The Study On The Vestibular Hair Cell Regeneration Of Adult Rat Induced By Math1 Gene Transfer In Vivo

Vestibular dysfunction patients increase gradually because of congenital abnormality,viral disorders,ototoxic drugs and aged degeneration in modern society.Although there are a diverse series of underlying causes for balance disorders,loss of vestibular hair cells represents a common cause of balance dysfunction.At presently,the peripheral vestibular dysfunction alleviation is depended on mostly central vestibular function compensation instead of vestibular sensory epithelium local restoration.So,the peripheral vestibular dysfunction patients maybe gain more effective therapy if we can find a method to replace the loss or damaged hair cells substantially.Many of researches previous demonstrated that mature mammalian hair cells of Corti organ were lack of capability of regeneration absolutely,but preserved a limit regeneration capacity still in vestibular sensory epithelium. Regeneration of vestibular hair cells can be induced in a variety of model systems including guinea pig and rat macular organs and the chinchilla crista ampullaris.One of the genes involved in the development of hair cells is the mammalian atonal homologue math1.Mice carrying a homozygous knockout of math1 failed to develop auditory and vestibular hair cells.Delivery of a plasmid vector expressing math1 to neonatal organ of Corti cultures produced supernumerary hair cells in vitro.These results were repeated using the human homologue of math1(hath1) and an adenovector(Ad) as the delivery vehicle. Most recently,hair cells were shown to be regenerated in adult mice vestibular neuroepithelium and restored vestibular function by math1 transfer in vivo after ototoxic damaged.So,it is possible that vestibular dysfunction will be alleviate by gene therapy.But some problems still exsist in the field of vestibular hair cell regeneration,for instances,property,resouce and function of new hair cells,and so on.These problems hamper vestibular dysfunction gene therapy in clinic presently.Our experiment carried out a series of researches,including preparation of vestibular dysfunction animal model and the approaches of gene transfer into vestibule and the effection of math1 in vestibular hair cells regeneration after aminoglycoside ototoxic insult.The present study can be divided into following three parts:â… .Research of ototoxicity of neomycin to rat vestibular hair cellObjective:To research neomycin ototoxicity for vestibule hair cell of rat, and provide the reference for the pharmacal vestibular dysfunction animal model developement.Methods:Fivteen mature wistar rats were divided into normal control group,neomycin group and artificial perilymph group(n=5).Right ear of the neomycin group rat was administrated 5ul 0.1%(g/100ml)neomycin through the way of drilling scale tympanic of cochlear basal turn.The artificial perilymph group did the same way.As a control,the normal group do nothing to inner ear. To evaluate animal vestibular function,the click-evoked potentials on the surface of the cervical dura mater(CDM-CEP) and swimming time were recorded in all rats after treatment 3 days,and then histological and morphologic observation were carried out after animals sacrificed.Results:Vestibular hair cells were destroied and vestibular function was severely injuried in neomycin group after treatment 3 days.The swimming time was 4.0±0.71s in normal control group, 10.2±1.64s in neomycin group,and 4.4±1.14s in artificial perilymph group.The threshold and latency of CDM-CEP was 85±3.54 dB(SPL) and 6.59±0.41s in normal control group,90±5.0 dB(SPL) and 6.68±0.31 s in artificial perilymph group,but could not induce CDM-CEP at 120dB(SPL) level still in neomycin group.Conclusions:The neomycin possess the powerful capability of eleminating vestibular hair cells of adult rat;Neomycin intracochlear administration via scale tympani can develop an ideal vestibular dysfunction animal model.â…¡.Evaluation of the best way of Gene Transfer into Vestibule and safety of Ad-Math1 to inner earObjective:To explore the method that transfer gene into vestibule and observe the safety of Ad-Math1,in order to provide the base data for vestibular dysfunction gene therapy.Methods:Twenty-five mature wistar rats were divided into normal control group,the group of adenovirus(E1,E3-Deleted and carried math1 and enhanced green fluorescent protein report gene, Ad-Math1-EGFP) scale tympanic transfering group,Ad-Math1-EGFP scale vestibular transfer group.Right ears of the Ad-Math1-EGFP transfering group rats were deliveried 5ul Ad-Math1-EGFP(physical tite 2.1×10¹¹v.p./ml) into cochleas through the way of drilling scale tympanic or scale vestibular of cochlear basal turn.As a control,the normal group do nothing to inner ear.In order to estimate functional condition of vestibule and cochlea,the click-evoked potentials on the surface of the cervical dura mater(CDM-CEP) and threshold of auditory brain-stem response(ABR) and swimming time were recorded in all rats after treatment 7 days,animals were sacrificed after treatment 3,7 days respectively, and then histologic and morphologic observation were carried out after animals sacrificed.Results:All the vestibular end organs were transfected after treatment 3 days in the group of Ad-Math1-EGFP scale vestibular transfer,but could not observe the transfection in Ad-Math1-EGFP scale tympanic transfering group until 7 days after treatment,exception of cochlea.All animals' morphologic observation showed that inner ear hair cells were normal after transfer;After treatment 7 days,the swimming tim ewas 4.0±0.71s in normal control group, 4.8±0.84s in Ad-Math1-EGFP scale tympanic transfer group,and 5.0±0.71s in scale vestibular delivery group.The threshold and latency of CDM-CEP was 85±3.54 dB(SPL) and 6.59±0.41s in normal control group,91±5.48 dB(SPL) and 6.76±0.26s in Ad-Math1-EGFP scale tympanic transfering group,and 89±6.52 dB(SPL) and 6.78±0.26s in Ad-Math1-EGFP scale vestibular delivery group;ABR(auditory brain-stem response) threshold was 37±4.47 dB(SPL), 42±2.74 dB(SPL) and 40±3.54 dB(SPL) respectively in the groups above. Conclusions:The Ad(E1,E3-Deleted) is safe for vestibular and cochlea hair cells and can be used as an ideal vector of gene transfer.The cochleotomy of basal turn through scale vestibular can be served as effective way of transfering gene into vestibule.â…¢.Vestibular hair cell regeneration of adult rat induced by Math1 gene transfer in vivoObjective:To investigate effects and mechanisms of Math1 gene in vestibular hair cell regeneration of rats.Methods:Thirty five adult wistar rats wre divided into normal control group,neomycin group and Math1 gene transfer group.The rats of neomycin group did not transfer Math1 gene into inner ear followed neomycin intracochlear administration via scale tympani and the Math1 gene transfer group animals deliveried Math1 gene 5ul into vestibule through scale vestibuli after neomycin intracochlear injection via scale tympani two days and an adenovirus(E1,E3-Deleted and physical tite 2.1×10¹¹v.p./ml) as the delivery vector.As a control,the normal group do nothing to inner ear.In order to estimate functional condition of vestibule,the click-evoked potentials on the surface of the cervical dura mater(CDM-CEP) and swimming time were recorded in all rats after treatment 3 months.All animals were sacrificed respectively at three time points one,two and three months followed treatment, and then vestibular organs were dissected out and prepared for histological and morphologic examination.Results:Vestibular sensory epithelium did not occur hiar cell regeneration after Math1 gene transferred one month,but observe new hair cell in damaged vetibular sensory epithelium followed Math1 gene transfered two months and the similar phenomenon occured after Math1 gene transferred three months.The new hair cells were determined typeâ… nearly entire and formed intact synapsis with afference nerves by using transmission electron microscopy(TEM).Furthermore,mitosis was detected in renewal vetibular sensory epithelium.After treatment three months,the swimming time was 4.0±0.71s in normal control group,11.2±1.64s in neomycin group,and 10.4±1.52s in math1 gene transfer group.The threshold and latency of CDM-CEP was 85±3.54 dB(SPL) and 6.59±0.41s in normal control group,but could not induce CDM-CEP at 120dB(SPL) level still in neomycin and math1 gene transfer group. Conclusions:Hair cell could regenerate induced by Math1 gene transfered in ototoxicity damaged vetibular sensory epithelium of adult rat.The new hair cells is identified as typeâ… nearly entire and formed intact synapsis with afference nerves.Mitosis is involved in vestibular hair cell regeneration procedure.Conclusions1.The neomycin possess the powerful capability of eliminating vestibular hair cell of rat;Neomycin intracochlear administration via scale tympani can develop as an ideal vestibular dysfunction animal model.2.The Ad(E1,E3-Deleted) is safe to vestibular and cochlea hair cell and can be used as an ideal vector for gene transfer.The cochleotomy of basal turn through scale vestibuli can be served as effective way of transfer gene into vestibule system.3.Hair cell could regenerate induced by Math1 gene transfered in ototoxicity damaged vetibular sensory epithelium of adult rat.The new hair cells were determined with typeâ… nearly entire and formed intact synapsis with afference nerves.Mitosis is involved in vestibular hair cell regeneration procedure.The results above demonstrated that vestibular hair cells of rats could regeneration by math1 gene transfer and an advirus(E1,E3-Deleted) as delivery vector,and show the possibility as gene therapy for vestibular dysfunction.