Study On Connexin26 Related Late-onset Non-syndromic Genetic Hearing Loss Mouse Model And It's Susceptibility To Acoustic Trauma

PART IEstablishment of Cx26 knocked down mouse models at postnatal day 18Object:To establish Cx26 knocked down mouse models at postnatal day 18.Methods:The Cx26loxP/loxP transgenic mice were crossed with the Rosa26CreER mice to generate the Cx26loxP/WT;Rosa26CreER mice. The Cx26loxP/WT;Rosa26CreER mice were mated with each other to generate Cx26loxP/loxP;Rosa26CreER mice and Cx26loxP/1oxP transgenic mice.At postnatal day 18 (P18), the Cx26loxP/loxP;Rosa26CreER mice were intraperitoneal injected by one dose of tamoxifen to obtain the Cx26 knocked down mice and the Cx26loxP/loxP mice received the same injection were used as control. Western Blot was used to detect the Cx26 expression in these mice. Auditory threshold were evaluated by measuring the auditory brainstem responses (ABR).Results:Western Blot confirmed that Cx26 protein in cochlear was down regulated in Cx26loxP/loxP;Rosa26CreER mouse models. ABR testing indicated there was a significant hearing loss in Cx26 knocked down mice of five month old while no hearing loss at the age of one month.Conclusion:We have successfully established the Cx26 knocked down mouse models at postnatal day 18.Part IIThe effect of Cx26 knocked down in the late postnatal days on acoustic deafnessObject:To investigate the susceptibility of Cx26 knocked down mouse at P18 to acoustic trauma.Methods:Mice were divided into four groups named Control group, Knocked down group(KD group),Noise group and KD+Noise group. All the mice received intraperitoneal injection of TMX at P18.Cx26loxP/loxP mice were used in Control and Noise group, and Cx26 was only down regulated in Cx26loxP/loxP;Rosa26CreER mice in KD and KD+Noise group. Mice in Noise group and KD+Noise group were exposed to consecutive white band noise 110dB SPL from P25,8 hours per day,for 5 days. Western Blot and immunofluorescence were used to detect the cx26 expression in the cochlear of these four groups mice at P30. Auditory threshold were evaluated by measuring the auditory brainstem responses (ABR) at P30 and P45.Results:Comparing to the mice in Control group, the Cx26 protein was much lower in the mice of KD and KD+Noise group. ABR testing indicated mice in Control and KD group showed normal hearing at P30 and P45. After noise exposure, mice in Noise group and KD+Noise exhibited a similar severe hearing loss at the frequency 8-48KHz at P30. At P45, the threshold shift of mice in Noise group decreased at the frequency 8-32KHz but failed to decrease in KD+Noise group.Conclusion:Before the postnatal day 45, mice Cx26 knocked down at P18 showed normal hearing the same as conrol but showed more severe hearing loss after acoustic trauma comparing to Noise group.Part ?The pattern of cellular degeneration in the cochlea of the cx26 knocked down mouse after acoustic traumaObject:To explore the pattern of cellular degeneration in the cochlea of the cx26 knocked down mouse after acoustic traumaMethods:Mice were divided into four groups named Control group, Knocked down group(KD group),Noise group and KD+Noise group. All the mice received intraperitoneal injection of TMX at P18.Cx26loxP/loxP mice were used in Control and Noise group, and Cx26 was only down regulated in Cx26loxP/loxP;Rosa26CreER mice in KD and KD+Noise group. Mice in Noise group and KD+Noise group were exposed to consecutive white band noise 110dB SPL from P25,8 hours per day,for 5 days.Hair cell loss at P30 and P45 was estimated quantitatively by cytocochleogram using whole-mount cochlear preparations. The cochlear morphology were checked by resin plastic sections at P45.Transmission electronic microscope was used to observe the utralstructure of the cochlear cells.Results:Rarely loss of HCs was observed in the cochlear of mice in Control and KD group at P30.Up to 17.8% of the OHCs were absent in the basal turn in the cochlear of the mice in Noise group,while 17.0%-56.1% in the middle and basal turns in the cochlear of the mice in the KD+Noise group.And IHCs in these two groups showed no significant loss. At P45,rarely HCs were absent in the mice in Control and KD group.Loss of OHCs extended mildly in the mice of Noise group,and up to 25.5% of the OHCs disappeared in the basal turn.17.6%-60.0% of the OHCs were absent in the middle and basal turns in the cochlear of the mice in KD+Noise group. Resin plastic sections implied that the lateral wall,the stria vascularis and the supporting cells showd no significant abnormity in all four groups.Abnormal mitochondrials were observed in the intermediate cells of the stria vascularis in the mice of KD+Noise group by the transmission electronic microscope.Conclusion:HCs were survived till P45 in the mice Cx26 knocked down in the late postnatal day. Exposure to consecutive high intensive noise resulted in minor OHCs loss in the basal turn of the normal mice. And part of OHCs in the middle and basal turns degenerated in the mice Cx26 knocked down in the late postnatal days after exposed to consecutive high intensive noise.Part IVConnexin26 knocked down after birth effectted the microtubules in the pillar cellObject:To explore the Connexin26 knocked down after birth effect on the microtubules in the pillar cell.Methods:Cx26loxP/loxP; Rosa26CreER mice and Cx26loxP/loxPmice were injected TMX subcutaneous at PI. Imunofluorescence was used to detect the Cx26 expression in the mice of the two groups at P4 and P9. Resin plastic sections were sliced to observe the morphology of the cochlear at P9 and P18.Transmission electronic microscope was used to observe the utralstructure of the pillar cell.Results:Immunofluorenscence reveald that the Cx26 expression decreased in KD group comparing to the Control group at P4 and P9. Resin plastic sections showed the developed organ of Corti,the opened tunnel of Corti and the formed space of Nuel in all the three turns in the cochlear of the mice in control group at P9. But the KD group performed contrary: the tunnel of Corti was not opened and the space of Nuel wasn't formed.At P45 in the KD group,the tunnel of Corti was still not opened,while the space of Nuel was still closed.Supporting cells and HCs loss were observed in the middle turn and basal turn in KD group,but the lateral wall and stria vascularis were normal.Bundles of microtubules in the pillar cell in control group could be observed by transmission electronic microscope at P9,while rarely could be observed in KD group.Conclusion:The tunnel of Corti could not open and microtubules decreased in the mice Cx26 knocked down at P1.