An optimized, clinically relevant mouse model of cisplatin-induced ototoxicity

Abstract

Cisplatin-induced ototoxicity results in significant, permanent hearing loss in pediatric and adult cancer survivors. Elucidating the mechanisms underlying cisplatin-induced hearing loss as well as the development of therapies to reduce and/or reverse cisplatin ototoxicity have been impeded by suboptimal animal models. Clinically, cisplatin is most commonly administered in multi-dose, multi-cycle protocols. However, many animal studies are conducted using single injections of high-dose cisplatin, which is not reflective of clinical cisplatin administration protocols. Significant limitations of both high-dose, single-injection protocols and previous multi-dose protocols in rodent models include high mortality rates and relatively small changes in hearing sensitivity. These limitations restrict assessment of both long-term changes in hearing sensitivity and effects of potential protective therapies. Here, we present a detailed method for an optimized mouse model of cisplatin ototoxicity that utilizes a multi-cycle administration protocol that better approximates the type and degree of hearing loss observed clinically. This protocol results in significant hearing loss with very low mortality. This mouse model of cisplatin ototoxicity provides a platform for examining mechanisms of cisplatin-induced hearing loss as well as developing therapies to protect the hearing of cancer patients receiving cisplatin therapy.

Keywords: Auditory brainstem response (ABR); Cisplatin; Distortion product otoacoustic emissions (DPOAE); Mouse; Ototoxicity; Vestibular sensory evoked potential (VsEP).

Figure 1:. A multi-cycle model of cisplatin administration.

A: Time course for audiometric evaluations and three cycles of cisplatin administration. Each cycle consists of a 4-day cisplatin injection period followed by 10 days of recovery. B: Changes in mouse weight are shown across the entire cisplatin protocol. Weight and body conditioning scores (BCS) were recorded daily. Mean± SEM, n=4-9 mice per group. Mice lost weight with each injection period and regained weight during each of the recovery periods. No animals met the criteria for euthanasia. C: Twice-daily nutrition and hydration support was provided throughout the protocol.

Figure 2:. Cisplatin causes dose-dependent loss of cochlear outer hair cell function and hearing sensitivity.

Cochlear OHC function was assessed using distortion product otoacoustic emissions (DPOAEs). A: Relative to control mice, all cisplatin-treated mice showed significantly reduced DPOAE amplitudes or absent DPOAEs. As (cumulative) cisplatin dose increases, DPOAE amplitudes decrease. Mice treated with 3.5 mg/kg cisplatin (green) showed a nearly complete loss of DPOAEs across all frequencies, whereas mice treated with 3.0 mg/kg (red) showed near-total loss in the high frequencies, moderate loss in the mid-frequencies, and no change in the low frequencies. Low-dose cisplatin (2.5 mg/kg, blue) resulted in absent or reduced DPOAEs at high frequencies only. Grey shaded region denotes the mean biological noise floor. B: Auditory sensitivity was measured using auditory brainstem response (ABR) recordings. ABR threshold is defined as the lowest stimulus intensity that elicits a repeatable response. Control mice did not have changes in their ABR thresholds. Cisplatin resulted in significant threshold elevations in the high frequency region (>32 kHz) for all cisplatin-treated mice. As cisplatin dose increases, the hearing loss becomes more severe and spreads to include lower frequencies. 3.0 mg/kg cisplatin resulted in hearing loss across frequencies that was more severe in the higher frequencies. Mean± SEM, n=4-9 mice per group. Statistical analysis consisted of a 2-way ANOVA with Dunnett’s multiple comparisons, *p<0.05, **p<0.001.

Figure 3:. Cisplatin ototoxicity progresses after the cessation of cisplatin administration.

A second cohort of mice was treated with 3.0 mg/kg (controls received saline). Auditory tests were again conducted before cisplatin and repeated after cycle 3. A subset of animals was re-tested 4 months later to assess progression of cisplatin-induced hearing loss. A: Changes in DPOAE amplitudes at the end of cycle 3 (red) were similar to those seen in the first cohort. No significant differences in DPOAEs were observed between male and female mice. Four months after cycle 3 (blue) a significant progressive decrease in DPOAE amplitudes was observed in the mid-frequency region relative to end of cycle 3. This progression of OHC dysfunction is evident in both male and female mice. B: ABR threshold shifts at the end of cycle 3 in the second cohort were comparable to those seen in the first cohort. No differences in ABR threshold shifts were observed between sexes. Mice evaluated 4 months after cycle 3 demonstrated a significant, progressive loss in hearing sensitivity, shown here as a greater threshold shift, relative to the end of cycle 3 in both sexes (blue). Mean± SEM, n=4-8 mice per group. Statistical analysis consisted of a 2-way ANOVA with Dunnett’s multiple comparisons, *p<0.05, **p<0.001.

Figure 4:. Cisplatin results in significant loss of outer hair cells.

Microdissected cochlear turns immunostained for myosin-VIIa (blue) and CtBP2 (white) were imaged to assess IHC and OHC loss across the cochlea. A: IHCs were intact in both control (upper row) and cisplatin-treated mice (lower row). In contrast, cochleas from cisplatin-treated mice showed significant loss of OHCs in the middle and basal turns relative to controls at the end of Cycle 3 with significant subsequent deterioration 4 months later in the mid-frequency region. Panels B and C show quantification of IHC and OHC numbers, respectively, and further illustrate no loss of IHCs and significant loss of OHCs in cisplatin-treated mice in the middle and basal regions of the cochlea. Means±SEM, n=4-8 per group. Statistical analysis consisted of Multiple t-tests, ***p<0.001, ****p<0.0001, n.s. = no significance.

Figure 5:. Cisplatin did not result in vestibulotoxicity.

Mouse utricles were immunostained for myosin-VIIa (hair cell marker, white). No observable (A) or quantifiable (B) loss of utricular hair cells was present within the extrastriolar region. C: VsEP amplitudes were measured to assess vestibular function at the end of cycle 3 (red) and again 4 months later (blue). No significant differences in VsEP thresholds were observed between control and cisplatin-treated mice at either time point. Means±SEM, n=4-8 mice per group (1 ear per mouse). Statistical analysis consisted of a one-way ANOVA with Tukey’s multiple comparisons test (B) and paired t-test (C), n.s. = no significance.