TISSUE PREPARATION AND MICROSCOPIC EXAMINATION

4.3.1 Tissue Harvesting in Vivo

Animals were killed in a carbon dioxide (CO2) chamber or anaesthetized with 6%

pentobarbitone sodium (Rhone Merieux Ltd) following required procedures laid down by the British Home Office. The animals were decapitated and both bullae were removed. The bullae were widely opened to expose the vestibular parts and the cochlea. Holes were made in the apex o f the cochlea and the stapes was removed from the oval window, or the specimens were cut in two parts, then perfused directly with fixative by gentle injection

before the whole bulla was immersed in the fixative for further processing. The time from anaesthesia overdose, decapitation, perfusion and fixation was less than five minutes for each animal on average. Each time group at least three ears from gentamicin-treated guinea pigs were prepared.

4.3.2 Tissue Harvesting in Vitro

When the required incubation period was completed for in vitro studies, the cultured explants remained in situ on the coverslip were rinsed in cold buffered HBSS to wash out the residual culture medium, then processed for various microscopic examinations.

4.3.2 Fluorescence Light Microscopy

Fluorescence light microscopy was applied for whole mount examination o f the in vivo and in vitro specimens (Raphael and Altschuler, 1991a; 1991b; Meiteles and Raphael, 1994b; Goodyear and Richardson, 1992). The specimens from in vivo experiments for phalloidin staining o f actin were fixed in 4% paraformaldehyde containing 0.5% glutaraldehyde in O.IM sodium phosphate buffer, pH 7.3. Following two hours fixation with gentle rotation at room temperature, the bullae were washed in 0.0 IM phosphate buffered saline (PBS) and then the vestibular and cochlear tissues were dissected out under the dissecting microscope. The utricles and saccules were further dissected to remove the otoconia and reveal the apical surfaces. The cupula o f the cristae and the tectorial membrane o f the organ o f Corti were also removed carefully, if possible.

Phalloidin-FITC (Sigma Chemical Ltd) was prepared as a stock solution o f 0.1 mg / ml in methanol. Before use, it was diluted 1:100-500 in O.OIM phosphate buffer saline (PBS) or in Hank's Balanced Salt Solution (HBSS) with 5 mM HEPES at pH 7.3. The final staining

concentration varied between 0 .2 -lp g / ml. The cristae, the utricular and saccular maculae, and the organ o f Corti were immersed in 0.3% triton X-100 (Sigma Chemical Ltd) for 15 minutes before they were incubated in phalloidin-FITC staining solution. The phalloidin- FITC solution contained 3% bovine serum albumin (Sigma Chemical Ltd) to reduce background staining. The incubation time was two hours at room temperature. After rinsing in PBS three times, phalloidin-FITC labelled specimens were mounted on glass slides using Citifluor (Agar Scientific Ltd) or in glycerol with 0.1% paraphenyldiamine (Sigma Chemical Ltd). The preparations were examined and photographed with a Zeiss universal microscope or Nikon Optiphot-2 microscope equipped for epifluorescence. Fluorescence images were obtained using FITC-filters and photographs were taken on Kodak Tmax 400 or Ilford HP5 400 films.

4.3.3 Electron Microscopy

The specimens for electron microscopy were fixed in 2.5% glutaraldehyde in O.IM sodium cacodylate buffer with 3 mM calcium chloride (CaClz), pH 7.4, total osmolarity 550 mOsm. Fixation was continued for 1.5-2 hours at room temperature with constant slow rotation. After washing in the O.IM cacodylate buffer, specimens were post-fixed in 1% O.IM cacodylate buffered osmium tetroxide (OSO4) for another 1.5 hours at room

temperature. The cristae, the utricular and saccular maculae, and the organ o f Corti were dissected out in buffer or 70% ethanol under the stereo dissecting microscope. Some specimens were kept in buffer or in 70% ethanol at 4°C overnight for group processing.

The cultured vestibular explants were also processed for electron microscopy using the routine procedures. For transmission electron microscopy, the cultured explants were fixed

in 2.5% glutaraldehyde in O.IM sodium cacodylate buffer with 3 mM calcium chloride (CaClz), which was supplemented with 0.3% tannic acid (Goodyear and Richardson, 1992) for thin sectioning.

Specimen Preparation fo r Scanning Electron Microscopy

Samples for scanning electron microscopy were processed with double thiocarbohydrazide (TCH) and osmium procedure (Hunter-Duvar, 1978; Davis and Forge, 1987; Forge et al., 1992). Dissected specimens were placed in a processing basket and incubated in a freshly prepared and filtered saturated solution o f TCH for 20 minutes at room temperature. Following six washes in distilled water, the samples were incubated in 1% aqueous OSO4

for one hour. The whole procedure was then repeated. After dehydration through a graded series o f ethanol, critical point drying was carried out in Polaron critical point dryer using liquid CO2 The specimens were mounted on the surface o f a stub and sputter coated with

a thin layer o f gold before examination using JEOL JSM 35C SEM. Observation o f the specimens was usually carried out at low magnification first and increased to high magnification.

Data Analysis o f SEM Photographs

The number o f hair bundles was counted from SEM pictures taken along the striolar region o f the treated and control utricular and saccular maculae after systemic and topical treatment with gentamicin. Separate areas along the striolar regions where hair bundles were oriented in different directions to confirm the accurate selection were photographed under SEM (the number o f fields photographed from each specimen varied). Counting was carried out and repeated either from original negatives using a light box or from contact prints o f photographs taken at a nominal magnification o f 2 0 0 0x only, providing a field

o f 3200 \xm^ real area. Hair bundle numbers in the individual field o f the striolar regions from the different post-treatment survival groups and from control groups were compared using one-way analysis o f variance (ANOVA). ANOVA is a widely used statistical technique to test statistical significance o f many different trials which examines the variability o f the observations within each group as well as the variability between the group means. Duncan’s test for ANOVA was used to compare multiple group data with each other to determine which group means are significantly different from others and the difference between each tw o groups is considered as significance at p level 0.05. The ANOVA results from each experiment are reported in Chapter 5 in the simplified tables with the number o f samples, the mean ± s.d. and the median only.

Thin Sections fo r Transmission Electron Microscopy

F or transmission electron microscopy, the inner ear tissues were incubated en bloc in 1% uranyl acetate in 70% ethanol for one hour at room temperature. Then, the dehydrated specimens were infiltrated in the plastic resin (Agar Scientific Ltd) in three changes each time over tw o hours (2 parts propylene oxide : 1 part resin; 1 part propylene oxide : 2 parts resin; 100% resin). Finally, specimens were embedded in fresh resin using a flat mould and the plastic resin was polymerised at 60°C for 24 hours. Semi-thin sections were cut at 0.5-1 pm thickness on a Reichert-Jung ultramicrotome and stained with toluidine blue for light microscopy. Ultra-thin sections were cut at 90-110 nm thickness and efforts were made to cut sections as closely as possible perpendicular to the lumenal surface o f each epithelium. For most o f the embedded tissue blocks, sections o f the entire width o f the epithelium were taken at three different levels across the tissue. Serial thin sections w ere collected at each level and mounted on 200 mesh copper grids. The grids were stained in 4% uranyl acetate and Reynold’s lead citrate in an enclosed petri dish containing IM sodium hydroxide (NaOH) to prevent lead precipitate on the tissue sections, then examined by JEOL 1200 EX II TEM at an accelerating voltage o f 80 kV.