Henry C. Ou

Cisplatin-induced hair cell loss in zebrafish (Danio rerio) lateral line

We have used time-lapse imaging to study cisplatin-induced hair cell death in lateral line neuromasts of zebrafish larvae in vivo. We found that cisplatin-induced hair cell death occurred much more slowly than had been shown to occur in aminoglycoside-induced hair cell death. By prelabeling hair cells with FM1-43FX, and assessing hair cell damage, it was established that cisplatin causes hair cell loss in the lateral line in a dose-dependent fashion. The kinetics of hair cell loss during exposure to different concentrations of cisplatin was also assessed and it was found that the onset of hair cell loss correlated with the accumulated dose of cisplatin. These data demonstrate the feasibility and repeatability of cisplatin damage protocols in the zebrafish lateral line and set the stage for future evaluations of modulation of cisplatin-induced hair cell death.

Identification of FDA-approved drugs and bioactives that protect hair cells in the zebrafish (Danio rerio) lateral line and mouse (Mus musculus) utricle.

The hair cells of the larval zebrafish lateral line provide a useful preparation in which to study hair cell death and to screen for genes and small molecules that modulate hair cell toxicity. We recently reported preliminary results from screening a small-molecule library for compounds that inhibit aminoglycoside-induced hair cell death. To potentially reduce the time required for development of drugs and drug combinations that can be clinically useful, we screened a library of 1,040 FDA-approved drugs and bioactive compounds (NINDS Custom Collection II). Seven compounds that protect against neomycin-induced hair cell death were identified. Four of the seven drugs inhibited aminoglycoside uptake, based on Texas-Red-conjugated gentamicin uptake. The activities of two of the remaining three drugs were evaluated using an in vitro adult mouse utricle preparation. One drug, 9-amino-1,2,3,4-tetrahydroacridine (tacrine) demonstrated conserved protective effects in the mouse utricle. These results demonstrate that the zebrafish lateral line can be used to screen successfully for drugs within a library of FDA-approved drugs and bioactives that inhibit hair cell death in the mammalian inner ear and identify tacrine as a promising protective drug for future studies.

Drug screening for hearing loss: Using the zebrafish lateral line to screen for drugs that prevent and cause hearing loss

Several animal models have been used for the study of mechanosensory hair cells and hearing loss. Because of the difficulty of tissue acquisition and large animal size, these traditional models are impractical for high-throughput screening. The zebrafish has emerged as a powerful animal model for screening drugs that cause and prevent hair cell death. The unique characteristics of the zebrafish enable rapid in vivo imaging of hair cells and hair cell death. We have used this model to screen for and identify multiple drugs that protect hair cells from aminoglycoside-induced death. The identification of multiple drugs and drug-like compounds that inhibit multiple hair cell death pathways might enable the development of protective cocktails to achieve complete hair cell protection.

Screening for chemicals that affect hair cell death and survival in the zebrafish lateral line.

The zebrafish lateral line is an efficient model system for the evaluation of chemicals that protect and damage hair cells. Located on the surface of the body, lateral line hair cells are accessible for manipulation and visualization. The zebrafish lateral line system allows rapid screens of large chemical libraries, as well as subsequent thorough evaluation of interesting compounds. In this review, we focus on the results of our previous screens and the evolving methodology of our screens for chemicals that protect hair cells, and chemicals that damage hair cells using the zebrafish lateral line.

Endoscopic electrocauterization of pyriform fossa sinus tracts as definitive treatment.

OBJECTIVE To (1) update the technique of endoscopic electrocauterization of patients with pyriform fossa sinus tracts and (2) evaluate its effectiveness as a definitive treatment. METHODS Retrospective case series with nine patients (age range, 3.3-16.1 years) who were diagnosed with pyriform fossa sinus tracts between 2000 and 2007 at a single tertiary care childrens hospital and underwent endoscopic electrocauterization of the sinus tract. Data collected including age of diagnosis, presenting symptoms, time from presentation to diagnosis, diagnostic studies, prior and subsequent treatments, length of hospital stay, and recurrence, were reviewed and analyzed with descriptive statistics. RESULTS All nine patients presented with recurrent left or midline neck masses or abscesses. Seven patients (78%) had at least one previous incision and drainage procedures for abscess treatment. All patients had a computed tomography scan with findings suspicious for left-sided pyriform fossa sinus tract. The diagnosis was confirmed with laryngoscopy. For seven patients (78%), endoscopic electrocauterization was definitive treatment with no recurrences to date. Two patients (22%) had recurrent left neck abscesses after endoscopic treatment; these patients ultimately underwent excision of sinus tract with left thyroid lobectomy without complications or further recurrences. CONCLUSIONS Endoscopic electrocauterization of pyriform fossa sinus tracts is a safe and definitive treatment for most patients. We advocate this minimally invasive procedure as first line of treatment for pyriform fossa sinus tracts, reserving open excision with or without thyroid lobectomy for failures.

Fish in a Dish: Drug Discovery for Hearing Habilitation.

The majority of hearing loss is caused by the permanent loss of inner ear hair cells. The identification of drugs that modulate the susceptibility to hair cell loss or spur their regeneration is often hampered by the difficulties of assaying for such complex phenomena in mammalian models. The zebrafish has emerged as a powerful animal model for chemical screening in many contexts. Several characteristics of the zebrafish, such as its small size and external location of sensory hair cells, uniquely position it as an ideal model organism for the study of hair cell toxicity, protection, and regeneration. We have used this model to screen for drugs that affect each of these aspects of hair cell biology and have identified compounds that affect each of these processes. The identification of such drugs and drug-like compounds holds promise in the future ability to stem hearing loss in the human population.

Hair Cell Toxicity in Anti-cancer Drugs: Evaluating an Anti-cancer Drug Library for Independent and Synergistic Toxic Effects on Hair Cells Using the Zebrafish Lateral Line

Inner ear hair cell loss is the most common pathology seen after ototoxic drug injury. While certain drugs such as aminoglycosides and cisplatin are well-known to have dramatic ototoxic effects, it is probable that there are other drugs that cause occult degrees of hair cell loss and lesser degrees of hearing loss. Anti-cancer drugs are particularly strong candidates due to their general cytotoxicity. We have screened a library of 88 anti-cancer drugs (National Cancer Institute Approved Oncology Drugs Set) for drugs that damage hair cells of the zebrafish lateral line. The screen identified four out of five known ototoxic drugs. The screen also identified four out of seven suspected ototoxic drugs (drugs that have isolated case reports of patients developing hearing loss after administration). Five additional drugs with no known ototoxicity were identified as potentially novel ototoxins. Additional dose–response curves were performed to evaluate relative toxicity. Since anti-cancer drugs are often used clinically in combination, we also performed dose–response curves for a variety of anti-cancer drug combinations and demonstrated synergistic toxicity in five out of ten drug combinations that we tested. These findings support the use of the zebrafish lateral line as a screening tool to detect ototoxic effects in drugs and also suggest that ototoxicity should be considered in terms of drug regimens rather than individual drugs.

Mitochondrial calcium uptake underlies ROS generation during aminoglycoside-induced hair cell death

Exposure to aminoglycoside antibiotics can lead to the generation of toxic levels of reactive oxygen species (ROS) within mechanosensory hair cells of the inner ear that have been implicated in hearing and balance disorders. Better understanding of the origin of aminoglycoside-induced ROS could focus the development of therapies aimed at preventing this event. In this work, we used the zebrafish lateral line system to monitor the dynamic behavior of mitochondrial and cytoplasmic oxidation occurring within the same dying hair cell following exposure to aminoglycosides. The increased oxidation observed in both mitochondria and cytoplasm of dying hair cells was highly correlated with mitochondrial calcium uptake. Application of the mitochondrial uniporter inhibitor Ru360 reduced mitochondrial and cytoplasmic oxidation, suggesting that mitochondrial calcium drives ROS generation during aminoglycoside-induced hair cell death. Furthermore, targeting mitochondria with free radical scavengers conferred superior protection against aminoglycoside exposure compared with identical, untargeted scavengers. Our findings suggest that targeted therapies aimed at preventing mitochondrial oxidation have therapeutic potential to ameliorate the toxic effects of aminoglycoside exposure.

Quinoline Ring Derivatives Protect Against Aminoglycoside-Induced Hair Cell Death in the Zebrafish Lateral Line

We have previously published results from a screen of 1,040 FDA-approved drugs and bioactives (NINDS Custom Collection) for drugs that protect against neomycin-induced hair cell death (Ou et al., J Assoc Res Otolaryngol 10:191–203, 2009). Further evaluation of this drug library identified eight protective drugs that shared a common quinoline scaffold. These drugs were tested further in terms of their protection against other aminoglycosides, as well as their effect on aminoglycoside uptake. All of the eight quinolines that protected against neomycin were found to protect against short- and long-term gentamicin damage protocols. We then tested the structurally related compounds quinoline, isoquinoline, naphthalene, and indole for protective effects. Of these compounds, indole demonstrated a small but significant amount of protection against neomycin, while quinoline and isoquinoline partially protected against long-term gentamicin damage. We examined whether the protective activity of this group of compounds was related to known targets of the quinoline derivatives. The protective effects did not seem linked to either the cholinergic or histaminergic pathways that are regulated by some members of the quinoline family. However, all eight protective drugs were found to reduce the uptake of aminoglycosides into hair cells. Subsequent experiments suggest that reduction of uptake is the primary mechanism of protection among the quinoline drugs.

Otoplasty: An alternative approach to management of the deep conchal bowl.

INTRODUCTION Numerous otoplasty techniques have been developed to optimize the appearance and position of the pinna.1–3 The most common auricular abnormality is the prominent ear deformity associated with a poorly formed or absent antihelix resulting in increased projection of the pinna. In such cases, the shape and position of the ear is corrected by creating the antihelix and superior crus of the triangular fossa. Numerous techniques have been described to create the antihelix.2–5 The deep or excessive conchal bowl is another common abnormality that may contribute to increased projection of the pinna. The deep conchal bowl has been assumed to result from excessive cartilage. A bowl depth of greater than 1.5 cm is considered abnormally deep. Surgical techniques have been developed to reduce the projection of the conchal cartilage using concha mastoid sutures6 or direct excision of conchal cartilage with or without overlying skin.7,8 Concha mastoid sutures may result in anterior displacement of the anterior edge of the conchal cartilage resulting in collapse of the cartilaginous external auditory meatus. Collapse or stenosis of the external auditory meatus compromises egress of cerumen and otoscopic examination.6,9 Furthermore, these sutures may obliterate the postauricular sulcus. Direct excision of conchal cartilage may result in alteration of the normal contour of the pinna. The senior author (K.S.) has observed that, in some cases, the appearance of a “deep” conchal bowl results from a poorly defined helical crus. In a normal ear, the helical crus extends into the concha, dividing it into the concha cavum inferiorly and the concha cymba superiorly (Fig. 1). Absence of the helical crus creates a concha that lacks the separation of the concha cymba and concha cavum. This is analogous to the absence of the antihelix creating prominence of the superior pinna (Fig. 2). Standard techniques for creating the antihelix, using cartilage scoring and mattress sutures, can be applied to the conchal bowl to create the helical crus. Creation or accentuation of the helical crus decreases projection of the ear by rotating the superior–posterior edge of the pinna toward the mastoid cortex. Placement of these mattress sutures also pulls the anterior edge of the conchal carti-