) and Schlemm's canal (SC) primary cell cultures by Western blot analysis. The Rho kinase-specific inhibitor (Y-27632)-induced changes in actin stress fibers, focal adhesions, and protein phosphotyrosine status were evaluated by staining with rhodamine-phalloidin, anti-paxillin, and anti-phosphotyrosine antibodies, respectively. Myosin light-chain phosphorylation was determined by Western blot analysis. Y-27632-induced changes in SC cell monolayer permeability were quantitated using a colorimetric assay to evaluate horseradish peroxidase diffusion through SC cell monolayers grown in transwell chambers. Aqueous humor outflow facility was measured using enucleated porcine eyes and a constant-pressure perfusion system. RESULTS: Treatment of HTM and SC cells with Y-27632 (10 microM) led to significant but reversible changes in cell shape and decreases in actin stress fibers, focal adhesions, and protein phosphotyrosine staining. SC cell monolayer permeability increased (by 80%) in response to Y-27632 (10 microM) treatment, whereas myosin light-chain phosphorylation was decreased in both HTM and SC cells. Aqueous humor outflow facility increased (40%-80%) in enucleated porcine eyes perfused with Y-27632 (10-100 microM), and this effect was associated with widening of the extracellular spaces, particularly the optically empty area of the juxtacanalicular tissue (JCT). The integrity of inner wall of aqueous plexi, however, was observed to be intact. CONCLUSIONS: Based on the Rho kinase inhibitor-induced changes in myosin light-chain phosphorylation and actomyosin organization, it is reasonable to conclude that cellular relaxation and loss of cell-substratum adhesions in HTM and SC cells could result in either increased paracellular fluid flow across Schlemm's canal or altered flow pathway through the JCT, thereby lowering resistance to outflow. This study also suggests Rho kinase as a potential therapeutic target for the development of drugs to modulate intraocular pressure in glaucoma patients.

[Influence of the blight of Morinda officinalis How on microscopic structure and constituents of the host].

The results showed that microscopic structure in biennial roots, the contents of B and Mn in nutritive organ and beta-sitosterol content in roots, stems of Morinda officinalis were affected markedly. The total sugar content in the roots and aerial stems was decreased by 5.42%-15.29%.

Liver involvement in epidemic haemorrhagic fever: in situ hybridization, immunohistochemical and pathological studies.

In the present study, liver biopsy specimens from 37 patients with epidemic haemorrhagic fever (EHF) were investigated by using light and electron microscopy, immunohistochemistry for the EHF virus envelope protein G2 and in situ hybridization (ISH) for EHF viral RNA. Immunostaining and in situ hybridization were both positive, and a few of the aetiological agents, the EHF virus (EHFV) particles, were found individually within the dilated Golgi cisternae and vesicles, and the dilated rough endoplasmic reticulum of the hepatocyte. The pathological alterations of the liver cells infected with EHFV were: hepatocellular degeneration, cytoplasmic vacuolation, and spotty necrosis; moreover, zonal necrosis was present and adjacent to the narrowed and occluded sinusoids. In degenerated and spotty necrotic cells, the positive signals from of ISH for EHFV RNA and those from immunostaining for the virus protein were both stronger than those in the zonal liver cell necrosis area. The results showed that hepatocellular degeneration and spotty necrosis might be directly related to the virus and its duplication inside the liver cells, while the zonal necrosis was ischaemic and thought to be caused by microcirculation dysfunction.