Inner ear drug delivery via a reciprocating perfusion system in the guinea pig

Abstract

Rapid progress in understanding the molecular mechanisms associated with cochlear and auditory nerve degenerative processes offers hope for the development of gene-transfer and molecular approaches to treat these diseases in patients. For therapies based on these discoveries to become clinically useful, it will be necessary to develop safe and reliable mechanisms for the delivery of drugs into the inner ear, bypassing the blood-labyrinthine barrier. Toward the goal of developing an inner ear perfusion device for human use, a reciprocating microfluidic system that allows perfusion of drugs into the cochlear perilymph through a single inlet hole in scala tympani of the basal turn was developed. The performance of a prototype, extracorporeal reciprocating perfusion system in guinea pigs is described. Analysis of the cochlear distribution of compounds after perfusion took advantage of the place-dependent generation of responses to tones along the length of the cochlea. Perfusion with a control artificial perilymph solution had no effect. Two drugs with well-characterized effects on cochlear physiology, salicylate (5 mM) and DNQX (6,7-Dinitroquinoxaline-2,3-dione; 100 and 300 microM), reversibly altered responses. The magnitude of drug effect decreased with distance from the perfusion pipette for up to 10 mm, and increased with dose and length of application.

Fig. 1

Top panel: System diagram of the recirculating delivery system. Note the intentional use of a long length of compliant PE tubing on the return line. Dimensions are not to scale, but the tubing widths do indicate relative diameters. Bottom panel: Photograph of principal components of the system. Not shown are the electronic controller, the stainless steel sample loop and some components of the injection valve.

Fig. 2

Lumped circuit model simulating the fluid network. Units of resistance are psi min/μl, units of capacitance are μl/psi. The pump is modeled as a current source, the rigid PEEK and silica tubing as resistors, and the compliant PE tubing as capacitors.

Fig. 3

Experimental and simulation data showing the volume output of the reciprocating system when pumping four strokes at 0.5 Hz in 5-min cycles. Note the inlet tube length for this experiment was 3.0 cm rather than 28.5 cm as used for all guinea pig experiments. Of the 2 μl pumped in each cycle, approximately 750 nl is released from the outlet and then withdrawn back into the recirculating loop. The pressure at the outlet of the pump, upstream from the manifold, is also shown. Discrepancies between the data and the simulation probably result because the simulation neglects nonlinear capacitance arising from air bubbles.

Fig. 4

(Bottom) Measured displaced volume over one cycle into empty capillary, and (top) time-step images of output of fluorescein-solution into reservoir tube prefilled with AP. The arrow indicates the location of the outlet of the reciprocating system.

Fig. 5

Net quantity of fluorescein transferred into a cylindrical reservoir of AP during the first eight reciprocating cycles.

Fig. 6

Surgical preparation of the cochlea and perfusion of the control artificial perilymph solution produces minimal changes in low-level CAPs and DPOAEs. Preoperative data were collected after anesthesia, but before any surgery. Post cochleostomy data were taken following drilling of the cochleostomy and placement of the perfusion pipette into the hole. Post AP data were recorded following 33 min perfusions with the control artificial perilymph solution. On this and subsequent figures, DPOAEs are plotted relative to the stimulus level and frequency of the f2 primary required to produce a criterion DPOAE amplitude (iso-response) of 0 dB SPL. The low-frequency differences in the shape of the tuning curves for CAPs vs. DPOAEs are likely related to middle ear filtering of the acoustic DPOAE responses. Data are plotted as means ± S.E. (n = 11 animals).

Fig. 7

Time dependent changes in CAP thresholds (upper panel) and iso-DPOAEs (lower panel) are plotted as functions of time before, during, and after perfusion of 100 μM DNQX, a glutamate receptor antagonist that blocks transmission between the hair cell and the auditory nerve. Artificial perilymph or DNQX was introduced at the times indicated with the bar between the panels. Times during which data were taken differ slightly because CAPs and DPOAEs were recorded sequentially. Data are plotted as means ± S.E. (n = 5 animals).

Fig. 8

Perfusion of DNQX (100 μM for 85 min) elevated ABR thresholds without affecting iso-DPOAE contours, consistent with its action to block afferent transmission. Effects were greatest at highest frequencies and were reversible with washing. Threshold shifts are indicated in the lower panels, where data for 100 μM DNQX (taken from the panel above) are compared to those with perfusion with 300 μM DNQX. With the higher concentration (300 μM) effects were larger and extended to lower frequency regions of the cochlea (5.7 kHz). Data are plotted as means ± S.E. (n = 5 animals).

Fig. 9

Perfusion of salicylate. Salicylate (5 mM) reversibly elevated CAP thresholds and low-level iso-DPOAE contours. Highest frequencies were affected most and earliest, consistent with the basal site of drug entry. The magnitude of the high frequency shifts in DPOAEs are underestimated in this plot, because post-drug responses in many animals reached the stimulus level ceiling for frequencies above 24 kHz. Data are plotted as means ± S.E. (n = 6 animals).

Fig. 10

CAP thresholds and DPOAE iso-response values (re: 0 dB SPL) are shown before and after perfusion of control and 5 mM salicylate as functions of frequency. Data plotted are mean ± S.E. for 6 animals.