Role Of CTR1, ATP7A And ATP7B In Cisplatin Induced Ototoxicity Of Wistar Rat And The Protective Effect Of Copper Sulfate

Objective:To investigat the functional and morphological changes of cochlea in wistar rat caused by different delivery methods of cisplatin, and seek an effective way of building the animal model of cisplatin-induced ototoxicity.Method:Male Wistar rats were randomly divided into three groups:round window niche saline infusion group (group Ⅰ), round window niche cisplatin infusion group (groupⅡ) and intraperitoneal injection cisplatin group (group Ⅲ).The auditory brainstem response, cochlear axis slices with HE staining, basement membrane hematoxylin staining and hair cell counting were used to evaluate the functional and morphological changes of wistar rat.Result:1. Auditory brainstem response:the hearing threshold shift (HTS) of group Ⅰ, group Ⅱ and group Ⅲ were1.66±4.08dB SPL,56.67±17.51dB SPL and41.67±14.72dB SPL respectively. The HTS of group I compared with group II and group III respectively, the differences were significant statistically significant (P<0.01). The HTS of group Ⅱ compared with group Ⅲ, the difference was not statistically significant (p>0.05).2. Cochlea axis slice HE staining:The morphous of Corti's organ, spiral ganglion cells and stria vascularis of group Ⅰ were normal. The Corti's organ of group Ⅱ and group Ⅲ shrinked and the outer hair cells ruptured or disappeared. The numbers of spiral ganglion cells of group Ⅱ and group Ⅲ were reduced and the nucleuses were decrescent with the nerve fibres disorderly arranged. The structure of stria vascularis were breakdown.4. Cochlea basilar membrane stretched preparation and hair cell counting:Three lines of outer hair cells in group Ⅰ were kept well and dyed uniformity and one line of the inner hair cells were all intact. Three lines of outer hair cells in group Ⅱ and group Ⅲ arranged disorderly and some of them were lost, but the inner hair cells were nearly intact. The average loss rate of outer hair cell in group Ⅰ of each turn were rather lower than in group Ⅱ and Ⅲ, showing statistically significant difference (p<0.05). The losses of outer hair cell in group Ⅱ and Ⅲ were mainly observed in the base turn. There were not significant losses of outer hair cell in the apical turn of each group. Compared the average loss rates of outer hair cell of group Ⅱ and Ⅲ of each turn, showing no statistically significant difference (p>0.05) The losses of inner hair cell in each turn were slodemly.Conclusion:1. The surgical procedure of round window niche drugs administration does not cause functional and morphological changes of the cochlea in wistar rat.2. The round window niche cisplatin (1mg/ml) infusion can successfully create the animal model of cisplatin-induced hearing loss. It is a simple way which can keep a healthy status of the animal body with a low death rate. Objective:To explore the spatial distributions of copper transport proteins CTR1, ATP7A and ATP7B in the cochlea, and to investigate the expressions changes of CTR1, ATP7A and ATP7B proteins after copper ions or cisplatin round window niche administration, providing the theoretical basis for the prevention of cisplatin-induced ototoxicity.Method:Male Wistar rats were randomly divided into four groups:, normal control group (group Ⅰ); the round window copper sulfate(0.02mg/kg) infusion group(group Ⅱ); the round window copper sulfate(0.04mg/kg) infusion group (Group Ⅲ); the round window cisplatin infusion group (Group Ⅳ). The cochleas were paraffin-embedded. The CTR1, ATP7A and ATP7B protein were detected by the immunohistochemical (IHC) techniques. Total protein was extracted. The expression levels of CTR1, ATP7A and ATP7B proteins were detected by Western-blot technique. Extracting cochlear tissues total RNA by RT-PCR and the CTRlmRNA, ATP7A mRNA and ATP7B mRNA expression levels were detected by RT-PCR technique. Result:1. IHC detections of CTR1, ATP7A and ATP7B proteins:The expressions of CTR1, ATP7A and ATP7B proteins were observed in the cytoplasm and cell membrane of Corti's organ cells, spiral ganglion cells and stria vascularis of very groups. Analyze the average optical densities (AOD) of the expressions of CTR1, ATP7A and ATP7B protein by the IPP6.0software. The AOD of CTR1protein was a downward trend, but the AODs of ATP7A and ATP7B proteins were upward trends.2. Western-blot. detection of CTR1, ATP7A and ATP7B proteins.:The expressions of CTR1, ATP7A and ATP7B proteins were observed in four different groups. The optical density analysis of CTR1showed that the optical densities were0.532±0.031,0.394±0.024,0.234±0.030and0.191±0.015respectively. There had a downward trend and were statistically differences between groups (P<0.05). The optical densities of ATP7A expression were0.149±0.012,0.274±0.026,0.420±0.030and0.515±0.021respectively. It had an upward trend and were statistically differences between groups (P<0.05). The optical densities of ATP7B expressions were0.146±0.013,0.273±0.027,0.423±0.027and0.513±0.053respectively. It had an upward trend and were statistically difference between groups (P<0.05).3. RT-PCR detection of CTR1mRNA, ATP7A mRNA and ATP7B mRNA expressions:The CTR1mRNA, ATP7A mRNA and ATP7B mRNA were observed in four different groups. The optical density analysises of CTR1mRNA showed that the optical densities were0.508±0.035,0.391±0.022,0.240±0.02and0.186±0.021respectively. It had a downward trend and were statistically differences between groups (P<0.05). The optical densities of ATP7AmRNA expressions were0.148±0.012,0.262±0.025,0.423±0.029and0.510±0.056respectively. It had an upward trend and were statistically differences between groups (P<0.05). The optical densities of ATP7B mRNA expressiones were0.144±0.013,0.267±0.023,0.416±0.01and0.509±0.059respectively. It had an upward trend and were statistically differences between groups (P<0.05).Conclusion:1. The CTR1, ATP7A and ATP7B proteins are abundantly expressed in Corti's organ, spiral ganglion cells and stria vascularis of the cochlea, suggesting that CTR1, ATP7A and ATP7B proteins involve in the cisplatin and the copper ion transporter in the inner ear.2. Round window copper ions infusion contributes to the decreased expression of CTR1protein and increased expressions of ATP7A and ATP7B proteins, suggesting that CTR1, ATP7A and ATP7B proteins play roles in maintaining the balance of the copper ions in the inner ear cells.3. The decreased expression of CTR1protein and the increased expressions of ATP7A and ATP7B proteins were observed in the rat ototoxicity model established by round window cisplatin administration, suggesting that inner ear cells may reduce the absorption of cisplatin by adjusting the expression level of CTR1, ATP7A and ATP7B proteins, which may be a feedback mechanism of inner ear cells to antagoniz the cisplatin ototoxicity. Objective:To investigate the protective effect of copper sulfate round window administration in cisplatin-induced ototoxicity of rat and provide a theoretical basis for the prevention of cisplatin-induced ototoxicity.Method:Male Wistar rats were randomly divided into three groups, round window administration of saline30minutes plus cisplatin20mg/kg intraperitoneal injection (group I), copper sulfate round window administration30minutes plus cisplatin20mg/kg intraperitoneal injection (group II), copper sulfate round window administration4hours plus cisplatin20mg/kg intraperitoneal injection(group Ⅲ). To evaluate the protective effect of copper sulfate by auditory brainstem response testing, HE staining of cochlea paraffin sections, the basilar membrane stretched and hair cell counting.Result:1. Auditory brainstem response:the hearing threshold shift(HTS) were53.33±15.06,15.00±5.48and18.33±7.53in group Ⅰ, group Ⅱ and group Ⅲ respectively. HTS had statistically differences between Group I and group Ⅱ, group Ⅰ and group Ⅲ respectively (P<0.01). HTS had no statistically difference between group II and group III (P>0.05).2. Cochlea axis slice HE staining:the losses of the outer hair cells were obivious and the structures were unclear in group Ⅰ. The number of Spiral ganglion cells was reduced and the nucleus were shrinked with confuse arranged nerve fibers in group Ⅰ. The morphous of Corti's organ, spiral ganglion cells and stria vascularis in group Ⅱ and group Ⅲ were normal.3. Cochlea basilar membrane stretched preparation and hair cell counting:There lines of outer hair cells in group Ⅰ arranged disorderly and some of them lost, but the one line of the inner hair cells was nearly intact. Three lines of outer hair cells in group Ⅱ and group Ⅲ were kept well and dyed uniformity and one line of the inner hair cells were intact. The results of hair cell counting indicated that the losses of outer hair cells in group Ⅰ were obivious and were mainly observed in base turn. Compared the average loss rates of outer hair cells of each turn of group Ⅰ and group Ⅱ, group Ⅰ and group Ⅲ respectively, showing statistically significant differences (p<0.01). The average loss rates of outer hair cells of group Ⅱ and group Ⅲ showed no statistically difference (p>0.05). The losses of inner hair cell were slodemly in each turn.Conclusion:Copper sulfate round window administration can reduce cisplatin induced ototoxicity, which may play the protective role by reducing the uptake of cisplatin of inner ear cells.