The Effects Of FGF,BMP,WNT Signal Pathways On The Development Of Inner Ear Neuron And Glutamate-induced Neurotoxicity In The Postnatal Mice Cochlea

PartⅠExpression of signal pathway genes in the postnatal micecochleaObjective Signal pathway genes (FGF,BMP,WNT et al) areclosely connected with the function of nerve system. They not only playimportant roles in the regulation of development, maturity, proliferation,but also have strong ability to sustain neuron survival. Previous evidenceindicates that they may also be involved in several aspects of the peripheralauditory system. The aim of this study was to understand the expression ofFGF,BMP,WNT signal pathways and other associated genes in thepostnatal mice cochlea.Methods The cochlea from postnatal 1 day (P1) mice, 20 pieces eachgroup, were removed and cultured in DMEM/F-12 culture medium(containing 2％fetal serum) for short term. Total RNA of the cochlea wasextracted by Trizol one step way. RT-PCR was performed to approve theexpression of these genes before sequencingResults The distinct expression of Nudt6,BMP4,Wnt7a,six1,six4,fas,bcl-2,β-actin were found in each group, and Nudt6 was proved to beexpressed in inner ear for the first time.Conclusions The expression of signal pathways and apoptotic geneswere found in the P1 cochlea, even the antisense gene of bfgf-Nudt6,which suggested that these genes could be involved in auditory function. PartⅡPreliminary studies on the relations between signalpathway and the development of inner ear neuronObjective Signal pathways play important functions during thedevelopment of central nerve system, but its function in auditory sensoryorgan is still unknown. In this study, BMP and WNT signal of culturedcochleas in vitro will be blocked, and the changes of the gene expressionand the tissue configuration will be observed. We expect that this studymay provide foundation for further studies to understand the fuction ofthese genes.Methods Noggin and WIF-1 were used to block BMP, and WNTsignal in vitro, bFGF was added to the culture media. After two days ofculture, total RNA extraction were made, real-time RT- PCR was carried,and apoptotic cochlear neurons were identified with TUNEL labelingtechnique.Results The expression of Six1 (3.3061±0.1454),Six4(6.6179±0.5069),bcl-2 (3.8766±0.3112) significantly raised aftertreatment, and Nudt6 (0.4672±0.0430) was lower than control. However,there were no significant changes in fas gene expression and neuronapoptosis in the histochemical study.Conclusions The expression of Nudt6, Six1, Six4, fas, bcl-2 were changed after using BMP, WNT signal blocker, that neuron apoptosis wasinhibited by signal pathway blocking, which suggested that the signalpathways were important in the development of cochlear neuron.PartⅢThe influence of signal pathways on the glutamateinduced neurotoxicity in the cochleaObjective Glutamate is an important regulator of nerve system.However, excess glutamate has a toxic effect on cochlear neuron. Therole of signal pathways in this process is still unknown. Therefore, thegene expression of the cochlear neuron (P1) treated by additionalglutamate in vitro was quantified, and morphologic changes wereobserved to understand the influence of signal pathways to glutamateinduced neurotoxicity.Methods The cochlea were treated with glutamate (200 uM) beforeBMP, WNT signal were blocked by special antagonists, and bFGF(10ng/ml) was added to the culture medium. After short term culture,total RNA were extracted for real time RT- PCR, and TUNEL was used toidentify the apoptotic neurons.Results After glutamate treatment, apoptotic cochlear neuronincreased, the expression of fas (76.0933±7.2652) was significantlyhigher than bcl-2 (9.6117±1.5023), Nudt6 (7.3138±0.3302) and six (six4 17.4846±0.6854, six1 6.8887±0.3675). But there was no significantchange in BMP and WNT antagonist groups. However, bFGF amelioratedthe effects of glutamate ototoxicity.Conclusions Excess glutamate, even in low concentration, has ancochlear neurotoxicity, which can be aggravated by BMP, WNT signalblocker and be ameliorated by bFGF protein. These foundings providefurther evidences of glutamate induced neurotoxicity.