Toxic Effects Of Bilirubin On Mice Cochlear Tissue In Vitro And Hydrogen Antagonism

PART 1 The effects of different concentrations of bilirubin on mice's cochlear tissueObject To observe the damage effect of different concentrations of bilirubin on mice's cochlear tissue in vitro culture.Method The cochlear basilar membranes containing the spiral ganglions were gently isolated from P5 mice under the dissecting microscope.After 48 hours in vitro culture,under the inverted microscope,the well-grown cochlear basilar membranes were selected for experiment.The selected basilar membranes of the cochlea were randomly divided into four groups: control group,25?mol / L bilirubin group,50?mol / L bilirubin group,100?mol / L bilirubin group.According to grouping concentration,the bilirubin was added to the culture media in each dish in the experimental group and the control group was added with the corresponding volume of physiological saline.After 24 hours in drug treatment,all the samples in the four groups were fixed and underwent immunofluorescence staining.The morphology of the nerve fibers and hair cells was observed under laser confocal microscope.The numbers of the nerve fibers and hair cells was counted and underwent statistical analysis.Result The primary culture of basilar membrane was successful.The quantity and activity is good enough to be examined.In the 25?mol/L bilirubin group,the number of nerve fibers reduced,nevertheless,the morphology and quantity of the hair cells didn't change.In the 50?mol/L bilirubin group,the number of nerve fibers reduced obviously,and yet the morphology and quantity of the hair cells didn't change.In the 100?mol/L bilirubin group,the nerve fibers almost disappeared,but,the morphology and quantity of the hair cells didn't change.The data were analyzed by Kruskal-Wallis test.The number of the nerve fibers in each experimental group was significantly different from that in the control group(P<0.05),and the number of the nerve fibers between the experimental groups was also statistically different(P <0.05).Conclusion The nerve fibers injury by bilirubin in a concentration-dependent manner.That is,the number of the nerve fibers reduced apparently with the increasing bilirubin concentration.When the bilirubin concentration does not exceed 100?mol/L,the bilirubin does not cause damage to the inner hair cells and outer hair cells of the cochlear tissue cultured in vitro.PART 2 The rich hydrogen saline resist the effect of bilirubin on mice's cochlear tissueObject To observe whether hydrogen can antagonize the neurotoxicity of bilirubin on mice's cochlear tissue.Method The cochlear basilar membranes containing the spiral ganglions were gently isolated from P5 mice under the dissecting microscope.After 48 hours in vitro culture,under the inverted microscope,the well-grown cochlear basilar membranes were selected for experiment.The selected basilar membranes of the cochlea were randomly divided into two groups: control group(50?mol/L bilirubin+ physiological saline),experimental group(50?mol/L bilirubin+ rich hydrogen saline).According to grouping concentration,the bilirubin was added to the culture media in each dish.At the same time,the rich hydrogen saline was added to the culture media in each dish in the experimental group and the control group was added with the corresponding volume of physiological saline.After 24 hours in drug treatment,all the samples in the two groups were fixed and underwent immunofluorescence staining.The morphology of the nerve fibers was observed under laser confocal microscope.The numbers of the nerve fibers was counted and underwent statistical analysis.Result The number of the nerve fibers in the hydrogen-rich group was significantly higher than that in the control group.The data were analyzed by Group T test.The number of the nerve fibers in the experimental group was significantly different from that in the control group(P <0.05).Conclusion The rich hydrogen saline antagonizes the toxic effects of bilirubin on cochlear nerve fibers tissue.