Alan G. Cheng

All Publications

• Direct cellular reprogramming and inner ear regeneration. Expert opinion on biological therapy Atkinson, P. J., Kim, G. S., Cheng, A. G. 2018

  Abstract

  INTRODUCTION: Sound is integral to communication and connects us to the world through speech and music. Cochlear hair cells are essential for converting sounds into neural impulses. However, these cells are highly susceptible to damage from an array of factors, resulting in degeneration and ultimately irreversible hearing loss in humans. Since the discovery of hair cell regeneration in birds, there have been tremendous efforts to identify therapies that could promote hair cell regeneration in mammals. Areas covered: Here, we will review recent studies describing spontaneous hair cell regeneration and direct cellular reprograming as well as other factors that mediate mammalian hair cell regeneration. Expert opinion: Numerous combinatorial approaches have successfully reprogrammed non-sensory supporting cells to form hair cells, albeit with limited efficacy and maturation. Studies on epigenetic regulation and transcriptional network of hair cell progenitors may accelerate discovery of more promising reprogramming regimens.

  View details for DOI 10.1080/14712598.2019.1564035

  View details for PubMedID 30584811

• Aminoglycoside ribosome interactions reveal novel conformational states at ambient temperature. Nucleic acids research O'Sullivan, M. E., Poitevin, F., Sierra, R. G., Gati, C., Dao, E. H., Rao, Y., Aksit, F., Ciftci, H., Corsepius, N., Greenhouse, R., Hayes, B., Hunter, M. S., Liang, M., McGurk, A., Mbgam, P., Obrinsky, T., Pardo-Avila, F., Seaberg, M. H., Cheng, A. G., Ricci, A. J., DeMirci, H. 2018

  Abstract

  The bacterial 30S ribosomal subunit is a primary antibiotic target. Despite decades of discovery, the mechanisms by which antibiotic binding induces ribosomal dysfunction are not fully understood. Ambient temperature crystallographic techniques allow more biologically relevant investigation of how local antibiotic binding site interactions trigger global subunit rearrangements that perturb protein synthesis. Here, the structural effects of 2-deoxystreptamine (paromomycin and sisomicin), a novel sisomicin derivative, N1-methyl sulfonyl sisomicin (N1MS) and the non-deoxystreptamine (streptomycin) aminoglycosides on the ribosome at ambient and cryogenic temperatures were examined. Comparative studies led to three main observations. First, individual aminoglycoside-ribosome interactions in the decoding center were similar for cryogenic versus ambient temperature structures. Second, analysis of a highly conserved GGAA tetraloop of h45 revealed aminoglycoside-specific conformational changes, which are affected by temperature only for N1MS. We report the h44-h45 interface in varying states, i.e. engaged, disengaged and in equilibrium. Third, we observe aminoglycoside-induced effects on 30S domain closure, including a novel intermediary closure state, which is also sensitive to temperature. Analysis of three ambient and five cryogenic crystallography datasets reveal a correlation between h44-h45 engagement and domain closure. These observations illustrate the role of ambient temperature crystallography in identifying dynamic mechanisms of ribosomal dysfunction induced by local drug-binding site interactions. Together, these data identify tertiary ribosomal structural changes induced by aminoglycoside binding that provides functional insight and targets for drug design.

  View details for DOI 10.1093/nar/gky693

  View details for PubMedID 30113694

• Molecular therapy for genetic and degenerative vestibular disorders. Current opinion in otolaryngology & head and neck surgery Sayyid, Z. N., Kim, G. S., Cheng, A. G. 2018

  Abstract

  PURPOSE OF REVIEW: The primary purpose of this review is to summarize current literature in the field of vestibular regeneration with a focus on recent developments in molecular and gene therapies.RECENT FINDINGS: Since the discovery of limited vestibular hair cell regeneration in mammals in the 1990s, many elegant studies have improved our knowledge of mechanisms of development and regeneration of the vestibular system. A better understanding of the developmental pathways of the vestibular organs has fueled various biological strategies to enhance regeneration, including novel techniques in deriving vestibular hair cells from embryonic and induced pluripotent stem cells. In addition, the identification of specific genetic mutations responsible for vestibular disorders has opened various opportunities for gene replacement therapy.SUMMARY: Vestibular dysfunction is a significant clinical problem with limited therapeutic options, warranting research on biological strategies to repair/regenerate the vestibular organs to restore function. The use of gene therapy appears promising in animal models of vestibular dysfunction.

  View details for DOI 10.1097/MOO.0000000000000477

  View details for PubMedID 30045104

• Sox2 haploinsufficiency primes regeneration and Wnt responsiveness in the mouse cochlea JOURNAL OF CLINICAL INVESTIGATION Atkinson, P. J., Dong, Y., Gu, S., Liu, W., Najarro, E., Udagawa, T., Cheng, A. G. 2018; 128 (4): 1641–56

  Abstract

  During development, Sox2 is indispensable for cell division and differentiation, yet its roles in regenerating tissues are less clear. Here, we used combinations of transgenic mouse models to reveal that Sox2 haploinsufficiency (Sox2haplo) increases rather than impairs cochlear regeneration in vivo. Sox2haplo cochleae had delayed terminal mitosis and ectopic sensory cells, yet normal auditory function. Sox2haplo amplified and expanded domains of damage-induced Atoh1+ transitional cell formation in neonatal cochlea. Wnt activation via β-catenin stabilization (β-cateninGOF) alone failed to induce proliferation or transitional cell formation. By contrast, β-cateninGOF caused proliferation when either Sox2haplo or damage was present, and transitional cell formation when both were present in neonatal, but not mature, cochlea. Mechanistically, Sox2haplo or damaged neonatal cochleae showed lower levels of Sox2 and Hes5, but not of Wnt target genes. Together, our study unveils an interplay between Sox2 and damage in directing tissue regeneration and Wnt responsiveness and thus provides a foundation for potential combinatorial therapies aimed at stimulating mammalian cochlear regeneration to reverse hearing loss in humans.

  View details for DOI 10.1172/JCI97248

  View details for Web of Science ID 000431958600039

  View details for PubMedID 29553487

  View details for PubMedCentralID PMC5873847

• Mind Your Ears: A New Antidote to Aminoglycoside Toxicity? JOURNAL OF MEDICINAL CHEMISTRY O'Sullivan, M. E., Cheng, A. G. 2018; 61 (1): 81–83

  Abstract

  Aminoglycoside antibiotics are known toxins to cochlear hair cells, causing permanent hearing loss. Using the zebrafish lateral line system as a platform for drug screen and subsequent validation in the rat cochlea in vivo, Chowdhury et al. characterized a novel otoprotectant working against aminoglycoside-induced hearing loss.

  View details for DOI 10.1021/acs.jmedchem.7b01645

  View details for Web of Science ID 000422810800004

  View details for PubMedID 29256598

• Towards the Prevention of Aminoglycoside-Related Hearing Loss Frontiers in Cellular Neuroscience O'Sullivan, M. E., Perez, A., Lin, R., Ricci, A. J., Cheng, A. G. 2017; 11: 325

  Abstract

  Aminoglycosides are potent antibiotics deployed worldwide despite their known side-effect of sensorineural hearing loss. The main etiology of this sensory deficit is death of inner ear sensory hair cells selectively triggered by aminoglycosides. For decades, research has sought to unravel the molecular events mediating sensory cell demise, emphasizing the roles of reactive oxygen species and their potentials as therapeutic targets. Studies in recent years have revealed candidate transport pathways including the mechanotransducer channel for drug entry into sensory cells. Once inside sensory cells, intracellular targets of aminoglycosides, such as the mitochondrial ribosomes, are beginning to be elucidated. Based on these results, less ototoxic aminoglycoside analogs are being generated and may serve as alternate antimicrobial agents. In this article, we review the latest findings on mechanisms of aminoglycoside entry into hair cells, their intracellular actions and potential therapeutic targets for preventing aminoglycoside ototoxicity.

  View details for DOI 10.3389/fncel.2017.00325

  View details for PubMedCentralID PMC5651232

• Profiling Specific Inner Ear Cell Types Using Cell Sorting Techniques. Methods in molecular biology (Clifton, N.J.) Jan, T. A., Jansson, L., Atkinson, P. J., Wang, T., Cheng, A. G. 2016; 1427: 431-445

  Abstract

  Studies of specific tissue cell types are becoming increasingly important in advancing our understanding of cell biology and gene and protein expression. Prospective isolation of specific cell types is a powerful technique as it facilitates such investigations, allowing for analysis and characterization of individual cell populations. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and limited cell markers. In this chapter, we describe methods for selectively labeling and isolating different inner ear cell types from the neonatal mouse cochlea using fluorescence-activated cell sorting.

  View details for DOI 10.1007/978-1-4939-3615-1_23

  View details for PubMedID 27259940

• Intraoperative acupuncture for posttonsillectomy pain: A randomized, double-blind, placebo-controlled trial LARYNGOSCOPE Tsao, G. J., Messner, A. H., Seybold, J., Sayyid, Z. N., Cheng, A. G., Golianu, B. 2015; 125 (8): 1972-1978

  Abstract

  To evaluate the effect of intraoperative acupuncture on posttonsillectomy pain in the pediatric population.Prospective, double-blind, randomized, placebo-controlled trial.Patients aged 3 to 12 years undergoing tonsillectomy were recruited at a tertiary children's hospital between February 2011 and May 2012. Participants were block-randomized to receive acupuncture or sham acupuncture during anesthesia for tonsillectomy. Surgeons, staff, and parents were blinded from treatment. Tonsillectomy was performed by one of two surgeons using a standard technique (monopolar cautery), and a single anesthetic protocol was followed. Study endpoints included time spent in the postanesthesia care unit, the amount of opioids administered in the perioperative period, and pain measures and presence of nausea/vomiting from postoperative home surveys.Fifty-nine children aged 3 to 12 years were randomized to receive acupuncture (n = 30) or sham acupuncture (n = 29). No significant demographic differences were noted between the two cohorts. Perioperative data were recorded for all patients; 73% of patients later returned home surveys. There were no significant differences in the amount of opioid medications administered or total postanesthesia care unit time between the two cohorts. Home surveys of patients but not of parents revealed significant improvements in pain control in the acupuncture treatment-group postoperatively (P = 0.0065 and 0.051, respectively), and oral intake improved significantly earlier in the acupuncture treatment group (P = 0.01). No adverse effects of acupuncture were reported.This study demonstrates that intraoperative acupuncture is feasible, well tolerated, and results in improved pain and earlier return of diet postoperatively.1b. Laryngoscope, 2015.

  View details for DOI 10.1002/lary.25252

  View details for Web of Science ID 000358379700048

• Sensory hair cell development and regeneration: similarities and differences DEVELOPMENT Atkinson, P. J., Najarro, E. H., Sayyid, Z. N., Cheng, A. G. 2015; 142 (9): 1561-1571

  Abstract

  Sensory hair cells are mechanoreceptors of the auditory and vestibular systems and are crucial for hearing and balance. In adult mammals, auditory hair cells are unable to regenerate, and damage to these cells results in permanent hearing loss. By contrast, hair cells in the chick cochlea and the zebrafish lateral line are able to regenerate, prompting studies into the signaling pathways, morphogen gradients and transcription factors that regulate hair cell development and regeneration in various species. Here, we review these findings and discuss how various signaling pathways and factors function to modulate sensory hair cell development and regeneration. By comparing and contrasting development and regeneration, we also highlight the utility and limitations of using defined developmental cues to drive mammalian hair cell regeneration.

  View details for DOI 10.1242/dev.114926

  View details for Web of Science ID 000353591300002

  View details for PubMedID 25922522

  View details for PubMedCentralID PMC4419275

• Making sense of Wnt signaling-linking hair cell regeneration to development FRONTIERS IN CELLULAR NEUROSCIENCE Jansson, L., Kim, G. S., Cheng, A. G. 2015; 9

  Abstract

  Wnt signaling is a highly conserved pathway crucial for development and homeostasis of multicellular organisms. Secreted Wnt ligands bind Frizzled receptors to regulate diverse processes such as axis patterning, cell division, and cell fate specification. They also serve to govern self-renewal of somatic stem cells in several adult tissues. The complexity of the pathway can be attributed to the myriad of Wnt and Frizzled combinations as well as its diverse context-dependent functions. In the developing mouse inner ear, Wnt signaling plays diverse roles, including specification of the otic placode and patterning of the otic vesicle. At later stages, its activity governs sensory hair cell specification, cell cycle regulation, and hair cell orientation. In regenerating sensory organs from non-mammalian species, Wnt signaling can also regulate the extent of proliferative hair cell regeneration. This review describes the current knowledge of the roles of Wnt signaling and Wnt-responsive cells in hair cell development and regeneration. We also discuss possible future directions and the potential application and limitation of Wnt signaling in augmenting hair cell regeneration.

  View details for DOI 10.3389/fncel.2015.00066

  View details for Web of Science ID 000352399400001

  View details for PubMedID 25814927

  View details for PubMedCentralID PMC4356074

• Protein-engineered hydrogel encapsulation for 3-d culture of murine cochlea. Otology & neurotology Chang, D. T., Chai, R., DiMarco, R., Heilshorn, S. C., Cheng, A. G. 2015; 36 (3): 531-538

  Abstract

  Elastin-like protein (ELP) hydrogel helps maintain the three-dimensional (3-D) cochlear structure in culture.Whole-organ culture of the cochlea is a useful model system facilitating manipulation and analysis of live sensory cells and surrounding nonsensory cells. The precisely organized 3-D cochlear structure demands a culture method that preserves this delicate architecture; however, current methods have not been optimized to serve such a purpose.A protein-engineered ELP hydrogel was used to encapsulate organ of Corti isolated from neonatal mice. Cultured cochleae were immunostained for markers of hair cells and supporting cells. Organ of Corti hair cell and supporting cell density and organ dimensions were compared between the ELP and nonencapsulated systems. These culture systems were then compared with noncultured cochlea.After 3 days in vitro, vital dye uptake and immunostaining for sensory and nonsensory cells show that encapsulated cochlea contain viable cells with an organized architecture. In comparison with nonencapsulated cultured cochlea, ELP-encapsulated cochleae exhibit higher densities of hair cells and supporting cells and taller and narrower organ of Corti dimensions that more closely resemble those of noncultured cochleae. However, we found compromised cell viability when the culture period extended beyond 3 days.We conclude that the ELP hydrogel can help preserve the 3-D architecture of neonatal cochlea in short-term culture, which may be applicable to in vitro study of the physiology and pathophysiology of the inner ear.

  View details for DOI 10.1097/MAO.0000000000000518

  View details for PubMedID 25111520

• Designer aminoglycosides prevent cochlear hair cell loss and hearing loss. journal of clinical investigation Huth, M. E., Han, K., Sotoudeh, K., Hsieh, Y., Effertz, T., Vu, A. A., Verhoeven, S., Hsieh, M. H., Greenhouse, R., Cheng, A. G., Ricci, A. J. 2015; 125 (2): 583-592

  Abstract

  Bacterial infections represent a rapidly growing challenge to human health. Aminoglycosides are widely used broad-spectrum antibiotics, but they inflict permanent hearing loss in up to ~50% of patients by causing selective sensory hair cell loss. Here, we hypothesized that reducing aminoglycoside entry into hair cells via mechanotransducer channels would reduce ototoxicity, and therefore we synthesized 9 aminoglycosides with modifications based on biophysical properties of the hair cell mechanotransducer channel and interactions between aminoglycosides and the bacterial ribosome. Compared with the parent aminoglycoside sisomicin, all 9 derivatives displayed no or reduced ototoxicity, with the lead compound N1MS 17 times less ototoxic and with reduced penetration of hair cell mechanotransducer channels in rat cochlear cultures. Both N1MS and sisomicin suppressed growth of E. coli and K. pneumoniae, with N1MS exhibiting superior activity against extended spectrum β lactamase producers, despite diminished activity against P. aeruginosa and S. aureus. Moreover, systemic sisomicin treatment of mice resulted in 75% to 85% hair cell loss and profound hearing loss, whereas N1MS treatment preserved both hair cells and hearing. Finally, in mice with E. coli-infected bladders, systemic N1MS treatment eliminated bacteria from urinary tract tissues and serially collected urine samples, without compromising auditory and kidney functions. Together, our findings establish N1MS as a nonototoxic aminoglycoside and support targeted modification as a promising approach to generating nonototoxic antibiotics.

  View details for DOI 10.1172/JCI77424

  View details for PubMedID 25555219

• Making sense of Wnt signaling-linking hair cell regeneration to development. Frontiers in cellular neuroscience Jansson, L., Kim, G. S., Cheng, A. G. 2015; 9: 66-?

  Abstract

  Wnt signaling is a highly conserved pathway crucial for development and homeostasis of multicellular organisms. Secreted Wnt ligands bind Frizzled receptors to regulate diverse processes such as axis patterning, cell division, and cell fate specification. They also serve to govern self-renewal of somatic stem cells in several adult tissues. The complexity of the pathway can be attributed to the myriad of Wnt and Frizzled combinations as well as its diverse context-dependent functions. In the developing mouse inner ear, Wnt signaling plays diverse roles, including specification of the otic placode and patterning of the otic vesicle. At later stages, its activity governs sensory hair cell specification, cell cycle regulation, and hair cell orientation. In regenerating sensory organs from non-mammalian species, Wnt signaling can also regulate the extent of proliferative hair cell regeneration. This review describes the current knowledge of the roles of Wnt signaling and Wnt-responsive cells in hair cell development and regeneration. We also discuss possible future directions and the potential application and limitation of Wnt signaling in augmenting hair cell regeneration.

  View details for DOI 10.3389/fncel.2015.00066

  View details for PubMedID 25814927

• Lgr5+ cells regenerate hair cells via proliferation and direct transdifferentiation in damaged neonatal mouse utricle. Nature communications Wang, T., Chai, R., Kim, G. S., Pham, N., Jansson, L., Nguyen, D., Kuo, B., May, L. A., Zuo, J., Cunningham, L. L., Cheng, A. G. 2015; 6: 6613-?

  Abstract

  Recruitment of endogenous progenitors is critical during tissue repair. The inner ear utricle requires mechanosensory hair cells (HCs) to detect linear acceleration. After damage, non-mammalian utricles regenerate HCs via both proliferation and direct transdifferentiation. In adult mammals, limited transdifferentiation from unidentified progenitors occurs to regenerate extrastriolar Type II HCs. Here we show that HC damage in neonatal mouse utricle activates the Wnt target gene Lgr5 in striolar supporting cells. Lineage tracing and time-lapse microscopy reveal that Lgr5+ cells transdifferentiate into HC-like cells in vitro. In contrast to adults, HC ablation in neonatal utricles in vivo recruits Lgr5+ cells to regenerate striolar HCs through mitotic and transdifferentiation pathways. Both Type I and II HCs are regenerated, and regenerated HCs display stereocilia and synapses. Lastly, stabilized ß-catenin in Lgr5+ cells enhances mitotic activity and HC regeneration. Thus Lgr5 marks Wnt-regulated, damage-activated HC progenitors and may help uncover factors driving mammalian HC regeneration.

  View details for DOI 10.1038/ncomms7613

  View details for PubMedID 25849379

  View details for PubMedCentralID PMC4391285

• Spontaneous hair cell regeneration in the neonatal mouse cochlea in vivo (vol 141, pg 816, 2014) DEVELOPMENT Cox, B. C., Chai, R., Lenoir, A., Liu, Z., Zhang, L., Duc-Huy Nguyen, Chalasani, K., Steigelman, K. A., Fang, J., Rubel, E. W., Cheng, A. G., Zuo, J. 2014; 141 (7): 1599

  View details for DOI 10.1242/dev.109421

  View details for Web of Science ID 000333184500019

• Spontaneous hair cell regeneration in the neonatal mouse cochlea in vivo DEVELOPMENT Cox, B. C., Chai, R., Lenoir, A., Liu, Z., Zhang, L., Nguyen, D., Chalasani, K., Steigelman, K. A., Fang, J., Cheng, A. G., Zuo, J. 2014; 141 (4): 816-829

  Abstract

  Loss of cochlear hair cells in mammals is currently believed to be permanent, resulting in hearing impairment that affects more than 10% of the population. Here, we developed two genetic strategies to ablate neonatal mouse cochlear hair cells in vivo. Both Pou4f3(DTR/+) and Atoh1-CreER™; ROSA26(DTA/+) alleles allowed selective and inducible hair cell ablation. After hair cell loss was induced at birth, we observed spontaneous regeneration of hair cells. Fate-mapping experiments demonstrated that neighboring supporting cells acquired a hair cell fate, which increased in a basal to apical gradient, averaging over 120 regenerated hair cells per cochlea. The normally mitotically quiescent supporting cells proliferated after hair cell ablation. Concurrent fate mapping and labeling with mitotic tracers showed that regenerated hair cells were derived by both mitotic regeneration and direct transdifferentiation. Over time, regenerated hair cells followed a similar pattern of maturation to normal hair cell development, including the expression of prestin, a terminal differentiation marker of outer hair cells, although many new hair cells eventually died. Hair cell regeneration did not occur when ablation was induced at one week of age. Our findings demonstrate that the neonatal mouse cochlea is capable of spontaneous hair cell regeneration after damage in vivo. Thus, future studies on the neonatal cochlea might shed light on the competence of supporting cells to regenerate hair cells and on the factors that promote the survival of newly regenerated hair cells.

  View details for DOI 10.1242/dev.103036

  View details for Web of Science ID 000331460900009

  View details for PubMedID 24496619

  View details for PubMedCentralID PMC3912828

• Transient, afferent input-dependent, postnatal niche for neural progenitor cells in the cochlear nucleus (vol 110, pg 14456, 2013) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Volkenstein, S., Oshima, K., Sinkkonen, S. T., Corrales, C., Most, S. P., Chai, R., Jan, T. A., van Amerongen, R., Cheng, A. G., Heller, S. 2013; 110 (42): 17160

  View details for DOI 10.1073/pnas.1317787110

  View details for Web of Science ID 000325634200095

• Transient, afferent input-dependent, postnatal niche for neural progenitor cells in the cochlear nucleus. Proceedings of the National Academy of Sciences of the United States of America Volkenstein, S., Oshima, K., Sinkkonen, S. T., Corrales, C. E., Most, S. P., Chai, R., Jan, T. A., Cheng, A. G., Heller, S. 2013; 110 (35): 14456-14461

  Abstract

  In the cochlear nucleus (CN), the first central relay of the auditory pathway, the survival of neurons during the first weeks after birth depends on afferent innervation from the cochlea. Although input-dependent neuron survival has been extensively studied in the CN, neurogenesis has not been evaluated as a possible mechanism of postnatal plasticity. Here we show that new neurons are born in the CN during the critical period of postnatal plasticity. Coincidently, we found a population of neural progenitor cells that are controlled by a complex interplay of Wnt, Notch, and TGFβ/BMP signaling, in which low levels of TGFβ/BMP signaling are permissive for progenitor proliferation that is promoted by Wnt and Notch activation. We further show that cells with activated Wnt signaling reside in the CN and that these cells have high propensity for neurosphere formation. Cochlear ablation resulted in diminishment of progenitors and Wnt/β-catenin-active cells, suggesting that the neonatal CN maintains an afferent innervation-dependent population of progenitor cells that display active canonical Wnt signaling.

  View details for DOI 10.1073/pnas.1307376110

  View details for PubMedID 23940359

  View details for PubMedCentralID PMC3761577

• Tympanic border cells are Wnt-responsive and can act as progenitors for postnatal mouse cochlear cells DEVELOPMENT Jan, T. A., Chai, R., Sayyid, Z. N., van Amerongen, R., Xia, A., Wang, T., Sinkkonen, S. T., Zeng, Y. A., Levin, J. R., Heller, S., Nusse, R., Cheng, A. G. 2013; 140 (6): 1196-1206

  Abstract

  Permanent hearing loss is caused by the irreversible damage of cochlear sensory hair cells and nonsensory supporting cells. In the postnatal cochlea, the sensory epithelium is terminally differentiated, whereas tympanic border cells (TBCs) beneath the sensory epithelium are proliferative. The functions of TBCs are poorly characterized. Using an Axin2(lacZ) Wnt reporter mouse, we found transient but robust Wnt signaling and proliferation in TBCs during the first 3 postnatal weeks, when the number of TBCs decreases. In vivo lineage tracing shows that a subset of hair cells and supporting cells is derived postnatally from Axin2-expressing TBCs. In cochlear explants, Wnt agonists stimulated the proliferation of TBCs, whereas Wnt inhibitors suppressed it. In addition, purified Axin2(lacZ) cells were clonogenic and self-renewing in culture in a Wnt-dependent manner, and were able to differentiate into hair cell-like and supporting cell-like cells. Taken together, our data indicate that Axin2-positive TBCs are Wnt responsive and can act as precursors to sensory epithelial cells in the postnatal cochlea.

  View details for DOI 10.1242/dev.087528

  View details for Web of Science ID 000315445800006

  View details for PubMedID 23444352

  View details for PubMedCentralID PMC3585657

• Integrity and Regeneration of Mechanotransduction Machinery Regulate Aminoglycoside Entry and Sensory Cell Death PLOS ONE Vu, A. A., Nadaraja, G. S., Huth, M. E., Luk, L., Kim, J., Chai, R., Ricci, A. J., Cheng, A. G. 2013; 8 (1)

  Abstract

  Sound perception requires functional hair cell mechanotransduction (MET) machinery, including the MET channels and tip-link proteins. Prior work showed that uptake of ototoxic aminoglycosides (AG) into hair cells requires functional MET channels. In this study, we examined whether tip-link proteins, including Cadherin 23 (Cdh23), regulate AG entry into hair cells. Using time-lapse microscopy on cochlear explants, we found rapid uptake of gentamicin-conjugated Texas Red (GTTR) into hair cells from three-day-old Cdh23(+/+) and Cdh23(v2J/+) mice, but failed to detect GTTR uptake in Cdh23(v2J/v2J) hair cells. Pre-treatment of wildtype cochleae with the calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) to disrupt tip-links also effectively reduced GTTR uptake into hair cells. Both Cdh23(v2J/v2J) and BAPTA-treated hair cells were protected from degeneration caused by gentamicin. Six hours after BAPTA treatment, GTTR uptake remained reduced in comparison to controls; by 24 hours, drug uptake was comparable between untreated and BAPTA-treated hair cells, which again became susceptible to cell death induced by gentamicin. Together, these results provide genetic and pharmacologic evidence that tip-links are required for AG uptake and toxicity in hair cells. Because tip-links can spontaneously regenerate, their temporary breakage offers a limited time window when hair cells are protected from AG toxicity.

  View details for DOI 10.1371/journal.pone.0054794

  View details for Web of Science ID 000314023600111

  View details for PubMedID 23359017

  View details for PubMedCentralID PMC3554584

• A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis. PloS one Herget, M., Scheibinger, M., Guo, Z., Jan, T. A., Adams, C. M., Cheng, A. G., Heller, S. 2013; 8 (6)

  Abstract

  Mechanosensitive hair cells and supporting cells comprise the sensory epithelia of the inner ear. The paucity of both cell types has hampered molecular and cell biological studies, which often require large quantities of purified cells. Here, we report a strategy allowing the enrichment of relatively pure populations of vestibular hair cells and non-sensory cells including supporting cells. We utilized specific uptake of fluorescent styryl dyes for labeling of hair cells. Enzymatic isolation and flow cytometry was used to generate pure populations of sensory hair cells and non-sensory cells. We applied mass spectrometry to perform a qualitative high-resolution analysis of the proteomic makeup of both the hair cell and non-sensory cell populations. Our conservative analysis identified more than 600 proteins with a false discovery rate of <3% at the protein level and <1% at the peptide level. Analysis of proteins exclusively detected in either population revealed 64 proteins that were specific to hair cells and 103 proteins that were only detectable in non-sensory cells. Statistical analyses extended these groups by 53 proteins that are strongly upregulated in hair cells versus non-sensory cells and vice versa by 68 proteins. Our results demonstrate that enzymatic dissociation of styryl dye-labeled sensory hair cells and non-sensory cells is a valid method to generate pure enough cell populations for flow cytometry and subsequent molecular analyses.

  View details for DOI 10.1371/journal.pone.0066026

  View details for PubMedID 23750277

  View details for PubMedCentralID PMC3672136

• A simple method for purification of vestibular hair cells and non-sensory cells, and application for proteomic analysis. PloS one Herget, M., Scheibinger, M., Guo, Z., Jan, T. A., Adams, C. M., Cheng, A. G., Heller, S. 2013; 8 (6)

  View details for DOI 10.1371/journal.pone.0066026

  View details for PubMedID 23750277

• Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chai, R., Kuo, B., Wang, T., Liaw, E. J., Xia, A., Jan, T. A., Liu, Z., Taketo, M. M., Oghalai, J. S., Nusse, R., Zuo, J., Cheng, A. G. 2012; 109 (21): 8167-8172

  Abstract

  Inner ear hair cells are specialized sensory cells essential for auditory function. Previous studies have shown that the sensory epithelium is postmitotic, but it harbors cells that can behave as progenitor cells in vitro, including the ability to form new hair cells. Lgr5, a Wnt target gene, marks distinct supporting cell types in the neonatal cochlea. Here, we tested the hypothesis that Lgr5(+) cells are Wnt-responsive sensory precursor cells. In contrast to their quiescent in vivo behavior, Lgr5(+) cells isolated by flow cytometry from neonatal Lgr5(EGFP-CreERT2/+) mice proliferated and formed clonal colonies. After 10 d in culture, new sensory cells formed and displayed specific hair cell markers (myo7a, calretinin, parvalbumin, myo6) and stereocilia-like structures expressing F-actin and espin. In comparison with other supporting cells, Lgr5(+) cells were enriched precursors to myo7a(+) cells, most of which formed without mitotic division. Treatment with Wnt agonists increased proliferation and colony-formation capacity. Conversely, small-molecule inhibitors of Wnt signaling suppressed proliferation without compromising the myo7a(+) cells formed by direct differentiation. In vivo lineage tracing supported the idea that Lgr5(+) cells give rise to myo7a(+) hair cells in the neonatal Lgr5(EGFP-CreERT2/+) cochlea. In addition, overexpression of β-catenin initiated proliferation and led to transient expansion of Lgr5(+) cells within the cochlear sensory epithelium. These results suggest that Lgr5 marks sensory precursors and that Wnt signaling can promote their proliferation and provide mechanistic insights into Wnt-responsive progenitor cells during sensory organ development.

  View details for DOI 10.1073/pnas.1202774109

  View details for Web of Science ID 000304445800053

  View details for PubMedID 22562792

  View details for PubMedCentralID PMC3361451

• Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Jan, T. A., Chai, R., Sayyid, Z. N., Cheng, A. G. 2011

  Abstract

  Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae. The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown. To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

  View details for DOI 10.3791/3432

  View details for Web of Science ID 000209222300024

  View details for PubMedCentralID PMC3369666

• Dynamic Expression of Lgr5, a Wnt Target Gene, in the Developing and Mature Mouse Cochlea JARO-JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY Chai, R., Xia, A., Wang, T., Jan, T. A., Hayashi, T., Bermingham-McDonogh, O., Cheng, A. G. 2011; 12 (4): 455-469

  Abstract

  The Wnt signaling pathway is a recurring theme in tissue development and homeostasis. Its specific roles during inner ear development are just emerging, but few studies have characterized Wnt target genes. Lgr5, a member of the G protein-coupled receptor family, is a Wnt target in the gastrointestinal and integumentary systems. Although its function is unknown, its deficiency leads to perinatal lethality due to gastrointestinal distension. In this study, we used a knock-in reporter mouse to examine the spatiotemporal expression of Lgr5 in the cochlear duct during embryonic and postnatal periods. In the embryonic day 15.5 (E15.5) cochlear duct, Lgr5-EGFP is expressed in the floor epithelium and overlapped with the prosensory markers Sox2, Jagged1, and p27(Kip1). Nascent hair cells and supporting cells in the apical turn of the E18.5 cochlear duct express Lgr5-EGFP, which becomes downregulated in hair cells and subsets of supporting cells in more mature stages. In situ hybridization experiments validated the reporter expression, which gradually decreases until the second postnatal week. Only the third row of Deiters' cells expresses Lgr5-EGFP in the mature organ of Corti. Normal cochlear development was observed in Lgr5(EGFP/EGFP) and Lgr5(EGFP/+) mice, which exhibited normal auditory thresholds. The expression pattern of Lgr5 contrasts with another Wnt target gene, Axin2, a feedback inhibitor of the Wnt pathway. Robust Axin2 expression was found in cells surrounding the embryonic cochlear duct and becomes restricted to tympanic border cells below the basilar membrane in the postnatal cochlea. Both Lgr5 and Axin2 act as Wnt targets in the cochlea because purified Wnt3a promoted and Wnt antagonist suppressed their expression. Their differential expression among cell populations highlights the dynamic but complex distribution of Wnt-activated cells in and around the embryonic and postnatal cochlea.

  View details for DOI 10.1007/s10162-011-0267-2

  View details for Web of Science ID 000292047900004

  View details for PubMedID 21472479

  View details for PubMedCentralID PMC3123443

• Functional Hair Cell Mechanotransducer Channels Are Required for Aminoglycoside Ototoxicity PLOS ONE Alharazneh, A., Luk, L., Huth, M., Monfared, A., Steyger, P. S., Cheng, A. G., Ricci, A. J. 2011; 6 (7)

  Abstract

  Aminoglycosides (AG) are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET) channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR) uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride) significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss.

  View details for DOI 10.1371/journal.pone.0022347

  View details for Web of Science ID 000293175100021

  View details for PubMedID 21818312

  View details for PubMedCentralID PMC3144223

• Intrinsic regenerative potential of murine cochlear supporting cells Scientific Reports Sinkkonen ST, Chai R, Jan, T, Hartman B, Laske R, Gahlen F, Sinkkonen W, Cheng AG, Oshima K, Heller 2011; 1 (26): DOI:10.1038/srep0002
• Mechanisms of aminoglycoside ototoxicity and targets of hair cell protection. International journal of otolaryngology Huth, M. E., Ricci, A. J., Cheng, A. G. 2011; 2011: 937861-?

  Abstract

  Aminoglycosides are commonly prescribed antibiotics with deleterious side effects to the inner ear. Due to their popular application as a result of their potent antimicrobial activities, many efforts have been undertaken to prevent aminoglycoside ototoxicity. Over the years, understanding of the antimicrobial as well as ototoxic mechanisms of aminoglycosides has increased. These mechanisms are reviewed in regard to established and potential future targets of hair cell protection.

  View details for DOI 10.1155/2011/937861

  View details for PubMedID 22121370

• Decompression of the Orbital Apex An Alternate Approach to Surgical Excision for Radiographically Benign Orbital Apex Tumors ARCHIVES OF OTOLARYNGOLOGY-HEAD & NECK SURGERY Almond, M. C., Cheng, A. G., Schiedler, V., Sires, B. S., Most, S. P., Jian-Amadi, A. 2009; 135 (10): 1015-1018

  Abstract

  To study the outcome of patients with orbital apex lesions treated with endoscopic decompression alone.Retrospective medical chart review with a mean follow-up of 25.6 months.Departments of Ophthalmology and Otolaryngology, University of Washington, Seattle.Five individuals seen at the University of Washington Medical Center from November 2003 through December 2005 with visual disturbance caused by orbital apex lesions as documented by preoperative magnetic resonance imaging or computed tomographic scan.All patients underwent endoscopic decompression of the medial wall of the orbital apex with incision of the periorbita.Postoperative visual acuity, presence or absence of a relative afferent pupillary defect, color vision, and visual field were recorded.All 5 patients presented with visual field deficits, 4 of whom improved postoperatively. Three patients had dyschromatopsia preoperatively, 2 of whom improved postoperatively. Visual acuity improved or stabilized in 4 of 5 patients postoperatively. One patient had progressive visual loss during the course of her follow-up, which, after obtaining postoperative imaging, was attributed to inadequate decompression of the apex at its most posterior aspect. This same patient also developed postoperative sinusitis that resolved with antibiotic treatment. Two patients developed diplopia, 1 in primary gaze requiring treatment with prismatic lenses. All patients presented with and maintained normal intraocular pressures.Orbital apex lesions can often be effectively and relatively safely treated by endoscopic decompression alone.

  View details for Web of Science ID 000270777800010

  View details for PubMedID 19841341

• Sensorineural hearing loss in patients with cystic fibrosis OTOLARYNGOLOGY-HEAD AND NECK SURGERY Cheng, A. G., Johnston, P. R., Luz, J., Uluer, A., Fligor, B., Licameli, G. R., Kenna, M. A., Jones, D. T. 2009; 141 (1): 86-90

  Abstract

  To determine the prevalence of sensorineural hearing loss (SNHL) in cystic fibrosis (CF) patients and its relationship to antibiotic use.Case series with chart review.Tertiary care pediatric hospital.We reviewed the medical records of CF patients seen in our children's hospital between March 1994 and December 2007. Data collected included patient demographics, audiograms, tympanograms, genotype, and use of potentially ototoxic antibiotics.Seven of 50 (14%) patients had SNHL. Three percent of patients who received 10 courses (P<0.01). No patients who received five or fewer courses of nasal irrigation with aminoglycosides had SNHL versus 23 percent of those who received more than five courses (P<0.05). Nine percent of patients who received five or fewer courses of macrolides had SNHL versus 60 percent of those who received more than five courses (P=0.079).CF patients receiving aminoglycosides are at high risk for developing SNHL.

  View details for DOI 10.1016/j.otohns.2009.03.020

  View details for Web of Science ID 000267404900019

  View details for PubMedID 19559964

• Airway management in Nager Syndrome LARYNGOSCOPE Ho, A. S., Aleshi, P., Cohen, S. E., Koltai, P. J., Cheng, A. G. 2009; 119: S179-S179

  View details for DOI 10.1002/lary.20468

  View details for Web of Science ID 000207862500179

• Airway management in Nager Syndrome INTERNATIONAL JOURNAL OF PEDIATRIC OTORHINOLARYNGOLOGY Ho, A. S., Aleshi, P., Cohen, S. E., Koltai, P. J., Cheng, A. G. 2008; 72 (12): 1885-1888

  Abstract

  Nager acrofacial dysostosis is a rare congenital syndrome characterized by malformed mandibulofacial structures and pre-axial upper limbs. Trismus and glossoptosis from mandibular abnormalities predisposes infants to life-threatening respiratory distress. A case of a Nager Syndrome mother delivering a similarly afflicted fetus is presented, with approaches to maintaining both tenuous airways described. Distinguishing this condition from similar syndromes is critical for care and prognosis.

  View details for DOI 10.1016/j.ijporl.2008.09.007

  View details for Web of Science ID 000261543000021

  View details for PubMedID 18947886

• Melanoacanthoma of the external auditory canal: a case report and review of the literature AMERICAN JOURNAL OF OTOLARYNGOLOGY Cheng, A. G., Deubner, H., Whipple, M. E. 2007; 28 (6): 433-435

  View details for DOI 10.1016/j.amjoto.2006.11.006

  View details for Web of Science ID 000251068600016

  View details for PubMedID 17980780

• Cerebrospinal fluid leak in the neck: A rare complication of glomus vagale excision OTOLARYNGOLOGY-HEAD AND NECK SURGERY Cheng, A. G., Maronian, N. C., Futran, N. D. 2006; 134 (2): 334-335

  View details for DOI 10.1016/j.otohns.2005.07.032

  View details for Web of Science ID 000235293600027

  View details for PubMedID 16455388

• Mechanisms of hair cell death and protection. Current opinion in otolaryngology & head and neck surgery Cheng, A. G., Cunningham, L. L., Rubel, E. W. 2005; 13 (6): 343-348

  Abstract

  Sensory hair cells are mechanotransducers of the inner ear that are essential for hearing and balance. Hair cell death commonly occurs following acoustic trauma or exposure to ototoxins, such as the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Loss of these inner ear sensory cells can lead to permanent sensorineural hearing loss, balance disturbance, or both. Currently, the only effective clinical intervention is prevention from exposure to known ototoxic insults. To help improve therapeutic strategies, a better understanding of the molecular mechanisms underlying hair cell degeneration is required. Current knowledge of these cell death mechanisms and potential therapeutic targets are discussed in this review.Studies have shown that caspase-9 and caspase-3 are key mediators of hair cell death induced by noise, aminoglycosides, and cisplatin. The Bcl-2 family consists of a group of proapoptotic and antiapoptotic molecules that act upstream of and regulate caspase activation. Recent studies have shed light on the roles of molecules acting more upstream, including mitogen-activated protein kinases and p53.The mechanisms of sensory hair cell degeneration in response to different ototoxic stimuli share a final common pathway: caspase activation. Inhibition of caspases prevents or delays hair cell death and may preserve hearing/balance function. Inhibition of mitogen-activated protein kinases protects against noise-induced and aminoglycoside-induced but not cisplatin-induced hair cell death, which suggests divergent upstream regulatory mechanisms.

  View details for PubMedID 16282762

• Auramine orange stain with fluorescence microscopy is a rapid and sensitive technique for the detection of cervical lymphadenitis due to mycobacterial infection using fine needle aspiration cytology: a case series OTOLARYNGOLOGY-HEAD AND NECK SURGERY Cheng, A. G., Chang, A., Farwell, D. G., Agoff, S. N. 2005; 133 (3): 381-385

  Abstract

  We sought to evaluate the effectiveness of the auramine orange (AO) stain in diagnosing mycobacterial cervical adenitis (MCA) from fine needle aspiration (FNA) cytology.A retrospective review of 19 patients evaluated at 2 urban hospitals from 2000 to 2003 for suspected MCA. FNA specimens were inoculated to culture media and had direct smears stained by the auramine acid fast method.Mycobacteria were identified in 16 (84.2%) of 19 AO-stained FNA specimens, with results available within 4 hours. Corresponding cultures were positive for mycobacteria in 12 specimens, 9 tuberculous and 3 nontuberculous, and grew Mycobacterium tuberculosis from the 3 AO-negative specimens. Three of the 4 patients with negative cultures had previously taken anti-mycobacterial medications.The AO stain with fluorescence microscopy is a sensitive and rapid method for detecting tuberculous and nontuberculous mycobacteria. It is a valuable tool for the otolaryngologists and pathologists in the diagnosis of MCA.

  View details for DOI 10.1016/j.otohns.2005.04.027

  View details for Web of Science ID 000231748100015

  View details for PubMedID 16143186

• Neomycin-induced hair cell death and rapid regeneration in the lateral line of zebrafish (Danio rerio) JARO-JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY Harris, J. A., Cheng, A. G., Cunningham, L. L., MacDonald, G., Raible, D. W., Rubel, E. W. 2003; 4 (2): 219-234

  Abstract

  Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 microM neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after neomycin treatment were able to give rise to new hair cells by 24-48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.

  View details for DOI 10.1007/s10162-002-3022-x

  View details for Web of Science ID 000184781600008

  View details for PubMedID 12943374

• Hair cell death in the avian basilar papilla: Characterization of the in vitro model and caspase activation JARO-JOURNAL OF THE ASSOCIATION FOR RESEARCH IN OTOLARYNGOLOGY Cheng, A. G., Cunningham, L. L., Rubel, E. W. 2003; 4 (1): 91-105

  Abstract

  Caspases are a family of proteases that have been implicated as key mediators of cell death. Although nonspecific inhibition of caspase activation has been reported to prevent mammalian sensory hair cell death, the exact roles of individual caspases during hair cell death are unclear. In other systems, the activation of initiator caspases, such as caspase-8 and caspase-9, can lead to the activation of the effector caspase-3. We have begun to systematically characterize hair cell death in an in vitro system by examining the activation of these specific caspases in degenerating hair cells after acutely damaging the whole avian basilar papilla with gentamicin. Basilar papillae (BP) displayed a dose-dependent hair cell loss after a 24-h treatment with gentamicin at concentrations of 0.1, 0.5, and 2.0 mM. When treated with 0.5 mM gentamicin for 6, 12, or 24 h, hair cells first began to degenerate in the basal third of the BP and damage progressed apically. Supplementation of z-VAD-fmk, a general caspase inhibitor, provided short-term protection against gentamicin-induced hair cell death. Treatment with gentamicin for 6 or 12 h promoted the expression of active caspase-3 and active caspase-9 in many hair cells along the BP as shown by immunohistochemistry. At these time-points, specific fluorescent-labeled peptide substrates detected more active caspase-3, caspase-8, and caspase-9 in gentamicin-treated hair cells relative to controls. Our data indicate that auditory hair cells degenerate as a result of gentamicin exposure in a caspase-dependent manner. Specifically, the upstream caspases, caspase-8 and caspase-9, and the downstream caspase-3 are activated in aminoglycoside-damaged hair cells.

  View details for DOI 10.1007/s10162-002-3016-8

  View details for Web of Science ID 000181329000008

  View details for PubMedID 12417974

• Caspase activation in hair cells of the mouse utricle exposed to neomycin JOURNAL OF NEUROSCIENCE Cunningham, L. L., Cheng, A. G., Rubel, E. W. 2002; 22 (19): 8532-8540

  Abstract

  Aminoglycoside exposure results in the apoptotic destruction of auditory and vestibular hair cells. This ototoxic hair cell death is prevented by broad-spectrum caspase inhibition. We have used in situ substrate detection, immunohistochemistry, and specific caspase inhibitors to determine which caspases are activated in the hair cells of the adult mouse utricle in response to neomycin exposure in vitro. In addition, we have examined the hierarchy of caspase activation. Our data indicate that both upstream caspase-8 and upstream caspase-9, as well as downstream caspase-3 are activated in hair cells exposed to neomycin. The inhibition of caspase-9-like activity provided significant protection of hair cells exposed to neomycin, whereas the inhibition of caspase-8-like activity was not effective in preventing neomycin-induced hair cell death. In addition, caspase-9 inhibition prevented the activation of downstream caspase-3, whereas the inhibition of caspase-8 did not. These data indicate that caspase-9 is the primary upstream caspase mediating neomycin-induced hair cell death in this preparation.

  View details for Web of Science ID 000178246000021

  View details for PubMedID 12351727

• Oxidative stress-induced apoptosis of cochlear sensory cells: otoprotective strategies INTERNATIONAL JOURNAL OF DEVELOPMENTAL NEUROSCIENCE Huang, T., Cheng, A. G., Stupak, H., Liu, W., Kim, A., Staecker, H., LEFEBVRE, P. P., Malgrange, B., Kopke, R., Moonen, G., Van de Water, T. R. 2000; 18 (2-3): 259-270

  Abstract

  Apoptosis is an important process, both for normal development of the inner ear and for removal of oxidative-stress damaged sensory cells from the cochlea. Oxidative-stressors of auditory sensory cells include: loss of trophic factor support, ischemia-reperfusion, and ototoxins. Loss of trophic factor support and cisplatin ototoxicity, both initiate the intracellular production of reactive oxygen species and free radicals. The interaction of reactive oxygen species and free radicals with membrane phospholipids of auditory sensory cells creates aldehydic lipid peroxidation products. One of these aldehydes, 4-hydroxynonenal, functions as a mediator of apoptosis for both auditory neurons and hair cells. We present several approaches for the prevention of auditory sensory loss from reactive oxygen species-induced apoptosis: 1) preventing the formation of reactive oxygen species; (2) neutralizing the toxic products of membrane lipid peroxidation; and 3) blocking the damaged sensory cells' apoptotic pathway.

  View details for Web of Science ID 000086197800017

  View details for PubMedID 10715580

• Calpain inhibitors protect auditory sensory cells from hypoxia and neurotrophin-withdrawal induced apoptosis BRAIN RESEARCH Cheng, A. G., Huang, T., Stracher, A., Kim, A., Liu, W., Malgrange, B., LEFEBVRE, P. P., Schulman, A., Van De Water, T. R. 1999; 850 (1-2): 234-243

  Abstract

  Inhibitors of calpain have been shown to protect nerve growth factor (NGF)-deprived ciliary ganglion neurons and hypoxic cortical neurons. Calpains have been identified in the cochlea and are active during ischemic injury. Since apoptosis can be initiated by loss of neurotrophic support, hypoxia, and ototoxins (e.g., cisplatin, CDDP), the role of calpain inhibitors under these conditions was examined in auditory hair cells and neurons. Dissociated spiral ganglion neuron (SGN) cell cultures and organ of Corti explants from P3 rats were used to test the efficacy of calpain inhibitors as otoprotective molecules. Our results indicate that calpain inhibitor I, calpain inhibitor II, and leupeptin all provided significant protection of SGNs against neurotrophin-withdrawal and hypoxia-induced apoptosis. The increase in neuronal survival ranged from 2.16 to 2.31 times greater than in untreated neurotrophin-withdrawn SGN cell cultures. BOC-Asp(Ome)-Fluoromethyl Ketone (B-D-FMK), a general caspase inhibitor, increased neuronal survival 2.16 times more. Neuronal survival rates were from 1.88 to 2.27 times greater than in untreated, hypoxic neurons and hair cell survival rates were from 1.98 to 2.03 times greater than untreated, hypoxic organ of Corti explants. However, protection of auditory hair cells and neurons from CDDP-induced damage (10 and 6 micrograms/ml, respectively) was limited with any of these calpain inhibitors. Apoptotic pathways initiated by neurotrophin-deprivation and ototoxic stress (e.g., CDDP) have been shown to be different. Our results agree with this finding, with neurotrophin-withdrawal and hypoxia, but not CDDP damage-induced apoptosis being calpain-dependent.

  View details for Web of Science ID 000084222800027

  View details for PubMedID 10629769

• LATERAL VENTRICULAR EFFACEMENT AS AN ISOLATED SONOGRAPHIC FINDING IN PREMATURE-INFANTS - PREVALENCE AND SIGNIFICANCE AMERICAN JOURNAL OF ROENTGENOLOGY Patel, M. D., Cheng, A. G., Callen, P. W. 1995; 165 (1): 155-159

  Abstract

  The sonographic finding of effaced lateral ventricles in premature infants, defined as the absence of visible CSF within the lateral ventricles on both coronal and sagittal sonograms, may be cause to suspect diffuse cerebral edema, especially as published reference standards do not address this phenomenon. This investigation was undertaken to determine the prevalence and significance of effaced lateral ventricles without associated parenchymal abnormality (isolated lateral ventricular effacement, or ILVE) in premature infants.Sonographic records of 398 consecutive newborns examined from January 1 to December 31, 1993, were reviewed retrospectively to identify those premature infants (< 36 weeks of gestational age) whose initial sonograms showed no evidence of intracranial hemorrhage, ventriculomegaly, structural abnormality, or abnormal parenchymal echogenicity. We identified 142 neonates who met these criteria. Patients were separated into two groups on the basis of whether they had at least one sonographic study in which CSF was not visible within both lateral ventricles on coronal and sagittal images. Medical records were reviewed to assess neurologic outcome.Forty patients (28%) had at least one sonogram demonstrating ILVE, with neurologic follow-up in 33 (representing group A). One hundred two patients (72%) never demonstrated ILVE, with neurologic follow-up established in 86 (representing group B). A comparison of the two groups showed no significant difference in the development of ischemic injury (one patient in each group). ILVE was first detected on the initial sonogram obtained (mean, 4 days) in 30 of the 33 neonates in group A. ILVE was demonstrated beyond the seventh day of life in 30%. Of the 89 patients whose initial sonograms showed CSF in the lateral ventricles (86 in group B and three in group A), three (3%) subsequently had sonograms that showed ILVE; all three were normal at follow-up.ILVE in premature infants is common and not associated with neurologic deficits indicative of hypoxic-ischemic encephalopathy. By itself, ILVE is not a significant finding.

  View details for Web of Science ID A1995RE24000036

  View details for PubMedID 7785575