2017 Stanford OHNS Basic and Translational Research Report (excerpts from)
Contributions by the individual laboratory heads.
Summary by Research Vice Chair Stefan Heller, PhD.
Summary of our achievements in 2016.
Last year was another highly successful year for the Research Division. It is very obvious that our department’s research scope has grown far beyond the initial focus on inner ear regeneration. The latitude of projects cover: surgical simulation, improving audiologic training for patients, immune response to cancer, advanced imaging, African elephant communication, novel non-ototoxic aminoglycoside antibiotics, stem cells, hair cell regeneration, voice disorders, hair cell mechano-transduction, synaptic and neural coding of sound information, as well as the impact of tobacco advertising – just to name a few. Clinical work has been focusing quite a bit on adapting principles of precision medicine, where patient-specific information is more and more taken into account. Of course, the core of the basic research group remains the goal of finding a cure for hearing loss and we are happy to report that fundraising for the Stanford Initiative to Cure Hearing Loss has been at the highest level since we started the initiative a couple of years ago. Funding through grants from federal and other entities continued to grow for the 11th year in a row. The number of grants towards research stands at a record number of 68. Productivity remains high and the quality of work has been recognized by many of our peers as outstandingly strong.
All faculty members who maintain an active endeavors have provided updates on their work and these updates are provided in the following pages. This report highlights basic and translational research in the department. Departmental faculty also contribute a sizable volume of clinically focused research.
CardinalSim – Development of a Virtual Surgical Rehearsal Platform
The CardinalSim research team, led by Nikolas Blevins and Kenneth Salisbury, continues to develop a platform to enable surgeons to rehearse complex cranial base surgical procedures based on preoperative anatomic imaging data. The primary hypothesis driving this project is that surgical outcomes will improve if surgeons are able to explore relevant anatomic data in an intuitive and surgically-relevant manner, prior to undertaking actual surgery. Clinicians currently have access to a wealth of preoperative imaging data (including both CT and MRI data) which are routinely examined only across-sectional abstractions of the complex 3-dimensional anatomic configurations they represent. The “CardinalSim” platform (http://med.stanford.edu/cardinalsim.html) addresses this, by allowing surgeons to load interact with clinical imaging data using haptic (touch) interfaces and stereoscopic displays (1). By exploring relevant anatomic relationships in a safe environment, the surgeon can be better prepared for otherwise unexpected challenges, thereby potentially minimizing risks.
Over the last year, our team has confirmed that surgical trainees using the workstation are capable of replicating key steps in a variety of temporal bone surgical procedures (2). The confirmation of our ability to accurately produce and display the surgically-relevant details that guide the course of a procedure is a critical step in validating our approach. Also this year, we completed a study demonstrating that the use of the simulation workstation in conjunction with actual anatomic dissections in a surgical laboratory could increase trainee confidence in performing experience-appropriate challenging segments of otologic surgery (3). This confirms that anatomy-specific rehearsal in our virtual environment can augment preparation for actual dissections.
Plans for the coming year include the facilitation of collaborative rehearsal through a shared database of cases, the incorporation of physics-based sound and deformable tissues into the virtual environment, and the development of effective methods to optimally displaythe insights from rehearsalwithin the operation room.
Chan S, Li P, Locketz G, Salisbury K, Blevins NH. (2016). High-fidelity haptic and visual rendering for patient-specific simulation of temporal bone surgery. Computer Aided Surgery.DOI: 10.1080/24699322.2016.1189966
Locketz GD, Lui JT, Chan, SK, Salisbury K, Dort JC, Youngblood P, Blevins NH. (2016). Replicating surgical procedures in patient specific virtual reality; presented at AAO/HNS Annual Meeting
Locketz GD, Lui JT, Chan, SK, Salisbury K, Dort JC, Youngblood P, Blevins NH. (Accepted 2016). Anatomy-specific virtual reality simulation in temporal bone dissection: impact on surgeon confidence and dissection performance. Otolaryngol Head Neck Surg.
Electrophysiology and the Classification of Auditory Stimuli
Our auditory electrophysiology research group has undertaken an investigating of non-invasive methods of assessing the efficacy of hearing restoration. We are using newclassification techniques of EEG data to determine if an individual with normal or altered hearing is able to differentiate between auditory stimuli. This approach may provide insights into the optimal means of presenting hearing signals to specific individuals, optimized for both their inner ears as well as their central auditory processing capacity. It is known that patients with hearing loss can respond quite differently to auditory signals. This is particularly the case for cochlear implant recipients in whom normal transduction of signals by the cochlea has been replaced by relatively few electrical contacts. These individuals oftenshow little ability to express the character of their experience – which is especially true for young children and those who have never experienced hearing. It is also clear that over time, such individuals can adapt and change in their response to stimuli, often being able to extract considerably more from the same signals. We hypothesize that a method to extract electrophysiologic data from EEG signals will provide insights into how to best present signals to a cochlear implant user to provide prognostic data and also optimize ultimate outcomes. Our preliminary data establishing these techniques in normal hearing individuals has been quite promising, and will be presented at the next meeting of the Association of Research in Otolaryngology.
Fitzgerald M, Losorelli S, Muscacchia G, Kaneshiro B, Blevins NH. Classification of auditory stimuli using event-related potentials. Accepted for presentation at ARO, 2017.
The Cheng laboratory has made important discoveries on several fronts in the past year. In the regeneration program, a new direction of our lab is to characterize the cells surrounding hair cells (the supporting cells). Since supporting cell loss leading to hair cell loss is the major cause of congenital hearing loss, we have been studying whether supporting cells can regenerate and how their loss causes secondary hair cell loss and eventually hearing loss. Moreover, we have been following up on our previous work that the immature mouse cochlea harbors progenitors (also supporting cells) that naturally regenerate hair cells after damage. Ongoing efforts focus on defining mechanisms that initiate or enhance this innate regenerative process.
In parallel, we continue to explore whether hair cell regeneration leads to a functional recovery in mammals. To better understand this process, we have been studying one of inner ear balance organs, the utricle, where a modest level of regeneration occurs. By establishing a time course of hair cell loss and subsequent regeneration and a whole animal vestibular function test, we have found significant differences between the young and mature utricle in terms of the degree and mechanisms of regeneration and also the degree of functional recovery. In the near future, we will be able to precisely describe the anatomy and function of a regenerating mammalian sensory organ, such that we can manipulate the degree of regeneration and assess how that affects functional recovery.
Our research program on cochlear development has been focusing one key signaling pathway, Wnt signaling, and how it regulates hair cell development. The complexity of this pathway lends itself to a step-by-step interrogation of individual components of the pathway. Thus far, we have found that this pathway affects both cell fate decision, maturation as well as organization of the developing cochlea, thus providing important insights into how to manipulate this pathway to promote regeneration.
Lastly, our group has been collaborating with Tony Ricci in developing novel non-ototoxic antibiotics. Using some of the latest technology in physics, we have visualized how our antibiotics interact with bacteria at the molecular level. This is critical information that will help guide us design additional versions of antibiotics that can prevent hearing loss.
Another mission of our group is foster the future generation of researchers to find a cure for hearing loss. We are extremely fortunate to have a talented of young, energetic and motivated scientists who work collaboratively. It has been a stellar year for them to have been awarded 2 grants from the NIH (Zahra Sayyid MSTP student, Tian Wang MD PhD research scientist), and 2 international grants from Australia (Patrick Atkinson PhD postdoctoral fellow) and European Union (Mary O’Sullivan PhD postdoctoral fellow).
Jan TA, Jansson L, Atkinson PJ, Wang T, Cheng AG. (2016). Profiling specific inner ear cell types using cell sorting techniques. Methods Mol Biol 1427:431-45. doi: 10.1007/978-1-4939-3615-1_23. PMCID: pending
In the Division of Audiology, our research efforts took three forms. First, we continued to investigate novel training procedures to facilitate learning and adaptation in individuals with hearing loss. We have demonstrated that individuals with hearing loss show different patterns of learning than individuals with normal hearing. This suggests that customized training programs may need to be developed for individuals with cochlear implants or hearing aids.
In our second line of research, we have laid the groundwork for a fundamental change in the audiologic test battery; making speech in noise the default clinical test of speech perception rather than word-recognition in quiet. We have collected data on over 1500 individuals which reveal that in most instances, speech in noise testing can replace word-recognition in quiet, and provide clear clinical guidelines for when to perform word-recognition in quiet.
Finally, we continue to investigate the mechanisms by which individuals with bilateral cochlear implants fuse information provided by each device. We have shown that some individuals are sensitive to mismatches in electrode insertion depth, while others are not. We have also demonstrated that self-selection of frequency tables has the potential to overcome any deficits in performance caused by between-ear mismatches in stimulation. Taken together, these lines of research have the potential to directly impact clinical practice, and to facilitate improvements in patient performance in the future.
Fitzgerald, M.B., Aaron, K.A., Tan, C-T., Glassman, E.K., and Svirsky, M.A. (in press). Self-selection of frequency tables with bilateral mismatches in an acoustic simulation of a cochlear implant. Journal of the American Academy of Audiology.
In 2016, the Grillet lab published three research papers in highly ranked journals, including two that conclude Dr. Grillet’s postdoctoral work. For each of them, new experiments were done at Stanford and involved members of the Grillet lab, Alix Trouillet (Postdoctoral Fellow) and Navid Zebarjadi (Research Assistant).
In the first publication, we characterized a gene responsible for deafness in a randomly mutated mouse strain. This gene, called Neuroplastin, codes for an adhesion molecule with two major splicing variants. These variants differ by the length of their extracellular domain. We showed that the shortest isoform is expressed specifically by the outer hair cells and is necessary for their function at 3 weeks of age. This work identified a new protein necessary for hair cell function in mice, and likely in humans as well.
In the second publication we demonstrated genetically that hair cells have two mechanical ionic channels: When the stereocilia bundle of hair cells is deflected in its normal direction of sensitivity, an electric current is generated. It is the initial current that codes for sound. When the stereocilia bundle is pushed in its reverse direction, no current is produced. Surprisingly, in newborn mice, once this mechanotransduction is abolished either by the rupturing of extracellular links that bridge the top of stereocilia to their taller neighbor, or by the genetic ablation of component of the sound mechanotransduction machinery, the hair cells show now an electric current when stimulated in the opposite direction. During the last five years a debate has emerged in the field about the origin of this current: Are both currents generated from the same ionic channel reconfigured to function in other directionalities or are they coming from distinct mechanosensitive channels? By studying the function the mechanosensitive channel Piezo2 that plays central role other sensory systems such as touch, we demonstrated that the “opposite” or “reverse” current requires Piezo2 while the normal current does not. Therefore, our work ends the debate and opens a new exciting question about the physiological role of this current that can be also triggered by local mechanical damage of the sensory epithelium.
In addition to these two publications, Dr. Grillet helped the research of Dr. Oghalai by providing his expertise in scanning electronic microscopy to image at nanometric resolution the stereocilia bundle of adult mice.
Next year looks very exciting for the Grillet lab as significant progress has already been made on the molecular requirement of a deafness gene. Also, Mattia Carraro joined the lab as a Postdoctoral Fellow upon graduating from the University of Toronto. Mattia is an expert in immunofluorescence staining of the entire inner ear at high resolution, as well as scanning electronic microscopy to image the blood vessels of the inner ear.
Wu Z*, Grillet N*, Zhao B, Cunningham C, Harkins-Perry S, Coste B, Ranade S, Zebarjadi N, Beurg M, Fettiplace R, Patapoutian A, Müller U. (2016). Mechanosensory hair cells express two molecularly distinct mechanotransduction channels. Nat Neurosci. Nov 28;* Shared first-authorship.
Zeng WZ*, Grillet N*, Dewey JB, Trouillet A, Krey JF, Barr-Gillespie PG, Oghalai JS, Müller U. (2016). Neuroplastin isoform np55 is expressed in the stereocilia of outer hair cells and required for normal outer hair cell function. J Neurosci. 36(35), 9201-16;* Shared first-authorship.
Lee HY, Raphael PD, Xia A, Kim J, Grillet N, Applegate BE, Ellerbee Bowden AK, Oghalai JS. (2016). Two-dimensional cochlear micromechanics measured in vivo demonstrate radial tuning within the mouse organ of corti. J Neurosci. 36(31), 8160-73.
2016 has been a transitional year for the Heller laboratory (http://hellerlab.stanford.edu). We have completed two major NIH R01 grants that mark the end of a period of research focusing on the functional assessment of a specific class of ion channels, which were once plausible candidates for playing a major role in hearing and balance. Very encouraging was that this planned transition went quite smoothly with the successful competition for a new R01 grant that focuses on assessing the molecular mechanisms by which mouse cochlear hair cells deal with noise-induced stress. This project is important because it can provide insights into the question why some people’s ears are more resistant to noise than others’. It also integrates well with a human genetics project pursued by Nicolas Grillet. We also implemented quite a lot of novel technologies with a major focus on single cells transcriptomics. This topic is a continuation of work that started a few years ago and I anticipate that we will have several major publications coming out next year as we apply this technology to study the triggers that initiate hair cell regeneration in the newborn mouse cochlea and in adult chickens.
We had 3 publications this year. The major one (Ealy et al., see below) describes 4 years of work on developing a way to guide human embryonic stem cells and induced pluripotent stem cells toward an early inner ear phenotype. The second paper is the result of a collaboration with Eri Hashino’s laboratory at the University of Indiana where we contributed tools and intellectual input to optimize the generation of sensory hair cells from mouse pluripotent stem cells. Finally, a previous postdoc of the Heller lab, who is now heading his own laboratory in China has published work on a project that started in the inner ear but concluded in muscle cells; this project begun almost a decade ago when our lab just had moved from Boston to Palo Alto.
Ealy M, Ellwanger DC, Kosaric N, Stapper AP, and Heller S. (2016) Single-cell analysis delineates a trajectory toward the human early otic lineage. Proc Natl Acad Sci U S A. 113: 8508-8513.
DeJonge RE, Liu XP, Deig CR, Heller S, Koehler KR, Hashino E. (2016) Modulation of Wnt Signaling Enhances Inner Ear Organoid Development in 3D Culture. PLoS One. 11:e0162508.
Cao JM, Cheng XN, Li SQ, Heller S, Xu ZG, Shi DL. (2016) Identification of novel MYO18A interaction partners required for myoblast adhesion and muscle integrity. Sci Rep. 6:36768.
Stanford Research Into the Impact of Advertising (SRITA) is an interdisciplinary research group which Dr. Jackler established over a decade ago. SRITA studies advertising, marketing, and promotion used by the tobacco industry to recruit and retain its customer. Our priority is research designed to inform regulators and legislators who are considering regulation of tobacco products.
The initial priority of SRITA was to create a digital repository of tobacco advertising material to support scholarship, advocacy, legal, and regulatory activity. As of 2016, the collection has grown to become the world’s largest repository with over 40,000 tobacco advertising images many of which are online in a searchable, meta-data rich, annotated database (tobacco.stanford.edu). The collection spans not only cigarette/cigar/pipe/snus/chew advertisements but also e-cigarettes, antismoking campaigns, with a new marijuana section in the to launch soon. As the historical collection is now comprehensive, recent emphasis has been acquiring contemporary tobacco advertising for the US and around the world (eg. “Be Marlboro” campaign).
As of December 2016, the online collection (tobacco.stanford.edu) includes 22,488 tobacco, 11,802 electronic cigarette, and 1152 anti-smoking advertisements. SRITA’s YouTube channel contains 178 tobacco and 157 electronic cigarette videos. Advertising comparison pairs (756) are available such as targeting women then versus now and African American versus mainstream advertisements. As of November 2016, the SRITA online collection has had 424,522 unique users with virtually every country in the world represented. The entire compendium of original tobacco advertisements, spanning 1890 to 2010+ have been donated to the National Museum of American History at the Smithsonian Institution.
My current research has focused upon the marketing of electronic cigarettes with special focus upon informing regulators about the advertising and promotional activities of the rapidly growing vapor industry. In 2016 my group has published on cessation imagery in e-cigarette advertising (AJPH), e-cigarette marketers utilizing of anti-smoking imagery (Tob Contol), exotic flavor based advertising (Tob Control) and we have papers submitted on alcohol flavored tobacco products and age gating on tobacco websites. A key collaboration is with Stanford pediatric professor Bonnie Halpern-Felsher who utilizes consumer perception methodology which compliments SRITA’s focus on content analysis. Perception studies extend and validate assumptions made via content analysis of advertisements in the SRITA collection.
In the coming year we are studying the marketing of e-cigarette flavors. The FDA banned flavors from combustible cigarette in 2009 with the exception of menthol. In 2016, the FDA solicited research into the impact of flavored vapor products on adult smoking cessation and youth initiation. We are studying the youth messaging of flavored e-cigarette advertising. We are also studying the 40 year marketing effort by the menthol brand Newport which led it to become a leading teen starter smoker brand.
Ramamurthi D, Gall PA, Ayoub N, Jackler RK. Leading-brand advertisement of quitting smoking benefits for e-cigarettes. Am J Public Health. 106: 2057-2063.
Jackler RK, Ramamurthi D. Unicorns cartoons: marketing sweet & creamy e-juice to youth. tob control. doi:10.1136/ tobaccocontrol-2016- 053206.
Jackler RK, Ramamurthi D, VanWinkle C, Bumanlag IM, Fayyaz P. Alcohol Flavored Tobacco Products: Tempting Teens to Transgress Two Adult Taboos with a Single Act. Submitted Tobacco Control Dec 2016.
Jackler RK. (2016). Testimony by otolaryngologists in defense of tobacco companies 2009-2014. Laryngoscope 125:2722–2729.
Ramamurthi D, Fadadu RP, Jackler RK. Electronic cigarette marketers manipulate anti-tobacco advertisements to promote vaping. Tob Control 2016; 25:720-722
The Mustapha laboratory published a collaborative study with Dr. Most from our department contributed to understanding the role of the classical complement pathway in recovery after facial nerve injury (Akdagli et al., 2016). We have two papers under revision. One is in PLOS Genetics describing the role of thrombospondins in afferent synapses maintenance and recovery after noise injury. A second paper is in under revision in Hearing Research describing the role of adrenergic receptors in cochlear function.
Akdagli, S., Williams, R., Kim H.J., Yan. Y., Mustapha, M and Most, S. Comparison of facial nerve recovery after injury in KbDb and C1q homozygous knockout mice: A preferential role for MHC-1 over the classical complement pathway in recovery after facial nerve injury. Plastic and Reconstructive Surgery – Global Open (in press).
Mendus D and Mustapha M. Contribution of beta1- and beta2-adrenergic receptors to cochlear function. Under revision in Hearing Research.
Wangsawihardja F, Balasubbu S, Sundaresan S, Leu R, Holt AG and Mustapha M. Accelerated noise-induced hearing loss and audiogenic seizure in mice lacking thrombospondins. Under revision in PLOS Genetics.
Mustapha M and Avril G. Holt. (2016). Genetics of deafness: in mice and men (auditory neuropathy). Perspectives from physics, Biology, Modeling, and Medicine. Chapter 5, 99-106
I developed a collaboration with Thomas Hildebrandt at the Institute for Zoo and Wildlife Research (IZW) in Berlin to coordinate and obtain permits to receive elephant temporal bone specimens in order to collaborate with Dr. Sunil Puria and Dr. Charles Steele in 2017 on their NIH grant