This work was supported by NIH Grants R01-DC010146 and R01-DC010397. Its content is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health.

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The authors declare no existing or potential conflict of interest.

בדוח זה אנו מתארים כיצד התרבות תאים HEI-OC1 ולהשתמש בהם כדי להעריך מנגנונים של רעילות סמים ולחקור תכונות פעילות של פרסטין, המנוע המולקולרי של OHCs שבלול. הנהלים הטכניים, עם זאת, הם כלליים מספיק כדי להתאים בקלות מחקרים שונים. כל הפרוטוק?...

אוזן בית מכון-איברים של 1 קורטי (HEI-OC1) תאים נגזרים מאיבר השמיעה של 1,2 עכבר מהונדס. דגירה של כל תא מעכבר מהונדס זה על 33 ° C / 10% CO 2 (תנאי מתירני) גרם ביטוי של גן מנציחה שמפעיל בידול דה ושגשוגם מואץ; הזזת תאים ל -39 ° C / 5% CO 2 (תנאים שאינם מתירני) להביא לפגיעה התפשטות, בידול, לפחות במקרה של HEI-OC1, מוות של תאים 2,3. תאי HEI-OC1 היו משובטים ומאופיינים במעבדה שלנו לפני למעלה מעשור, מחקרים ראשוניים הראו כי הם מביעים סמנים ספציפיים של תאי שיער שבלול, כגון פרסטין, 7 א שרירן, Atoh1, BDNF, calbindin ו calmodulin, אלא גם סמנים של תומכים תאים כמו connexin 26 ו הקולטן לגורם הצמיחה פיברובלסטים (FGF-R) 2. לכן, הוצע כי HEI-OC1 יכול לייצג common ובתאים עבור תאים חושיים ותמיכה של האיבר של קורטי 2. מחקרים מקבילים ספקו ראיות מוצקות לכך ארכיטיפי תרופות ototoxic כמו ציספלטין, גנטמיצין ו סטרפטומיצין מושרה הפעלת caspase-3 בתאים אלה, בעוד תרופות נחשבות בלתי ototoxic, כמו פניצילין, לא 2,3. לכן, שורת תא זו הוצעה כמערכת במבחנה כדי לחקור את המנגנונים התאיים ומולקולריים מעורבים ototoxicity ו להקרנה של ototoxicity הפוטנציאל או נכסי otoprotective של תרופות תרופתיות חדשות. ההערכה היא כי תאי HEI-OC1 שימשו יותר ממאה וחמישים מחקרים שפורסמו בעשר השנים האחרונות. בעוד מסתכל ההשפעה הפרו-אפופטוטיים הפוטנציאל של תרופות שונות היה המטרה העיקרית של רוב המחקרים שכללו את שורות תאים, תהליכים בתא חשובים אחרים כמו autophagy והזדקנות החלו רק כדי להיחקר בתאי HEI-OC1 4-7. אנינה מחקר שנערך לאחרונה במעבדה שלנו 8, השתמשנו תאים HEI-OC1 לאסוף מערך מקיף של נתונים על מוות של תאים, הישרדות, התפשטות, הזדקנות ו autophagy המושרה על ידי תרופות פרמקולוגיות שונות תדירים במרפאה. אנחנו גם בהשוואה כמה מהתגובות של תאי HEI-OC1 עם אלו של HEK-293 (תאי כליה עובריים אנושיים) והלה (תאי אפיתל אדם) מקבלים טיפול זהה. התוצאות שלנו הצביעו על כך HEI-OC1 תאים להגיב על כל תרופה בצורה אופיינית, עם dose- ייחודי ורגיש תלוי זמן לפחות אחד המנגנונים נחקרים. גם הדגשנו כי מחקר כי פרשנות נכונה של תוצאות הניסוי תדרוש ביצוע מחקרים מקבילים עם יותר מאחד טכניקה 8. במחקר אחר חקרנו את השימוש בתאי HEI-OC1 כדי להעריך את התגובה התפקודית של פרסטין, החלבון המנוע של תאי שיער חיצוניים שבלול (OHCs) 9 </sעד&gt;. דיווחנו cytometry זרימה ומחקרים מיקרוסקופיית לייזר confocal על דפוס הביטוי פרסטין, כמו גם קיבול קוי (NLC) ומחקרים ההידוק תא-תיקון כל בתאים HEI-OC1 בתרבית בשעה מתירנית (P-HEI-OC1) והלא תנאי היתר (NP-HEI-OC1). התוצאות שלנו הצביעו על כך הוא ביטוי פרסטין סך קרום פלזמת גידול לוקליזציה באופן תלוי-זמן בתאי NP-HEI-OC1. מעניין, מצאנו גם כי גידול לוקליזציה פרסטין על קרום הפלזמה של תאי NP-HEI-OC1 בקורלציה עם ירידה Na + K + ATPase, אשר translocated מן קרום הפלזמה אל הציטופלסמה ללא שינויים משמעותיים ביטוי תא כולל. בנוסף, הראינו כי P-HEI-OC1 יש לתאים NLC חזק הקשורים פרסטין תפקוד מוטורי, אשר ירד כאשר הצפיפות של מולקולות פרסטין נוכחי קרום הפלזמה גדלה. בסך הכל, תוצאות אלו תומכות התועלת של HEI-OC1 תאים לחקור חלבוני שמיעה. במאמר זה וידאו אנו מתארים כיצד תרבות תאי HEI-OC1, למה זה נוח להשתמש תאים גדלים בתנאי היתר (P-HEI-OC1) ללימודי cytotoxicity, איך להעריך את המנגנון / s של cytotoxicity-סמים ואיך לבצע מחקרים אלקטרו (למשל, תיקון- clamp, קיבול שאינו ליניארי (NLC)) לחקור תכונות פעילות של פרסטין, המנוע המולקולרי של OHCs שבלול.

<table><tbody><tr><td>HEI-OC1 cells</td><td></td><td></td><td>ALL THE ASSAY KITS, EQUIPMENTS&amp;nbsp;</td></tr><tr><td>Class II Biological Safety cabinet</td><td>The Baker Company</td><td>Sterilgard III</td><td>AND COMPANIES INDICATED IN THE</td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>5810R</td><td>PREVIOUS 2 COLUMNS ARE ONLY</td></tr><tr><td>Inverted microscope</td><td>Zeiss</td><td>Axiovert 25</td><td>EXAMPLES, AND ANY OTHER SIMILAR</td></tr><tr><td>Waterbath</td><td>Stovall</td><td>HWB115</td><td>PRODUCT COULD BE USED.</td></tr><tr><td>Cell counter</td><td>Nexcelom</td><td>Cellometer Auto T4</td><td></td></tr><tr><td>Two (2) Cell incubators, one at 33 &amp;deg;C/10% CO&lt;sub&gt;2&lt;/sub&gt; and other at 39 &amp;deg;C/5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Forma Scientific</td><td>3110</td><td></td></tr><tr><td>Cell culture dishes, PS, 100 mm x 20 mm with vents</td><td>Greinier Bio-One</td><td>664-160</td><td></td></tr><tr><td>Cell culture dishes , PS,&amp;nbsp; 60 mm x 15 mm with vents&amp;nbsp;</td><td>Greiner Bio-One</td><td>628160</td><td></td></tr><tr><td>Cellstar tissue culture flasks&amp;nbsp; 250 ml</td><td>Greiner Bio-One</td><td>658-175</td><td></td></tr><tr><td>Cellstar tissue cultur&amp;nbsp; flasks 550 ml</td><td>Greiner Bio-One</td><td>660-175</td><td></td></tr><tr><td>&amp;nbsp;6 well cell culture plate, with lid-Cellstar</td><td>Greiner Bio-One</td><td>657-160</td><td></td></tr><tr><td>Microtest Tissue culture plate, 96 well, flat bottom with lid</td><td>Becton Dickinson</td><td>353072</td><td></td></tr><tr><td>Micro-Assay-Plate, Chimmey, 96-well white, clear bottom</td><td>Greiner Bio-One</td><td>655098</td><td></td></tr><tr><td>50 ml Polypropylene conical tube with cap Cellstars</td><td>Becton Dickinson&amp;nbsp;</td><td>352070</td><td></td></tr><tr><td>15 ml Polypropylene conical tubes with cap-Cellstars</td><td>Greiner Bio-One</td><td>188-271</td><td></td></tr><tr><td>PBS pH 7.4 (1x)&amp;nbsp;</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>Life Technologies</td><td>11965-084</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Hyclone</td><td>SH10073.1</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium, no phenol red</td><td>Gibco/Invitrogen</td><td>21083-027</td><td></td></tr><tr><td>Trypsin, 0.25%&amp;nbsp;</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>TACS MTT Cell Proliferation Assay Kit</td><td>Trevigen</td><td>4890-25-K</td><td></td></tr><tr><td>Caspase-Glo 3/7 Assay&amp;nbsp; kit</td><td>Promega</td><td>&amp;nbsp; G8091&amp;nbsp;</td><td></td></tr><tr><td>BrdU Cell Proliferation Assay Kit&amp;nbsp;</td><td>Cell Signaling</td><td>6813</td><td></td></tr><tr><td>Non-enzymatic cell dissociation solution&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C5789</td><td></td></tr><tr><td>Cell-Tox Green Cytotoxicity Assay Kit</td><td>Promega</td><td>&amp;nbsp; G8741&amp;nbsp;</td><td></td></tr><tr><td>FACSAriaIII instrument&amp;nbsp;</td><td>BD Biosciences</td><td>&amp;nbsp;FACSAriaIII</td><td>With 488 nm excitation (blue laser)</td></tr><tr><td>Digital Blot Scanner</td><td>LI-COR</td><td>C-DiGit</td><td></td></tr><tr><td>Electrophoresis and Blotting Unit</td><td>Hoefer</td><td>SE300 miniVE</td><td></td></tr><tr><td>Spectra Max 5 Plate Reader with Soft Max Pro 5.2 Software</td><td>Molecular Devices</td><td>SpectraMax 5</td><td></td></tr><tr><td>Patch-clamp amplifier</td><td>HEKA</td><td>EPC-10</td><td></td></tr><tr><td>Puller for preparing patch electrodes</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr></tbody></table>

working with auditory hei-oc1 cells

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic drugs, these cells have been used to investigate drug-activated apoptotic pathways, autophagy, senescence, mechanism of cell protection, inflammatory responses, cell differentiation, genetic and epigenetic effects of pharmacological drugs, effects of hypoxia, oxidative and endoplasmic reticulum stress, and expression of molecular channels and receptors. Among other several important markers of cochlear hair cells, HEI-OC1 cells endogenously express prestin, the paradigmatic motor protein of outer hair cells. Thus, they can be very useful to elucidate novel functional aspects of this important auditory protein. HEI-OC1 cells are very robust, and their culture usually does not present big complications. However, they require some special conditions such as avoiding the use of common anti-bacterial cocktails containing streptomycin or other antibiotics as well as incubation at 33 &#176;C to stimulate cell proliferation and incubation at 39 &#176;C to trigger cell differentiation. Here, we describe how to culture HEI-OC1 cells and how to use them in some typical assays, such as cell proliferation, viability, death, autophagy and senescence, as well as how to perform patch-clamp and non-linear capacitance measurements.

בעוד כמה פרסומים האחרונים דיווחנו מערך מקיף של מחקרים שמטרתם להעריך את התגובות של תאי HEI-OC1 לסמים תרופתיים כמה נפוצים כמו גם חוקר פרסטין פונקצית 8,9. במחקרים אלה עשינו שימוש כל הפרוטוקולים מתוארים בסעיפים הקודמים. <p class="jove_content" fo:keep-together.within-page...

Watch this Scientific Journal Video about Working with Auditory HEI-OC1 Cells at JoVE.com

Working with Auditory HEI-OC1 Cells

House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

עבודה עם תאים השמיעתיים HEI-OC1

HEI-OC1 is one of the few mouse auditory cell lines available for research purposes. Originally proposed as an in vitro system for screening of ototoxic ...

Research

JoVE Journal

Biology

14.9K Views.  University of California, Los Angeles. House Ear Institute-Organ of Corti 1 (HEI-OC1) is one of the few mouse auditory cell lines currently available for research purposes. This protocol describes how to work with HEI-OC1 cells to investigate the cytotoxic effects of pharmacological drugs as well as functional properties of inner ear proteins.

Video: Working with Auditory HEI-OC1 Cells

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

הקלטות Postsynaptic ב דנדריטים מביא פנייה שבלול תאים שיער פנימית: שחרור Multivesicular ניטור ב סינפסה סרט

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

Neuroscience

Investigating Outer Hair Cell Motility with a Combination of External Alternating Electrical Field Stimulation and High-speed Image Analysis

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives from their characteristic apical bundle of stereocilia, a critical element for detection and transduction of sound energy 1. OHCs are able to change shape &#x2014;elongate, shorten and bend&#x2014; in response to electrical, mechanical and chemical stimulation, a motor response considered crucial for cochlear amplification of acoustic signals 2.
OHC stimulation induces two different motile responses: i) electromotility, a.k.a fast motility, changes in length in the microsecond range derived from electrically-driven conformational changes in motor proteins densely packed in OHC plasma membrane, and ii) slow motility, shape changes in the millisecond to seconds range involving cytoskeletal reorganization 2, 3. OHC bending is associated with electromotility, and result either from an asymmetric distribution of motor proteins in the lateral plasma membrane, or asymmetric electrical stimulation of those motor proteins (e.g., with an electrical field perpendicular to the long axis of the cells) 4. Mechanical and chemical stimuli induce essentially slow motile responses, even though changes in the ionic conditions of the cells and/or their environment can also stimulate the plasma membrane-embedded motor proteins 5, 6. Since OHC motile responses are an essential component of the cochlear amplifier, the qualitative and quantitative analysis of these motile responses at acoustic frequencies (roughly from 20 Hz to 20 kHz in humans) is a very important matter in the field of hearing research 7.
The development of new imaging technology combining high-speed videocameras, LED-based illumination systems, and sophisticated image analysis software now provides the ability to perform reliable qualitative and quantitative studies of the motile response of isolated OHCs to an external alternating electrical field (EAEF) 8. This is a simple and non-invasive technique that circumvents most of the limitations of previous approaches 9-11. Moreover, the LED-based illumination system provides extreme brightness with insignificant thermal effects on the samples and, because of the use of video microscopy, optical resolution is at least 10-fold higher than with conventional light microscopy techniques 12. For instance, with the experimental setup described here, changes in cell length of about 20 nm can be routinely and reliably detected at frequencies of 10 kHz, and this resolution can be further improved at lower frequencies. 
We are confident that this experimental approach will help to extend our understanding of the cellular and molecular mechanisms underlying OHC motility.

A reliable method to investigate outer hair cell (OHC) motile responses, including electromotility, slow motility and bending, is described. OHC motility is elicited by stimulation with an external alternating electrical field, and the method takes advantage of high-speed image recording, LED-based illumination, and last generation image analysis software.

חקירות תנועתיות החיצונית Cell שיער עם שילוב של גירוי חיצוני לסירוגין שדה חשמלי במהירות גבוהה ניתוח תמונה

OHCs are cylindrical sensorimotor cells located in the Organ of Corti, the auditory organ inside the mammalian inner ear. The name "hair cells" derives ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

גירוי optogenetic של עצב השמיעה

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

בשיטות אובו ואקס אובו לחקר התפתחות האוזן הפנימית של העופות

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

Developmental Biology

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

ההר שלם Dissection וImmunofluorescence של מבוגרי עכבר השבלול

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

גזע המוח השמיעתית תגובה והקלטה החיצוני תא שיער תיקון שלם-תא קלאמפ בחולדות כמחנכת

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

ניתוח שתל שבלולי והקלטות תגובה חשמלית עורר גזע המוח בעכברים C57BL/6

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

הכנה לפני שבלול בעכבר למבוגרים

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

In this study, we present a real-time imaging method using confocal microscopy to observe cells moving toward damaged tissue by ex vivo incubation with the cochlear epithelium containing the organ of Corti.

הפריה זמן לשגות הדמיה תא חי כדי לחקור את העברת תאים לכיוון האיבר של קורטי

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after ...

Medicine

טיפוח יעיל במבחנה של תאי שיער של שבלול העכבר

Isolation and Culture of Primary Cochlear Hair Cells from Neonatal Mice

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for hearing, within the osseous labyrinth of the inner ear. Cochlear hair cells consist of two anatomically and functionally distinct types: outer and inner hair cells. Damage to either of them results in hearing loss. Notably, as inner hair cells cannot regenerate, and damage to them is permanent. Hence, in vitro cultivation of primary hair cells is indispensable for investigating the protective or regenerative effects of cochlear hair cells. This study aimed to discover a method for isolating and cultivating mouse hair cells. 
After manual removal of the cochlear lateral wall, the auditory epithelium was meticulously dissected from the cochlear modiolus under a microscope, incubated in a mixture consisting of 0.25% trypsin-EDTA for 10 min at 37 &#176;C, and gently suspended in culture medium using a 200 &#181;L pipette tip. The cell suspension was passed through a cell filter, the filtrate was centrifuged, and cells were cultured in 24-well plates. Hair cells were identified based on their capacity to express a mechanotransduction complex, myosin-VIIa, which is involved in motor tensions, and via selective labeling of F-actin using phalloidin. Cells reached &gt;90% confluence after 4 d in culture. This method can enhance our understanding of the biological characteristics of in vitro cultured hair cells and demonstrate the efficiency of cochlear hair cell cultures, establishing a solid methodological foundation for further auditory research.

מחבר זרקור: התקדמות בטיפוח תאי שיער של עכברים למחקר שמיעתי 

Herein, we present a detailed protocol for isolating and culturing primary cochlear hair cells from mice. Initially, the organ of Corti was dissected from neonatal (aged 3-5 days) murine cochleae under a microscope. Subsequently, cells were enzymatically digested into a single-cell suspension and identified using immunofluorescence after several days in culture.

בידוד ותרבית של תאי שיער שבלול ראשוניים מעכברי יילודים

Cochlear hair cells are the sensory receptors of the auditory system. These cells are located in the organ of Corti, the sensory organ responsible for ...