Animals
Hartley strain guinea pigs weighing 350–400 g were purchased from Japan SLC, Inc. (Hamamatsu, Japan). The Animal Research Committee of the Graduate School of Medicine at Kyoto University approved all experimental protocols. Animal care was supervised by the Institute of Laboratory Animals of the Graduate School of Medicine at Kyoto University. All experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to limit the number of animals used and their suffering.
Noise exposure
Guinea pigs were exposed to a continuous pure tone at 6 kHz with a 130-dB SPL for 15 min under general anesthesia [23]. Each animal was immobilized in a noninvasive head holder. A speaker was positioned 10 cm in front of the animal’s head. Sound levels were monitored using a 1/2 inch condenser microphone (Sony, Tokyo, Japan) and a fast Fourier transform analyzer (Sony).
To clarify the cochlear damage before the drug application, cochlear specimens (n = 4) were collected on day 7 and were processed histologically.
To determine noise-induced damage, ABR recording was performed 7 days after noise exposure. Animals showing 40 dB and larger ABR threshold shifts in both ears and smaller than 10 dB differences in threshold shifts between left and right ears were provided following experiments. To determine symmetrical lesions in both ears of animals that matched this condition, we compared hair cell numbers in left ears with those of right ears (n = 4).
Local drug application
On day 7 after the noise exposure, the gamma-secretase inhibitor MDL (Sigma-Aldrich, St. Louis, MI) was locally applied to the perilymph. Under general anesthesia, a cochleostomy was performed 1 mm from the edge of the round window on the basal turn of the cochlea. A Tefron tube with an inner diameter of 180-μm (UT-03, Unique Medical Co., Ltd., Tokyo, Japan) was connected to a micro-osmotic pump (Alzet, Cupertino, CA) and inserted into the scala tympani of the basal turn of the left cochlea of each guinea pig. The mini pump pumped MDL at a rate of 0.25 μL/h for 14 days. The tip of a Tefrone tube positioned at distance of 3 mm from the round window membrane, which accords with the 85% region from the cochlea apex [24], in order to deliver MDL to a damaged region, 70–80% region from the apex securely.
The MDL was dissolved in dimethyl sulfoxide (DMSO) and diluted with a phosphate buffered saline (PBS) to give a final concentration of 1 mM containing 0.3% DMSO. This concentration was also used in our previous work [21]. For 14 days, the MDL solution was continuously injected through the micro-osmotic pump into the left cochlea of the guinea pigs (n = 7). The control animals (n = 5) received PBS (instead of MDL solution) into the left cochlea; the PBS contained 0.3% DMSO. In addition, to examine the effects of MDL treatment on the normal cochlear epithelia of mature guinea pigs, normal guinea pigs (n = 3) received local a MDL application through the micro-osmotic pump.
To examine cell proliferation in the cochlear sensory epithelium by MDL treatment, the assay for labeling with 5-ethynyl-2′-deoxyuridine (EdU) was performed. EdU (Click-iT® EdU Imaging Kits, Molecular Probe, Eugene, OR) was added to the MDL solution (n = 4) or the vehicle solution (n = 4). The final concentration of EdU was 10 μM.
Functional assessments
Auditory function was assessed by recording auditory brainstem responses (ABRs) and distortion-product otoacoustic emissions (DPOAEs). The baseline ABR thresholds were measured within 7 days before the noise exposure. The ABR thresholds were measured at frequencies of 8, 10 and 12 kHz, which correspond to the regions damaged by noise trauma used in this study [24]. The animals were anesthetized with an intramuscular injection of midazolam (10 mg/kg) and an intramuscular injection of xylazine (10 mg/kg). They were kept warm with a heating pad. The generation of acoustic stimuli and the subsequent recording of evoked potentials were performed using the Powerlab/4sp data acquisition system (ADInstruments, Colorado Springs, CO, USA), as described in our previous study [25]. Acoustic stimuli, consisting of tone-burst stimuli (0.1 ms cos 2 rise/fall and 1-ms plateau), were delivered monaurally through a speaker (ES1spc; Bioresearch Center, Nagoya, Japan) connected to a funnel fitted into the external auditory meatus. The thresholds were determined from a set of responses at varying intensities with 5-dB sound pressure level (SPL) intervals. The electrical signals were averaged over 1024 repetitions. The thresholds at each frequency were verified at least twice. ABR recordings were performed 14 days after drug application. In comparison ABR threshold shifts in MDL-treated cochleae with those in vehicle-treated cochleae, values that ABR threshold shifts in drug-treated cochleae (left) were subtracted those of contralateral cochleae (right) to reduce influences due to individual differences.
DPOAE recordings were made with an acoustic probe (ER-10C; Etymotic Research, Elk Grove Village, IL) using the DP2000 DPOAE measurement system version 3.0 (Starkey Laboratory, Eden Prairie, MN) before sacrifice. Two primary tones with an f2/f1 ratio of 1.2 were presented at intensity levels of 65 dB SPL (L1) and 55 dB SPL (L2). The f2 was varied in one-ninth-octave steps from 8 to 12 kHz. A peak at 2f1–f2 in the spectrum was recognized as a DPOAE. The DP/noise floor (NF) levels were calculated.
Histological analysis
On day 14 after drug application, the Tefron tube was gently removed from the cochlea under general anesthesia. Finally, MDL-treated (n = 3), vehicle-treated (n = 3), MDL + EdU-treated (n = 3) and vehicle + EdU treated cochleae (n = 3) were obtained. After removing the stapes from the oval window, followed by opening the cochlear apex, 4% of paraformaldehyde in PBS was perfused into the cochlea. The excised cochleae were then immersed in the same fixative at 4°C for 12 h. After decalcification with 0.1 M ethylenediaminetetraacetic acid for 14 days at 4°C, the cochleae were subjected to histological analysis of whole mounts. The cochlear specimens were permeabilized in 0.2% Triton X in PBS for 30 min at room temperature. To determine the location of the hair cells, immunohistochemistry was performed for myosin VIIa (anti-myosin VIIa rabbit polyclonal antibody; 1:500; Proteus Bioscience Inc., Romana, CA) and F-actin labeling by fluorescein-phalloidin (1:400; Molecular Probes). At the end of the staining procedures, nuclear staining was performed with 1 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probes) in PBS. For an EdU assay, a Click-iT® EdU Imaging Kit (Molecular Probes) was used. Staining was performed according to the supplier’s direction followed by immunostaining for myosin VIIa and DAPI staining. Specimens were viewed with a Leica TCS SP2 confocal microscope (Leica Microsystems Inc., Wetzlar, Germany).
Cell counts
Quantitative analyses for hair cell numbers were performed in the 70–80% region from the apex of the cochlea. The cells that were positive for myosin VIIa with phalloidin-labeled stereocilia and DAPI-positive nuclei were defined as surviving hair cells. The number of inner hair cells and outer hair cells was counted respectively. Considering individual differences between animals, we subtracted hair cell numbers of right cochleae from those of left cochleae that were locally applied drugs.
Statistical analyses
An unpaired t-test (one-tailed) was used to compare differences between the groups, and a paired t-test (one-tailed) was used for comparisons between left and right cochleae. The data are presented in the text and figures as the mean ± the standard error of the mean (SEM). A p value less than 0.05 was considered significant.