The Mechanisms Underlying The Effects Of Peroxynitrite- Elicited Damage And Apoptosis On Cochlear Hair Cells

Part ⅠThe Mechanisms Underlying the Effects of Peroxynitrite-elicited damage and apotosis on Cochlear Hair CellsObjectives:The present study was designed to investigate the damage and apotosis response of peroxynitrite (ONOO-) on cochlear Hair cells (HCs) of C57 healthy P3 rat on vitro culture, as well as to explore the apoptotic pathway of peroxynitrite induced damage in HCs and the mechanismsun underlying such effects.Methods:The organ of Corti in the whole cochlea from P3 C57 rats were isolated and cultured for 12 hours. Then, the cochlear organotypic cultures were treated with various concentrations and durations of peroxynitrite to induce oxidative stress. Samples in the control group received no peroxynitrite treatment. Confocal microscopy was used to evaluate the condition of HCs following immunohistochemical staining and the apoptosis of HCs following TUNEL labeling staining. In addition, the ultrastructure of the stereocilia bundle, hair cells were examined by transmission electron microscopy. The basilar membrane was cultured in vitro for 24 hours and were randomly selected into a control and 4 test groups. Samples in the first test groups were treated with peroxynitrite at 50 μM.100 μM,200 μM for 24 hours, which were then prepared for confocal microscope examination via immunofluorescence staining of Myosin7a. The second test group was treated with peroxynitrite at 100 μM for 24 hours, which were then prepared for detection by transmission electron microscopy. The drug treatment for the third test group was designed as the first test group preparing for the TUNEL labeling staining. The drug treatment for the fourth test group was designed as the second test group preparing for confocal microscope examination via immunofluorescence staining of Myosin7a, Caspase-3 and AIF. The fifth test group was designed for confocal microscope examination via immunofluorescence staining of Tuj-1 and Caspase-3 with culturing the middle turn of cochlear axis (keep complete conection between ANF and SGN). The number of hair cells within a 240 μm segment was counted on 15 sequently selected images and compared statistically using the SPSS 17.0 and Graphpad Prism 5.0 software.Result:The IHCs and OHCs in the control group were always arranged neatly in order with clear cell profile, with little or no OHC loss being seen after cultured for 48 hours. In the test group after 50 μM peroxynitrite treatment for 24 hours, the number of hair cells in the apical and middle turn showed no significant morphological changes, only with few hair cell loss and disorganized being observed in basal turn. The survival rate of cells was 86.99 ± 1.64% compared with normal control group. The hair cells presented an increasingly disorderly arrangement with the increase of drug concentration. The damage degree at the middle and basal turn was more severe than the 50 μM peroxynitrite treatment group than 100 μM group. Hair cell loss was quantified as 47.20 ± 2.24%. At the highest concentration (200 μM peroxynitrite). treatment for 24 hours resulted in approximately 19.81 ± 3.51% loss of hair cells. There were statistically significant differences in only samples treated with 200 μM peroxynitrite for apical turn. However, the number of HCs in the middle and basal turns was statistically different from the control group in all peroxynitrite treated samples.In the second test group, we can observe ultrastructural changes by transmission electron microscope after 100 uM peroxynitrite treatment:a fusion and loss of stereocilia bundles in hair cells and vacuolation in cytoplasm and degradation of the nuclear. While, control hair cells cultured for 24 hours revealed neatly stereocilia and complete cell strcture with normal outer, inner, and cristal membranes.Apoptosis was determined by TUNEL staining in the third test group. The result showed that the apoptotic rate of hair cells in normal control group and the 50 uM Peroxynitrite group was 4.36 ± 1.47% and 6.31 ± 0.99% respectively, the difference had no significance (P= 0.078). However, the difference was both significant while comparing with the treatment group of 100μM peroxynitrite (40.27 ± 3.26%) and 200 μM peroxynitrite (60.55 ± 3.54%). In morphological changes, the inner and outer hair cells had different levels of deletions, especially in the outer hair cells, the remnants of the outer hair cells are in disorder and deleted a lot, while the inner hair cells didn’t delete much, and morphological changes are visible, such as the nucleus concentration, fragmentation, cytoplasm had visible contraction, moreover, the volume of cells was shrank compare with normal hair cells and irregular, the damaged hair cells show bright green.In the forth test group, immunofluorescence was further applied to detect the expression of apoptosis relevant factors such as Caspase-3 and AIF after the treatment of 100μM Peroxynitrite. It found out that the hair cells in the experimental group had no Caspase-3 positive, while AIF appeared point-like red fluorescent on the position of hair cell nuclei, showing nuclear translocation phenomenon.Immunofluorescence was used in the fifth test group to detect the expression of anti-Casepase-3 and Tuj-1 antibody which marked ANF and SGN after the treatment of 100 μM Peroxynitrite. It found out that the hair cells in the experimental group showed both Caspase-3 and Tuj-1 marked positively in SGN, while negatively in control group.Conclusions:To the best of our knowledge, this is the first study on the damage effects of peroxynitrite and the apoptotic mechanism underlying such effects on basilar membrane hair cells in vitro cultured for P3 mice. These findings from the present work prove that peroxynitrite could markedly reduce the cell viability of hair cells, induce apoptosis and oxidative damage, and that the hair cells present an increasingly disorderly arrangement with the increase of drug concentration and the more serious injury tendency from apical turn to basal turn in cochlea hair cells. Then it is also suggested that the translocation of AIF to the nucleus without any involvement of Caspase-3 activation indicats that the Caspase-independent pathway is, indeed, operated in the apoptosis reaction when hair cells exposed to peroxynitrite. While, the result indicated that the Caspase-dependent pathway participates in the apoptosis reaction of SGN induced by peroxynitrite.Part ⅡThe Regulation of Wnt signaling pathway in the processing of Peroxynitrite induced damage on Cochlear Hair CellsObjectives:To study the regulation function of Wnt3a (Wnt signal agonist) and IWP-2 (Wnt signal inhibitor) on Peroxynitrite induced damage of mouse cochlear hair cells, and to further explore the possible mechanism of Wnt signaling pathway regulating hair cells apoptosis.Methods:Cochlear basilar membrane dissection in P3 healthy C57 mice was conducted with the application of cochlear micromanipulative technique, then, was subjected to the primary culture in vitro for 24 hours. There was no drug treatment in the normal control group, keeping on culturing for 24 hours; on the other hand, the experimental group was further divided into four subgroups according to appropriate concentration of Peroxynitrite. Wnt3a+Peroxynitrite, IWP-2 and IWP-2 plus Peroxynitrite, respectively, keeping on culturing for 24 hours. Immunohistochemical staining and TUNEL staining were exployed for the further experiment, with the primary antibody Myosin7a for marking hair cells. Confocal microscopy was used to observe the damage of hair cells and to select images for hair cells count and with SPSS 17.0 and Graphpad Prism 5.0 software for statistical analysis.Results:The inner and outer hair cells of cochlear basilar membrane in control group with no drug treatment were arranged in neat, evenly colored with clear structure displaying intact blue nuclei and green fluorescence endochylema. In the first group after 24 hours of 100 μM Peroxynitrite on cochlear hair cells, both the inner and outer hair cells had different levels of deletions, especially in the outer hair cells. The remnants of the outer hair cells arranged in disorder with a loss of some outer hair cells while a little loss of inner hair cells. The morphological changes were visible, such as nucleus concentration, fragmentation, visible cytoplasm contraction. Moreover, the volume of damaged cells, which showed a bright green fluorescence, was shrank and irregular comparing with normal hair cells. The total survival rate of hair cells was 48.50 ± 2.349% after the treatment of 100 μM Peroxynitrite. The second group was added with a lower concentration of Wnt3a (25 ng/ml), and the immunofluorescence result showedthat both the inner and outer hair cells were arranged in neat, evenly colored, with clear structure, and the damage to hair cells was significantly reduced, the survival rate of hair cells was 92.50±1.62% compared with normal control group, the decreasing of survival cell was not significant (P= 0.065) while the difference was statistically significant (P< 0.05) comparing with the survival rate in Peroxynitrite treatment group. TUNEL staining showed that the apoptotic rate of hair cells in normal control group and the Wnt3a protection group was 2.25 ± 0.84% and 5.00 ± 1.18% respectively, the difference is not significant (P= 0.078), however, the difference was significant while comparing with the treatment group of 100 μM Peroxynitrite(45.28 ± 1.67%). The result in the third group showed that with Wnt signaling inhibitor of IWP-2, the total survival rate of hair cell was 98.33 ± 3.54%, no significant decreasing compared with the normal control group (P> 0.05), but the survival rate was statistically significant compared with the Peroxynitrite treatment group (P< 0.05). In the fourth group, the result showed that the survival rate of hair cells in IWP-2 plus Peroxynitrite treatment group had reduced to 43.75 ± 2.85% of normal control group, and the number of hair cells was significantly reduced compared with normal control group and IWP-2 treatment group, the difference was statistically significant (P< 0.01). while change of the survival rate of hair cells in Peroxynitrite treatment group was not significant compared with IWP-2+Peroxynitrite treatment group. (P= 0.618).Conclusion:In this study, we explore the regulation function of related factors in Wnt signaling pathway namely Wnt3a and IWP-2 on Peroxynitrite induced cochlear hair cells damage. It demonstrates that Peroxynitrite can reduce the survival rate of cochlear hair cells in suckling mice, inducing oxidative damage and apoptosis for the hair cells; Wnt3a has a protective effect on the cochlear hair cells damage caused by Peroxynitrite in mice, suggesting that Wnt signaling pathway plays a positive regulatory role during the process of Peroxynitrite induced cochlear hair cells damage: IWP-2 does not appear to increase the damage of Peroxynitrite induced hair cells damage, in addition no significant damage or inhibitory effect on the growth of hair cells invitro culture. Wnt3a can be used as a potential anti-oxidant with a role in prevention of sensorineural hearing loss due to the injury of hair cells caused by oxidative stress.