In vivo real-time imaging reveals megalin as the aminoglycoside gentamicin transporter into cochlea whose inhibition is otoprotective

Abstract

Aminoglycosides (AGs) are commonly used antibiotics that cause deafness through the irreversible loss of cochlear sensory hair cells (HCs). How AGs enter the cochlea and then target HCs remains unresolved. Here, we performed time-lapse multicellular imaging of cochlea in live adult hearing mice via a chemo-mechanical cochleostomy. The in vivo tracking revealed that systemically administered Texas Red–labeled gentamicin (GTTR) enters the cochlea via the stria vascularis and then HCs selectively. GTTR uptake into HCs was completely abolished in transmembrane channel-like protein 1 (TMC1) knockout mice, indicating mechanotransducer channel-dependent AG uptake. Blockage of megalin, the candidate AG transporter in the stria vascularis, by binding competitor cilastatin prevented GTTR accumulation in HCs. Furthermore, cilastatin treatment markedly reduced AG-induced HC degeneration and hearing loss in vivo. Together, our in vivo real-time tracking of megalin-dependent AG transport across the blood–labyrinth barrier identifies new therapeutic targets for preventing AG-induced ototoxicity.

An in vivo biomarker to characterize ototoxic compounds and novel protective therapeutics

“…Previous in vivo evaluations utilizing GTTR demonstrated the intracochlear trafficking pattern of aminoglycosides, including trans-strial uptake into the endolymph before entering hair cells (Wang and Steyger, 2009). Indeed a recent study demonstrated via real-time imaging using GTTR that aminoglycoside enters the cochlea via the stria vascularis (Kim and Ricci, 2022). Consistent with this analysis, we observe a transient increase in stria vascularis fluorescence before fluorescence returns to near baseline levels by 48 hours post injection.…”

“…Despite the variety of mechanisms described for aminoglycoside-induced ototoxicity, there is consensus that a fundamental property of susceptible cells is their uptake and retention of aminoglycosides. Previous studies demonstrated that proximal convoluted tubule cells within the kidney and inner ear mechanosensory hair cells show aminoglycoside uptake and retention following in vivo systemic aminoglycoside administration, and there appears to be good correlation between aminoglycoside uptake and cell death (Imamura and Adams, 2003; Dai et al, 2006; Kim and Ricci, 2022).…”

An in vivo Biomarker to Characterize Ototoxic Compounds and Novel Protective Therapeutics

“…Previous in vivo evaluations utilizing GTTR demonstrated the intracochlear trafficking pattern of aminoglycosides, including trans- strial uptake into the endolymph before entering hair cells ( Wang and Steyger, 2009 ). Indeed a recent study demonstrated via real-time imaging using GTTR that aminoglycoside enters the cochlea via the stria vascularis ( Kim and Ricci, 2022 ). Consistent with this analysis, we observe a transient increase in stria vascularis fluorescence before fluorescence returns to near baseline levels by 48 h post-injection.…”

“…Despite the variety of mechanisms described for aminoglycoside-induced ototoxicity, there is consensus that a fundamental property of susceptible cells is their uptake and retention of aminoglycosides. Previous studies demonstrated that proximal convoluted tubule (PCT) cells within the kidney and inner ear mechanosensory hair cells show aminoglycoside uptake and retention following in vivo systemic aminoglycoside administration, and there appears to be good correlation between aminoglycoside uptake and cell death ( Imamura and Adams, 2003 ; Dai et al, 2006 ; Kim and Ricci, 2022 ).…”

The Hair Cell Analysis Toolbox: A machine learning-based whole cochlea analysis pipeline

“…Varied data are required for the training of generalizable deep learning models. In addition to data collected in our lab, we sourced generous contributions from the larger hearing research community from previously reported 6,30,[35][36][37][38][39][40][41][42] , and in some cases unpublished, studies. Bounding boxes for hair cells seen in maximum intensity projected z-stacks were manually annotated using the labelImg 43 software and saved as an XML file.…”