Study Towards The Mechanism Of Connexin26 And Connexin30 In Genetic Deafness With Gene-knockout Mice Models

PARTⅠGeneration of connexin30 and inducible conditional connexin26 nullmouse modelsObject: To generate connexin30 （Cx30） and inducible conditionalconnexin26 （Cx26） null of transgenic mouse models.Methods: We back crossed the Cx30 knock out （KO） mice with a C57/BLgenetic background mice with a CBA/CaJ background for eight generations.Cx26 inducible KO (Cx26^flox/flox;Rosa26Cre^ER/+) mice were generated by onedose injection of tamoxifen （0.1mg/g of body weight, I.P.） to pregnant motherwith at embryonic day 19 （E19）. The tamoxifen-induced Cre recombinaseactivities were checked by using a reporter mouse (Rosa26^{CreER/LacZ-stop}).Genotyping, western blot and immunolabeling were used to confirm thatCx26 and Cx30 were knock out in these Cx mutant mouse models. Hearingthreshold changes of Cx KO mice were assessed by measuring the auditorybrainstem responses （ABR）. Results: The reporter mouse result confirmed that strong Cre activities wereinduced in the sensory epithelial and lateral wall of the cochlea by theinjections of tamoxifen （TMX） . Genotyping, western blot andimmunolabeling confirmed that Cx26 and Cx30 were knock out in these Cxmutant mice models. ABR testing revealed severe to profound hearing lossacross all frequencies in these mice.Conclusion: We have obtained two valid mice model for studying congenitalnonsyndromic deafness caused by Cxs null mutation.PartⅡConnexin30 and inducible conditional connexin26 mice display distinctpattern and time course of cellular degeneration in the cochleaObject: We have used Cx30 and inducible conditional Cx26 （cCx26） nullmice to investigate the time course and regional pattern of degeneration ofhair cells and the secondary death of spiral ganglion neurons （SGNs） in thecochlea.Methods: Hearing threshold changes of Cx KO mice were assessed bymeasuring the auditory brainstem responses （ABR） elicited by tone bursts inthe frequency range of 4-32 kHz. Postnatal day 18 （P18） to 18 month-old Cx30 KO mice （n=5）, and P18 to 5 month-old Cx26 inducible KO mice （n=5）were tested. Hair cell loss was quantitatively estimated by cytocochleogramusing whole-mount cochlear preparations stained with DAPI （n=6）. Theneuronal cell death was quantified by point-counting the remaining SGNsfrom 5μm plastic resin sections （n=6）.Results: In the Cx30 KO mice, ABR testing revealed severe to profoundhearing loss across all frequencies at P18. The hearing progressively get worseand the mice were totally deaf at 12 months of age. In the cochlea of Cx30null mice, survival of most inner HCs, supporting cells and SG neurons wasobserved for up to eighteen months. The most severe degeneration was foundin apical SG neurons and OHCs. OHC loss followed a slow time course and abase to apex gradient. Similar to the results shown by the Cx30KO mice,severe hearing loss across all frequencies was observed for the inducible Cx26KO mice at P18. Cochleograms showed that all types of cells in the organ ofCorti disappeared in the middle turn of the cochlea. The hair cell loss thenextended to apical and basal turns with aging. Most SGNs in the middle andbasal turns disappeared in the first three months, while significant amounts ofapical SGNs survived. Gross structures of the endolymphatic space and striavascularis observed at the light microscope level were unchanged in either Cxnull mouse models. Conclusion: The radically different pathogenesis processes displayed bycCx26 and Cx30 null mice suggest heterogeneous underlying deafnessmechanisms, despite of the coassembly of Cx26 and Cx30 in forming gapjunctions in the cochlea.PartⅢConnexin26 play important roles in cochlear morphogenesis andendolymphatic potential generationObject: To explore the role of Cx26 in the endolymphatic potential （EP）generation and cochlear morphogenesis.Methods: We injected TMX to reduce the expression of Cx26 in the organ ofCorti at different stages in the induciable cCx26 null mice during the EPgeneration. The time points are E19, P2, P4, P6, P8, P10, P12, P16. Thetamoxifen-induced Cre recombinase activities were checked by using areporter mouse (Rosa26^{CreER/LacZ-stop}). To quantify the change in Cx26 proteinlevels in various regions of the cochlea resulted from the TMX injections inCx26^loxP/loxP; Rosa26^CreER mice, we performed western blots using cochlearsamples obtained separately from the stria vascularis, spiral ligament and organ of Corti. The morphology changes during P4, P8, P10, P12, P14, P18,P24, P30 were check by plastic resin sections （5μm）. Hearing threshold wereassessed by measuring the ABR. EP was measeured at P30 with all groups.Results: LacZ staining patterns in the cochlear of reporter mice showed noobvious difference between TMX injected at P4 and P16. Western blotsconformed that Cx26 was knock down at all TMX injected groups. Thedeafness phenotype and moderate reduction in EP was readily induced if theCx26 expression in the organ of Corti was substantially reduced before P4.Reducing Cx26 expression after P4, however, generated a phenotype moreconsistent with symptoms of early-onset age-dependent hearing loss. Whenwe injected TMX before P4, the postnatal development of the organ of Cortiwas stalled as the tunnel of Corti and the Nuel space were never opened incCx26 null mice. Starting at around P14, epithelial cells in the organ of Cortistarted to degenerate and all types of cells in the middle turn were completelylost by P30.Conclusion: Cx26 plays essential roles in the EP generation and postnatalmaturation of the organ of Corti, also is required for the survival of all types ofcells resided on the basilar membrane. PartⅣProtection SGN degeneration by small molecular agonist of TrkBreceptor 78-DHFObject: To promote the survival of SGNs using a novel small molecularagonists of TrkB receptor 78-DHF in cCx26 null mouse model.Methods: cCx26 null mice were generated by one dose injection of tamoxifen（0.1mg/g of body weight, I.P.） to pregnant mother with at embryonic day 19（E19）. From P2 to P30, the new born Cx26^loxP/loxP; Rosa26^CreER mice wereinjected with 78-DHF（1μg/ml, 5μl/g of body weight, I.P.） as experimentalgroup（n=6）. The Cx26^loxP/loxP; Rosa26^CreER mice injected with 20%DMSO/PBS used as blank control （n=5）. Rosa26^CreER mice used as littermatecontrol （n=5） . The morphology changes were check by resin plastic sections（5μm） at P30. Hair cell loss was estimated using whole-mount cochlearpreparations stained with myosinⅥ（n=6）. Hearing threshold were assessed bymeasuring the ABR.Results: All the mice were killed at P30, in the experimental group all thecells in the organ of Corti were disappeared while the SGNs in the middle turnwere completed rescued compared with blank control. The ABR resultsshowed at P30 hearing threshold were significantly elevated across afrequency range of 4-32 kHz while no obvious difference between experimental treatment group and blank control group. The littermate controlmice have normal hearings.Conclusion: Small molecular agonists of TrkB receptor 78-DHF can dramaticpromote the survival of SGNs in ccx26 mice.