The Transport Mechanism Of Ca²⁺ In The Isolated OHCs Of Guinea Pig

Objective:To observed the auditory brainstem Auditory(ABR) threshold level of the developing guinea pig. Exploring fast separation method of the outer hair cells (Outer Hair Cells, OHCs) from guinea pig cochlear and observing the vitality of the OHCs to provide fast experimental methods for electrophysiological experiments. To observed calcium ion transport phenomena of developing cochlear OHCs and explore the transport mechanism. The ion transport mechanism of the auditory system will hoped to provide a theoretical basis for clinical prevention and treatment of the auditory system disease.Method:Detected the ABR threshold of guinea pig in different ages with audiological testing equipment; Using mechanism combined with Collagenaseâ…£digestion to isolate OHCs quickly from cochlea and observing the vitality of them under the inverse microscopy; Non-invasive micro-test technology was used to detecte calcium ions flow of isolated cochlear OHCs in resting state and observed the changes of calcium flow treated with nimodipine.Result:1,Postpartum 1 day guinea pigs have about 8dB SPL higher ABR threshold than 3 days, 7 days and adult guinea pigs(P <0.05 ANOVA).ABR threshold of Postpartum 3 days, 7 days guinea pigs and adult guinea pigs was no significant difference (P> 0.05 ANOVA), so ABR threshold level of 3 days guinea pigs has reached the hearing level of adult guinea pigs .2. The methods used in this experiment can rapid obtained a fair amount of living isolated OHCs. Cochlear outer hair cells adhere after a short time. Put them aside for 7 minutes after the separation then coherence can be observed . Different patterns of OHCs have different surviving time. The shorter cells soma can survive about 3 hours, and the longer can survive 6 to 7 hours.3. Four types of Ca²⁺ transport were identified in isolated OHCs and OHCs on basilar membrane tissue fragments: influx at the head of with efflux at the bottom (type 1): efflux at the head of OHCs with influx at the bottom (type 2); influx at the both head and bottom (type 3); and efflux at the both head and bottom (type 4). However, only type 1 and type 3 of Ca²⁺ ion transport were detected in the cochlea. Ca²⁺ flow in OHC can be significantly inhibited by Nimodipine in resting state, inhibition rate of 76.78% at head and 100% at bottom.4,Ca²⁺ flow gradually increased in the isolated OHCs of the developing guinea pigs. Ca²⁺ flow of OHCs which from postnatal 1 day guinea pig was significantly less than that the normal adult guinea pigs(P <0.05 ANOVA). Most of the isolated OHCs from postnatal 3 days and 7 days guinea pigs have the Ca²⁺ flow which was close to that of adult guinea pigs (P> 0.05 ANOVA).Conclusion:1,Using mechanism combined with Collagenaseâ…£digestion to isolate OHCs quickly from cochlea could obtain a sufficient number of good shape and activity OHCs.They could survive for a long time and meet for most of the electrophysiological experiments.2,Ca²⁺ ion transport exists in adult guinea pig cochlear OHCs in resting state and is variable.But the dominant trend is not clear, still need further study.3,Ca²⁺ flow gradually increased in the isolated OHCs of the developing guinea pigs. Most postpartum 3 days have reached the levels of normal adult guinea pigs, establishing a foundation for us further study in the mechanisms of ion transport.