All OAE recordings were performed by Dr A. Moulin (Institute o f Laryngology and Otology, University College London, UK) using an Otodynamics IL092 system, in a soundproof room, using a neonatal ILO probe fitted in the outer ear canal. The animals were anaesthetised using the same combination o f intra-muscular buprenorphine and intraperitoneal ketamine chlorhydrate. Body temperature was controlled using a thermostatic warming blanket during each recording.
Transiently Evoked Otoacoustic Emissions (TEOAEs)
TEOAEs were recorded using a non-linear click stimulus with a presentation rate o f 80Hz. Two hundred responses were averaged with a recording window o f 12.5ms. The stimulus intensity varied fi-om 80dB SPL to 46 dB SPL. Post-hoc analysis consisted o f windowing the TEAOE traces using a cosine fimction, between 1.2 and 8ms. The total echo amplitude and amplitude over frequency bands centred at 1.5kHz, 3kHz, 4.6kHz, 6.1kHz and 7.6kHz were calculated.
The crossed efferent system (COCB) was tested using the suppressive effect o f an ipsilateral broadband noise (BBN) on click evoked TEOAEs, according to a method similar to Berlin et al. (1995). A 52ms long BBN stimulus o f 65dB SPL, followed by a 5ms silent gap, was sent to the ear prior to a set o f 4 identical clicks eliciting the TEOAEs. The OAE response was recorded over a 12.5ms window, and the response elicited by each o f the 4 clicks was averaged separately in a different buffer. Four different stimulus intensities ranging from 70 dB pSPL to 52 dB pSPL in 6 dB steps were used in a randomised manner. For each stimulus intensity, 200 epochs were averaged without BBN, and 200 epochs with a BBN. Post-hoc analysis consisted o f windowing the OAE traces over 1.5 to 9ms. TEOAE amplitude was obtained for each
o f the 4 clicks o f the stimulus set, and for the sum o f the 4 clicks. TEAOE amplitude was compared when recorded in the presence, or absence, o f an ipsilateral BBN, at the several intensities recorded, in order to calculate the attenuation o f the ipsilateral BBN (Equivalent attenuation (EA, see Chery-Croze et al. (1994) for greater detail)).
Acoustic Overexposure.
Two animals per group were overexposed to sound (1 16dB SPL broad band) by Dr D. Hill (Institute o f Laryngology and Otology, University College London) as part o f his own investigations. Tissue was used to examine NC expression. Following exposure, animals were allowed to survive for 1, 3, 9 or 21 days.
Tissue Preparation fo r Light Microscopy and Immunofluorescence.
Guinea pigs were deeply anaesthetised with an intraperitoneal injection o f sodium pentobarbital (Reckitt and Coleman, Hull, UK, 0.05mg kg'* body weight). Once the rib cage was completely exposed bone scissors were used to cut each side o f the ribcage, lateral to the midline, allowing the mid-portion o f the ribcage to be raised, exposing the underlying organs. A needle was inserted into the left ventricle o f the heart. This was held in place using a butterfly clip. A small incision was made in the right auricle to allow blood to escape.
Rubber tubes used in perfusion were attached to the needle at one end and a blood pressure meter at the other. The meter was used to determine the rate o f perfusion, generally at, or ju st above the natural blood pressure o f the animal (approximately 80hg). In this way, animals were perfused via the aortic arch with a phosphate buffered saline (PBS) containing heparin (1ml o f lOOOunits/ml, pH 7.3), followed by 4%
paraformaldehyde in PBS (pH 7.3). Following perfusion, animals were decapitated, prior to tissue dissection.
Tissue Dissection,
The brain was removed from the skull and coronal cuts made rostral to the inferior colliculus and caudal to the cochlear nucleus. The brain tissue was immersed in 4% paraformaldehyde for further fixation overnight at 4°C. The auditory bullae were removed and cochleae were dissected out, post fixed with paraformaldehyde for 2 hours, washed in PBS, then decalcified in 4.15% ethylene diaminotetracteic acid (EDTA; Sigma Chemicals, Dorset, UK) for 24-48 hours at room temperature. Following décalcification, the bony otic capsule and modiolus were carefully removed. Tissues were then treated in one o f two ways:
Tissue Preparation fo r Wax Sectioning.
Following washing in PBS for 1 hour, brain tissue was dehydrated through graded alcohols (Sigma Chemicals, Dorset, UK). Specimens were placed in 70% ethanol for 6- 8 hours, 90% ethanol for 12-16 hours, three changes in 100% ethanol (2 hours), before being transferred to chloroform for 12-16 hours and embedding. Each specimen was then placed in three changes o f paraffin wax (Solmedia, Essex, UK) at 60°C, before embedding in fresh wax. Serial 8|im coronal sections were cut on a Reichert rotary microtome. Individual sections were mounted on poly-L-lysine coated slides (BDH, Dorset, UK) and allowed to air dry, before storage at 4°C.
Cryostat Sectioning.
Dissected cochleae were cryoprotected in sucrose (30% in PBS), mounted in OCT compound on a small piece o f cardboard and snap frozen in isopentane cooled to
-180°C in liquid nitrogen, prior to cryostat sectioning. Serial Sp-m midmodiolar sections were cut using a Leica CM 1900 cryostat at -23°C . Sections were collected onto poly-L-lysine coated slides and placed in an oven at 37°C for 2-10 hours to adhere.
N issl Staining,
Wax sections were dewaxed in 2 changes o f histoclear for 2-3 minutes (BDH, Dorset, UK), followed by rehydration in a graded series o f alcohol (2 changes o f 100% ethanol for 2 minutes. 70% ethanol for 2 minutes). Wax and frozen sections were then placed in distilled water for 2 minutes, followed by cresyl fast violet staining for 5-20 minutes. Sections were washed in distilled water for 1 minute, the stain differentiated in 95% ethanol containing 0.05% acetic acid (Sigma Chemicals, Dorset, UK) for 2-3 seconds, followed by washing in 95% ethanol for 2-3 seconds. Specimens were then dehydrated in 100% ethanol (2 changes, each for 2 minutes), and placed in two changes o f histoclear (2 minutes). Coverslips were mounted with DPX (BDH, Dorset, UK) and viewed using a Nikon light microscope.
Immunofluorescence.
All tissue sections were processed simultaneously. Prior to immunolabelling paraffin wax sections o f brain tissue were dewaxed by two washes in Histoclear (BDH, Dorset, UK), followed by rehydration through a graded series o f alcohol to water. These sections, together with frozen cochlea sections were rinsed in PBS. Immunolabelling was carried out in a humidified box. Sections were blocked with normal goat serum (5% in PBS) for 1 hour at room temperature. The sections were then incubated with the primary antibody (anti-nociceptin 1:1000-1:2000, Biotrend Chemikalien GmbH, Koln)
1% BSA/0.04% triton-xlOO/1.5% normal goat serum (pH 7.3). Following three washes in PBS, bound antibody was detected using fluorescein isothiocyanate- (FITC) - conjugated goat anti-rabbit IgG (Sigma chemicals, Dorset, UK) diluted 1:100 in the above phosphate buffer solution (90mins). Sections were then washed in PBS ( 4 x 5 min) prior to mounting w ith Citifluor (Agar Scientific Ltd., Essex, UK).
As a further control all tissue specimens were prepared and immunolabelled simultaneously. Every effort was made to label the same area o f each auditory tissue. For example, basal turns o f the cochlea were examined in each case.
Immunolabelled specimens were examined by epifluorescent microscopy using a N ikon Optiphot microscope or by scanning confocal microscopy. All images were collected in Adobe Photoshop 4.0.
3.3 RESULTS
Control Sections,
For secondary antibody control, the primary antibody was replaced with phosphate buffer solution, prior to incubation with the secondary antibody. Alternatively, primary antibody controls were performed by pre-incubation o f the tissue with nociceptin overnight, prior to immunolabelling (5pM, Tocris-Cookson Chemicals, Bolwin, MO). No immunostaining was observed under these control conditions.
The normal histology in the guinea pig cochlea, spiral ganglion and AVON at postnatal day 7 (P7) is shown in figure 3.1. These figures will be used as control references for further results in this section. No appreciable differences in appearance were observed in older animals. Figures 3.1a and 3.1b show the organ o f Corti, 3.1c and 3. Id the spiral ganglion cells and 3 .le and 3 .I f the anteroventral cochlear nucleus. The sections have been stained with cresyl fast violet (Nissl stained. Figures 3.1a, c and e) or immunolabelled with an anti-nociceptin antibody (Figures 3.1b, d and f).
(I) Unilaterally Deafened Animals,
In the results presented here to show the effects o f unilateral deafening on the gross histology and pattern o f nociceptin labelling in the AVCN, spiral ganglion cells and organ o f Corti, the guinea pigs had been deafened on the left side.