The Role Of Connexin26in The Late Developmental Stage Of Mouse Cochlea

PART IEstablishment and identification of connexin26knocked down mouse models at different postnatal time pointsObject:To establish and identify connexin26(Cx26) knocked down mouse models at different postnatal time points.Methods:By genetic approaches, we crossbred the Cx26loxp/loxp transgenic mice with the Rosa26CreER mice to generate Cx26loxp/WT;Rosa26CreER as first filial generation. Interbreeding the first filial generation mice, Cx26loxp/loxp;Rosa26CreER mice were acquired. Connexin26was knocked down in Cx26loxp/loxp;Rosa26CreER mouse at different postnatal time points by a single subcutaneous injection of tamoxifen (TMX) on the postnatal day1(P1),6(P6) and12(P12) respectively. Genotyping, Western Blot and immunofluorescence were used to detect the genetic background and the connexin26expression in these mouse models. Auditory threshold in these mice models were checked by measuring the auditory brainstem responses (ABR).Results:The Cx26loxp/loxp; Rosa26CreER mice reproduced stably, with normal weight and development as well as bright coat color, and no obvious degeneration phenomenon were observed. Western Blot and immunofluorescence confirmed that connexin26were all knocked down in the cochlea of these mice received injections at three different time points. ABR testing indicated that P1and P6knocked down (KD) groups appeared middle to severe hearing loss in the early stage, while P12KD group showed normal hearing in the early stage and progressive hearing damage in the late stage.Conclusion:We have successfully established connexin26knocked down mouse models at different postnatal time points. Connexin26knocked down mice display distinct long term injury patterns in the cochlea at different postnatal time pointsObject:To investigate and compare the degeneration of cochlea hair cells, morphological changes in the organ of Corti (OC) and the secondary death of spiral ganglion cells (SGCs) in connexin26KD mice at different postnatal time points.Methods:Cochlear hair cell loss and OC morphological changes in P1, P6and P12KD groups were observed by whole-mount cochlear preparation (n=3) and axial sections of cochlea (n=3) at1,3and5month after birth. The level of SGNs death in experimental and control groups at various time points was compared by spiral ganglion cell counting (n=3).Results:Whole-mount cochlear preparation showed massive hair cell loss in the middle turn of the cochlea in P1and P6KD group at one month after birth, and gradually expanded to the basal turn of the cochlea, with increasing damage in the middle turn. P12KD group appeared small amount of cell loss in the basal turn of the cochlea at three month after birth. Sections with HE staining showed the tunnel of Corti (TC) was failed to open and gradually appeared massive supporting cell (SC) death in P1KD group, while OC could opened normally in P6and P12KD groups. There was no obvious changes in the structure of stria vascularis, lateral wall and other parts in above three groups. Spiral ganglion cell counting suggested secondary SGC death was found in the corresponding place of HC loss in P1and P6KD group, while no distinct SGC death was found in P12KD group.Conclusion:The degree and range of HC, SC and SGC loss were significantly worse in the early connexin26KD group than in the late connexin26KD group, indicating that connexin26plays an important role in the early stage of postnatal cochlea development, while its function in the late stage is replaceable. Investigation on cochlear cell injury and glucose transport disorder in connexin26KD mice at the early postnatal time pointObject:To explore the relationship between cochlear cell injury and glucose transporter (GLUT) changes as well as cellular energy metabolism in connexin26KD mice at early postnatal time point.Methods:Western blot was used to test the expression of phosphorylation AMPK protein of cochlea in P1KD mice (n=5). GLUT changes were estimated by real-time quantitative PCR in these mice.Results:There was no significant difference between the expression of phosphorylation AMPK protein of cochlea in P1KD group and in control group. The mRNA expressions of GLUT1, GLUT8and GLUT10showed no significant differences between P1KD group and control group.Conclusion:There may have no obvious correlation between cochlear cell injury and cellular energy metabolism disorder in P1KD mice. The knocking down of connexin26has no significant effect on the regulation of GLUT in cochlear.