# Evaluation of Ototoxic Effects of Cisplatin in a Rat Model: A Dose-Response Study
## 4 Discussion
Although acknowledging the potential occurrence of sectioning angle effects that could be analyzed by the 3D reconstruction [47; 48], in our study, we counted hair cell nuclei in multiple sections (spaced 5 microns apart) adjacent to the modiolar region. This method demonstrated comparable data accuracy, although with increased labor intensity [49]. The cochlea was meticulously oriented during paraffinembedding and microtome sectioning. The collected data were subjected to inferential statistical analysis, consistent with methodologies in similar studies [50; 51; 52]. Notably, this counting technique offers a viable alternative to reduce animal sacrifices [52]. Given that cochleogram analyses typically require one cochlea per animal, this approach enables comprehensive evaluations of all cochleae, including biometric and immunohistochemical analyses. Furthermore, adhering to the principle of the Three Rs (Replacement, Reduction, and Refinement) is paramount when seeking permits for animal experimentation, with a particular emphasis on minimizing animal sacrifice. Administration of CDDP led to apoptosis in the basal region of the cochlea as its primary site, in both the OHCs of the ODC and the spiral ganglion and throughout the width of the stria vascularis [53]. Previous studies have demonstrated morphological changes and cell death in all tissues of the inner ear [50; 54]. In human and animal models, it has been documented that CDDP primarily causes the degeneration of the basal cochlea turns in OHCs, which can extend to IHCs in some cases [55; 50]. Spiral ganglion neurons have also been shown to have decreased neuronal density when CDDP is administered [47; 55], and loss of myelin sheath has been observed in type I cells [50]. Lesions reported in the stria vascularis included edema, blister formation, rupture and compression of marginal cells [56], or atrophy due to intermediate cell narrowing [57]. Unlike other studies, we did not observe any damage to the spiral ganglion or changes in neuronal density in the three regions of the cochlea after four days of treatment [58; 47; 50]. The results may be attributed either to the dose of CDDP used or to the observation time. The direct effect of CDDP on neurons includes the reduction in the myelin sheath, causing detachment of the neuron body, or because of hair cell loss. The loss of the ability of HCs to transmit trophic stimuli to neurons results in neuron degeneration [50; 59; 60]. However, this process is slow: damage to spiral ganglion neurons has been documented in rats, at cumulative doses greater than 14 mg/kg or even at lower doses, but only after 15 days of treatment [50; 58]. Marginal cells are believed to be the primary site of CDDP-induced ototoxic damage in the stria vascularis [61; 62]. The stria vascularis deteriorated in a time-dependent manner over 3 to 14 days after intradynamic injection of CDDP in C57 BL/6 mice, while rats exposed to CDDP at 16 mg/kg administered intraperitoneally did not show atrophy [56; 63; 64]. Our study agrees with the latest findings as there have been no noticeable changes in the stria vascularis (data not reported). The differences in previous studies could be attributed to the animal model's sensitivity or, more likely, to the different injection sites: the local administration of the chemotherapy agent through intrathecal injection results in immediate and direct action on the organ; while, with IP administration, CDDP takes longer to get absorbed and reach the inner ear, which reduces the ototoxic effect. Arguably, IP administration represents a more accurate model for studying the ototoxic effects observed in humans. Nephrotoxicity is a common and harmful side effect of CDDP administration. Dose- and time-dependency was present in CDDP-induced nephrotoxicity, as demonstrated in patients [16] and in vivo experimentation in animal models [30; 31; 33; 38; 40].