Study Of The Injury Mechanism Of Cultured Spiral Ganglion Cells From Mouse Cochlea Induced By Gentamicin

【Objective】To explore the injury mechanism in spiral ganglion cells (SGC) of postnatal mouse cochlear in vitro caused by gentamicin.【Methods】SGCs were isolated by using a combinatorial approach of enzymatic digestion and mechanical separation from P2～6 Kunming mouse cochlear.After 4 days,cultured SGCs were fixed by 4﹪paraformaldehyde in room temperature and then immunocytochemical identification was performed by methods of S-P and the monoclonal antibody of mouse neurofilament protein(Neurofilament-68/200Kda,NF-L+H).SGCs of the 4th day in vitro were randomly divided into blank control group and three Gentamicin experimental groups(the concentrations of gentamicin in the medium were 50mg/L,100mg/L,150mg/L),and cultured for 48h.Collect SGCs from different groups respectively and observe the cells of each group with transmission electron microscopy.The expression of apoptotic executive protein Caspase-3 in each group was tested by immunocytochemical method and the staining result was analyzed quantitativly by image analysis system and SPSS statistical software.【Results】The primary culture of SGCs was successful. SGCs usually have two neurites growing relatively to each other and are classical bipolar neurons.The cytoplasm and neurites of SGCs were dyed brownish yellow with the monoclonal antibody of NF-L+H by immunocytochemistry methods. The ultrastructure of SGCs were observed by transmission electron microscopy.It was found that compared to blank control group,SGCs of the experimental groups changed obviously in morphology,especially in the morphology and structure of cell nucleus and mitochondria.These morphological changes may concern with apoptosis.The apoptotic executive protein Caspase-3 masculine reaction staining appeared obviously in the experimental groups,which was higher than that in control group statistically.With the increase of gentamicin concentration,the degree of Caspase-3 masculine reaction staining was getting deeper.【Conclusion】Gentamicin has direct toxic effect on SGCs from mouse cochlear and could induce the apoptosis of SGCs.The damage of mitochondria may play an important role in the process.And the activation of Caspase-3 is involved in the apoptotic process.