whole-neonatal-cochlear-explants-as-an-in-vitro-model

Whole Neonatal Cochlear Explants as an <em>In vitro</em> Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

University of Basel Hospital

The process of gentamicin-induced hair cell damage includes the activation of oxidative stress processes. Sestrins, as stress-responsive proteins, protect cells against oxidative stress. Sestrins, particularly Sestrin-2, suppress excessive reactive oxygen species (ROS) accumulation and inhibit mammalian target of rapamycin complex 1 (mTORC1). Thus, we addressed the role of Sestrin-2 in the regulation of sensory hair cell survival after gentamicin exposure. Here, we show that Sestrins were expressed in the inner ear tissues, and Sestrin-2 immunolocalized in sensory hair cells and spiral ganglion (SG). The expression of Sestrin-2 was unchanged, and later downregulated, in gentamicin-treated explants from wild-type mice . Compared with wild-type mice, Sestrin-2 knockout mice exhibited significantly greater hair cell loss in gentamicin-treated cochlear explants. Significant downregulation of phosphorylation of AMP-activated protein kinase alpha (AMPKα) and upregulation of the 70-kDa ribosomal protein S6 kinase (p70S6K) were measured in wild-type cochlear explants exposed to gentamicin compared with their untreated controls. Such regulatory effect was not observed between explants from untreated and gentamicin-treated knockout mice. The gentamicin effect on mTOR signaling was rapamycin-sensitive. Thus, our data provide evidence that Sestrin-2 plays an important role in the protection of hair cells against gentamicin, and the mTOR signaling pathway appears to be modulated by gentamicin during hair cell death.

Sesn2 gene ablation enhances susceptibility to gentamicin-induced hair cell death via modulation of AMPK/mTOR signaling.

Brimonidine, an alpha-2 adrenergic receptor (α2-AR) agonist, has neuroprotective effects in the visual system and in spiral ganglion neurons. Auditory hair cells (HCs) express all 3 α2-AR subtypes, but their roles in HCs remain unknown. This study investigated the effects of brimonidine on auditory HCs that were also exposed to gentamicin, which is toxic to HCs. Organ of Corti explants were exposed to gentamicin in the presence or absence of brimonidine, and the α2-AR protein expression levels and Erk1/2 and Akt phosphorylation levels were determined. Brimonidine had a protective effect on auditory HCs against gentamicin-induced toxicity that was blocked by yohimbine. This suggested that the protective effect of brimonidine on HCs was mediated by the α2-AR. None of the treatments altered α2-AR protein expression levels, and brimonidine did not significantly change the activation levels of the Erk1/2 and Akt proteins. These observations indicated that brimonidine, acting directly via α2-AR, protects HCs from gentamicin-induced toxicity. Therefore, brimonidine shows potential for preventing or treating sensorineural hearing loss.

Brimonidine Protects Auditory Hair Cells from in vitro-Induced Toxicity of Gentamicin.

Telmisartan is an angiotensin II receptor blocker that has pleiotropic effects and protective properties in different cell types. Moreover, telmisartan has also shown partial agonism on the peroxisome proliferator-activated receptor γ (PPAR-γ). Auditory hair cells (HCs) express PPAR-γ, and the protective role of PPAR-γ agonists on HCs has been shown.

Telmisartan Protects Auditory Hair Cells from Gentamicin-Induced Toxicity in vitro.

Hearing loss affects many people worldwide and occurs often as a result of age, ototoxic drugs and/or excessive noise exposure. With a growing number of elderly people, the number of people suffering from hearing loss will also increase in the future. Despite the high number of affected people, for most patients there is no curative therapy for hearing loss and hearing aids or cochlea implants remain the only option. Important treatment approaches for hearing loss include the development of regenerative therapies or the inhibition of cell death/promotion of cell survival pathways. The mammalian target of rapamycin (mTOR) pathway is a central regulator of cell growth, is involved in cell survival, and has been shown to be implicated in many age-related diseases. In the inner ear, mTOR signaling has also started to gain attention recently. In this review, we will emphasize recent discoveries of mTOR signaling in the inner ear and discuss implications for possible treatments for hearing restoration.

mTOR Signaling in the Inner Ear as Potential Target to Treat Hearing Loss.

Hearing loss affects millions of people worldwide. Yet, there are still no curative therapies for sensorineural hearing loss. Frequent causes of sensorineural hearing loss are due to damage or loss of the sensory hair cells, the spiral ganglion neurons, or the synapses between them. Culturing the organ of Corti allows the study of all these structures in an experimental model, which is easy to manipulate. Therefore, the in vitro culture of the neonatal mammalian organ of Corti remains a frequently used experimental system, in which hair cell survival is routinely assessed. However, the analysis of the surviving hair cells is commonly performed via manual counting, which is a time-consuming process and the inter-rater reliability can be an issue. Here, we describe a deep learning approach to quantify hair cell survival in the murine organ of Corti explants. We used StarDist, a publicly available platform and plugin for Fiji (Fiji is just ImageJ), to train and apply our own custom deep learning model. We successfully validated our model in untreated, cisplatin, and gentamicin treated organ of Corti explants. Therefore, deep learning is a valuable approach for quantifying hair cell survival in organ of Corti explants. Moreover, we also demonstrate how the publicly available Fiji plugin StarDist can be efficiently used for this purpose.

Article	Year
Sesn2 gene ablation enhances susceptibility to gentamicin-induced hair cell death via modulation of AMPK/mTOR signaling. Cell death discovery\| PubMed ID: 28580173	2017
Brimonidine Protects Auditory Hair Cells from in vitro-Induced Toxicity of Gentamicin. Audiology & neuro-otology\| PubMed ID: 28889125	2017
Telmisartan Protects Auditory Hair Cells from Gentamicin-Induced Toxicity in vitro. Audiology & neuro-otology\| PubMed ID: 32369826	2020
mTOR Signaling in the Inner Ear as Potential Target to Treat Hearing Loss. International journal of molecular sciences\| PubMed ID: 34198685	2021
A deep learning approach to quantify auditory hair cells. Hearing research\| PubMed ID: 34343849	2021

Maurizio Cortada

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