An In Vitro Investigation Of The Molecular Mechanism Of The Regulation To The Blood-labyrinth Barrier Permeability Of Guinea Pig

Objective: To investigate the molecular mechanism of the regulation to the blood-labyrinth barrier permeability of guinea pig by the in vitro model .Methods: 1. Aseptically, the cochlear stria vascularis was acquired from normal guinea pigs with micradissection and the endothelial cells were isolated by Collagenase â…  digestion. Then, they were cultured and purified by using conditioned medium and mechanical curettement. The cultured cells were detected with SP immunohistochemical test on its specific Factor â…§-related antigen. To establish in vitro model of the blood-labyrinth barrier by using small pools, which is composed of inner pool and outer pool. The bottom of the inner pool (Millicell) is covered by confluent inner ear capillary endothelial cell monolayer and the outer pool is the cultivation well of six-well culture plate. 2. We exposed guinea pig inner ear capillary endothelial cell monolayers to the substance known to increase cyclic AMP :Cholera toxin (CT) for three hours and measured its effect on endothelial permeability to albumin and Evan's blue. 3. We exposed guinea pig inner ear capillary endothelial cell monolayers to CT or fuluvamide for four hours and measured their effect on the endothelial permeability curve to albumin and Evan's blue. 4. We exposed guinea pig inner ear capillary endothelial cell monolayers to CT or Fuluvamide for three hours and measured their effect on endothelial cell cyclic AMP and F-actin concentrations. Results: 1. We found that several cell islands being made up of a few or tens of short Fusiform cells or blunt round cells on the wall of the culture bottle on the seventh day after primary culture. Then, these cell islands graduallyproliferated and extended. The cultured cells appeared likes"Paving stone"while the dense cell monolayer had formed. According to immunohistochemical detection on Factor â…§-related antigen, over 99% of the cultured cells possessed Factor â…§-related antigen, which indicated their endothelium origin. The cultured endothelial cell, derived from the stria vascularis from guinea pig inner ear, preservers many features of the in vivo the stria vascularis capillary endothelium, and hence is of interest as a potential in vitro model of the blood-labyrinth barrier. We successfully created an in vitro model of the blood-labyrinth barrier. 2. CT led to a marked reduction in albumin and Evan's blue transfer across endothelial monolayers(P<0.01). 3. the endothelial permeability curve to albumin and Evan's blue rose gradually. Compared with that of the control group, the curve of the CT group rose more slowly and the curve of the Fuluvamide group rose more rapidly. 4. CT led to a marked increase in cyclic AMP concentration(P<0.01) and a marked reduction in F-actin concentration (P<0.05); In contrast, Fuluvamide led to a marked reduction in cyclic AMP concentration(P<0.01) and a marked increase in F-actin concentration (P<0.01). It implied that cyclic AMP and F-actin were invovled in the molecular regulation to the blood-labyrinth barrier permeability of guinea pig.Conclusion: The results indicated that cyclic AMP is an important determinant of the blood-labyrinth barrier in vitro.The barrier function changes was associated with apparent endothelial shape changes and qualitative and quantitative changes in F-actin,suggesting that influences on cytoskeletal assembly may be involved in this process.