Serum Prestin After Ototoxin Exposure Is Not Dependent on Outer Hair Cell Loss.

HYPOTHESIS: Cyclodextrin (CDX)-induced serum prestin burst is not dependent on outer hair cell (OHC) loss. BACKGROUND: Serum prestin has been proposed as a biomarker for ototoxicity. We recently used an automated Western approach to quantify serum prestin changes in a newly introduced model of CDX ototoxicity. To gain insights into prestin as a biomarker, here we further characterize serum prestin in the CDX model. METHODS: Guinea pigs were treated with 750, 3,000, or 4,000 mg/kg CDX, and serum samples were obtained through up to 15 weeks after exposure. Serum prestin levels were quantified using automated Western, and hair cell counts were obtained. RESULTS: All three doses induced an N -glycosylated ~134-kDa prestin burst; however, only the 3,000 and 4,000 mg/kg resulted in robust OHC loss. Prestin levels returned to baseline where they remained up to 15 weeks in the absence of OHCs. CONCLUSION: The ~134-kDa prestin burst induced after CDX administration is N -glycosylated, representing a posttranslational modification of prestin. Serum prestin seems to be a promising biomarker when using therapeutics with ototoxic properties because it is not dependent on OHC loss as a necessary event, thus affording the opportunity for early detection and intervention.

Automated Western Blot Analysis of Ototoxin-Induced Prestin Burst in the Blood after Cyclodextrin Exposure.

HYPOTHESIS: Ototoxin cyclodextrin (CDX) will induce a burst in serum prestin when quantified with automated Western blot analysis. BACKGROUND: In the clinical realm, we primarily rely on audiological measures for diagnosis and surveillance of sensorineural hearing loss (SNHL) and have limited therapeutic options. We have proposed a blood-based biomarker approach to overcome this challenge by measuring the outer hair cell's (OHC) electromotile protein, prestin, in the blood. Previously, we demonstrated a burst in serum prestin after cisplatin exposure using enzyme-linked immunosorbent assayELISA. METHODS: Guinea pigs were treated with either 3,000 or 4,000 mg/kg CDX, and serum samples were obtained through 3 days after exposure. Serum prestin levels were quantified using automated blot analysis, western and hair cell counts were obtained. RESULTS: Both 3,000 and 4,000 mg/kg resulted in robust OHC loss, although more variability was seen at the lower dose. Automated Western blot analysis demonstrated that the prestin profile after CDX exposure is different than baseline. Specifically, a new ~134- kDa band accounted for the prestin burst after ototoxin ablation of OHCs at both doses. CONCLUSIONS: We reproduced the prestin burst seen after cisplatin administration using CDX. Automated Western blot western analysis revealed that a ~a ~ 134- kDa species of prestin is responsible for the burst. We suggest that the induced band may be a prestin dimer, which could serve as a biomarker for early detection of ototoxicity in the clinical setting. These results add further promise to the potential of serum prestin to serve as an ototoxicity biomarker when using therapeutics with ototoxic properties.

'We don't need another Scottish vet school'.

Will McLean argues that the SRUC's plans to open its own vet school is not an appropriate solution to the problems the profession faces with regards to a shortage of vets working in rural and public health roles.

Improved Speech Intelligibility in Subjects With Stable Sensorineural Hearing Loss Following Intratympanic Dosing of FX-322 in a Phase 1b Study.

OBJECTIVES: There are no approved pharmacologic therapies for chronic sensorineural hearing loss (SNHL). The combination of CHIR99021+valproic acid (CV, FX-322) has been shown to regenerate mammalian cochlear hair cells ex vivo. The objectives were to characterize the cochlear pharmacokinetic profile of CV in guinea pigs, then measure FX-322 in human perilymph samples, and finally assess safety and audiometric effects of FX-322 in humans with chronic SNHL. STUDY DESIGNS: Middle ear residence, cochlear distribution, and elimination profiles of FX-322 were assessed in guinea pigs. Human perilymph sampling following intratympanic FX-322 dosing was performed in an open-label study in cochlear implant subjects. Unilateral intratympanic FX-322 was assessed in a Phase 1b prospective, randomized, double-blinded, placebo-controlled clinical trial. SETTING: Three private otolaryngology practices in the US. PATIENTS: Individuals diagnosed with mild to moderately severe chronic SNHL (≤70 dB standard pure-tone average) in one or both ears that was stable for ≥6 months, medical histories consistent with noise-induced or idiopathic sudden SNHL, and no significant vestibular symptoms. INTERVENTIONS: Intratympanic FX-322. MAIN OUTCOME MEASURES: Pharmacokinetics of FX-322 in perilymph and safety and audiometric effects. RESULTS: After intratympanic delivery in guinea pigs and humans, FX-322 levels in the cochlear extended high-frequency region were observed and projected to be pharmacologically active in humans. A single dose of FX-322 in SNHL subjects was well tolerated with mild, transient treatment-related adverse events (n = 15 FX-322 vs 8 placebo). Of the six patients treated with FX-322 who had baseline word recognition in quiet scores below 90%, four showed clinically meaningful improvements (absolute word recognition improved 18-42%, exceeding the 95% confidence interval determined by previously published criteria). No significant changes in placebo-injected ears were observed. At the group level, FX-322 subjects outperformed placebo group in word recognition in quiet when averaged across all time points, with a mean improvement from baseline of 18.9% (p = 0.029). For words in noise, the treated group showed a mean 1.3 dB signal-to-noise ratio improvement (p = 0.012) relative to their baseline scores while placebo-treated subjects did not (-0.21 dB, p = 0.71). CONCLUSIONS: Delivery of FX-322 to the extended high-frequency region of the cochlea is well tolerated and enhances speech recognition performance in multiple subjects with stable chronic hearing loss.

Approaches to Treat Sensorineural Hearing Loss by Hair-Cell Regeneration: The Current State of Therapeutic Developments and Their Potential Impact on Audiological Clinical Practice.

Sensorineural hearing loss (SNHL) is typically a permanent and often progressive condition that is commonly attributed to sensory cell loss. All vertebrates except mammals can regenerate lost sensory cells. Thus, SNHL is currently only treated with hearing aids or cochlear implants. There has been extensive research to understand how regeneration occurs in nonmammals, how hair cells form during development, and what limits regeneration in maturing mammals. These studies motivated efforts to identify therapeutic interventions to regenerate hair cells as a treatment for hearing loss, with a focus on targeting supporting cells to form new sensory hair cells. The approaches include gene therapy and small molecule delivery to the inner ear. At the time of this publication, early-stage clinical trials have been conducted to test targets that have shown evidence of regenerating sensory hair cells in preclinical models. As these potential treatments move closer to a clinical reality, it will be important to understand which therapeutic option is most appropriate for a given population. It is also important to consider which audiological tests should be administered to identify hearing improvement while considering the pharmacokinetics and mechanism of a given approach. Some impacts on audiological practice could include implementing less common audiological measures as standard procedure. As devices are not capable of repairing the damaged underlying biology, hair-cell regeneration treatments could allow patients to benefit more from their devices, move from a cochlear implant candidate to a hearing aid candidate, or move a subject to not needing an assistive device. Here, we describe the background, current state, and future implications of hair-cell regeneration research.

Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells.

Death of cochlear hair cells, which do not regenerate, is a cause of hearing loss in a high percentage of the population. Currently, no approach exists to obtain large numbers of cochlear hair cells. Here, using a small-molecule approach, we show significant expansion (>2,000-fold) of cochlear supporting cells expressing and maintaining Lgr5, an epithelial stem cell marker, in response to stimulation of Wnt signaling by a GSK3ß inhibitor and transcriptional activation by a histone deacetylase inhibitor. The Lgr5-expressing cells differentiate into hair cells in high yield. From a single mouse cochlea, we obtained over 11,500 hair cells, compared to less than 200 in the absence of induction. The newly generated hair cells have bundles and molecular machinery for transduction, synapse formation, and specialized hair cell activity. Targeting supporting cells capable of proliferation and cochlear hair cell replacement could lead to the discovery of hearing loss treatments.

Distinct capacity for differentiation to inner ear cell types by progenitor cells of the cochlea and vestibular organs.

Disorders of hearing and balance are most commonly associated with damage to cochlear and vestibular hair cells or neurons. Although these cells are not capable of spontaneous regeneration, progenitor cells in the hearing and balance organs of the neonatal mammalian inner ear have the capacity to generate new hair cells after damage. To investigate whether these cells are restricted in their differentiation capacity, we assessed the phenotypes of differentiated progenitor cells isolated from three compartments of the mouse inner ear - the vestibular and cochlear sensory epithelia and the spiral ganglion - by measuring electrophysiological properties and gene expression. Lgr5+ progenitor cells from the sensory epithelia gave rise to hair cell-like cells, but not neurons or glial cells. Newly created hair cell-like cells had hair bundle proteins, synaptic proteins and membrane proteins characteristic of the compartment of origin. PLP1+ glial cells from the spiral ganglion were identified as neural progenitors, which gave rise to neurons, astrocytes and oligodendrocytes, but not hair cells. Thus, distinct progenitor populations from the neonatal inner ear differentiate to cell types associated with their organ of origin.

Distribution of Na,K-ATPase α subunits in rat vestibular sensory epithelia.

The afferent encoding of vestibular stimuli depends on molecular mechanisms that regulate membrane potential, concentration gradients, and ion and neurotransmitter clearance at both afferent and efferent relays. In many cell types, the Na,K-ATPase (NKA) is essential for establishing hyperpolarized membrane potentials and mediating both primary and secondary active transport required for ion and neurotransmitter clearance. In vestibular sensory epithelia, a calyx nerve ending envelopes each type I hair cell, isolating it over most of its surface from support cells and posing special challenges for ion and neurotransmitter clearance. We used immunofluorescence and high-resolution confocal microscopy to examine the cellular and subcellular patterns of NKAα subunit expression within the sensory epithelia of semicircular canals as well as an otolith organ (the utricle). Results were similar for both kinds of vestibular organ. The neuronal NKAα3 subunit was detected in all afferent endings-both the calyx afferent endings on type I hair cells and bouton afferent endings on type II hair cells-but was not detected in efferent terminals. In contrast to previous results in the cochlea, the NKAα1 subunit was detected in hair cells (both type I and type II) but not in supporting cells. The expression of distinct NKAα subunits by vestibular hair cells and their afferent endings may be needed to support and shape the high rates of glutamatergic neurotransmission and spike initiation at the unusual type I-calyx synapse.

BK channels mediate cholinergic inhibition of high frequency cochlear hair cells.

BACKGROUND: Outer hair cells are the specialized sensory cells that empower the mammalian hearing organ, the cochlea, with its remarkable sensitivity and frequency selectivity. Sound-evoked receptor potentials in outer hair cells are shaped by both voltage-gated K(+) channels that control the membrane potential and also ligand-gated K(+) channels involved in the cholinergic efferent modulation of the membrane potential. The objectives of this study were to investigate the tonotopic contribution of BK channels to voltage- and ligand-gated currents in mature outer hair cells from the rat cochlea. METHODOLOGY/PRINCIPAL: Findings In this work we used patch clamp electrophysiology and immunofluorescence in tonotopically defined segments of the rat cochlea to determine the contribution of BK channels to voltage- and ligand-gated currents in outer hair cells. Although voltage and ligand-gated currents have been investigated previously in hair cells from the rat cochlea, little is known about their tonotopic distribution or potential contribution to efferent inhibition. We found that apical (low frequency) outer hair cells had no BK channel immunoreactivity and little or no BK current. In marked contrast, basal (high frequency) outer hair cells had abundant BK channel immunoreactivity and BK currents contributed significantly to both voltage-gated and ACh-evoked K(+) currents. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that basal (high frequency) outer hair cells may employ an alternative mechanism of efferent inhibition mediated by BK channels instead of SK2 channels. Thus, efferent synapses may use different mechanisms of action both developmentally and tonotopically to support high frequency audition. High frequency audition has required various functional specializations of the mammalian cochlea, and as shown in our work, may include the utilization of BK channels at efferent synapses. This mechanism of efferent inhibition may be related to the unique acetylcholine receptors that have evolved in mammalian hair cells compared to those of other vertebrates.

Distribution of the Na,K-ATPase alpha subunit in the rat spiral ganglion and organ of corti.

Processing of sound in the cochlea involves both afferent and efferent innervation. The Na,K-ATPase (NKA) is essential for cells that maintain hyperpolarized membrane potentials and sodium and potassium concentration gradients. Heterogeneity of NKA subunit expression is one mechanism that tailors physiology to particular cellular demands. Therefore, to provide insight into molecular differences that distinguish the various innervation pathways in the cochlea, we performed a variety of double labeling experiments with antibodies against three of the alpha isoforms of the NKA (NKA alpha 1-3) and markers identifying particular subsets of neurons or supporting cells in whole mount preparations of the organ of Corti and spiral ganglion. We found that the NKA alpha 3 is abundantly expressed within the membranes of the spiral ganglion somata, the type I afferent terminals contacting the inner hair cells, and the medial efferent terminals contacting the outer hair cells. We also found expression of the NKA alpha 1 in the supporting cells that neighbor the inner hair cells and express the glutamate transporter GLAST. These findings suggest that both the NKA alpha 1 and NKA alpha 3 are poised to play an essential role in the regulation of the type I afferent synapses, the medial efferent synapses, and also glutamate transport from the afferent-inner hair cell synapse.