Effect Of Wnt Signal Pathway On Paclitaxel-induced Damage On Spiral Ganglion Cells Of Rat Cochlea

BackgroundSensorineural deafness is a relatively common disorder with acoustic disturbance and it's usually severe deafness.Lesions are usually implicated in hair cells,auditory nerve and auditory centers at all levels.There are barriers of patients'feeling of sound and the transmission of nerve impulses,leading to the hearing loss.Sensorineural deafness reduces the patient's quality of life badly.Deafness caused by loss of hair cells is called sensory deafness(cochlear deafness)while deafness associated with auditory nerve and its pathway called neural deafness(retrocochlear deafness).It's hard to cure the retrocochlear deafness as the lack of nerve cell's ability to repair itself.As the recovery strategy for sensorineural deafness,a new exploration suggests that stem cells restore hearing is a potential treatment.Stem cells have the potential to differentiate into new hair cells,neurons and support cells.Research has shown that Wnt signal pathways play a decisive role for the proliferation of neural stem cells.The activation of Wnt signal pathways may turn the non-nerve cells into the cell cycle again to transform into neurons,and this makes it possible to treat the retrocochlear deafness.Wnt signal pathway is a highly conserved signal pathway in evolution,which plays an important role in many biological processes.Proteins of the pathway bind to receptors on the cell membrane by autocrine or paracrine,activate the downstream signaling pathways,and regulate the expression of target genes,thereby affecting the process of cell proliferation,differentiation and apoptosis of cells such as polar cells and stem cells.The abnormal activation of the pathways is often associated with the occurrence of tumor.Wnt signaling pathway includes the classic Wnt signaling pathways and the non-classic Wnt signaling pathway.The concentration and transportation into the nucleus of ?-catenin declare the activation of the classical Wnt signal pathways.After transported into the nucleus,Beta-catenin combines with the transcription factors to regulate the expression of downstream target genes.The non-classic Wnt signal pathways which do not depend on ?-catenin,are made up of other proteins or ions.The lack of cochlear spiral ganglion cells can cause neural deafness.A large number of studies have shown that many common injuries,such as ototoxic drugs,noise,and anoxia,can cause apoptosis of cochlear hair cells and spiral ganglion cells in mice cochlear and cause deafness.Current researches of ototoxic drugs chiefly focus on cisplatin.The ototoxicity of paclitaxel is often ignored due to the drug combination with cisplatin.Studies also show that paclitaxel has indeed neurotoxicity,so we can guess ototoxicity of paclitaxel is associated with nerve cells of the ear.As the first level of auditory pathway,spiral ganglion neurons are suspected to be the target point of the injury.The purpose of this work is to investigate whether or not the paclitaxel can cause the injury of spiral ganglion cells in rat cochlea as well as the possible effect of Wnt signal pathway in this course.ObjectiveTo investigate whether or not the paclitaxel can cause the injury of spiral ganglion cells in cochlea of C57BL/6 mice as well as the possible effect of Wnt signal pathway in this course.Methods1.Cochlea organotypic cultures from postnatal day three rats of C57BL/6 mice were achieved.2.The cochlea were treated with different concentrations of paclitaxel and the cell survival rate was measured by the MTT method to select appropriate concentration for further experiments.The cochlear transits were randomly divided into four groups and treated with the prudential,paclitaxel,paclitaxel plus Wnt3a,an agonist of Wnt signal pathway,and paclitaxel plus IWP2,an inhibitor of the Wnt signal pathway,Immunofluorescent staining and TUNEL staining were employed after 48 h to observe the changes of spiral ganglion and nerve fibers.Western blot was employed to verify the result of the immunofluorescent staining.3.One-month-old mice were treated with paclitaxel by intraperitoneal injection.The mice were dissected after ABR audiometry.The cochlear were made into frozen sections and immunofluorescence staining was employed to observe the changes of spiral ganglion.Results1.The result of MTT showed that the reduction of the cell survival rate occurred at concentration as low as 1 ?l paclitaxel and the spiral ganglion injury depended on the concentration of paclitaxel.A concentration of 30?l was chosen for following experiments.Immunofluorescence staining showed that the structure of spiral ganglions in paclitaxel group was destroyed,and the number of cells and nerve fibers was decreased.2.According to the immunofluorescence staining,the SGN cells were destroyed.There were manifestation of apoptosis such as nuclear pyknosis and apoptotic bodies.The existence of apoptosis was confirmed by TUNEL staining.3.The staining of Caspase-3 was positive in paclitaxel group.The increased expression of Caspase-3 was confirmed by Western blot.4.The damage of cells in group treated with Wnt3a fell between the control group and the paclitaxel group while the damage of cells in IWP2 group was severity.5.There was no significant difference between the ABR results of the intraperitoneal injection mice and the normal one-month-old mice,but the frozen section showed the injury and apoptosis of spiral ganglion neurons in rat cochlea.Conclusion1.Paclitaxel leads to the injury of the spiral ganglion cells in rat cochlea.2.Paclitaxel induces spiral ganglion apoptosis,which is probably dependent on a capase-3-activated pathway.3.The activation of Wnt signal pathway protects spiral ganglion cells in rat cochlea from paclitaxel-induced damage in certain degree.