Novel in vivo imaging analysis of an inner ear drug delivery system in mice: comparison of inner ear drug concentrations over time after transtympanic and systemic injections

Abstract

Objective: Systemic steroid injections are used to treat idiopathic sudden-onset sensorineural hearing loss (ISSHL) and some inner ear disorders. Recent studies show that transtympanic (TT) steroid injections are effective for treating ISSHL. As in vivo monitoring of drug delivery dynamics for inner ear is lacking, its time course and dispersion of drugs is unknown. Here, we used a new in vivo imaging system to monitor drug delivery in live mice and to compare drug concentrations over time after TT and systemic injections.

Methods: Luciferin delivered into the inner ears of GFAP-Luc transgenic mice reacted with luciferase in GFAP-expressing cells in the cochlear spiral ganglion, resulting in photon bioluminescence. We used the Xenogen IVIS® imaging system to measure how long photons continued to be emitted in the inner ear after TT or systemic injections of luciferin, and then compared the associated drug dynamics.

Results: The response to TT and IP injections differed significantly. Photons were detected five minutes after TT injection, peaking at ~20 minutes. By contrast, photons were first detected 30 minutes after i.p. injection. TT and i.p. drug delivery time differed considerably. With TT injections, photons were detected earlier than with IP injections. Photon bioluminescence also disappeared sooner. Delivery time varied with TT injections.

Conclusions: We speculate that the drug might enter the Eustachian tube from the middle ear. We conclude that inner-ear drug concentration can be maintained longer if the two injection routes are combined. As the size of luciferin differs from that of therapeutics like dexamethasone, combining drugs with luciferin may advance our understanding of in vivo drug delivery dynamics in the inner ear.

Figure 1. Strong bioluminescence around the ear of GFAP-luc derived from cochlear spiral ganglion.

Previous bioimaging study with IVIS reported luminescence around the ear of GFAP-luc. We first reveal here that the signal results from spiral ganglion. (A) Signal observed after local injection of luciferin via tympanic membrane. (B–D) Bioluminescence image of mouse head overlaid with a corresponding micro-CT image. (B) Micro-CT image of the head. (C) Merged image of A and C. (D) Photon bioluminescence image of the head. Note the signal was overlapped with temporal bone and not with superficial skins. (E) Ex-vivo bioluminescence in a freshly dissected temporal bone. Strong signal in the cochleae was observed (arrows), while no signal was detected in the skin and ear cartridge (right). (F) Immnohistochemistry for luciferase in the cochlea showed the expression was high in spiral ganglion (arrows).

Figure 2. Photon bioluminescence in GFAP-luc mice.

(A) Differences in photon bioluminescence of round windows with and without obstructions. The left panel shows photon bioluminescence in a mouse's left ear, in which the round window has been obstructed. The right panel shows the lack of bioluminescence in the mouse's right ear, in which the round window remains intact. (B) Time course showing photon bioluminescence in ears of mice with ip, with and without round window obstruction. ip;intraperitoneal injection, RWM; round window membrane.

Figure 3. Time course of photon bioluminescence.

(A) Time course of photon bioluminescence in five groups of animals: Luc ip, luciferase-expressing mice receiving intraperitoneal injections; Luc TT, luciferase-expressing mice receiving transtympanic injections; Luc TT RW closure, luciferase-expressing mice that received transtympanic injections after round window membrane obstruction; WT ip, wild-type mice receiving intraperitoneal injections. Background, background (no photons). (B) Bar graphs showing the highest photon counts by region. ip, intraperitoneal injection; TT, transtympanic injection; RW cl/TT, transtympanic injection after round window membrane obstruction. (C) Delivery time into GFAP-expressing cells of inner ear. Average time when the signal was maximum in each group was shown (mean ± SEM). Same conventions as in Fig. 2. *p<0.05.

Figure 4. Quantification of photons in different groups of mice with TT injections.

(A) Time course of photon bioluminescence for each group of mice. (B) Peak photon counts.