Female albino National Institutes of Health Swiss mice (Harlan Sprague Dawley, Inc., Indianapolis, IN), weighing from 20 to 24 g, were used. Prior to surgery and sacrifice, each mouse was deeply anesthetized with a mixture of ketamine hydrochloride (50 mg/kg), xylazine hydrochloride (5 mg/kg), and acepromazine maleate (1 mg/kg) intramuscularly. All the animal work was done in accordance with the animal use committee of the Veterans Affairs Medical Center.
The KLH (Pacific Biomarine Laboratories, Port Hueneme, CA) was an ammonium sulfate precipitate and prepared for use as follows: The slurry was suspended in phosphate-buffered saline (PBS), pH 6.4, dialyzed aseptically against PBS, and then centrifuged for 20 min at 20,000g. The supernatant was recentrifuged for 90 min at 87,000g. The pellet (associated KLH) was dissolved in PBS for systemic sensitization and intracochlear injection.
Secondary inner ear immune response
An inner ear immune response was generated by directly challenging the inner ear with KLH. Animals were first systemically sensitized to KLH by subcutaneous injection of the antigen in the back (0.2 mg KLH emulsified in Freund's complete adjuvant). Two weeks later they were boosted (0.2 mg KLH in Freund's incomplete adjuvant). Ten days later the right cochlea was injected with KLH (0.1 mg in 5 µl PBS, pH 6.4) through a microhole drilled through the cochlear capsule. The left cochlea served as a normal, nonsurgical control. The right cochlea was approached using an operating microscope and sterile technique. A postauricular incision was made and a hole was drilled into the bulla to allow good visualization of the promontory of the cochlear basal turn, stapedial artery, and round window niche. The stapedial artery was cauterized and removed and a hole (25 µm) was drilled through the cochlear capsule into the scala tympani of the lateral basal turn. The overflow of perilymph was gently removed by suction. KLH was very slowly injected using pressure from an air-filled Hamilton syringe coupled with plastic tubing to a glass micropipette containing the antigen. The hole was sealed with bone wax after injection and any excess fluid was removed. Five mice were sacrificed at each of the following time points after KLH injection: 3, 6, 24, 48, and 168 h (7 days). Seven mice were sacrificed after 12 h.
To determine the effects of surgery and fluid injection into the cochlea on cytokine expression, 5 mice were sacrificed 3 and 12 h after injection of sterile PBS (5 µl) into the cochlea. Two mice were sacrificed after 24 and 168 h.
Blockage of TNFα
Fourteen KLH-sensitized mice were divided randomly into one of two groups of 7 animals each. Etanercept, EnbrelTM (recombinant human TNF receptor: human IgG1 Fc fusion protein, rhuTNFR:Fc; Immunex, Seattle, WA) (2.5 mg in 0.1 ml), purchased through a pharmacy, was intraperitoneally (IP) injected into one group of 7 mice 10 min before KLH injection into the cochlea. Three days after the first etanercept injection, the same dose of etanercept was injected again. The other 7 mice did not receive the drug treatment, only intracochlear KLH. All the animals were sacrificed 7 days after cochlear KLH injection. The number of inflammatory cells in the cochlea and endolymphatic sac and IL-6 expression in these cells was compared between the two groups.
Tissue preparation and immunohistochemistry
At the designated survival time the animals were deeply anesthetized as described above and sacrificed by intracardiac perfusion of heparinized warm saline, followed by about 40 ml of periodate-lysine-paraformaldehyde (4% paraformaldehyde final concentration) fixative. The temporal bones were immediately removed and decalcified with buffered 5% ethylenediaminetetraacetate (EDTA) (pH 7.2) for 14 days (4°C). After decalcification, temporal bones were immersed sequentially in increasing concentrations (50%, 75%, 100%) of Optimal Cutting Temperature (O.C.T.) compound (Tissue-Tek; Miles Inc., Elkhart, IN), snap-frozen (-70°C), and cryostat-sectioned (4 µm) parallel to the modiolus. Sections were collected for hematoxylin and eosin (H & E) staining and immunohistochemistry.
IL-1β, IL-6, and TNFα expression was investigated using affinity-purified goat anti-mouse IL-1β (25 µg/ml), IL-6 (25 µg/ml), and TNFα (6.25 µg/ml) polyclonal antibodies (R&D Systems, Minneapolis, MN) in PBS with 0.1% BSA and 0.1% NaN3. The sections were incubated with 0.6% H2O2 in methanol for 20 min at room temperature (RT) to inactivate endogenous peroxidases and then incubated with 9% normal rabbit serum with 0.1% NaN3 for 60 min at 37°C to inhibit nonspecific binding of secondary antibody. Sections were incubated at RT for 1 h with primary antibody. Control sections were incubated with normal goat IgG (Sigma, St. Louis, MO). Following incubation the sections were washed with PBS for 30 min. Biotinylated rabbit anti-goat IgG (Vector Laboratories, Burlingame, CA) was used for 30 min at RT as secondary antibody. Antibody binding was detected using an Elite ABC Kit (Vector) for 30 min at RT with 3,3′-diaminobenzidine–H2O2 as chromagen. The specimens were dehydrated in graded ethanol and immersed in Citrisolv (Fisher Scientific, Tustin, CA) before mounting in Permount (Fisher).
KLH distribution in the endolymphatic sac was evaluated immunohistochemically using guinea pig anti-KLH serum (1:2000) as the primary antibody and affinity-purified biotinylated goat anti-guinea pig IgG (Vector) as the secondary antibody. The same detection system was used as described above. Nonimmune guinea pig serum was used as a reagent control.
For the development of the immunohistochemical assays, we used lipopolysaccharide (LPS)-stimulated spleen as positive control tissue. Three mice were injected intraperitoneally with 800 µg/kg of LPS (Sigma) and sacrificed 2, 4, and 6 h after injection. Spleen tissue was processed through the same preparative steps as the cochlea. The antibodies for IL-lβ, TNFα, and IL-6 identified the cytokine-expressing cells among the cells of the red pulp.
For each primary antibody, randomly chosen cochlear sections (more than 10 in most cochleas) and sections containing the endolymphatic sac (n = 4/cochlea) were immunolabeled. For each antibody, the number of labeled cells was counted in each section and an average was calculated. The total number of inflammatory cells was also counted within the same sections using Nomarski differential interference contrast optics. Inflammatory cells were identified by their spherical shape as opposed to the spindle-shaped fibrocytes or cuboidal-shaped epithelial cells of the endolymphatic sac. The percentage of labeled inflammatory cells/section was then calculated. The cells in the cochlear scalae and those in the perisaccular connective tissue and endolymphatic sac lumen were evaluated separately. The adjacent section was sometimes stained with H&E, if necessary, for an accurate assessment of the total number of inflammatory cells.
For the evaluation of the ability of etanercept to reduce inflammation, cochlear sections (usually more than 8) and sections containing the endolymphatic sac (n = 4) were randomly chosen and stained with H&E, the number of inflammatory cells was counted in each section, and an average was calculated.