Study On Hearing Loss Induced By Co-administration Of Kanamycin With Furosemide And Gene Therapy In Guinea Pigs

Hearing loss is divided into three types:conductive,sensorineural and mixed deafness.Sensorineural deafness influences the human being severely.There are still no clinical useful means for curing sensorineural hearing loss at present while regeneration of hair cells is the key point of treatment of sensorineural deafness. Gene therapy technology has improved in recent years,making it a promising technology for treating inner ear disorders.Our study is divided into three parts as following.Part one:Evaluation of hearing loss induced by co-administration of kanamycin with furosemide in guinea pigsCo-administration of kanamycin(KM)with the loop diuretic ethacrynic acid has previously been shown to produce a rapid and profound hearing loss in guinea pigs.In the present study,we described a modified technique for developing a profound hearing loss in guinea pigs.Guinea pigs were received subcutaneous injection of KM followed 2h later by intravenous infusion of furosemide. Auditory brainstem responses(ABRs),compound action potential(CAP)and cochlear microphonic(CM)were recorded to monitor the animal's hearing at the 3th,7th day and 2 month after the infusion.ABR monitoring showed that profound hearing loss was both bilateral and permanent.There was no difference between ABR threshold of the 3th day,7th day and 2 month(p＞0.05).CM results showed it was more serious than that of CAP.In conclusion,co-administration of KM and furosemide effectively produces a profound hearing loss in guinea pigs.It is an effective deafening method for acute animal experiments. Part two:Ototoxicitic observation of co-administration of kanamycin with furosemide in guinea pigsTo explore the ototoxicity of co-administration of kanamycin with furosemide in guinea pigs,immunohistochemical and histopathological changes of guinea pigs with profound hearing loss(ABR＞95dBSPL)were observed under light microscope and scanning electron microscope.The results showed absence of hair cells(HCs)and absense of OHCs predominated in the cochlea.Absense of HCs was more severely near the round window.There was loss of neurofibra 1 week sooner after the deafening procedure.The extent of loss of neurofibra was dependent on their distance from the round window and the period of survival following the deafening procedure.The loss of neurofibra was found in the neurofibra of OHCs.Both apoptotic- and necrotic-like morphology was evident during the period of ongoing cell death.However,TUNEL could not be detected positive stained in all cochlear hair cells.Part Three:Effect of Hath1 gene therapy in deaf guinea pigs induced by co-administration of kanamycin with furosemideHere we report that Hath1,a gene also known as Math1 which is the key regulator of hair cell development and induces regeneration of hair cells.Our results showed the number of HCs increased in deaf animals by delivery Ad.Hath1-EGFP gene to cochlea.But hearing threshold of 4w and 8w were not changed neither through intact round window nor througn scala tympani.Transfer effecacy through scala tympani was advantageous while the intact round window method was less invasive.In contrast,the number of HCs didn't change in all controls(include empty group,Ad-EGFP group and artificial perilymph group). Conclusion:Delivery Hath1 gene to cochlea can generate new HCs in mature guinea pigs.The effect of through scala tympani is better than that of through intact round window.Multiple gene therapy may be necessary to improve hearing.