Platinum/Iridium alloy (Pt–Ir 90%/10%) wire (0.0055″) coated with Teflon was used to make the electrode (A-M System Inc, Carlsborg, WA). The end intended for implantation was melted into a ball (300–400um) using a propane/oxygen torch (). Silicone (Dow Corning Corporation, Midland, MI) was used to coat the bare part of the electrode in an hour glass configuration () to help position and stabilize the agarose/fibroblast matrix near the electrode ball. Care was taken to insure the ball was left uncoated. The total length of the electrode was 4 cm.
Figure 1 A schematic drawing showing the design and construction stages of the cochlear implant electrode: (a) Platinum iridium wire end formed in to ball (arrow points to Teflon coating of platinum iridium wire). (b) The bare end of wire coated in silicone in (more ...)
Coating electrodes with agarose and cells
Electrodes were coated using allogeneic fibroblasts. Guinea pig footpad fibroblast cell culture were established (see below) and expanded to eight confluent flasks (passaged 5 times). The fibroblasts were transduced with a viral vector 24 hrs prior to being seeded on the electrode.
On the day of electrode coating, a 6.25% low melt agarose solution (Boehringer-Mannheim, Mannheim, Germany) in MEM (GIBCO) was prepared. The solution was heated to 80°C and 200μl were transferred to microcentrifuge tubes, taking care to remove air bubbles. The tubes were then autoclaved. Only one tube is required for the coating. Occasionally however the agarose will “boil” due to expansion of air bubbles and therefore 2 or 3 spare tubes were prepared. Because the agarose must be kept in liquid state, the microcentrifuge tubes were transferred to a hotplate at 40°C immediately after being removed from the autoclave, ensuring that they were not allowed to cool down to room temperature.
The fibroblast culture flasks were passaged with the exception that all the fibroblasts from eight confluent flasks were resuspended in 10ml MEM. The fibroblasts were centrifuged (5min, 1000RPM, 400g) and resuspended in 50μl of MEM. This suspension was transferred to a microcentrifuge tube on the hotplate at 40°C. Once the fibroblasts warmed to 40°C, they were added to the 200μl low melt agarose and mixed.
The ball end of the previously prepared and autoclaved electrodes was dipped into the agarose/cell mixture with slow up and down movement. The ball end was held within the sterile laminar flow hood for a few seconds to allow the agarose to cool and solidify. With practice, a small quantity of agarose will fill the area in the “waist” part of the hour glass zone of silicone ( and ). If the amount of agarose was too small, the electrode could be re-dipped into the agarose. It was not possible to remove excess agarose. The coated electrodes were transferred to a large Petri-dish with 20 ml of fresh culture media. The coated end was fully immersed in the media.
Figure 2 a. A light micrograph of an electrode coated with cell/agarose matrix. b. An epi-fluorescence micrograph showing reporter gene expression in cells (arrows) within agarose matrix retrieved from coated electrode after implantation for 7 days. The bracket (more ...)
Primary cell culture
In order to coat the electrode with fibroblasts, a primary cell culture was established. All cell culture handling was carried out under sterile conditions within a laminar flow hood. A donor guinea pig was deeply anaesthetized (xylazine 10 mg/kg, i.m., ketamine 40 mg/kg, i.m.). The footpad of the posterior left limb was prepped with 10% povidine-iodine (Triad Disposables, Brookfield, WI) and 70% isopropyl alcohol (Humco, Texarkana, TX) solution. A 4 mm by 3 mm ellipse of skin (epidermis and dermis) was removed. The wound was closed with 4/0 Ethilon (Ethicon, Somerville, New Jersey) and the foot bandaged. The ellipse of skin was cleaned three times by immersion in Earl’s Balanced Salt Solution (GIBCO-BRL, Rockville, MD) with 1% penicillin/streptomycin (10,000 units penicillin G sodium and 10,000 microgram/ml streptomycin sulphate, GIBCO) and 0.5 μg Amophotericin B (GIBCO) for 5 min. The skin was cut into six pieces and placed dermis down in a 25 cm2 culture flask (Corning Incorporate, Corning, NJ). Two ml of Dulbecco modified Eagle medium (DMEM) (GIBCO) containing 10% fetal calf serum (Summit, Fort Collins, CO) and 1% penicillin/streptomycin was added. The flask was placed in a 37° C incubator with 5% CO2. After 10 days the fibroblasts had migrated onto the flask bottom and the cells were passaged as follows. Cells were resuspended with 0.05% Trypsin, spun at 1000RPM for 5 min (800g) and the pellet resuspended in 4 ml of medium and replated in four 25 cm2 flasks. Once cells were confluent (3–7 days), further passaging was performed. Cells from the primary cultures were later used for preparation of the cell/agarose matrix, coated on to the cochlear implant electrode and implanted into guinea pig ears.
Adenoviral vectors and cell transduction
We used recombinant adenoviruses (serotype 5 human adenovirus) with E1A, E1B and E3 regions deleted. Gene inserts were driven by the CMV promoter. The BDNF vector (Ad.BDNF) was a gift from Dr. Adriana Di Polo (Montreal, Canada). The vector was amplified by the University of Michigan Viral Vector Core. Ad.empty and Ad.LacZ were purchased from the Vector Core. All vectors were at a titer of 1×1012 plaque forming units (PFU) per ml. Viral stocks were aliquoted and stored in 10% glycerol at −80°C until use. After thawing, 25 μl of the appropriate viral suspension was added to each confluent 25 cm2 flask (0.1–2 × 1010 cells). The flask was returned to the incubator. After 1 hr the supernatant was discarded and the flask rinsed twice with media. The flask was returned to the incubator and the transduced fibroblasts were used to coat electrodes after 24 hrs.
Testing Ad.BDNF activity in vitro
To assay for Ad.BDNF activity, we transduced guinea pig firbroblasts as described above. We exposed the cells to the viral vector by adding 25 μl of Ad.BDNF to 4 ml of DMEM medium for 1 hr. Four days later, the medium was sampled and prepared for testing with a BDNF ELISA kit to determine the amount of BDNF produced by the fibroblast cells. Medium from non-transfected cells served as control. BDNF levels in the medium were determined using ChemiKine™ BDNF Sandwich ELISA kit, following kit instructions. The experiment was done twice.
Hair cell elimination and spiral ganglion counts
On day 0 the animals were deafened with kanamycin (420 mg/kg s.c.) and ethacrynic acid (52.5mg/kg i.v.). After confirming successful deafening (n=14) with acoustically-evoked auditory brainstem response audiometry (a minimum threshold shift of 60 dB) the animals were divided into two groups. On day 7, 8 animals were implanted (left ear) with electrodes coated with agarose and fibroblasts transduced with Ad.BDNF (designated BDNF group) and 6 animals were implanted (left ear) with an electrode coated with agarose and fibroblasts transduced with Ad.empty (control group).
Outbred pigmented guinea pigs (Elm Hill Laboratories, Chelmsford, MA) were used in this experiment and were 300–400g at the onset of the experiments. The University Committee for the Use and Care of Animals approved the animal experiments. The University of Michigan is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.
The guinea pigs were deeply anaesthetized (xylazine 10 mg/kg, i.m., ketamine 40 mg/kg, i.m.) and given chloramphenicol succinate (30mg/kg, i.m.) as a prophylactic antibiotic. The postauricular tissues were infiltrated with 250 μl of 1% lidnocaine and 1/80000 epinephrine. The bulla was exposed by means of a postauricular approach and opened to reveal the middle ear cavity. An electric drill with 1.5 mm drill bit was used to create a cochleostomy by enlarging the round window inferiorly. This allowed a straighter insertion angle compared to that via the unmodified round window, and longer insertion length than a separate cochleostomy. The coated electrode was inserted and small pieces of muscle used to seal the cochleostomy. The electrode was secured to the bulla with carboxylate cement (Durelon ESPE America, Norristown, PA). The subcutaneous tissues and skin were closed in two layers with 3/0 Vicryl and 4/0 Ethilon. Ten milliliters of warmed saline were administered subcutaneously and the animal recovered.
Retrieval of the electrodes and inner ear tissues
Guinea pigs were deeply anaesthetized (xylazine 10 mg/kg, i.m., ketamine 40 mg/kg, i.m.) and decapitated. The temporal bones were removed, taking care not to open the bulla at this stage. The anterior bulla was then opened and the electrode traversing the middle ear cut with a microscissor. The rest of the bulla was opened, the basal turn of the cochlea revealed and the electrode removed carefully (ensuring the agarose coating remains on the electrode), and placed in 2% paraformaldehyde fixative.
Histology, spiral ganglion counts and statistics
On day 48, animals were euthanized, the inner ears harvested and prepared for spiral ganglion counts. Briefly, tissues were decalcified in 2% EDTA and 0.25% glutaraldehyde for three weeks, and the electrode was removed by pulling from the round window, leaving the agarose coating within the basal turn of the cochlea. Ears were embedded in JB4 (Electron Microscopy Scientific, Washington, PA) and sectioned at 3 μm with glass knives at the near mid-modiolar plane. Section spacing, selection and counting methods were performed by a blinded assessor as previously described (Kanzaki et al., 2002
). The area of Rosenthal’s canal was measured using Metamorph (Universal Imaging Corporation, Downingtown, PA). The neuron density (number of cells/10000μm2
) for each cochlea’s lower six Rosenthal’s canal sections was determined using Metamorph image analysis software.
For statistical analysis, the spiral ganglion density for all six sections was summed for each animal and the difference in the total was compared between the BDNF group and control group using the univariate F-test. The spatial distribution of cells was tested by computing the mean values for each half turn and evaluating the rank order correlation (Spearman’s rho, SPSS v. 13); linear regression was not used because turn position is not a linear function of distance. Differences between means for each half turn were also evaluated by t-test, and the sequential Bonferroni criterion for multiple comparisons was applied.
Reporter gene expression and cell survival
To test in vivo the survival of transduced fibroblasts on the implanted electrodes, we implanted four guinea pigs with electrodes coated with agarose and fibroblasts transduced with Ad.LacZ. Animals were euthanized on day 7 (N=1), 19 (N=1), or 28 (N=2) days after implantation, and the electrodes were retrieved and assessed for reporter gene expression in cells embedded in the agarose. Coated electrodes were fixed for 2 hrs, rinsed in PBS, incubated with Triton X-100 for 10 min, then incubated with a rabbit polyclonal antibody against β-galactosidase (Chemicon, Temecula, CA) diluted 1:1000 in PBS, for one hour. Following three rinses with PBS the electrodes were incubated with rhodamine-conjugated goat anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA) diluted 1:100 in PBS, for 30 min. After a final rinse in PBS the electrodes were viewed with a Leica DMRB epifluorescence microscope.
In order to assess cell survival the agarose was carefully removed from the electrode after fixation, rinsed twice in PBS and placed in 30% sucrose solution for 12 hrs. The agarose was transferred to a specimen holder. To aid identification of specimen during cryosectioning the agarose bead was immersed in 2% alcian blue (Sigma-Aldrich, St. Louis, MO) for 1 min, placed in OCT (VWR International, West Chester, PA) and frozen in a mixture of dry ice and 100% ethanol. The OCT was sectioned in a Leica CM3000 cryostat. Ten μm sections were air dried and stained in hematoxylin and eosin (Sigma-Aldrich). Fibroblast survival was quantified by counting cells with nuclei as a percentage of all cells.