Evaluation of Ototoxic Effects of Cisplatin in a Rat Model: A Dose–Response Study

Abstract

Cisplatin (cis-diclorodiamminoplatin, CDDP) is a common chemotherapeutic agent for solid tumors, but its use is limited by severe side effects such as ototoxicity and nephrotoxicity. Variability in CDDP dosing and administration, along with high mortality and sensitivity in animal models, complicates experimental approaches. This study aimed to evaluate ototoxic damage in rats by comparing a single bolus versus three divided CDDP injections, also considering nephrotoxic effects. Twenty-four Sprague-Dawley rats were divided into three groups: eight received a single intraperitoneal injection of CDDP (14 mg/kg), eight received three injections (4.6 mg/kg/day), and eight were untreated controls. All CDDP-treated rats showed significant high-frequency hearing loss and morphological damage, including cochlear outer hair cell loss and renal glomerular atrophy with proximal tubule necrosis. Oxidative stress markers (nitrotyrosine and SOD1 expression) confirmed cochlear and renal alterations. Notably, the single bolus group had a 25% mortality rate and significant weight loss among survivors, unlike the other groups. This study introduces the novel finding that cumulative administration of three doses reduces mortality and weight loss while maintaining similar ototoxic and nephrotoxic effects. Therefore, cumulative administration is recommended for future studies to mitigate ototoxic and nephrotoxic damage, offering a potentially improved protocol for the administration of cisplatin.

1. Introduction

In 2022, approximately 20 million new cancer cases and 9.7 million deaths occurred worldwide [1]. Despite well-documented side effects, cisplatin (cis-diclorodiamminoplatin, CDDP) chemotherapy is widely used as single-agent or combination therapy for induction and neoadjuvant therapy in several solid tumors. The therapeutic and side effects of CDDP are influenced by various pharmacological factors, including dose and method of administration (single and cumulative dose) [2,3,4]. In particular, the therapeutic effects of CDDP can be achieved through cytotoxic lesions in cancer cells, as it can form bonds with DNA and possibly also interact with proteins and RNA, leading to the destruction of cell functions and induction of apoptosis [5,6]. Nausea and vomiting (currently treated with an antiemetic regimen) are the main side effects followed by neurotoxicity, nephrotoxicity, and ototoxicity, as well as less frequent but notable liver, respiratory, and hematological effects [2,5,7].

CDDP ototoxicity manifests itself in vertigo, tinnitus, and hearing loss. In severe cases, hearing loss can become bilateral either temporarily or permanently [3,8]. The onset of hearing loss occurs at high frequencies within the cochlea and then progresses to the frequency range of spoken language [4]. The use of animal models has validated that this type of hearing loss is caused by CDDP ototoxicity, as it is capable of interacting and forming adducts with DNA strands, causing an inflammatory response and generating reactive oxygen species (ROS) [3,9,10,11]. ROS production in the basal turn of the cochlea causes the loss of outer hair cells, resulting in an influx of calcium in the cochlear cells, which ultimately leads to cell apoptosis [3]. In mammals, hair cell loss is not repairable due to the lack of stem cell niches in the cochlea to replace damaged sensory tissue [12,13].

CDDP-induced nephrotoxicity is common and frequently severe due to tubular dysfunction. The development of acute kidney injury usually occurs after a few days of treatment. The occurrence of hypomagnesemia is possible, especially after multiple doses of CDDP [14,15]. Risk factors for CDDP nephrotoxicity include single and cumulative dose, frequency of administration [16], female sex, advanced age, smoking and hypoalbuminemia, as well as preexisting kidney problems [17,18]. Nephrotoxicity caused by cisplatin is complex and involves multiple processes that are directed toward cell death. In vitro experiments conducted in the primary culture cells of the mouse proximal tubule have shown that low concentrations of CDDP lead to apoptosis, while high concentrations lead to necrosis. However, in vivo administration causes death by both necrosis and apoptosis [17].

To date, it has become clear that toxicity is due to oxidative stress in both cases of nephrotoxicity and ototoxicity [6,8,19]. Oxidative stress occurs when there is a discrepancy between the amount of ROS in an organism and its ability to counteract them through detoxification or damage repair. ROS are divided into two categories: radical molecules such as superoxide anion (O²⁻) and hydroxyl radical (OH-) and non-radical molecules such as hydrogen peroxide (H₂O₂) [20]. The superoxide radical is very reactive, so it has a very short half-life (~1 ns), while H₂O₂ has such low reactivity that the molecule can enter the cell nucleus, generating peroxide ions and hydroxyl radicals that can cause serious damage to DNA, membrane proteins, and lipids [20]. H₂O₂ has already been shown to be a dangerous reactive species in in vitro cells derived from the organ of Corti (ODC) [21].

ROS-induced oxidative stress can be addressed by cells that produce certain molecules with defense functions, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) [20]. Antioxidants become active when reduced and are reactivated by specific enzymes. Superoxide dismutase regulates the process of turning superoxide anions into H₂O₂. In response to high levels of hydrogen peroxide, SOD1 can enter the nucleus. The enzyme in the nucleus interacts with DNA and binds to the promoter sequence of target genes that regulate their expression at the transcriptional level [20,22,23].

Nitrotyrosine is also known to be a marker of oxidative stress in cells and tissues. Oxidative stress caused by reactive nitrogen species (RNS) is also called “nitrative stress”, with the first term referring to excess production of the NO radical and the second term indicating excess formation of peroxynitrite. Nitration of tyrosine by RNS, including peroxynitride and nitrogen dioxide, results in the production of 3-nitrotyrosine. This compound is found in numerous pathological conditions and is, therefore, considered a marker of NO-dependent oxidative stress. It is recognized as a marker of cell damage and inflammation and also serves as an indicator of nitric oxide (NO) production. The 3-nitrotyrosine is involved in protein nitration at the level of tyrosine residues, an irreversible and potentially harmful process that involves conformational modification of the protein and consequent loss of its functionality [24].

This study is based on previous research that shows that giving cisplatin (CDDP) in three daily doses, rather than one dose, results in comparable hearing loss but significantly reduces mortality in rats [11]. This administration protocol closely mirrors human chemotherapy regimens, making it more suitable for long-term preclinical studies on ototoxicity prevention. Specifically, the goal is to develop an in vivo model of cisplatin-induced ototoxicity and nephrotoxicity that exhibits noticeable but not life-threatening damage. This study aims to examine the mechanisms of ototoxicity caused by drug exposure through functional, morphological, and immunohistochemical analyses, while nephrotoxic damage will also be documented to provide a reference for systemic effects in future ototoxicity prevention studies. The findings of this study will serve as a basis for future research on protective compounds that can mitigate or prevent cisplatin-induced ototoxicity in healthy animals, i.e., those without oncological diseases, with nephrotoxic damage serving as an internal control.

2. Materials and Methods

2.1. Animals and Treatment Groups

Twenty-four male Sprague Dawley rats (150–200 g; Charles River, Milan, Italy) were used in this study. The Italian guidelines DL 116/92 and the directive of the European Economic Community 86/609 were strictly followed by treating the animals. The Animal Use Ethics Committee of the University of Ferrara, with registration number 15599, approved all experiments, and the University of Padova Ethics Committee (Padova, Italy), with registration number 192013, also approved all experiments. The animals were randomly divided into the following groups: A: control group (NT, n: 8); B: treated with an intraperitoneal injection (IP) of CDDP 14 mg/kg on day 1 (1-IP, n: 8); C: treated with an IP of CPT 4.6 mg/kg for 3 consecutive days (from day 1 to day 3, cumulative dose 14 mg/kg) (3-IP, n: 8). Rats treated with CDDP received a daily subcutaneous injection of 1 mL of saline solution from the beginning of treatments until the day of sacrifice. The rats were anesthetized and sacrificed on day 5 (Figure 1). Cisplatin (1 mg/mL) was obtained from Accord Healthcare Italia Srl, Monza, Italia. Each animal was monitored daily for general health conditions and body weight. There were no significant differences in weight between each group and the initial average weight of the rats was 184 ± 18 g. The animals were sacrificed painlessly with decapitation on day 5 or earlier, if they showed significant distress signs such as unusual bleeding and dyspnea.

2.2. Auditory Brainstem Responses

The auditory threshold was assessed using auditory brain responses (ABRs) as previously described [11,25]. All ABR tests were performed in a 1 m³ soundproof chamber with the animals placed on a homoeothermic blanket to maintain constant body temperature at 37.5 °C. Three platinum–iridium needle electrodes were used to record the animal’s ABRs by placing them subdermally over the vertex (positive), mastoid (negative), and dorsum area (reference/ground). The sound was transmitted by a tweeter sound transducer that had a flat response of ±1.5 dB at 4.0 to 35 kHz through a plastic ear speculum placed in the external ear canal. The ABR was amplified 20,000 times and filtered from 20 to 5000 Hz. An average of 1000 individual responses was used in each recording. The ABR was generated in response to 2, 4, 8, 16, and 32 kHz tone pips (1 ms rise–fall time; 10 ms plateau) from 100 to 30 dB in 5 dB intervals. In two average runs, the threshold was established as the lowest intensity at which the wave III ABR could be observable. To prevent binaural stimulation at the highest stimulus intensities, the contralateral ear was occluded with earplugs. Auditory function was assessed by ABRs on day 1 and day 5 in all animals. Data analysis was based on dividing the frequencies into low, medium, and high frequencies, as we know that the cochlea regions (apical, medial, and basal) correspond to the perception of low (2–4 kHz), medium (8 and 16 kHz), and high frequencies (32 and 64 kHz) [26]. The global response is represented by the click spectrum, which encompasses all frequencies.

2.3. Histology

Both the cochleae and kidneys had been explanted and processed for paraffin embedding on day 5 (96 h after the first treatment) as previously described [27]. Cochlea and kidney samples were placed in Shandon GlyoFixx^TM solution (Thermo Scientific, Milan, Italy) for overnight incubation followed by washing in 0.1 M PBS pH 7.4 (Lonza, Milan, Italy). Only the cochleae were decalcified overnight at 4 °C by incubating them with Surgipath’s Decalcifier I^® solution (Leica Microsystems, Wetzlar, Germany) and again incubating them with Surgipath’s Decalcifier II^® solution (Leica) for 4 h at room temperature. All samples were processed using the automatic tissue processor MTP (Slee, Mainz, Germany) and embedded in paraffin with wax embedding center type MPS/P1 (Slee) and colding plate MPS/C (Slee). Briefly, after drying the samples through an increased series of histalcool (Diapath, Bergamo, Italy), they were clear in xylene (Diapath) and then embedded in Diawax paraffin (melting point 56–58 °C, Diapath). The semiautomatic microtome CUT 5062 (Slee) was used to cut the samples into 5 μm sections which were then sequentially collected on Superfrost^® Plus microscope slides (Diapath). For each sample, at least 12 sections were analyzed for each antibody and biometric investigation. To analyze the entire sample, each section was separated by 50 μm from the next one. Hematoxylin-eosin staining was performed for each sample. Samples were examined with the ECLIPSE 50i optical microscope (Nikon Instruments, Melville, NY, USA), and Nis Elements D 3.2 software was applied to capture and analyze the images.

2.4. Biometric Investigations

Biometric investigations were carried out by dividing the cochlear duct into several cross-sections containing the organ of Corti (OC) and related spiral ganglion (SG). These sections (Figure 2a) are numbered from 1 to 4 (in some sections there are also 1b and 4b) and correspond to the cochlear apex (sections 1b and 1); middle turn (sections 2 and 3); and base (sections 4 and 4b).

In all sections of the cochlear duct where the complete organ of Corti was visible, hair cells were counted if their nucleus was visible, and those whose nucleus was not visible were considered missing. The counted cells were classified into inner hair cells (IHCs) and outer hair cells (OHCs) (Figure 2b), and the ratio between observed and expected cell numbers was calculated for each type of cochlear region, each type of hair cell, and each animal.

Neuronal density was evaluated for each animal in four histological sections of the cochlear modiolus by measuring the section area of the Rosenthal canal, which contains the spiral ganglion (the area delimited by a pink line in Figure 2c). Neuronal density was obtained as the ratio (multiplied by 10,000) of the neurons counted that show a nucleus (Figure 2c) over the Rosenthal canal section area (expressed in μm²). Neuronal density was calculated for each animal and each cochlear region, i.e., apical, middle, and basal turns (Figure 2c).

The stria vascularis was quantified for each animal in 4 histological sections of the cochlea identified as described above (Figure 2a). The thickness of the stria vascularis was measured at least in 3 different points for each section (Figure 2d).

For each animal in all sections of the kidney stained with hematoxylin-eosin, the area of at least 10 glomeruli in their equatorial section (identifiable by the presence of the open proximal twisted tubule and the arteriole at the poles of the glomerulus) has been measured without considering the Bowman capsule (Figure 2e). The density of the glomeruli corresponds to the average number of glomeruli counted in ten images taken at 100X magnification per sample for each treatment. The areas in the basal turn of the stria vascularis were measured for each animal in four histological sections of the cochlea near the modiolus. Additionally, the thickness in the basal turn of the stria vascularis was evaluated by taking at least three measurements at different points within the same structure. The values obtained were normalized to the mean value obtained in the untreated group, allowing us to appreciate the variation in terms of percentage.

2.5. Immunohistochemistry

A 0.1% Tween-20 solution in PBS (Sigma-Aldrich, St. Louis, MO, USA) was used to permeabilize the rehydrated sections, a 1% hydrogen peroxide solution was applied to block endogenous peroxidase, and the blocking buffer Vecstain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) was used to saturated non-specific sites. The primary antibody was incubated with the sections overnight at 4 °C. Nitrotyrosine antibody was used at the concentration of 1:700 and was provided by Santa Cruz Biotechnology (Dallas, TX, USA); SOD1 was used at the concentration of 1:800 for cochlear samples and 1:700 for kidney samples and was provided by GeneTex (Irvine, CA, USA). After 24 h, the secondary antibody Vecstain Elite ABC kit (Vector) was incubated. The signal was then amplified and detected with the Vector SG VIP Peroxidase Substrate Kit (Vector). Samples were examined with the ECLIPSE 50i optical microscope (Nikon Instruments), and Nis Elements D 3.2 software was applied to capture and analyze the images.

2.6. Statistical Analysis

For every experimental group, the average with the standard deviation and the median with the interquartile range were computed. To determine significant differences among groups, we used parametric one-way ANOVA (post hoc Bonferroni) or nonparametric Kruskal–Wallis tests to analyze ABRs and biometric data. Normality tests (Shapiro–Wilk and Kolmogorov–Smirnov) and Bartlett’s test were applied to the data set. Significant p values were those between 0.05 and 0.001, and highly significant p values were those under 0.001. GraphPad Prism 8.0.1 (GraphPad Software, Inc., San Diego, CA, USA) was used to process all the data collected from ABR recordings and biometric investigations.

3. Results

3.1. Body Weight

The control group (NT) did not show mortality during all the experiments carried out for this study and remained in an excellent state of health with an increase in weight of 12 ± 4%. On the contrary, mortality increased to 25% after 4 days of treatment (on day 5) in animals who received only one dose of CDDP 14 mg/kg (group 1-IP). Furthermore, the animals that survived showed signs of suffering, resulting in a highly significant weight loss of 11 ± 5% (p-value < 0.001). The mortality rate of the animals in group 3-IP was zero, despite a significant weight loss of 14 ± 5 percent (p-value < 0.05). The weight loss observed in the treated groups was significantly higher compared to the NT group after 4 days, while there was no significant difference between the two treatment groups (Figure 3a).

3.2. Auditory Function

The results of the ABR (auditory brainstem response) test, performed as previously described [11,25], are shown in Figure 3b, where the threshold shift (TS) values depend on the frequency ranges tested and for each experimental group. Compared to the NT group, both cisplatin-treated groups have had a highly significant hearing loss at both the click and at the different frequencies analyzed (p < 0.001) (Figure 3b); the greatest threshold shift was observed at high frequencies (basal region) (Figure 3b). It should be noted that despite the fact that both models of cisplatin administration cause significant hearing loss, in the basal region, the 1-IP group appeared more severe than the 3-IP group (Figure 3b).

3.3. Morphological Damage to Cochlear Structures

Upon examining the histological sections stained with hematoxylin-eosin, it was evident that the organ of Corti (ODC) of the NT group present in the three regions of the cochlea was well preserved: the three rows of outer hair cells (OHCs) and the row of inner hair cells (IHCs) have been clearly detected (Figure 4, NT group). In both groups, 1-IP and 3-IP, hair cells were lost in the middle and basal regions of the ODC, while in the apical region results appeared similar to the NT group (Figure 4). Furthermore, it appeared that the cisplatin treatments have had a greater impact on OHCs than IHCs (Figure 4, medial and basal regions).

The cell count results have confirmed that cisplatin treatment causes, compared to the NT group, a highly significant reduction (p < 0.001) in the number of cells present in the middle cochlear region (the average number of cells decreased by 36.46% for 1-IP and 4.85% for 3-IP) and an even greater and significant reduction in the basal region (68.32% for 1-IP and 25.65% for 3-IP) (Figure 5a). It can be concluded that CDDP injections, single or repeated, can cause cell damage, mainly at the base of the cochlea (p < 0.001), without significant differences between the two treatment methods (Figure 5a). In detail, it has been confirmed that OHCs were the cells that were more affected by cisplatin treatment, especially in the basal region of the cochlea. Again, the 1-IP group had significantly fewer OHCs (p < 0.001) than the 3-IP group in the medial and basal regions (Figure 5b). Despite the significant damages (p < 0.01) detected in the basal region of the cochlea, the IHC count did not show significant differences between the two cisplatin treatments (Figure 5b).

The investigation of neuronal density and the area and thickness of the stria vascularis did not show significant differences among the three experimental groups (

Table 1. Neuronal density. Data were obtained by multiplying the ratio of neuronal nuclei to ganglia area by 10,000 for each treatment group and cochlear region.

	NT	1-IP	3-IP
Apical region	19.3 ± 4.1	16.4 ± 5.5	18.8 ± 4.0
Medial region	17.7 ± 3.3	18.7 ± 2.1	17.8 ± 2.8
Basal region	12.8 ± 2.0	15.1 ± 5.8	12.7 ± 2.4

Data are expressed in terms of mean ± standard deviation. No differences on neuronal density were detected among groups by using one-way ANOVA and Bonferroni’s multiple comparison test.

and

Table 2. Stria vascularis. Data were obtained by multiplying the ratio of neuronal nuclei to ganglia area by 10,000 for each treatment group and cochlear region.

	NT	1-IP	3-IP
Area	100 ± 22.73	105.2 ± 2.73	105.7 ± 349
Thickness	97 (77.6; 105)	106 (93; 114)	97 (91.4; 106.9)
Basal region	12.8 ± 2.0	15.1 ± 5.8	12.7 ± 2.4

Stria vascularis area values are expressed in terms of mean ± standard error of the mean, and differences were measured among groups by using one-way ANOVA (F (2, 34) = 0.4617, p-value = 0.634). Stria vascularis thickness values are expressed in terms of the median (1st percentile; 3rd percentile), and differences were measured among groups by using the Kruskal–Wallis test (H = 4.699, p-value = 0.095).

).

3.4. Morphological Changes in Renal Structures

In the NT group, it was possible to appreciate the normal morphology of the kidney, with clearly identifiable cortical and medulla regions, normal glomeruli, intact proximal convoluted tubules (PCTs), and distal convoluted tubules (DCTs) and Henle’s loop (Figure 6, NT group). In the 1-IP group, the damaged areas extend to almost all of the cortex and part of the medulla. The tissue in the areas of necrosis and damage is less compact and is evidenced by a lower hematoxylin-eosin stain (Figure 6, 1-IP group, cortex, and medulla). Damages included glomerular atrophy (Figure 6, cortical region), necrosis of the PCT epithelium with extensive infiltration of inflammatory cells (Figure 6, cortex/medulla), and the presence of hyaline casts in the DCT of the medulla (Figure 6, medulla region). In the 3-IP group, necrotic areas are less extended and restricted to the boundary between the cortex and the medulla (Figure 6, 3-IP group) compared to 1-IP rats. Dilation of the DCT in the medulla and the presence of few inflammatory cells in the DCT lumen (Figure 6, medulla region) were also observed. It should be noted that the identification of inflammatory cells was based on H&E staining, which is not definitive without specific inflammatory markers. On the contrary, although cisplatin treatment caused a significant reduction in the glomerular area compared to the NT group (17%), no significant differences were observed between the two treated groups, and no significant variations were detected among all groups in terms of glomerular density (Figure 7a,b). The study of DCT and PCT lumen thickness revealed significant dilatation for both tubules in both CDDP treatments, with comparable results (Figure 7c,d).

3.5. 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 (Figure 8 and Figure 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 (Figure 8 and Figure 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 Figure 8 and Figure 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).

4. Discussion

For almost 45 years, CDDP has been the cornerstone of chemotherapy for various solid cancers. However, its use was often limited by its side effects, such as ototoxicity and nephrotoxicity [28]. Investigating these effects in animal models remains crucial in developing better prevention protocols for oncological patients. The high mortality rate in these models, which was determined by the dose used and attributed to the systemic toxic effects of CDDP, remains an important obstacle in the experimental settings of CDDP [29,30]. Therefore, our objective was to develop an in vivo model of cisplatin ototoxicity and nephrotoxicity that exhibited measurable damage and achieved the lowest to absent mortality. Our study found that in the 1-IP group, a single intraperitoneal injection group had a mortality rate of 25%, while the 3-IP group had a mortality rate of zero, which was achieved by giving CDDP 14 mg/kg in three daily doses of 4.6 mg/kg. The physical condition of the animals in the IP-3 group was still optimal after four days after the first injection, except for a predictable weight loss. The results are consistent with those of He and collaborators [31], who treated CBA/J mice with CDDP at 15 mg/kg in a single dose or four daily doses, with a mortality rate of 70% to 10%, respectively. Weight loss was between 9% and 16% in our Sprague Dawley rat model, with no significant differences between the two drug administration modalities. Similar findings were found in other studies, where weight loss was dose-dependent and not influenced by the mode of administration of CDDP [32,33,34]. The causes of CDDP-induced weight loss can be called gastrointestinal toxicity, which leads to decreased appetite and reduced food intake, as well as reduced water absorption, resulting in excessive sodium secretion by tubular cells in the kidney, leading to polyuria, dehydration, and, ultimately, weight loss [35,36].

Ototoxicity is a common adverse effect associated with CCDP administration, with an estimated incidence of up to 36% among adult patients [37]. In both animal and human populations, frequency and severity were observed to be dose-dependent [29,31,38,39,40]. ABR data confirm that CDDP at 14 mg/kg causes a shift in the threshold at all frequencies (Figure 4). This agrees with previous findings from previous experimental studies, particularly in the medial (8–16 kHz) and basal (32–64 kHz) regions of the cochlea [32,41,42]. It is worth noting that not only the dose of CDDP administered affects the extent of auditory damage, but also the way of administration [3,39]. As we showed, compared to CDDP administered in three daily doses, a single dose of CDDP results in a reduction in hearing loss in the basal region of the cochlea (ABR: 3-IP 19.7 vs. 1-IP 34.7 dB, p = 0.011). Although, the differences in the apical and medial regions were not significant between the two treated groups.

The permanent hearing loss caused by CDDP was caused by irreversible damage to neurosensorial tissue [2,11,41,42]. The cells most affected were OHC, with increasing damage from the apical cochlear region to the base [31,38,43,44,45]. This damage depends on both the dose and time, and the number of missing hair cells is correlated with increasing doses of CDDP [2,39,44,46]. Our study confirmed these results. The 3-IP group had significantly higher cell loss than the NT group but was lower than the 1-IP group (1-IP 68.32% vs. 25.65% 3-IP). The observed damage, as expected, mainly affects hair cells (especially OHCs) in the basal region, with less extensive damage in the medial region and no damage in the apical region. Cochleograms obtained by scanning electron microscopy (SEM) or confocal TUNEL analysis facilitate accurate cell counting by correlating cell integrity with the presence of stereocilia and nuclei, respectively. 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 paraffin embedding 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 [50,55]. 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 [47,50,58]. 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 intratympanic injection of CDDP in C57BL/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]. CDDP was mainly excreted through glomerular filtration and tubular secretion, with a smaller amount being excreted through tubular secretion [35]: within the first 24 h after administering cisplatin, more than 50% of the drug was excreted in the urine and the concentration achieved in the kidney cortex is several times higher than that in plasma and other organs, which explains the high incidence of nephrotoxicity [17,65,66,67]. The proximal tubular cells, and especially segment S3, were specifically damaged by the direct cytotoxic effects of CCDP. Histopathology suggests that tubular–interstitial lesions were caused by necrosis and apoptosis, particularly in cells that have more proliferative activity [68,69]. In vivo studies have revealed atrophy of the glomeruli and damage to the renal tubules, leading to the formation of hyaline casts and areas of tubular necrosis [70,71]. In vivo studies on CDDP nephrotoxicity in rats have been carried out using various protocols, with 7.5 mg/kg IP injected as a single dose, to cumulative doses of 15 mg/kg (three IP doses of 5 mg/kg) [30,33,70]. In our investigation, a dose of 14 mg/kg led to atrophy of the renal glomeruli, with a decrease in their area of approximately 17%, without significant differences between the treatment groups. Despite this, both treatment methods showed significant alterations in tubular damage compared to control. In general, both PCTs and DCTs were affected, with epithelial dilation, detachment, and necrosis of PT cells. These findings were consistent with the results previously reported in studies that examined nephrotoxicity in mice and rats that received a lower dose of cisplatin [72,73,74]. Furthermore, we identified inflammatory cells in necrotic areas and hyaline urinary casts (mainly in DCTs), which agrees with the findings of previous studies [30,40,70,71]. Both the 1-IP and 3-IP groups showed damage throughout their cortical regions, accompanied by extensive infiltration of inflammatory cells and hyaline urinary casts, indicating a decrease in diuresis and solute concentration during DCT. The boundary zone between the cortical and medullary regions was the area most affected by CDDP, and it appeared that CDDP accumulates there first, as demonstrated in rat models [75,76]. The physiological mechanisms have not been fully explained, but the hypothesis is that basolateral drug transporters and organic cation transporters play a role in it [77,78,79,80,81]. CDDP nephrotoxicity has been suggested to be caused by inflammation and oxidative stress [17,70]. The inflammatory response caused by CDDP administration can cause or worsen AKI by producing TNF-α and other cytokines in epithelial cells themselves, eventually leading to activation of NF-kB and iNOS, resulting in the release of NO and peroxynitrite and leading to damage caused by oxidative stress.

CDDP targets mitochondria, and they play a greater role in mediating chemotherapy-induced cell death than DNA damage [82]. Cell sensitivity to CDDP was related to the number of mitochondria [83]. This could explain why CDDP had a negative impact on PCT epithelial cells in the kidney, stria vascularis, spiral ligament, organ of Corti, and spiral ganglion neurons in the cochlea, as cells in these tissues were reported to be rich in mitochondria, making them highly sensitive to oxidative stress [84,85]. Although the mechanism behind CDDP cellular toxicity has been extensively studied, it is still incompletely understood. The presence of oxidative stress could have a significant impact, with decreased levels of endogenous antioxidant enzymes (such as SOD, catalase, glutathione peroxidase, and glutathione reductase) and higher levels of stimulating enzymes, such as NADPH oxidase, which could increase lipid peroxidation and accumulate ROS and reactive nitrogen species (RNS) in tissues [30,33,39,63,64,70,71,86]. When ROS and RNS interact, peroxynitrite (ONOO-) is formed, which is a nitrogen radical that nitrates protein tyrosine. The product of this nitration, 3-nitrotyrosine, is considered a biological marker of oxidative stress, more specifically termed “nitrative stress” [24,82].

The expression of nitrotyrosine was evaluated in both organs involved in our investigation, the cochlea and the kidney. Although protein nitration could occur under basal physiological conditions, it increases significantly after ROS and RNS elevation, and it could only be observed in regions that experienced oxidative stress [24,87]. In control rats, nitrotyrosine was observed in certain areas of the cochlea, such as the spiral ligament and the organ of Corti cells, while absent in the stria vascularis and kidney. After CDDP administration, nitrotyrosine expression was observed to increase in all tissues, particularly in the stria vascularis, renal tubular epithelium, and cells detached from the tubular lumen. Previous studies in the cochleae and kidneys of animals treated with CDDP have documented an increase in nitrotyrosine expression, suggesting a key role for protein nitration in CDDP-induced ototoxicity and nephrotoxicity [39,63,64,71,88].

SOD1 is a Cu/Zn enzyme that is generally found in the cellular cytoplasm [89] but has been observed to move into the nucleus when ROS levels increase, allowing it to regulate genes involved in the control of oxidative stress [23]. In the NT group, we found that SOD1 was present in the stria vascularis, almost everywhere in the spiral ligament, and in the cytoplasm of spiral ganglion neurons, as indicated by other studies, which also noticed high expression in the Corti organ [90]. Treatment with CDDP resulted in the presence of SOD1 in the nuclei of hair cells instead. The different distribution of SOD1 in cochlear tissues might suggest a varying sensitivity to oxidative stress. Treatment with CDDP caused a significant reduction in this protein throughout the spiral ligament and localized labeling in the basal marginal cells of the stria vascularis. Due to its highly vascularized tissue, the stria vascularis is the first to be affected by CDDP treatment and oxidative stress, ahead of all other cochlear structures, but it was also the tissue with the highest activity of SOD1 [90], which could explain its readiness to respond to oxidative stress. Furthermore, the stria vascularis has a high level of CAT and GSH, which prevents the formation of superoxides when SOD1 levels are reduced [91,92]. However, hair cells could be more susceptible to oxidative stress. In rats treated with CDDP, nuclear staining disappeared in hair cells damaged by exposure to chemotherapy. This suggests that CDDP caused oxidative stress in hair cells, which caused the depletion of endogenous antioxidant defenses, leading to morphological and functional damage and cell death. Previous data have shown that SOD1 was crucial for hair cell survival, which was consistent with this hypothesis [84,93].

In the kidney, SOD1, which has two isoforms, has been studied in different tissues of rat [94]. These experiments revealed that SOD was mainly active in the glomeruli and has reduced activity in the epithelial cells of the DCT, collecting ducts, Henle’s loops, and the brush border of PCT epithelial cells. During these studies, it was discovered that SOD activity and immunolocalization of the two isoforms differed significantly, with SOD1 and SOD2 staining appearing mild in all nephron structures but absent in glomeruli [94]. It has been suggested that the differences may be attributed to the presence of another extracellular SOD, SOD3. Recent studies on the nephrotoxic effects of cisplatin confirmed a reduction in SOD expression in rats treated with the drug [76,95,96]. Our investigation revealed that SOD1 was absent from the glomeruli but was present in the nuclei of PT cells, especially in the region that borders the medulla. The terminal part of the PT was more sensitive to harmful stimuli than the initial part, which was closer to the glomerulus and the middle part [97]. It was probable that the border region, which encompasses the terminal portion of PCT, was more susceptible to ototoxic agents, and the medulla and cortex can stimulate other cells to generate SOD1 in the nucleus, allowing epithelial cells to be prepared to counteract potential oxidative damage. In rats treated with CDDP, nuclear staining was present in PCT cells throughout the cortex, while detached and necrotic cells were not detected in the damaged region; although, degenerate nuclei and necrotic nuclear fragments were observed. Stress-induced cells located in the border region between the medulla and cortex can stimulate other cells to generate SOD1 to be prepared for oxidative stress, and cells in areas with negative staining may have lost their mechanism of protection against oxidative stress and died through apoptosis or necrosis.

Comparable results were observed in other animal models treated with cisplatin, where the investigation focused on the role of cisplatin in the induction of drug-induced liver injury (DILI), a significant concern due to its potential to precipitate acute liver failure [98,99]. In mice, it was shown that the hepatotoxic effects of cisplatin are predominantly mediated through the generation of reactive oxygen species (ROS), such as the superoxide anion (O²⁻), which induce oxidative stress, apoptosis, and subsequent liver damage [98]. Additionally, the expression levels of superoxide dismutase 1 (SOD1) and glutathione peroxidase, another antioxidant enzyme, were found to be reduced. Furthermore, histopathological analyses revealed that exposure to cisplatin results in hepatocellular necrosis, particularly in the centrilobular regions of the liver [99].

In cisplatin-induced ototoxicity, both the Haber–Weiss and Fenton reactions are involved. These reactions contribute to the generation of reactive oxygen species (ROS), which play a significant role in oxidative stress leading to cochlear cell damage [3,4,5,6]. Regarding CYI isoforms, the involvement of cytochrome P450 enzymes (CYPs) in cisplatin-induced ototoxicity has been studied, but specific CYI isoforms (such as CYP1A1 and CYP1A2) are typically not highlighted in the context of ototoxicity [100,101]. The focus is more on oxidative stress and inflammatory pathways. The Haber–Weiss reaction produces hydroxyl radicals (OH-) from hydrogen peroxide (H₂O₂) and superoxide (O²⁻). The Fenton reaction generates hydroxyl radicals (OH-) from hydrogen peroxide (H₂O₂) and ferrous ions (Fe²⁺). We describe the ROS involvement investigating nitrotyrosine and superoxide dismutase 1 (SOD1). Nitrotyrosine formation indicates nitrosative stress, resulting from the reaction of tyrosine with peroxynitrite (ONOO-). SOD1 catalyzes the dismutation of superoxide (O²⁻) into oxygen (O₂) and hydrogen peroxide (H₂O₂), mitigating oxidative stress.

In summary, the cumulative dose of CDDP of 14 mg/kg administered in three doses (IP-3) used in our experimental rat model was found to be the most effective way to study the ototoxicity and nephrotoxicity induced by exposure to this chemotherapeutic agent: due to the evidence of well-characterized induced ototoxic and nephrotoxic damage, along with the absence of mortality. Ototoxic damage in the IP-3 group was characterized by significant hearing loss, particularly at high frequencies, and loss of hair cells, especially OHCs, both related to the basal region of the cochlea. The stria vascularis and spiral ganglion did not show any morphological changes. CDDP causes oxidative stress in the cochlear area by increasing the number of nitrosylated proteins, specifically in the stria vascularis, spiral ligament, and organ of Corti. CDDP had a significant impact on SOD1 expression in OHCs, resulting in an increase in reactive nitrogen species, enzyme dysfunction, and cell death. The stria vascularis was not affected by these effects, probably due to the abundance of SOD and other antioxidant enzymes. Nephrotoxicity was characterized by glomerular atrophy and alteration of the epithelium of convoluted proximal and distal tubules, with a localized and well-distinguished area of necrosis at the boundary between the medullary and cortical regions. Oxidative stress could have a significant impact on nephrotoxicity, with an increase in nitrosylated proteins observed in necrotic areas and detached and shed epithelial cells from convoluted tubules proximal in the lumen of the tubule. The epithelial cell nuclei within the boundary between the convoluted tubules of the proximal region in control animals showed SOD1. In rats treated with CDDP, the drug caused an active response to oxidative stress, resulting in an increase in the expression of SOD1 in epithelial cells in the cortical region, while in areas already damaged by CDDP, we observed markedly reduced staining for SOD1 or present in fragmented or degenerate nuclei. Therefore, the decrease in this enzyme has led to an accumulation of RNS, causing cellular damage and subsequent necrotic cell death. Our data indicate that in animal models studying the ototoxic effects of cisplatin, administering the drug in multiple doses is preferable to a single dose. This approach allows for a more accurate analysis of substances that can counteract the adverse effects of cisplatin. Cisplatin-induced injury involves oxidative stress and immune responses, with inflammation triggered by release of cytokines from fibrocytes in the spiral ligament [102]. This leads to direct cytotoxic effects and the production of reactive oxygen species (ROS), causing cell damage in tissues such as the cochlea and kidneys [2,6]. Although the immune system contributes to damage, its role is believed to be secondary to direct cytotoxicity [6,103]. Steroids, such as corticosteroids, can reduce inflammation but do not prevent primary oxidative damage [104,105]. Dexamethasone is already used in certain chemotherapy regimens to manage side effects and improve treatment efficacy [2]. Additionally, dexamethasone-sparing antiemetic therapy with NK1RA and palonosetron effectively prevents nausea and vomiting in highly emetogenic chemotherapy [106]. Improving chemotherapy administration methods could improve the effectiveness of treatments aimed at mitigating oxidative and immune-mediated damage caused by cisplatin.

5. Conclusions

In the final analysis, our experiment with male Sprague Dawley rats, taking CDDP for a total of 14 mg/kg for 3 days via intraperitoneal injection (4.6 mg/kg/day), is a suitable and feasible model for studying CDDP toxicity. Lastly, our goal is to encourage other researchers to adopt this drug administration approach to meet the need for validated experimental models in the study of preventing cisplatin side effects. Proposing a new treatment protocol in an animal model for chemotherapy drugs involves optimizing dosages to maximize effectiveness while minimizing side effects. Increased survival in the animal model directly informs the study of the cellular and molecular mechanisms of chemotherapy, allows the evaluation of the efficacy of the protective drug, and may aid in the identification of predictive biomarkers for treatment. Ultimately, this provides valuable information for clinical research. Although preclinical results in human tissues are still needed, obtaining comparable data allows deeper investigations into the mechanisms behind potential prevention effects, potentially leading to new therapeutic boundaries.