Hair cell uptake of gentamicin in the developing mouse utricle

Abstract

Intratympanic injection of gentamicin has proven to be an effective therapy for intractable vestibular dysfunction. However, most studies to date have focused on the cochlea, so little is known about the distribution and uptake of gentamicin by the counterpart of the auditory system, specifically vestibular hair cells (HCs). Here, with a combination of in vivo and in vitro approaches, we used a gentamicin‐Texas Red (GTTR) conjugate to investigate the mechanisms of gentamicin vestibulotoxicity in the developing mammalian utricular HCs. In vivo, GTTR fluorescence was concentrated in the apical cytoplasm and the cellular membrane of neonatal utricular HCs, but scarce in the nucleus of HCs and supporting cells. Quantitative analysis showed the GTTR uptake by striolar HCs was significantly higher than that in the extrastriola. In addition, the GTTR fluorescence intensity in the striola was increased gradually from 1 to 8 days, peaking at 8–9 days postnatally. In vitro, utricle explants were incubated with GTTR and candidate uptake conduits, including mechanotransduction (MET) channels and endocytosis in the HC, were inhibited separately. GTTR uptake by HCs could be inhibited by quinine, a blocker of MET channels, under both normal and stressed conditions. Meanwhile, endocytic inhibition only reduced GTTR uptake in the CoCl2 hypoxia model. In sum, the maturation of MET channels mediated uptake of GTTR into vestibular HCs. Under stressed conditions, MET channels play a pronounced role, manifested by channel‐dependent stress enhanced GTTR permeation, while endocytosis participates in GTTR entry in a more selective manner.

Mechanisms of Aminoglycoside- and Cisplatin-Induced Ototoxicity

“…Exacerbation of Aminoglycoside-Induced Cochleotoxicity Experimentally induced sepsis (via parenteral injection of lipopolysaccharides [LPS]) potentiates the degree of aminoglycoside-induced hearing loss in mice (Jiang et al, 2019;Koo et al, 2015), and likely vestibulotoxicity (Qian et al, 2020). Pilot data suggest that inflammation (and fever) also increases the risk of aminoglycoside-induced hearing loss (Cross et al, 2015;Henry et al, 1983).…”

“…TRP vanilloid subtype 1 (TRPV1) and TRP vanilloid subtype 4 (TRPV4) expressed on the apical membranes of hair cells are also aminoglycoside-permeant channels (see Figure 2C ), and both are also expressed in the stria vascularis (see Figure 1 ; Jiang et al, 2019 ; Karasawa et al, 2008 ; Myrdal & Steyger, 2005 ; Zheng et al, 2003 ). Hair cell expression of TRPV1 is upregulated in the cochlea during inflammation that, in vitro, increases aminoglycoside entry into vestibular hair cells ( Jiang et al, 2019 ; Qian et al, 2020 ). The TRP ankyrin 1 (TRPA1) channel is another aminoglycoside-permeant channel that is presumptively expressed on the basolateral membranes of outer hair cells (OHCs; see Figure 2D ).…”

“…Another entry route into hair cells is via non–receptor-mediated endocytosis at the apical and synaptic membranes of hair cells and trafficking toward lysosomes (see Figure 2E ). However, blocking endocytosis (see Figure 2F ) or impeding intracellular trafficking of aminoglycoside-laden endosomes does not prevent hair cell death ( Alharazneh et al, 2011 ; Hailey et al, 2017 ; Hashino et al, 2000 ; Qian et al, 2020 ; Vu et al, 2013 ).…”

Apigenin alleviates neomycin-induced oxidative damage via the Nrf2 signaling pathway in cochlear hair cells

“…Gentamicin conjugated by Texas Red (GTTR) was diluted to the final working concentrations of 50 μmol/L as previously described [25]. Cochlear explants from the Atho1-EGFP mice were preincubated with 20 μmol/L apigenin for 2 h and then co-incubated with the GTTR for 30 min.…”

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

“…We examine intracellular accumulation of the aminoglycoside geneticin (G418) conjugated to the fluorophore Texas red (TR) in neonatal (P5) mouse hair cells following systemic injection. Building off recent studies ( Makabe et al, 2020 ; Qian et al, 2021 ), we performed experiments in neonatal mice because (I) neonatal mice can be readily obtained in large number and at low costs from a small number of breeding adults and (II) neonatal otic capsules are incompletely ossified, allowing for rapid tissue fixation and easy dissection of sensory epithelium. We demonstrate that neonates, like juvenile mice (P25-30) with mature hearing sensitivity, exhibit dose- and time-dependent uptake and accumulation of G418-TR in hair cells.…”