Infusion of fixed volumes of artificial perilymph (AP) and 10mM sodium salicylate (SAL) in AP were used to evaluate the efficiency of two surgical approaches for modulating cochlear function in the mouse model system. The cochleostomy infusion approach was similar to that described by
Chen and colleagues (2006) while the directed perfusion was a combination of this approach and the posterior semicircular canal canalostomy described by
Kawamoto and co-workers (2001) and
Nakagawa and co-workers (2003). The canalostomy was chosen over a second cochleostomy in scala vestibuli since it provides the least difficult surgical access. Cochlear function was evaluated via DPOAE thresholds, allowing an indirect physiological estimation of drug distribution within the cochlea. While potential non-uniform sensitivity to infused agents makes direct prediction of threshold shifts difficult, the technique allows for comparison of infusion protocols. A fixed flow rate of 16 nl/min (1 μl/hr) was used as this has been shown to have limited impact on cochlear function (
Chen et al., 2006). A higher flow rate at 32nl/min was also examined. For all experiments, long infusion tubing connected to a syringe pump was used with different fluids separated by ~10 nl air bubbles to avoid within-tube diffusion. Initial infusion of AP confirmed no surgical impact to auditory function, with subsequent SAL and then AP washout. Threshold shifts were calculated by subtraction of the pre-surgery baseline measurement at each frequency, with statistical analysis of differences between surgical approaches (methods). Statistical significance of peak response and recovery when compared to baseline were also performed.
Custom Surgical Equipment
To facilitate surgical consistency and limit cochlear damage, insertion stops were created on carbide microdrills (Drill Bit City, Prospect Heights, IL) used to make the cochleostomy and canalostomy openings. Polyimide coated fused silica capillaries of 353 μm OD, 201 μm ID (Polymicro Technologies, Phoenix, AZ) were cut into 2 mm lengths using a wafer saw, and cleaned with isopropyl alcohol. For each drill bit, a cut capillary section was manually positioned over the drill shank under a microscope using forceps. The insertion depth was controlled by careful length measurement of the exposed drill bit tip against stacks of shim stock of known thicknesses (Precision Brand, Downers Grove, IL). The capillary section was glued in place with cyanacrolyate adhesive (Loctite 4206) and dental cement (3M ESPE Durelon). Insertion depths of 102, 127, 153, and 178 μm were created on microdrills of 175 μm and 100 μm diameters.
The use of silicone insertion stops on the infusion tubing has been reported in the literature to limit the depth of penetration (
Kingma, et al., 1992;
Chen, et al., 2006;
Johnson et al., 2007). For the present study, more effective seals were produced by direct bonding to the polyimide cannulae without insertion stops, so the stops were not used. To mitigate risks associated with uncontrolled penetration into scala tympani, a micromanipulator was used to insert the tubing into the cochleostomy hole with a target insertion depth of 150 μm.
Animals and Surgical Procedures
A total of 28 young adult (age 2-4 months) CBA/CaJ mice, bred and raised in-house, were divided into two groups: CO and C+C. 16 animals were used for the 16nl/min flow rate and 12 animals for the 32nl/min flow rate. All animal experiments were approved by the University of Rochester Committee on Animal Resources, and were performed using accepted veterinary standards.
For the CO approach, animals were deeply anesthetized with a mixture of ketamine/xylazine (120 and 10 mg/kg body weight, respectively, intraperitoneal injection (IP)), and the left ventral surface of the neck was shaved and cleaned. For animals also receiving the canalostomy (C+C approach), the left post-auricular region was shaved and cleaned. The animal was positioned on a heated operative plane on their back under aseptic conditions. Surgery was performed on the left (ipsilateral) ear. Following the procedures initially developed by
Jero and colleagues (2001) and modified by
Chen and co-workers (2006), the tympanic bulla was exposed by a ventral approach. Under an operating microscope, an incision was made longitudinally along the ventral surface of the neck, extending from the angle of the mandible to the level of the clavicle. The submandibular gland was retracted laterally to expose the digastric muscle which was cut with an electrocautery to expose the bony tympanic bulla and the stapedial artery. The stapedial artery was carefully lifted from the surface of the bulla and cauterized at the entrance to the bulla with care taken to minimize cochlear heating. The surrounding tissue was removed to expose the inferior-medial aspect of the bulla which was carefully cleaned and dried. A cochleostomy was drilled by hand at a location approximately 300 μm below the stapedial artery stump using 175 μm diameter carbide micro drills modified to include insertion stops. Sequentially longer insertion depth 175 μm bits were used (153 and 178 μm), with cochlear entry determined by a subtle change in mechanical resistance.
Using a micromanipulator (MM3-3, World Precision Instruments, Sarasota, FL), a fine metal probe and adhesive (3M Repositionable 75 spray adhesive) loosely attaching the polyimide infusion tubing to the probe, the infusion tubing was inserted into the cochleostomy. Medical grade adhesive (Loctite 4206, Rocky Hill, CT) was used to temporarily secure the infusion tubing to the bulla opening, with subsequent application of dental cement (3M ESPE Duralon, St. Paul, MN) providing a more permanent and robust bond, sealing the cannula to cochleostomy site. Pilot experiments with infusion of dye confirmed no leakage around the cochleostomy site with this approach. The surgery site was loosely sutured closed for this acute experiment to provide strain relief for the infusion tubing.
For animals also receiving the canalostomy (C+C approach), following the cochleostomy procedure described above the animal was positioned on a heated operative plane on their stomach under aseptic conditions. Under an operating microscope, an incision was made behind the left pinna and the muscles separated to expose the posterior semicircular canal. A hole was drilled in the posterior semicircular canal with a 100 μm diameter drill bit modified with an insertion stop. Sequentially longer bits were used (102 and 127 μm), with canal entry determined by a subtle change in mechanical resistance. The canalostomy was loosely covered with muscle for the duration of the experiment.
During infusions, mice remained immobilized by anesthesia as described above, with supplementary doses (1/3 of the initial dose) administered as needed to maintain the proper levels of general anesthesia. Parameters such as foot or tail pinch, palpebral reflex and respiratory rate were monitored to indicate the need for supplemental doses.
Drug Infusion System and Solutions
AP and SAL solutions were delivered to the basal turn of scala tympani through a 30-40 cm length of U S Pharmacopoeia Class VI polyimide tubing (044-I; ID 110 μm; OD, 139 μm; Microlumen, Tampa, FL). The infusion tubing was connected to a 25 μl Hamilton syringe (1702 RNR 22S/2”) using a PEEK nanotight fitting (Upchurch Scientific, Oak Harbor, WA). The syringe was mounted in a syringe pump (UMP2, World Precision Instruments, Sarasota, FL) allowing precise control of infusion rates. A schematic illustration of the infusion setup is shown in .
The volume of the infusion tubing was sufficient to contain the initial AP and subsequent SAL solutions. The syringe was carefully filled with AP, with all trapped air removed via repeated rapid aspiration and ejection. The infusion tubing was then connected and filled via syringe ejection. Defined volumes of solution and air were then pre-loaded into the infusion tubing through aspiration of AP, air, and SAL. The final volumes were as depicted in , with the initial 1000 nl of AP providing ample volume for post-surgery auditory assessment prior to delivery of SAL. The subsequent AP provided washout of SAL, and confirmation that auditory thresholds returned to baseline conditions. The inclusion of ~10 nl air bubbles between solutions avoided within-tube diffusion, and in the present study had no impact on auditory measures. The tubing tip was left immersed in AP until being attached to the micromanipulator immediately prior to drilling the cochleostomy hole. At this time, infusion at 16 nl/min was started to ensure that evaporation at the tubing tip did not incorporate an air bubble of variable and uncontrolled size. Infusion continued during insertion of the cannula tube and the gluing process. For flow rate experiments, the flow rate was increased from 16 to 32 nl/min following post-surgery auditory assessment. While the exact amount of initial AP injected was variable, the point of SAL delivery was known via the dispensed volume.
The control solution and base for the SAL solution was AP with a composition (in mM) of: NaCl, 120; KCl, 3.5; CaCl2, 1.5; glucose, 5.5; and HEPES buffer, 20 (
Chen et al., 2006). The pH was adjusted to 7.5 with NaOH and the solution filter sterilized and stored for later use. 10 mM sodium salicylate (JT Baker, Phillipsburg, PA) solutions were mixed on the day of experiment using the prepared AP solution.
Auditory Function Assessment
Auditory function was assessed via automated DPOAE threshold measurements at F2 frequencies 8.94, 13.45, 17.89, 24.60, 35.78, and 49.19 kHz. Measurements were performed prior to surgery, immediately following surgery, and approximately every 12 minutes during infusion. Mice were anesthetized as described above with supplementary doses administered as needed. Prior to recordings, the ear canals and ear drums were inspected for signs of obstruction or infection, and only those animals with clear outer and middle ears were used. While under anesthesia, body temperature was maintained at 38°C with a heating pad. Recording sessions were completed in a soundproof acoustic chamber (IAC) with measurements performed on both ears. Contralateral ear DPOAE thresholds provided a control for non-infusion related auditory threshold shifts.
Tucker Davis hardware (TDT; Alachua, FL) was controlled via ActiveX from a custom Matlab r13 (Mathworks; Natick, MA) graphical user interface. Sound stimuli were generated and signals acquired using Tucker Davis RP2.1 processors running at a sample rate of 195312.5 Hz. All signals were played through two electrostatic speakers (TDT EC1) connected by 4 cm tubes to a probe containing an ER10B+ microphone (Etymotic; Elk Grove Village, IL); the entire speaker and probe assembly were mounted in an adjustable vibration-isolating frame on a micromanipulator arm. All recorded signals were loaded into Matlab r13 for analysis. Waveforms from each individual presentation were windowed using a Hamming window and high-resolution 390625-point FFTs (2x sample rate) were calculated. The resulting FFTs had a bin size of 0.5 Hz allowing for accurate measurement of signal level as a function of frequency. Frequency-domain averaging was used to minimize artifacts; FFTs for multiple repetitions of the same stimulus were averaged together before subsequent analysis. The probe microphone was calibrated relative to a ¼” B&K microphone (Type 4938, Bruel & Kjaer; Naerum, Denmark) using a 0.1 cc coupler (simulating the mouse ear canal).
DPOAE amplitudes were measured in the following manner: two primaries (F1 and F2) were generated at 65 and 50 dB SPL, respectively. The ratio of the two frequencies was 1.25. Waveforms of the output of the ER10B+ probe microphone were captured on a TDT RP2.1. FFTs for each presentation were averaged together and the signal level at five frequencies was sampled: F1, F2, DP (2F1-F2), and two noise bins above and below the DP frequency. Following FFT sampling, dBV was converted to SPL based on the ER10B+ microphone calibration.
DPOAE Thresholds were defined as the F1 level required to produce a DP of 0 dB SPL (+/- 1 dB). We developed an automatic threshold search algorithm implemented in Matlab r13 using TDT hardware and the Etymotic ER10B+ probe microphone. The measurement of DP threshold was interleaved with DP amplitude measures described above and began with two primaries as above at 65 and 50 dB SPL. Based on the distance of the DP from its target level of 0 dB SPL (distance henceforth: DP error), F1 and F2 level on the subsequent trial was incremented (or decremented) by 0.6 of this distance; e.g., if the DP was at 10 dB SPL, F1 F2 were decremented by 6 dB. F2 level was always F1–15 dB. The 0.6 “approach factor” was determined empirically to be an optimal rate of approach combining rapid acquisition of threshold and minimal oscillation around the target. Due to extremely steep DP I/O functions around 0 dB and the resulting overshoot of DP amplitude, occasionally on successive trials the DP amplitude may oscillate around 0 dB. In each case of oscillation, defined as three trials in which the sign of the DP error changes each trial, the approach factor was automatically made smaller by a factor of 1.5. This iterative procedure allowed rapid convergence on DP threshold while preventing overshoot. Once the DP was measured to be within 1 dB of 0 dB SPL, the identical F1 level was presented again for confirmation. Identification of thresholds requires two successive trials of F1 F2 levels that evoked a 0 dB SPL DP amplitude.
Modeling and Simulated Concentration Gradients
All simulations were performed with FluidSim V1.6i (
Salt, 2005) using the mouse template and perfusion/flow option. Parameter values within the model were: duration (65 min), output interval (20 sec), molecular weight of solute (137 g/mol), inflow chamber (scala tympani), inflow location to approximate the cochleostomy site (1.3 mm), infusion rate (16 nl/min or 32nl/min), interscala and scala-to-blood communication half-time (30 min). All other parameters remained at their default values. Selection of the fluidic exit within the model allowed simulation of the two delivery approaches. For the CO approach, the exit was placed in the basal turn of scala tympani (0.1 mm) to approximate the location of the cochlear aqueduct. In the C+C simulation, the exit was placed at the basal turn of scala vestibuli (0.1 mm) to approximate the vestibular system (not represented in the FluidSim model). Since the program allows for only a single fluidic exit, flow through the cochlear aqueduct was not modeled in the C+C simulation.
Model parameters were chosen to closely approximate the
in vivo experiments, with interscala communication modeled as a constant cross section with a half-time based on published measurements in guinea pig with trimethylphenylammonium (
Salt et al., 1991). Relative solute concentration was set arbitrarily at 1000, with results normalized to this starting concentration for all simulations. Predicted solute concentration versus position in scala tympani was extracted at the end of the experiment and compared to
in vivo DPOAE threshold shifts at the same time point for both surgical approaches (1000 nl SAL infusion). While non-linear sensitivity effects may make direct correlation of threshold shift to concentration difficult, this approach allows for investigation of general trends and comparison of surgical approaches. A physiological place-frequency map of the mouse cochlea (
Müller et al., 2005) allowed correlation of DPOAE responses with cochleotopic position. The characteristic frequency was related to the DPOAE F2 frequency, with a scala tympani length of 4.55 mm utilized (
Thorne et al., 1999).
The impact of flow rate on velocity profiles and fluid jet mixing was investigated using an idealized, linear cochlear model of scala tympani and numeric modeling using a commercial finite element program (COMSOL Multiphysics). In this two step simulation process, the fluid velocity and pressure field were first calculated via a steady form of the momentum equation. Subsequently, the transport of a soluble dye with prescribed concentration at the inlet was modeled with a time-dependent solution of diffusion and convection using the simulated velocity field. The diffusion constant was set to zero in order to isolate convective transport and allow visualization of flow and mixing mediated spread. The inlet velocity was set as 28μm/sec or 56μm/sec, corresponding to flow rates of 16nl/min and 32nl/min respectively. The outflow was maintained at ambient pressure and the no slip condition prescribed to all walls. The long tube modeling the cochlea was closed at its distal end (apex) and was not porous. The geometry was composed of ~80,000 tetrahedral elements.
The model assumed a circular cross-section of variable diameter with the cochlear aqueduct located at 0.1mm from the basal end. Diameters were extracted from scala tympani cross-sectional areas (
Thorne et al, 1999); diameters included in the model were 0.11 mm at the base, 0.48 mm at 0.7 mm from the base, 0.32 mm at 1.4 mm to 3.9 mm from the base, and 0.23 mm at 4.1 mm to 4.5mm from the base. Both velocity profiles and steady-state concentrations were evaluated at 16 nl/min and 32 nl/min flow rates, with apical spread qualitatively compared.
Statistical Analyses
DPOAE threshold shift data were collected from each experiment with three consecutive data points averaged for each animal approximating the half-way point of salicylate infusion, end of salicylate infusion, and washout. Two-way analysis of variance (ANOVA) with repeated measures and exclusion of pre-SAL threshold data allowed comparison between surgical approaches and flow rates (Prism, GraphPad, La Jolla, CA). Responses are graphically shown as a mean ± SEM for all animals receiving each surgical approach. Bonferroni post-hoc analysis for multiple comparisons was used to evaluate threshold shifts in response to SAL infusion for each frequency tested. These comparisons used the true peak for each animal rather than the three point average.
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