Vestibular Damage and Repair in Chronic Ototoxicity: Cellular Stages, Physiological Deficits and Molecular Mechanisms

Abstract

Progressive ototoxicity of the inner ear is prevalent in patients administered aminoglycoside antibiotics with little understanding of how this damage occurs and to what extent it can be recovered. Numerous in vitro and in vivo studies using acute methods have been completed to demonstrate various types of damage in vestibular and auditory tissue, including hair cell damage that results in apoptosis or necrosis, excitotoxic damage, and/or degeneration of their afferents. However, progressive damage has only just recently been studied utilizing a sub-chronic exposure rat model; this model takes into account the progressive exposure mirrored in aminoglycoside administration that is not implied in acute experimentation. With this in mind, the sub-chronic exposure model was adapted for a new mouse model to characterize the progressive damage taking place in vestibular sensory epithelia and ganglia, along with a preliminary characterization in cochlear sensory epithelia. Mice were exposed to 30 mM IDPN (3,3’- iminodipropionitrile) in regular drinking water for 8 weeks, and monitored for vestibular deficits using an established test battery; auditory deficits were recorded using auditory brainstem response (ABR) measurements. Various techniques for identifying functional, histological (scanning/transmission electron microscopy; immunoconfocal), and molecular (mRNA; protein) data were utilized to study alterations in the vestibular and auditory tissues after sub-chronic intoxication. In the vestibular tissue, SEM/TEM imaging demonstrated progressive damage with the loss of calyceal junctions between type I hair cells and their calyx afferents, the fragmentation and retraction of the afferents, stereociliary bundle coalescence, and the unique mechanism of hair cell extrusion, where the cell is ejected from the epithelia into the endolymphatic cavity. Immunoconfocal and qRT-PCR data demonstrated a loss of caspr1 and tenascin-c in the calyceal junctions of type I hair cells and their afferents. A loss of active synapses between hair cells and their afferents was also noted, where active synapses were defined by the pre-synaptic ribeye of the hair cells and the post-synaptic GluA2 receptor of the afferents. Synaptic scaffolding protein expression was upregulated (PSD95, Homer1), which translated into an increase in the protein level (PSD95), likely for hair cell-afferent synapse stabilization and compensation. Progressive damage was noted to be at least partially or completely recoverable up until stereocilia coalescence of the hair cells. Finally, the expression of numerous scaffolding and signaling proteins were shown to be downregulated (qRT-PCR; RNAseq) during the exposure in the vestibular epithelium and ganglion, leading to the hypothesis of a depression in cell-cell adhesion between hair cells and their afferents and a depression in afferent signaling, resulting in an overall depressed system. In the cochlea, profound hearing loss was observed in a tonotopic pattern during the exposure; higher frequencies were affected first with longer exposure times affecting lower frequencies. Outer hair cells were lost tonotopically due to prolonged exposure, followed by active synapse loss of the inner hair cells. Those intoxicated for the first two weeks demonstrated a capacity for recovery before any outer hair cell or active synapse losses were seen. A sub-chronic ototoxic IDPN model demonstrates the progressive damage of the inner ear, allowing for the study of this damage and its potential for recoverability, gaining a clearer understanding of the mechanisms affecting the tissues.

La ototoxicidad progresiva del oído interno prevalece en los pacientes a los que se les administraron antibióticos aminoglucósidos con poca comprensión de cómo se produce este daño y hasta qué punto se puede recuperar. Se han completado numerosos estudios in vitro e in vivo para demostrar diversos tipos de daño en el tejido vestibular y auditivo; recientemente, se ha estudiado el daño progresivo utilizando un modelo de intoxicación subcrónica en rata. Este modelo tiene en cuenta la exposición progresiva reflejada en la administración de aminoglucósidos que no está implícita en los experimentos agudos. El modelo de intoxicación subcrónica se adaptó a un nuevo modelo de ratón para describir como se caracteriza el daño progresivo que se produce en los epitelios sensoriales vestibulares y los ganglios, junto con una caracterización preliminar en los epitelios sensoriales cocleares. Los ratones se expusieron a IDPN 30 mM (3,3'-iminodipropionitrilo) en agua potable normal durante 8 semanas y se observaron los déficits vestibulares utilizando una batería de pruebas establecida; los déficits auditivos se registraron utilizando medidas de respuesta auditiva del tronco cerebral. Se utilizaron diversas técnicas para estudiar las alteraciones en los tejidos vestibular y auditivo después de una intoxicación subcrónica. En el tejido vestibular, demostró un daño progresivo con la pérdida de las uniones calíceas entre las células ciliadas tipo I y sus aferentes del cáliz, la fragmentación y retracción de los aferentes, la coalescencia estereociliar y el mecanismo único de extrusión de células ciliadas. También se observó una pérdida de sinapsis activas y se demostró que la expresión de numerosas proteínas de andamiaje y señalización estaba regulada a la baja durante la intoxicación. En la cóclea, se observó una pérdida auditiva profunda en un patrón tonotópico durante la exposición y las células ciliadas externas se perdieron tonotópicamente debido a la exposición prolongada, seguida de la pérdida activa de sinapsis de las células ciliadas internas. Un modelo de IDPN ototóxico subcrónico demuestra el daño progresivo del oído interno, lo que permite el estudio de este daño y su potencial de recuperación, obteniendo una comprensión más clara de los mecanismos que afectan a los tejidos.