| Glaucoma is a disease with the main feature of an optic nerve damage associated with progressive visual field defect. Today, glaucoma has become the world’s second disease causing blindness.Primary open-angle glaucoma(POAG) occupies a large proportion of the glaucoma.Currently, the etiology and pathogenesis of POAG has not yet fully elucidated. It is acceptted that high intraocular pressure is a major risk factor for optic nerve damage by glaucoma.Further,increased resistance of aqueous outflow by trabecular meshwork is one of the major reasons of high intraocular pressure in glaucoma.The position of increased resistance of aqueous outflow at trabecular meshwork is located in trabecular meshwork cells, JCT and endothelial cells of Schlemm’s canal.All these tissues express abundant actin.The structure and morphology of trabecular meshwork is determined by actin conformation,connection between cell-cell and connection between cell-extracellular matrix.If the structure and morphology of trabecular meshwork changes,the resistance of aqueous outflow changes too.In this research, Fasudil Hydrochloride,selective protein kinase inhibitor which can react with actin in cells and tissues is selected.With this background,we use Fasudil reacts with bovine eyes and bovine trabecular meshwork cells. Before and after treatment, on the one hand, changes of outflow facilities and morphology of anterior chamber angle in bovine eyes were studied by morphology methods.On the other hand, changes of morphology, distribution and expression of cytoskeletal proteins in bovine trabecular meshwork cells are also studied by immunohistochemistry, molecular biology methods. Combined with these two aspects,we evaluate whether Fasudil can reduce aqueous humor outflow resistance and its mechanism.We hope to provide new ideas and new directions for research and the treatment of primary open-angle glaucoma. Purpose:To investigate the effects of Fasudil on outflow facility and morphology of anterior chamber angle in bovine eyes.Whether Fasudil can reduce aqueous outflow resistance,thereby increasing the outflow of the aqueous humour is mainly discussed. The effects of Fasudil on trabecular meshwork cells are also investigated. The effects of Fasudil on regulating the cells morphology, cytoskeleton and connections between cells are focused chiefly.Methods:The first part:The bovine eyes were divided into two groups:GPBS was used in the control group,50uM Fasudil in experimental group.The first, GPBS was perfused into bovine eyes by15mmHg constant pressure,after that baseline and after treatment facility was measured.The second step,red beat solution was perfused to mark mode of aqueous humor outflow. Percent effective filtration length of aqueous humor marked by red beat solution was observed and measured by laser scanning confocal microscopy.We explored whether Fasudil can reduce aqueous outflow resistance.In addtion,whether the mode of aqueous humor outflow changes before and after treatment was investigated. The second part:Primary bovine trabecular meshwork cells were cultured and passaged.BTM cells were divided into control and Fasudil group. They were treated in1uM-1mM Fasudil solution for0.5-2h respectively. First,the cytotoxicity of Fasudil on cells was examined by MTT to investigate the safty of Fasudil. Second, morphological changes of the cells were observed under inverted microscope.Third,expression and arranging distribution of F-Actin and Vinculin were observed by laser scanning confocal microscopy.These are to investigated the effect of Fasudil on cytoskeletal proteins by morphology and immunohistochemistry methods.Results:The first part:The utflow facility of the control group increased0.34±0.03ul/min/mmHg,(18.69±3.44%)after treatment.Compared to the control group, the outflow facility of the Fasudil group increased0.87±0.13ul/min/mmHg,(48.00±9.33%) after treatment. The difference between these two groups is significant in statistics(P<0.01).Compared to the control group, the Fasudil group showed that red microspheres distributed more uniformly and a wider range of red beats marked along the inner wall. After treatment, PEFL in Fasudil group was56.56±4.55%.It was four times of the control group’s(P<0.01).The second part:Bovine trabecular meshwork cells were cultured successfully.After Fasudil intervened in bovine trabecular meshwork cells2h,MTT showed no obvious cytotoxicity.After treatment,Fasudil caused change in the morphological.In the cytoskeleton,Fasudil caused F-Actin depolymerization and rearrangment in cells. Otherwise,vinculin expression was decreased in the connections between cells.These effects depended on concentration and time.Conclusions:Fasudil can increase outflow facility of bovine eyes, thereby reducing aqueous outflow resistance. On bovine trabecular meshwork cells,Fasudil can reduce the phosphorylation of cytoskeletal proteins and rearrange cytoskeletal proteins,thus relaxing the cells and connections between cells.The effect is proved to be safe and no obvious cytotoxicity.Fasudil is likely a new anti-glaucoma drug that reduce aqueous outflow resistance through directly regulating the cytoskeleton of the trabecular meshwork pathway. |
PDF Full Text Request