Pathology Of The Postnatal Organ Of Corti In Conditional Cx26 Knock Out Mice And Therapy Exploring

PARTⅠValidation of the Cx26 Conditional Knockout Mice ModelBackground Deafness is one of the most prevalent sensory disturbances in human and neonatal deafness affects severely their verbal learning and communication ability. The deafness can be divided into congenital deafness and acquired deafness. Congenital deafness is a very frequent disorder occurring in approximately 1 in 500 live births and the remaining children inherit the tendency to develop hearing loss 1 in 1000 live births before the adult.Among those deafness cases,non-syndrome hereditary deafness accounted for more than 80%in hereditary deafness.The non-syndrome hereditary deafness caused by Cx26 gene mutations contribute to about 50%of inherited prelingual deafness cases.In the past several years,researches of the deafness caused by Cx26 mutation were numerous.However the molecular mechanismes of this kind of the deafness are still unknown and the corresponding mouse animal models are appropriate for studying.To investigate molecular mechanisms of deafness caused by Cx26 mutations,we generated two types of conditional Cx26(cCx26) null mice by cross mating.The genotypes of these mice are Foxgl-Cre cCx26ko and Pax2-Cre cCx26ko.Methods Cre reporter mice(Rosa26R mice) were used to monitor tissue specific Cre activation.Then the genotype was validated by regular PCR.Western blot was used to test the cx26 protein expression in cochlear of these mice.The immunohistochemisty of Cx26 and Cx30 was observed in order to locate the distribution of Cx26 and Cx30 in the cochlear of the mouse model.Results The expression of Cre started at as early as E11.5.The cochlear section showed that the Cre was expressed at sensory epithelial cell layer in the organ of Corti and spiral ganglion neurons.The PCR results revealed that the mice genotype as Cx26^loxP/loxP;pax2^cre/+ and Cx26^loxP/loxP;Foxg1^Cre/+.Western Blot results demonstrated that the Cx26 protein level was down-regulated only in the organ of Corti,not in the spiral ligament and Stria Vascularis.The cochlea frozen-section further displayed that the Cx26 was absent in the organ of Corti cell,but was still kept in the spiral ligament and spiral limbus.Conclusions The PCR,Western blot and immune labelling results demonstrated the Cx26 protein was absent in the epithelial cell layer of organ of Corti.In other words,the two conditional Cx26 null mice model were validated and can be further used in our following studiesPARTⅡPostnatal Pathology of the Cochlea in Cx26 Conditional Knockout MiceBackground Mutations in GJB2 gene are a major cause of autosomal recessive congenital heating loss.The general mutation rate of GJB2 is 12.2%(16/131) and the 235delC is the most common type of deafness-causing mutation in Chinese patients with congenital heating loss.Although there were some reports of Cx26 deafness cases,most of them focused on genetic analysis and family studies.The mechanism underlying Cx26 mutation-induced deafness remains unclear.Gene knockout animal models provide a powerful ways and means.Through studying the pathological changes and investigate the auditory function of the cochlea of the conditional Cx26 gene knockout mice,we can try to understand indirectly the cause of the deafness induced by Cx26 mutations in human being.Methods tmmunohistochemistry and resin sections in cCx26KO mice and control mice were used to determine the morphological differences in the organ of Corti.Through the auditory brainstem evoked potential and the determination of endolymphatic potential,the hearing of cCx26KO mice model were evaluated.Results We found no difference between the cCx26 null mice and the WT mice in the Proxl and P75 cochlear staining,indicating that the supporting cell can differentiate normally in the cCx26 null mice cochlear.The cochlear morphology was then observed in the cochlear resin section.At P4,no difference was observed in the cochlea.From P8,the tunnel of Corti started to open in WT cochlea,however,the cCx26 null mice stalked and never finished opening of tunnel of Corti.Starting at around P14,the out hair cell in the middle turn of Cochlea started to die and all of the epithelial cells in the organ of Corti including HC,supporting cells were totally lost at around P21.In contrast,the apical turn epithelial cells were spared.The neuron fiber degenerate at P18 and some of the SGN started to die at around P21.Most of the SGNs were dead at P1m and only few condensed SGNs were found in the P2m cochlear.The endolymph potential was measured in the WT and Cx26 null mice model respectively.In the WT,the EP is about 90±1 Mv from P15 to adult level.In contrast,the EP in the age corresponding cCx26 null mice displayed two distinct groups,the lower group has a low EP at about 35±1 mv and the higher group has a EP of 66.5±1.3mv.The ABR heating test showed that the two cCx26 null mice models were profoundly deaf and the threshold was significantly Higher than the WT at each tested frequency(4K,8K,12K,18K,24K and 32K)Conclusions The cCx26 null mice were severely deaf and the EP was impaired.The cochlear development was delayed and the organ of Corti was abolished from middle turn to basal turn,the apical turn was spared.PARTⅢExperimental Treaments for Cx26 Conditional knockout miceBackground For the treatment of the severe sensotineural heating loss induced Cx26 mutation,the most effective way is cochlear implantation.Performance with cochlear implants is highly variable and depends on many factors.The existence of the spiral ganglion is one of the important reasons.So increase the survival of spiral ganglion is an effective method.Another possible treatment options is overexpressing the related Cxs by a novel transgenic strategy;however,the translation of the finding into clinical applications is not applicable so far.Based on our finding,there is time window for the cell death in the organ of Corti.Therefore,timely intervene by injection drugs and gene therapy may prevent the organ of Corti cell death.Methods The conditional Cx26 gene knockout mice were treated by the 7,8-Dihydroxyflavone from P4 till P64.Then the ABR is tested and the cochlear of cCx26ko mouse was observed to evaluate the morphological changes.Another method is gene delivery.Ad5-EGFP was injected into the scala media through a cochleostomy in C57 mouse,three days later,the pattern of EGFP in the cochlear was observed.Results After the 78DH injection at a dose of 5ug/g/d,the mice did not show much improvement of hearing threshold(P＞0.05).However,all of the epithelial cells in the organ of Corti and the SGNs were kept intact after treatment although the tunnel of Corti was still unopened.The expression of GFP was firstly detected after 3 days injection of Ad5-Egfp in the supporting cells of cochlear.Conclusions 78DH can effectively protect the cell death in the organ and SGN in the Cx26 null mice model.Ad5-Egfp can be expressed in the supporting cells and can be a potential method for the treatment for cCx26 null deafness mice model.