Research On Effects Of Acid Sensitive Ion Channel 1a On Bilirubin-induced Cochlear Toxicity

Objective: To explore the role of acid-sensing ion channel 1a in bilirubin-induced toxicity of cochlear hair cells and spiral ganglion neurons in vitro.Methods: The cochlear organotypic culture of newborn 3-day Sprague-Dawley rats was used as the verification system.After the animal was disconnected,the basement membranes were placed on the surface of the rat tail gel covered with serum-free medium.After 12 hours culture in37 ℃ 5% CO2 incubator,group experiment was carried out.The basement membranes were randomly divided into control group,bilirubin group,acid-sensing ion channel 1a blocker group and acid-sensing ion channel 1a blockers + bilirubin co-treatment group.After 24 hours of culture,samples were taken for results analysis and validation.To verify the expression of acid-sensing ion channel 1a in the basement membrane of cochlear by immunoblotting and immunofluorescence staining.The morphological changes,number changes and apoptotic signals of cochlear hair cells and spiral ganglion neurons were observed by using cochlear basement membrane preparation and immunofluorescence staining.Results: Under the condition of pH=7.4,the morphology of cochlear hair cells and spiral ganglion neurons were basically normal after 50 umoL/L bilirubin treatment,and no cell loss was found.Under the condition of pH=6.8,after 50 umoL/L bilirubin treatment,the morphology of cochlear hair cells was abnormal,no cell loss was found,but the spiral ganglion neurons began to appear different degrees of damage,some spiral ganglion neurons appeared cytoplasmic shrinkage,accompanied by nuclear pyknosis or fragmentation.After 100 umoL/L bilirubin treatment,the volume of spiral ganglion neurons decreased significantly,accompanied by nuclear pyknosis or fragmentation and the number of cells decreased significantly.The hair cells began to show ciliary abnormalities and loss of number in varying degrees.And the toxicity of bilirubin was stronger in pH=6.8 than in pH=7.4.After the treatment of acid-sensing ion channel 1a blockers + 100 umoL/L bilirubin,the morphological abnormality and quantity loss of spiral ganglion neurons and the hair cells were alleviated,and the number of Caspase-3 positive spiral ganglion neurons was decreased.Conclusions: In cochlear organotypic culture,bilirubin damages hair cells and spiral ganglion neurons in a dose-dependent manner,and the toxic effect of bilirubin on basement membrane is stronger in acidic culture environment.Blocking Acid-sensing ion channel 1a can attenuate bilirubin-induced injury of hair cells and spiral ganglion neurons.