A Preliminary Investigation Of The Molecular Mechanism Of The Regulation To The Blood-labyrinth Barrier Permeability Of Guinea Pig Inner Ear Affected By Nitric Oxide In Vitro

Objective: To investigate the molecular mechanism of the regulation to the blood-labyrinth barrier permeability of guinea pig inner ear affected by nitric oxide in vitro.Methods:1.Aseptically,the cochlear stria vascularis was acquired from normal guinea pig with micradissection and the endothelial cells were isolated by Collagenase I digestion.Then,they were cultured and purified by using conditioned medium and mechanical curettement.The cultured cells were checked by 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 different concentration tumor necrosis factor(TNF) for 30rnin,60min,90min and measured the permeability to the Eve' s blue.3. We exposed guinea pig inner ear capillary endothelial cell monolayers to the 0.2ng/ml TNF for 30min,60min and 90min.And we each added different concentration L-arginine(L-Arg) for incubating 2 hours and measured the permeability to the Eve' s blue.4.We exposed guinea pig inner ear capillary endothelial cell monolayers to the 0.2ng/ml TNF for 30min, 60min and 90min.And we each added different concentration N -monomethyl-L-arginine (L-NMMA)for incubating 2 hours and measured the permeability to the Eve' s blue.5.Following the in vitro model of the blood-labyrinth barrier of guinea pig inner ear,we established the in vitro model of the lung miciovessl endothelial cell monolayer of rabbit.We added 0.2ng/ml TNF to each group for incubating 90 min.Selecting the groups ofL-Arg and L-NMMA,we added lml 2mmol/L L-Arg and O.2mmol/L #J L-NMMA partly for working 2 hours and measured the permeability to the Eve s blue.6. We exposed guinea pig inner ear capillary endothelial cell monolayers to the different concentration TNF for 90min and measured F-actin concentrations.And then we added different concentrtion L-Arg and L-NMMA partly after we exposed guinea pig inner ear capillary endothelial cell monolayers to the 0.2ng/ml TNF for 90min.After working 2 hours,we measured its 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 gradually proliferated and extended.The cultured cells appeared likes "Paving stone" while the dense cell monolayer had formed.According to the immunohistochemical detection on Factor Vffl-related antigen,over 99% of the cultured cells possessed Factor VHI-related antigen,which indicated their endothelium origin.2.TNF led to a marked increase in Even' s blue transfer across endothelial monolayers(P<0.01). And the filtration rate of Even s transfer across endothelial monolayers was increased as the time was elongation or the concentration of TNF was step-up.3.After worked by the 0.2ng/ml TNF, the NO donor of the L-Arg led to a marked increase in Even s blue transfer across endothelial monolayers(P<0.01).It offered experimental evidence of a direct relationship between the filtration rate of Even' s transfer across endothelial monolayers and the concentration of L-Arg.4. After worked by the 0.2ng/ml TNF, the NO inhibitor of the L-NMMA led to a marked increase in Even' s blue transfer across endothelial monolayers(P<0.01).It offered experimental evidence of an inverse relationship between the filtration rate of Even' s transfer across endothelial monolayers and the concentration of L-NMMA.5.After worked on the same conditions,we compared the filtration rate of the endothelial monolayers of the guinea pigs inner ear and the lung of rabbits.The former was lower than the latter obviously(P<0.01).6. TNF led to a marked reduction in the content of F-actin.It appeared an inverse relationship between the content of F-actin and the concentration of TNF.And L-Arg alsoled to a marked reduction in the content of F-actin seriouly.But L-NMMA led to a marked recovery increase in the content of F-actin.Conclusion:l.As an inflammation stimulating factor,TNF can induce NOS of the cochlear stria vascularis microvessel endothelial cells to produce NO that can regulate the blood-labyrinth barrier.2.It is the important factor that NO produced by NOS of the cochlear stria vascularis microvessel endothelial cells on the pathological conditions can affect the permeability of the cochlear stria vascularis microvessel endothelial cells.It offers the reliable evidence to the clinic that the NO donor and the NO inhibitor can regulate the permeability of the cochlear stria vascularis microvessel endothelial cells.3. It is the important factor that the changes of cell junction which caused by the alteration of the density of F-actin can influence the permeability of the cochlear stria vascularis microvessel endothelial cells.