Caspase inhibitors promote vestibular hair cell survival and function after aminoglycoside treatment in vivo

Abstract

The sensory hair cells of the inner ear undergo apoptosis after acoustic trauma or aminoglycoside antibiotic treatment, causing permanent auditory and vestibular deficits in humans. Previous studies have demonstrated a role for caspase activation in hair cell death and ototoxic injury that can be reduced by concurrent treatment with caspase inhibitors in vitro. In this study, we examined the protective effects of caspase inhibition on hair cell death in vivo after systemic injections of aminoglycosides. In one series of experiments, chickens were implanted with osmotic pumps that administrated the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD) into inner ear fluids. One day after the surgery, the animals received a 5 d course of treatment with streptomycin, a vestibulotoxic aminoglycoside. Direct infusion of zVAD into the vestibule significantly increased hair cell survival after streptomycin treatment. A second series of experiments determined whether rescued hair cells could function as sensory receptors. Animals treated with streptomycin displayed vestibular system impairment as measured by a greatly reduced vestibulo-ocular response (VOR). In contrast, animals that received concurrent systemic administration of zVAD with streptomycin had both significantly greater hair cell survival and significantly increased VOR responses, as compared with animals treated with streptomycin alone. These findings suggest that inhibiting the activation of caspases promotes the survival of hair cells and protects against vestibular function deficits after aminoglycoside treatment.

Figure 1.

Direct infusion of zVAD into the vestibule promotes hair cell survival after concurrent treatment with streptomycin sulfate. A, Photomicrograph of hair cells stained for calretinin immunohistochemistry after treatment with 100 μm zVAD and 5 d of streptomycin (1200 mg/kg). B, Photomicrograph of calretinin-labeled hair cells after treatment with the carrier and streptomycin. C, Mean hair cell counts (±SEM) for the striolar and extrastriolar regions of the utricle after treatment with saline, streptomycin alone, carrier plus streptomycin, or 50 and 100 μm zVAD and streptomycin treatment. D, Mean hair cell counts for control, saline, carrier alone, 50 μm zVAD alone, or 100 μm zVAD alone treated animals. Calretinin-labeled cells were quantified in 10,000 μm ² regions of both the extrastriolar (6 regions per organ) and striolar (4 regions per organ) areas. n = 6–8 organs.

Figure 2.

Scanning electron micrographs of the horizontal crista ampullaris. A, Low magnification of the crista from an untreated animal with numerous hair cells throughout the sensory epithelium. B, High magnification of the central apical region. C, Low-magnification image from an animal treated with 5 d of streptomycin (1200 mg/kg) showed that surviving hair cells were present only at the edge of the epithelium. Hair cells with mature stereocilia are absent from the central apical region of the crista (C, D). E, Low-magnification image from an animal treated with 5 d of zVAD (1.5 mg/kg) and streptomycin. F, Central apical region of horizontal crista ampullaris shows only moderate hair cell loss. Scale bars: low magnification, 500 μm; high magnification, 10 μm.

Figure 3.

Scanning electron micrographs of utricles. A, Low-magnification image of a control utricle with densely populated hair cells. B, High-magnification image of the striolar region of the same utricle as shown in A. C, After 5 d of streptomycin treatment, hair cell damage is predominately localized to the striolar region with extensive stereociliary loss evident along the entire length of the striola (D). E, After 5 d of zVAD and streptomycin treatment, some hair cell loss is evident in the striola, but most hair cells are still present. Scale bars: low magnification, 500 μm; high magnification, 10 μm.

Figure 4.

Scanning electron micrographs of saccules. A, Low-magnification image of a control saccule, densely populated by hair cells. B, High-magnification image of the striolar region of the same saccule as shown in A. C, After 5 d of streptomycin treatment, hair cell damage is predominately localized to the striolar region with extensive stereociliary loss evident along the entire length of the striola (D). E, After 5 d of zVAD and streptomycin treatment, hair cell loss is evident in the striolar region (F), but many hair cells are still present when compared with animals treated with streptomycin alone. Scale bars: low magnification, 500 μm; high magnification, 10 μm.

Figure 5.

Systemic injections of zVAD promote hair cell survival. Chickens were given daily simultaneous injections of (1) zVAD (1.5 mg/kg) and streptomycin (1200 mg/kg), (2) streptomycin alone, or (3) saline alone for 5 d. Hair cells (either stereocilia bundles or calretinin ⁺ cells) were quantified in 10,000 μm² regions from the central region of the horizontal canal and both the extrastriolar and striolar areas of the utricle. For stereocilia bundle densities, means (±SD) represent three sampled regions per organ in the horizontal canal. In the utricle, two regions per organ of the extrastriolar region and one region per organ in the striolar region were sampled. For calretinin densities, means (±SD) represent two regions sampled in each horizontal canal. In the utricle, six regions per organ in the extrastriolar region and four regions per organ in the striolar region were sampled. More hair cells were present in both sensory organs after 5 d of treatment of zVAD/streptomycin, when compared with animals receiving streptomycin alone. Significantly fewer hair cells were present in all sampled regions of zVAD/streptomycin-treated animals when compared with animals receiving saline alone (p < 0.01). n = 3–5 organs.

Figure 6.

Rotation stimulation paradigms for horizontal vestibulo-ocular reflex (HVOR) and off-vertical axis rotation (OVAR). To stimulate hair cells in the horizontal canal, the chick was rotated around the EVA at frequencies ranging from 0.01 to 2 Hz with a constant peak velocity of 20°/sec, except for the 2 Hz stimulus, which was mechanically limited to 10°/sec. Compensatory eye movements were measured. To stimulate the utricle, OVAR was used to deliver low- to mid-frequency (0.0.0278–0.333 Hz) linear acceleration stimulation with the rotation axis tilted 11.7° (0.2 gm) relative to EVA. Both clockwise (CW) and counterclockwise (CCW) OVAR rotations were delivered.

Figure 7.

Three-dimensional eye-movement recordings from a normal chicken and a streptomycin-treated chicken rotated relative to EVA. HVOR responses are 1 Hz sinusoidal EVA rotations (20°/sec peak velocity). The top three traces represent torsional (E_tor), vertical (E_ver), and horizontal (E_hor) eye-position records with both slow and fast phase movements. The bottom three traces indicate torsional (Ω_tor), vertical (Ω_ver), and horizontal (Ω_hor) slow-phase eye velocity with the fast phases and post-saccadic eye oscillations removed. Upward deflection in the top three traces indicates counterclockwise, downward, and leftward eye movements, respectively. Dotted lines show zero eye velocity. The bottom trace represents head velocity (upward deflection equals leftward).

Figure 8.

Mean frequency response functions for the HVOR in streptomycin- and zVAD/streptomycin-treated animals. Mean (±SD) horizontal slow-phase eye velocity gain and phase values are plotted for control (filled circles), streptomycin only (open circles; n = 5 animals), and zVAD/streptomycin (star circles; n = 5 animals) treatment conditions. Each animal served as its own control. Gain values are presented as eye velocity/head velocity and phase values as degrees lead relative to head velocity.

Figure 9.

Frequency response functions for the HVOR in animals treated with zVAD alone. Slow-phase eye velocity gain and phase values are plotted for pretreatment performance (open symbols) and after 5 d of zVAD treatment (closed symbols; n = 2 animals). Gain values are presented as eye velocity/head velocity and phase values as degrees lead relative to head velocity. Treatment with zVAD had no effect on either the gain or phase.

Figure 10.

Mean (±SD) vertical and torsional slow-phase sensitivity and phase values to OVAR stimulation as a function of frequency in streptomycin- and zVAD/streptomycin-treated animals. Mean sensitivity values (degrees per second divided by gravity) were calculated as slow-phase eye velocity/head acceleration for four animals. Phase values (degrees) are shown relative to linear acceleration. Each animal served as its own control.