# Evaluation of Ototoxic Effects of Cisplatin in a Rat Model: A Dose-Response Study
## 3 Results
### Oxidative Stress  
To detect oxidative stress, immunohistochemical analysis was used to identify SOD1 and nitrotyrosine in the cochlear and renal tissues of each experimental group (Figures 8 and 9). In the NT group, SOD1 was detected in almost all spiral ligaments, in all stria vascularis, and in the cytoplasm of spiral ganglion neurons; in the ODC, staining was restricted to the nuclear and cytoplasmic regions of OHCs and IHCs, and only some nuclei of PCT epithelial cells were labeled in the kidney (Figures 8 and 9, SOD1-NT group). A marked reduction in SOD1 staining was observed in the spiral ligament in both the 1-IP and 3-IP results. Structural vascularis staining was localized in the basal marginal cells. In damaged ODCs, no staining was observed of the nuclei of the outer hair cells, and in the inner hair cells, the staining of the cytoplasm was reduced. No differences were observed in the spiral ganglion compared to the control (Figure 8, SOD1). An increase in the intensity of SOD1 staining and the number of stained nuclei was observed in the kidney areas of rats treated with CDDP (Figure 9, SOD1-cortex area). On the contrary, in the necrotic area, the staining was reduced, and positive cell fragments were visible in the PCT lumen (Figure 9, SOD1-cortex/medulla). Detectable expression of nitrotyrosine was shown in the spiral ligament and some neurons of the spiral ganglion in the ODC, but not in the stria vascularis and renal structures, as shown in Figures 8 and 9 (nitrotyrosine-NT group). Treatment with 1-IP and 3-IP results in an increase in the area and intensity of nitrotyrosine staining observed in the spiral ligament, ODC, and stria vascularis, while no significant variations in staining were observed in the spiral ganglion (Figure 8, nitrotyrosine). In the outside region of the renal cortex, anti-nitrotyrosine staining was absent in the glomeruli, while it was evident in the cytoplasm of PT epithelial cells (Figure 9, nitrotyrosine). In the necrotic area, the cells that stained the most were those that were detached and gathered in the tubular lumen (Figure 9, nitrotyrosine-cortex/medulla). Figure 7: Analysis of histological examination of kidney damage in the three experimental groups. (**a**) Glomerval area among the groups, where the mean and standard deviation are calculated for each group and expressed in the \(\upmu\)m\({}^{2}\); differences in glomerular area among the groups were analyzed using one-way ANOVA and [PERSON]’s multiple comparison test. (**b**) The number of glomeruli per area is reported as the median and quartile intervals for glomerular density. (**c**) The lumen thickness of proximal convoluted tubules (PCTs) is measured in \(\upmu\)m and reported in median and quartile intervals. (**d**) The lumen thickness of distal convoluted tubules (DCTs) is measured in \(\upmu\)m and reported in median and quartile intervals. The analysis of differences among treatment groups in glomerular density, PCT thickness, and DCT thickness was performed using Kruskal[PERSON]Wallis and Dunn’s multiple comparison tests. NT: not-treated animals; 1-IP: animals treated with one cisplatin bolus (14 mg/kg); 3-IP: animals treated with three consecutive cisplatin doses (4.6 mg/kg); ***: \(p<0.00\). Figure 8: Detection of oxidative stress by immunohistochemical analysis. Cochlear tissues in all three experimental groups were stained with anti-SOD1 and anti-nitrotyrosine, respectively, in the NT group, 1-IP group (14 mg/kg), and 3-IP group (14 mg/kg). Details of the cochlear basal region: (1) cochlear basal duct highlighting the stria vascularis with black arrows, the spiral ligament with the white asterisk, and the organ of Corti with the white arrows; the spiral ganglion is highlighted with the gray arrows; (2) stria vascularis highlighting the stained spiral ligament with asterisks, and the stained marginal cells of the stria vascularis are highlighted with arrowheads; (3) organ of Corti highlighting the hair cells (including those that are not present) with white arrowheads.