Experimental Study Of Cisplatin Ototoxicity In Vitro

Part 1 Effects of polybrene on lentivius transfection of cochlear cellsObjective: To detect the cytotoxic effects of polybrene, and the further effects thereof,on lentiviral transduction of cochlear cells, especially sensory hair cells(HCs).Material and Methods: Cochlear basilar membranes of newborn rats were cultured and treated with 0.1-10 μg/m L polybrene for 24 h to evaluate the loss and morphology of HCs to determine the safe dose of polybrene. TUNEL assay and PI staining were used to observe the apoptosis and necrosis of HC after cultured with 2 μg/m L polybrene. Various doses of lentivirus-GFP were added to cochlear organotypic cultures incubated for 24 h, and cultured(in the absence of the virus and polybrene)for a further 48 h to evaluate the transfection efficiency to determine the optimal dose of lentivirus. Then the optimal dose of lentivirus with safe polybrene were added to the cochlear explants to observe the effects of polybrene on transfection.Results: After cochlear explants cultured with 0.1 μg/m L polybrene for 24 h, HCs were very similar to control cultures. When the polybrene concentration was increased to 0.5 μg/m L, mild damage to HCs stereocilial bundles was evident, and a few cells were missing. When the concentration of polybrene was increased to 1μg/m L, the stereocilial bundles of both the inner hair cells(IHCs) and outer hair cells(OHCs) collapsed, and mild loss of HCs was evident. In contrast, polybrene at 2 and 5μg/m L severely damaged cochlear HCs; the higher polybrene concentration caused more damage. Moreover, in residual HCs, the stereocilial bundles were either partially or completely missing, and the HC bodies were shrunken and disorganized. Very few HCs were observed when the cochlear explants were exposed to 10 μg/m L polybrene.Averaged loss of hair cells showed that polybrene at 0.1 μg/m L caused little IHC or OHC loss(Kruskal–Wallis one-way ANOVA on ranks, pairwise comparisons,P>0.05). At 0.5 μg/m L, polybrene caused slight OHC loss, compared with the control group(Kruskal–Wallis one-way ANOVA on ranks, pairwise comparisons, P>0.05).When the polybrene concentration exceeded 0.5 μg/m L, significant increases in both IHC and OHC loss were observed(Kruskal–Wallis one-way ANOVA on ranks,pairwise comparisons, P<0.05). PI-positive nuclei debris were observed in the HC region, TUNEL-positive cells were not seen. Most nuclei in the supporting cells adjacent to HCs appeared normal. The average numbers of transductions field for each lentiviral suspension were as follows: 62.75±18.65(1×106 infectious units),196.83±22.34(5×106 infectious units), 205.42±5.74(1×107 infectious units), and247.44±13.51(1×107 infectious units+0.1 μg/m L polybrene). A significant increase in GFP-positive cell numbers was evident when 1×107 infectious units were combined with 0.1 μg/m L polybrene, compared with the polybrene-free sample(one-way ANOVA followed by the Bonferroni test; P<0.05. HCs(exposed to polybrene or not)showed no fluorescence.Conclusion: Polybrene at 0.1 μg/m L did not damage cochlear cells, but the 0.5-10μg/m L concentration destroyed hair cells in a dose-dependent manner which induced hair cell death by necrosis. However, supporting cells were not damaged. Lentiviral vector transduced into cochlear cells and 0.1 μg/m L polybrene enhanced transduction efficiency. However, hair cells could not be transfected with lentiviral vectors either alone or in the presence of 0.1 μg/m L polybrene. Polybrene could be used in cochlear cells, however, it required further attention.Part 2Role of autophagy in cisplatin-induced ototoxicityObjective: To detect the ototoxitic effect of different concentrations of cisplatin, to explore the machnisim in cisplatin-induced ototoxicity.Material and Methods: Cochlear basilar membranes of newborn rats were cultured and treated with various doses of cisplatin for 48 h to observe the morphology of hair cells(HCs) and spiral ganglion cells( SGCs) and to evaluate loss of HCs.Transmission electron and immunoblotting were applied to detect ultrastructures and expression of autophagy-related protein beclin 1 respectively. TUNEL detection was used to observe hair cell apoptosis at 24 h, 48 h and 72 h after cultured with 10 μM cisplatin.Results: After cochlear explants cultured for 48 h, no significant HCs damage was observed in control group, while 10 μM cisplatin destroyed HCs in disordered arrangement with a mean hair cell loss about 70%. HCs in 100 μM cisplatin were even worse with a mean hair cell loss about 90%, many cellular debris were seen.However, HCs morphology was good and no significant loss was observed in HCs numbers between 400 μM group and control group. Auditory nerve fibers(NFs) and SGCs showed normal appearance while most of them were lost after cultured with 10μM cisplatin. NFs and SGCs were even worse in 100 μM cisplatin which showed slender NFs and irregular cell bodies. Compared with control group celluar bodies of SGCs showed smaller but regular appearance but no significant loss was observed in400 μM group. Transmission electron micrographs showed significant neclei pyknosis and fragment which indicated apoptosis of HCs and SGCs in 10 μM group.Double-layed autophagosome were seen in the cytoplasm of outer hair cell both in control and 400 μM groups, in addition, slightly mitochondrial swelling was observed in the latter group. No significant spiral ganglion cell damage was detected in control and 400 μM groups. With the increase of cisplatin concentration, the expression of autophagy-related protein beclin 1 showed a descending and then elevating trend.After cultured with 10 μM cisplatin for 24 h, no obvious HC apoptosis was seen,however, lots of apoptosic HCs were seen after cultured for 48 h and nearly all the HCs suffered from apoptosis.Conclusion: In cultured condition, cisplatin-induced ototoxicity was not in a dose-dependent fashion, low doses of cisplatin resulted in significant damage of HCs and SGCs which induced by apoptosis, however, no significant damage was observed by high doses of cisplatin which may be associated with activation of autophagy.Autophagy activation allivated cisplatin-induced ototoxicity and had a protective effect on HCs and SGCs.Part 3Expression of copper transporters in cochleaeObjective: To observe the expression of copper transporter 2 in cochlear and to investigate the effect of cisplatin on copper transporter 1 and 2 expression.Material and Methods: Cochlear basilar membrane were separated from day 3-5 rat pups and fixed to conduct copper transporter 2 immunostaining and phalloidin staining. Using cochlear organic culture techniques, cochlear basilar membrane were cultured with 10 μM cisplatin for 48 h and then immunoblotting was used to detect expression of copper transporter 1 and copper transporter 2 in control group and 10μM cisplatin group.Results: Copper transporter 2 was observed in the cytoplasm of hair cell and marginal cell. Immunoblotting showed expression of copper transporter 1 both in control and10 μM cisplatin groups, and no significant change was seen in the expression of copper transporter 1 after cultured with 10 μM cisplatin group for 48 h. Copper transporter 2 expression was missing in both groups.Conclusion: Copper transporter 1 and copper transporter 2 were expressed in cochlear, and copper transporter 2 was located in the cytoplasm of hair cells and marginal cells.