Impact of Systemic versus Intratympanic Dexamethasone Administration on the Perilymph Proteome

Abstract

Glucocorticoids are the first-line treatment for sensorineural hearing loss, but little is known about the mechanism of their protective effect or the impact of route of administration. The recent development of hollow microneedles enables safe and reliable sampling of perilymph for proteomic analysis. Using these microneedles, we investigate the effect of intratympanic (IT) versus intraperitoneal (IP) dexamethasone administration on guinea pig perilymph proteome. Guinea pigs were treated with IT dexamethasone (n = 6), IP dexamethasone (n = 8), or untreated for control (n = 8) 6 h prior to aspiration. The round window membrane (RWM) was accessed via a postauricular approach, and hollow microneedles were used to perforate the RWM and aspirate 1 μL of perilymph. Perilymph samples were analyzed by liquid chromatography-mass spectrometry-based label-free quantitative proteomics. Mass spectrometry raw data files have been deposited in an international public repository (MassIVE proteomics repository at https://massive.ucsd.edu/) under data set # MSV000086887. In the 22 samples of perilymph analyzed, 632 proteins were detected, including the inner ear protein cochlin, a perilymph marker. Of these, 14 proteins were modulated by IP, and three proteins were modulated by IT dexamethasone. In both IP and IT dexamethasone groups, VGF nerve growth factor inducible was significantly upregulated compared to control. The remaining adjusted proteins modulate neurons, inflammation, or protein synthesis. Proteome analysis facilitated by the use of hollow microneedles shows that route of dexamethasone administration impacts changes seen in perilymph proteome. Compared to IT administration, the IP route was associated with greater changes in protein expression, including proteins involved in neuroprotection, inflammatory pathway, and protein synthesis. Our findings show that microneedles can mediate safe and effective intracochlear sampling and hold promise for inner ear diagnostics.

Keywords: 3D printing; dexamethasone; inner ear diagnostics; microneedle; perilymph sampling; proteomics; round window membrane; steroids.

Figure 1.. SEM image of hollow surgical microneedle designed for perilymph sampling at 134X magnification.

The hollow microneedle is mounted on a 30-gauge syringe needle. Outer diameter: 100 μm, Inner diameter: 35 μm. Lumen indicated by arrow.

Figure 2.. Composition of guinea pig perilymph proteome, by functional categories.

The 175 gene names were searched against the mouse gene list in PANTHER (http://www/pantherdb.org) to determine the distribution of proteins across functional classes. 146 proteins mapped to a functional class. A: The protein class fold-enrichment is displayed. B: The protein class −log10 FDR enrichment is displayed.

Figure 3.. Compound Action Potential (CAP) and Distortion product otoacoustic emissions (DPOAE).

A: Mean CAP threshold shifts at 72 h compared to baseline, for all tested frequencies. The shaded area is bounded by the upper and lower limits of the 95% confidence interval. There was no significant shift in CAP measured at 72 h after perforation, for all 18 frequencies (n=18). B and C: DPOAE at the frequency 2f1 – f2 in response to a 70 dB stimulus averaged over all survival experiments at 0 h after perforation (A, n=18) and 72 h after perforation (B, n=18). Solid gray lines show the average noise for the experiments; Solid blue lines show the mean measured baseline DPOAE signal; and dotted red lines show mean DPOAE signal at 0 h (B) or 72 h (C) post-perforation. Shaded areas are bounded by the upper and lower limits of the 95% confidence interval for the average noise, and mean DPOAE signals. All DPOAE signals remained out of noise.