Labeling L-type Calcium Channels With DMBODIPY-DHP And RH414 And Monitoring The Change Of Intracellular Ca～(2+) Concentrations Caused By Acoustic Overstimulation In Outer Hair Cells Of The Guinea-pig Cochlea

Calcium and calcium channels play a important role in the mechanic- electrical transduction in the inner ear. In the present study, the outer hair cells(OHC5) were isolated from various turns of the guinea pig cochlea .The calcium channels of OHCs of various turns were labeled with DMBODJPY-DHP and RH4 14. The change of intracellular Ca2 concentrations ([Ca²⁺]_i) in outer hair cells of the guinea-pig cochlea was obsreved after exposure to 4kHz narrow band noise.The relationship between the density of the calcium channels on OHCs among various turns and the tonotopic map in the guinea pig cochlea was investigated .The calcium overload in OHCs might be one of the possible pathogentic factors for acoustic trauma. Three parts are included in this article. I. Objective To probe the method of isolating outer hair cells from each of four turns of the guinea-pig cochlea. Methods In the light of the anatomical and histological characteristics of various turns of the cochlea , the organs of Corti were dissected accordingly. Then viable OHCs of various turns were isolated from the guinea pig cochlea by pipetting after treatment with enzyme. The fluorescent dyes FDA and P1 was used for the determination of cell viability. The lengths of OHCs were measured. Result (a) The viable QHCs of various turns were cylindrial in shape. A cuticular plate was seen at the apex from which the stereocillia protruded with the nuclei sitting at the base.The OHC was in a state of translucent under a phase contrast microscope. The viable OHCs exhibited green fluorescence of FDA and the deteriorated OHCs, red fluorescence of P1 which was most prominent in the nucleus under a fluorescent 3 Thesis of Military Post-^ratuate Medical CollaReABSTRA CT microscope. (b) The yield of OHCs isolated from the apical turns of the cochlea was higher than that from the basal turns. (c) The lengths of OHCs from each of four turns were 23.8 1,34.50,60.48 and 71.37 I^?m . Conclusions It is suggested that the key to success in isolating OHCs from each of four turns of the cochlea is to know very well the anatomical characteristics of each of four turns of the cochlea and the dissection is operated in accordance with normal rules. II. Objective To investigate the relationship between the density of L- type calcium channels on OHCs and the tonotopic map in the guinea pig cochlea. Methods The OHCs were labeled with DMBODIPY-DHP and RH414 at room temperature for 5 mm, then observed using laser scanning confocal microscope. L-type calcium channels labeled with DMBODIPY-DHP exhibited green fluorescence and the cellular membrane labeled with RH414 exhibited red fluorescence. The fluorescence ratio of DMBODIPY-DHP / RH414 revealed the distribution of L-type calcium channels on the cellular membrane. Result The results showed that the cellular membrane had lesser DMBODIPY-DHP staining when pretreated with nifedipine. It verified that calcium channels on OHCs shared the specificity of L-type calcium channels. The density of L-type calcium channels had a tendency of increasing gradually from low frequency areas to high frequency areas. Variance analysis showed that the difference of the density of L-type calcium channels on OHCs between the fourth turn and the second turn was significant. Conclusions The descripency of the distribution of L-type calcium channels on OHCs among various turns of the guinea pig cochlea may b...