In vivo study

Adult albino guinea pigs were used for this study. Guinea pigs have been proved to be a reliable animal model for hearing loss, in which a robust pathological response to aminoglycoside induced cochlear damage could be elicited.(Forge & Schacht, 2000) In addition, the historic role of guinea pigs in cochlear research is based on the easy surgical access to its cochlea, and well characterized cochlear anatomy and physiology. The ages of the animals were about 2~4 months old with body weight at about 350~550gm. The use and care of animals reported in the present study was approved by the Institutional Animal Care and Use Committee of the China Medical University (permission number:

97-60-N).

Animals were anesthetized by intramuscular injection of a mixture of Zoletil (30 mg/kg) and xylazine (10 mg/kg), which allowed for non–ventilator dependent oxygenation. A maintenance dose, 50% of the initial dose, was injected intramuscularly every 60 min thereafter. After sedation, atropine (0.05 mg/kg) and chloramphenicol (400 mg/kg) were given intramuscularly. For surgical accessibility and convenience, only the left ear was treated, but to avoid acoustical crossover from the cochlea of the right ear during auditory brainstem response (ABR) measurements, the latter was surgically destroyed.

6.2.2.1 Surgical procedures

After an animal had been anesthetized, the cervical hair was shaved. It was placed in the prone position, and 1% xylocaine was injected into the posterior auricular area of the right ear. The skin and subcutaneous myofascial plane of the ear were dissected to expose the mastoid bulla, which was then opened so that the cochlea could be directly destroyed by needle penetration and disruption.

The ventral approach to the labyrinthine branch of AICA has been described in details before (Perlman et al., 1959; Kimura & Perlman, 1958). The procedures were summarized as below. Electrodes were inserted subcutaneously into the left mastoid (anode), right mastoid (cathode), and the back (ground), and an earphone was inserted into the left ear canal to monitor the ABR perioperatively. The animal was then placed in

the supine position. The skin over the ventral neck was disinfected with 75% alcohol and draped with aseptic dressings. A submental incision about 2 to 3 centimeters in length was made medially to the mandibular edge. The submandibular gland was separated to expose the digastric muscle and the paracondylar process. Separation of the digastric muscle from the fractured paracondylar process exposed the tympanic bulla. The anterior wall of the tympanic bulla was opened using a rongeur so that the basal cochlear turns were visible. Drilling started at the petrous bone, continued medially to the basal turn and anteriorly to the inferior petrosal sinus. The dura and the inferior petrosal sinus were protected during drilling by placing a thin Silastic sheet over them. A fenestration about 1.5 × 3.0 mm was made at the base of the skull, so that the labyrinthine artery was visible under the dura. The dura was excised, and the area opened so that the labyrinthine artery was fully exposed. The labyrinthine artery was closed with V1 microclamps] (#00396-01, S&T Microsurgical Instruments, USA), and cochlear function was thereafter monitored by click ABR at a 120-dB sound pressure level (SPL) at least every 3 min. Compared with the pre-operative apparent ABR waveform, persistent absence of the ABR waveform indicated that the microclamps had successfully occluded the labyrinthine artery.

6.2.2.2 Hearing test

Tone burst ABR pre-operative and serial post-operative hearing tests were performed in a sound attenuated room. The pure tone bursts were generated with the amplitude specified by a real-time programmable attenuator (Intelligent Hearing Systems, IHC Smart EP version 3.97, Miami, Fla., USA) with ER2 insert earphone, with stimulus frequency at 1k, 2k, 4k, 8k, and 16k Hz (0.2ms rise/fall time and 1ms flat segment) with maximal output level 125, 123, 111, 117, and 98 dB SPL. The click/tone bursts were produced by IHS high frequency transducer in a closed acoustic system through the sound delivery system. Responses for 1024 sweeps were averaged at each intensity level around the threshold in 5 dB SPL steps. Threshold was defined as the lowest intensity at which a clear waveform was visible upon inspection of an evoked trace. At least two sequences of recordings were made at the threshold intensity to verify the reproducibility of the ABR

responses. Each ABR threshold was compared with the pre-operative threshold, which served as the baseline measurement.

For the sham operation (sham-op) and the treatment groups (described below), serial ABR measurements were performed pre-operatively, immediately after the operation (PODi), 1 and 3 days after the operation (POD1d, 3d,) and 1, 2, 3, and 4 weeks after the operation (POD1w, 2w, 3w, 4w).

6.2.2.3 Surface preparation of cochlea and hair cell counting

At the end of the study, the animals were deeply anesthetized and then sacrificed by decapitation. The left cochleae were fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) by perilymphatic perfusion and then immersed in 4%

paraformaldehyde in 0.1 M PBS for 1 day. Surface preparation of cochlea was performed as previous described(Kohonen and Tarkkanen, 1966). The bony modiolus with the organ of Corti was carefully detached at the base of the cochlea following removal of the bony capsule, lateral wall, and tectorial membrane. After permeabilization with 0.3% Triton X-100 in PBS for 10 min, the tissues were incubated at room temperature with rhodamine-coupled phalloidin (Molecular Probes, Eugene, OR, USA) diluted 1:200 with PBS for 30 min. After the tissues were rinsed with PBS, strips of the organ of Corti were divided into the four turns, which were mounted on glass slides and examined with a fluorescence microscope (Model Leitz DM RBE; Leica, Wetzlar, Germany) to count the number of hair cells (HCs) present at each cochlear turn, thereby determining the extent of HC loss. For each group of animals, the mean losses of inner HCs (IHCs) and at each row of outer HCs (OHCs) in each group were calculated.

6.2.2.4 Histopathological examination

Cochlear sectioning along the paramodiolar axis was done followed by hematoxylin/eosin staining. After fixation as described above, the cochleae were decalcified by immersion in 10% ethylenediamine tetra-acetic acid (EDTA) (in 0.1M PBS, pH 7.4) for 4 weeks, with gentle stirring at 4°C. The cochleae were then dehydrated,

embedded in paraffin, and serially sectioned (4 μm thick) parallel to the modiolar axis.

The sections were plated for hematoxylin/eosin staining and examined under a high-power light microscope.

6.2.2.5 Grouping

The animals were divided into the following groups. A1: Control (n = 6). No surgery.

A2: Gentamicin (GM) group (n=6). The animals received single dose of gentamicin (125 mg/kg) by intramuscular (i.m.) injection. B1: Sham operation (n = 6): The animals received surgery as described above until the step at which the labyrinthine artery was exposed. Although the overlying dura was excised, the labyrinthine artery was fully exposed only momentarily, and then the wound was closed. B2: Sham-op/GM group (n=6): The animals received single dose of i.m. gentamicin (125 mg/kg) after sham surgery. C1: 30-min ischemia group (n=6). The animals received surgery as described above until the step at which the labyrinthine artery was exposed. The labyrinthine artery was then temporarily occluded with microclamps for 30 min (6 animals per subgroup).

Then the microclamps was released, and the wounds were closed. During the time that the arteries were occluded, the effects that clamping had on hearing were monitored by serial click ABR at 120-dB SPL. C2: 30min-ischemia/GM group (n=6). The animals received single dose of i.m. gentamicin (125 mg/kg) after induction of transient cochlear ischemia for 30 minutes, described as above.

In each group of 6 sacrificed animals, 4 cochleae were prepared for cochlear surface preparation and HC counting. The cochleae of the other two animals were sectioned along paramodiolar axis and stained with hematoxylin/eosin.

6.2.3 Tracking gentamicin uptake using fluorescence