Measured dextran concentrations in perilymph samples.

<p>Dextran concentrations were measured for sequential fluid samples collected from the apex of implanted animals in which the cochleostomy was leaking during injection (A) or in which there was no detectable leak from the cochleostomy during the 2 hr injection period (B). Injections were performed at 100 nL/min either with a WPI Ultrapump (solid circles) or with iPRECIO SMP-200 pumps (solid triangles). Group average curves are shown in black. With leakage at the cochleostomy, the peak of the average marker concentration occurred in the 3^rd sample and averaged 271 μM (SD 103, n = 7) while with no leakage at the cochleostomy the peak of the average marker concentration occurred in the 4^th sample and averaged 529 μM (SD 138, n = 4). The predicted curve (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183374#pone.0183374.g003" target="_blank">Fig 3C</a>; 2 hr) is shown for comparison (gray diamonds). Measured concentrations were typically lower than predicted.</p

Measurements compared to simulations of the experiments.

<p>Open symbols: Calculated sample curves for 2 hr (green) and 24 hr (purple) injections using the same pharmacokinetic parameters. In these simulations we implemented a base-apex gradient of elimination (800 min half time at the base, transitioning to 30 min half time at the apex) and a base-apex gradient of volume flow (0.06 μL entering at the base, transitioning to zero at the apex). This scenario permitted the gradients along ST shown by samples 1–3 to be approximated after 2hr and 24 hr injections. Solid symbols: Measured group average curves. In the case of 24 hr injections the measured curve is also shown excluding one animal that had an abnormal decline in the later samples, which largely accounted for the difference between the measured and calculated curves in samples 6–10. The group mean for 7 day injections is also shown, but the calculated curve for 7 days of injection was identical to that at 24 hours (purple, open symbols) so it has been omitted.</p

Calculated gradients and sample concentrations.

<p>Calculated gradients along ST (upper row) and corresponding predicted sample concentrations (lower row) for FITC-dextran injections into ST for durations of 1 to 12 hours. The left column shows the results of 1 mM dextran injected at 100 nL/ min, and the right column shows the results of 10 mM dextran injected at 10 nl/min, with both injections occurring at 2.8 mm from the base of ST. The calculations use simulator parameters based on analysis of FITC-dextran data for 10 different injection-sampling conditions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183374#pone.0183374.ref030" target="_blank">30</a>], summarized in the text. Initially, a large gradient for dextran along ST is expected (red and blue curves in A and B) but the gradient is expected to decline as injection is prolonged and to disappear after 8–12 hours injection. The same is expected with the lower injection rate, but in this case the basal region of ST is not filled as effectively with dextran (B vs. A), so the peak of the sample curve occurs at sample 3 with 10 nL/min injection (D), rather than sample 4 with 100 nL/min injection (C).</p

Perilymph sample origins.

<p>(A) Exposed guinea pig cochlea with a cochlear implant inserted into the basal turn of scala tympani and with the apex prepared for sequential fluid sampling. The green substance surrounding the apex is silicone adhesive (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183374#sec002" target="_blank">methods</a>). The apex has been perforated and fluid efflux is accumulating on the hydrophobic surface, which can be collected with minimal contamination. (B) Calculated spatial origins for the first four 1 μL perilymph samples collected from the apex, based on the measured cross-sectional area (CSA) of the perilymphatic compartments. The first sample includes the apical portion of scala vestibuli where the samples are collected. Later samples take into account the unavailable volume occupied by the cochlear implant in the 1.3 to 8.3 mm region of scala tympani (ST). Fluid samples are also influenced by solute exchange with adjacent compartments as they travel up the cochlea (not represented in this figure).</p

Histological assessments of implanted cochleae.

<p>(A) 7 day; dissection opening scala vestibuli and removing the organ of Corti. Reddening on the wall of ST is apparent (arrow). (B) 7 day; implant contacting spiral prominence near basilar membrane (arrow). (C) 7 day; Reissner’s membrane normal (arrow), no indication of endolyphatic hydrops. (D) 24 hr; tight cochleostomy and unobstructed cannula outlet (arrow). (E) 24 hr; electrode at the region of cannula outlet near the middle of ST. Some particulate material is present in the fluid space of ST. (F) 24 hr; another specimen with plume of particulate material in the fluid of ST.</p

The implant-cannula-pump system ready for implantation.

<p>The pump reservoir was filled with 10 mM FITC-dextran solution in a bicarbonate-buffered artificial perilymph. The pump was coupled to the polyimide cannula of the implant with a 10 mm length of 21G stainless tubing, fixed to the polyimide tubing with cyanoacrylate. The stainless coupler was sutured to the implant to limit strain on the polyimide tubing.</p

Perilymph measurements at 3 time points compared.

<p>(A) Group mean curves for the three injection durations of 2hr, 24 hr or 7 days. The peak is higher for 24 hr injection than for 2 hr, but is lower after 7 days. Sample 1 concentration was lowest at 2 hrs and increased progressively with time. (B) Calculated ratio between sample 3 and sample 1 for individual animals in the study. The fitted regression line suggests that uniform distribution of dextran along ST (3:1 ratio of 1) would typically take about 11 days to achieve. Also shown on the curve is the change of 3:1 ratio with time initially predicted by simulations of the experiment.</p

Functional changes caused by implantation.

<p>Mean CAP threshold curves (A) and acoustic emission threshold curves (B) for 5 experimental groups with group numbers given below for CAP and emissions respectively. Unimplanted: (Green: 16, 7); Immediately after implantation (Brown: 9, 1); 1 day after implantation with injection (Purple, 5, 5); 7 days after implantation where injection failed (Blue, 4, 4); 7 days after implantation where injection was successful (Dark Blue, 3, 3). The implant location is shown on a frequency/distance plot for the guinea pig (C), adapted to show the relationship with distance along scala tympani (blue curve). The frequency range for significant CAP sensitivity loss corresponds to the middle region of the implant.</p

Dextran measurements at three time-points.

<p>Perilymph concentration of FITC-dextran in sequential samples collected from the apex after 2 hr (A), 24 hr (B) or 7 day (C) injections at 10 nL/min. Colored curves are individual experiments, black curves are the group mean and gray curves with open symbols are the simulator predictions. The mean 2 hr curve is lower, but similar to the predicted curve, with sample 3 having the highest concentration. Contrary to the prediction, dextran gradients were found to be still present even after 24 hours of injection, as seen in the low concentration found in sample 1 of all animals relative to sample 3. Even after 7 days, gradients still remained in some animals.</p