Study On Cochlea Hair Cell Regeneration In Presbycusis Mice By Math1 Gene Inner Ear Transfection

Presbycusis influences human being severely.Hair cell degeneration still on clinical impossible recovery present.So regenaration of hair cells is the key point of treatment of presbycusis.In order to test the probability of Math1 gene induces hair cell regeneration in cochlea of presbycusis mice,we prosecuted the study and divided into three parts as following.Part one:Age-related Hearing Loss and Morphological Study of Inner Ear in C57BL/6 Mice Inbred Mouse StrainsTo assess the dynamic age-related hearing loss of inbred mice C57BL/6 and investigate the morphological changes in the inner ear of varied age.In the present study,we tested ABR threshold to evaluate at 2,4,8,16,20kHz in C57BL/6 of 3,4,12,26 weeks old respectively.Morphological changes of inner ear hair cells were qualitatively determined by use of scanning electron microscopy and histochemistry staining of Myosinâ…¥with Neurofilament.The cochlea were sectioned at 6um and then stained with HE.ABR threshold of C57BL/6 gradually increased with age increasing.Contrasted that with 3 weeks old mice,ABR threshold of 26 weeks old mice were dramatically inecreased at 2,4,8,16,20kHz.In scanning electron microscopy and histochemistry staining,the hair cells at top turn and basal turn of cochlea missed and the stereocilia gradually became lossed,fussed,disorganised with age increasing.In 26 weeks old,the outer hair cells almost all missed.In HE section,both the SV and the SG were showed atendency for progressive atrophy to develop with age.Our study shown there is significant age-related hearing loss and hair cell missing in C57BL/6 inbred mouse strain.C57BL/6 inbred mouse strain could be served as animal models of age-related hearing loss for further study of hair cell regeneration. Part tow:The Study on Path Ways of Inner Ear Gene Transfection in MiceSection one:Adenoviral Vectors Mediate Inner Ear Gene Transfection by Postauricular Approach with Three Methods in C57BL/6 MiceTo assess the feasibility of adenoviral vectors mediate cochlear gene tranfer by postauricular microinjection through the round window and application of gelatin soaked with therapeutic agent to the round window membrane with or without puncture of the round window membrane in mouse.Three weeks old C57BL/6 mouse were used for this study.Group 1 mice were implanted with Ad-EGFP or artificial perilymphatic fluid by postauricular microinjection through the round window membrane.Group 2 mice were implanted by postauricular application of gelatin soaked with therapeutic agent to the round window.Group 3 mice were implanted by postauricular application of gelatin soaked with therapeutic agent to the round window with puncture of the round window membrane.On postoperative day 7,the animals were sacrificed after ABR test.The surface preparation was observed,and then stained with Myosinâ…¥.Other cochlea specimens were observed with SEM.Bright green fluorescence in the cochleae was observed in Ad-EGFP groups.Gene expression in groupl and group3 was higher than that in group 2.However,control groups were none any of fluorescence.The postauricular approach of mouse cochlear gene tranfer is simple for operation and with little side-effect.The technique of transgenic delivery into the inner ear through RWM by microinjection is feasible and effective,but damage to hearing transduction.Section two:Intact Tympanic Buila Cochlear Gene Delivery into the Scala Tympani of MouseTo assess the feasibility of adenoviral vectors mediate cochlear gene tranfer by ventral approach microinjection into the scala tympani of mouse with intact tympanic bulla and the expression of EGFP.Three weeks old C57BL/6 mice were used for the study and were microinjected with Ad-EGFP or artificial perilymphatic fluid in the scala tympani.Auditory brainstem response(ABR) thresholds were determined in all animals 7 days after surgery.Then the animals were sacrificed and the surface preparation and the section of cochleae were observed.The surface preparation was observed,and then stained with Myosinâ…¥.Other cochlea were observed with SEM.ABR measurement showed that the operation had no significant injury on the hearing.Bright green fluorescence in the cochleae was observed in Ad-EGFP groups.Artificial perilymphatic fluid and control groups were free of fluorescence.The technique of transgenic delivery into the inner ear by ventral approach microinjection into the scala tympani with intact tympanic bulla is feasible,atraumatic and effective.Part three:Cochlear on Hair Cell Like Cells Regeneration in Presbycusis C57BL/6 Mice after Math1 Gene Inner Ear TransfectionIn order to test the probability of Mathl gene induces hair cell like cells regeneration in cochlea of presbycusis C57BL/6 mice,8 mounths old C57BL/6 mice were used for this study and were microinjected with Ad-MathlorAd-EGFP in the scala tympani by ventral approach.Auditory brainstem response(ABR) thresholds was determined in all animals 3.5 mounths after surgery.Then the animals were sacrificed and the surface preparation and the section of cochleae stained by Myosinâ…¥were observed.Other cochlea were observed with SEM. Positive cells of Myosinâ…¥in the cochleae of Ad-Math1 groups were found.Some positive cells of Myosinâ…¥were proliferate and their karyons were dividing.This results show Math1 gene can induce regeneration of new hair cell like cells in cochlea of presbycusis C57BL/6 mice.But the hearing of the mice with Math1 transfection were not improved.So Math1 gene with other factors may be necessary to improve hearing.