Functional and structural changes in the chinchilla cochlea and vestibular system following round window application of carboplatin

Abstract

OBJECTIVE: In chinchillas, moderate doses of carboplatin administered systemically selectively destroy inner hair cells and type I vestibular hair cells; however, it is unclear whether this unique damage pattern persists if carboplatin is applied directly to the cochlea, how quickly the damage develops and what cell death pathways are involved. STUDY DESIGN: To address these questions, carboplatin (5 mg/ml, 50 µl) was applied to the round window. RESULTS: Carboplatin caused a rapid decline in distortion product otoacoustic emissions, significantly increased compound action potential thresholds and caused massive inner hair cell loss and less severe outer hair cell loss. Hair cell loss was initially more severe in the base than the apex of the cochlea, but by 28 days post-treatment most cochlear hair cells were missing and hair cell density in the utricle, saccule and lateral crista was greatly reduced. At one day post-treatment, many hair cell nuclei were condensed or fragmented indicative of apoptosis, and expressed initiator caspase-8 and executioner caspase-3, but not initiator caspase-9. Carboplatin-treated animals circled towards the treated ear and during the swim test rolled towards the treated ear. CONCLUSION: These results indicate that local application of carboplatin causes loss of hair cells that begins near the base of the cochlea and spreads towards the apex with increasing survival time. Hair cell loss is initiated by caspase-8 followed by executioner caspase-3.

Figure 1

DPOAE input/output functions (mean +/− SEM) at f2 = 2, 4, 8 and 16 kHz measured in control ears (n = 5) and carboplatin treated ears 1, 3, 7, 14 and 28 days after carboplatin treatment.

Figure 2

CAP thresholds (mean +/− SEM) in normal control ears (n = 5) and in carboplatin treated ears (n = 5) of the 28-day group. Thresholds that exceeded the maximum output of the system are indicated by arrows.

Figure 3

Photomicrographs of the organ of Corti stained with FITC labeled phalloidin from a control animal (A) and a carboplatin treated animal 1 day post-treatment (B). Some IHC were missing 1 day post-carboplatin (arrows) and the stereocilia on many OHC were missing (jagged arrow) or blurry (arrowhead). Outer hair cells (OHC), inner hair cells (IHC) and pillar cells (PC).

Figure 4

Surface preparations of organ of Corti stained with SDH. (A) Photomicrograph of cochlea from normal animal. Scale bar 10 µM. (B) Photomicrograph of cochlea obtained 1 day post-carboplatin; most OHC present, most IHC missing. (C) Photomicrograph of cochlea obtained 3 days post-carboplatin; most IHC missing and most OHC missing or damaged.

Figure 5

Mean cochleograms showing percent IHC and OHC cell loss as a function of percent distance from the apex of the cochlea in 3-day, 7-day, 14-day and 28-day groups. Cochlear frequency position maps for the chinchilla cochlea are shown on the upper abscissa.

Figure 6

Typical surface preparation views in the middle turn of the chinchilla cochlea obtained 24 h following round window application of carboplatin. Panels A, C and E show labeling with TRITC-phalloidin (green) and TOPRO3 (red); some TOPR03 labeled nuclei are condensed and fragmented (arrows) and in a few cells the labeling is condensed and unevenly dispersed (jagged arrow). The same three images as on left, but green labeling, showing caspase-8 (B), caspase-9 (D) and caspase-3 (F) plus TOPRO3 (red). Many hair cells have condensed and/or shrunken nuclei (arrows) and express caspase-8 and caspase-3 (panels B and F, respectively), but not caspase-9 (panel D). A few normal cells with large, round, evenly stained nuclei and absence of caspase labeling are present (arrowheads, panels C,D,F). Three rows of OHC (1, 2, 3) and row of pillar cells (P).

Figure 7

Typical surface preparation images of the macula of saccule stained with phalloidin in normal chinchillas (A) or those treated with carboplatin and allowed to survive for 1 day (B) or 3 days (C). Stereocilia bundles present on all vestibular hair cells in normal chinchillas (A). In contrast, a few stereocilia are present in the vestibular sensory epithelium at 1 day post-treatment (B). At 3 days post-carboplatin, no stereocilia are visible on the macula of saccule (C).

Figure 8

The number of hair cells per 0.01 mm² (mean +/− SEM) in indicated region of control (n = 5) and in carboplatin-treated chinchillas at survival times of 1, 3, 7, 14 or 28 days.