Study On The Ultrastructural Changes Of The Cochlear Cells In Connexin 26 Conditional Knockout Mice

Objective:Congenital deafness is one of the most common human genetic birth defects. Among a large number of the deafness genes,the gap junction protein Connexin 26 (GJB2) gene mutations result in hereditary deafness,which accounts for more than half of hereditary nonsyndromic deafness.The study on the pathogenesis of deafness caused by Connexin26 is still being explored,especially the pathological studies is very limited.By systematically observing the ultrastructural changes in cochlear cells of different developmental stages of Connexin26 gene knockout mice,this research will explore the pathogenesis of deafness caused by Connexin 26 gene mutations.Methods:Hybrid Foxg1-Cre cCx26ko conditional knockout mice model(referred as cCx26ko mice) was formed by Cx26 loxP/loxP and foxg1-cre transgenic mice,and wild mice served as controls.Experimental groups included 2 cCx26ko mice at postnatal age of 8(P8),10(P10),18(P18),30(P30),60(P60),90(P90),120(P120), 180(P180) days respectively,and 1 cCx26ko mouse aged 360 days.Control group included 2 wild mice at postnatal age of 10,18,30,360 days respectively.Firstly, separated cochlear specimens from all of the mice,made semithin sections for cochlear location,then processed ultrathin sections.Observed the samples by transmission electron microscope(TEM) and took photos last.The focus was on the pathological changes of hair cells,supporting cells and stria vascularis cells.Results:1.From P8,the formation of Corti tunnel of cCx26ko mice was inadequate and Nuel's space formed partly.Corti tunnel and Nuel's space narrowed significantly at P10.Both Corti tunnel and Nuel's space disappeared at P18.The number of microtubules of inner and outer pillar cells began to reduce after P10,and became less obvious after P18,till P30,the microtubules of the inner pillar cells almost disappeared.2.A small amount of vacuoles in inner hair cells of cCx26ko mice could be seen after P10,the number of lysosomes increased and mitochondria swelled after P18 and P30.At P60,the inner hair cells became deformed and a large number of spaces appeared.At P180,a large quantity of lysosomes appeared,mitochondria swelled and the volume of cell nucleus reduced.The volume of outer hair cell enlarged,cytoplasm was more abundant at P10,abnormal lysosomes and thinner cuticular plate was observed after P60,the outer hair cell became degenerated and necrosis after P90.3.Microglia-like cell appeared in Hensen cell region of cCx26ko mice after P180,which confirmed to be active supportive cells participating the repair and clean-up after damage.4.Little abnormal was observed in stria vascularis during early stage.From P120, the number of lysosomes increased.Large macrophages and pigment granules appeared after P180 and P360 respectively.Conclusions:The abnormal development of Corti tunnel and the inner and outer pillar cell during early stage of cCx26ko mouse may be one important cause of deafness.Hair cell metabolic disorder and absence of energy matter may result in regression and apoptosis.Microglia-like cell and macrophages appeared in organ of Corti and stria vascularis respectively on the later stage,whose function is to clean up cell fragments and participate in the repair process after tissue damage.