Name: neonatal murine cochlear explant technique as an in vitro screening tool in hearing research
Uploaded: 2017-06-08T15:00:00.000+00:00
Duration: 8 min 30 s
Description: Watch this Scientific Journal Video about Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research at JoVE.com

עבודה זו נתמכה על ידי המכון הלאומי של חירשות הפרעות תקשורת אחרות מעניקה R01DC015824 (KMS) ו T32DC00038 (תמיכה SD), משרד ההגנה מענק W81XWH-15-1-0472 (KMS), קרן ברטרלי (KMS), Nancy Sayles Day Foundation (KMS) ומרכז המחקר לאואר טיניטוס (KMS). אנו מודים לג &#39;סיקה E. Sagers, BA עבור הערות תובנה על כתב היד.

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החוקרים חייבים להשלים את הטכניקה לנתיחה לפני ביצוע ניסויים מעורבים explants cochlear. תאי שיער נפגעים בדרך כלל במהלך ניתוחים המבוצעים בשלב מוקדם בעקומת הלמידה, ורגע בעייתי במיוחד בשלמותם הוא הסרת הממברנה הטקטוריאלית, המחייבת ידיים יציבות, כלים מתאימים וניסיון. כדי לחסוך זמ...

אובדן שמיעה חושבי (SNHL) משקף נזק לאוזן הפנימית או מסלול שמיעתי עולה. בעוד אובדן שמיעה הוא הגירעון הסנסורי הנפוץ ביותר בבני אדם 1 , טיפולים מרפא עדיין לא קיים 2 . למרות שתלות שתלי המוח או השמיעה השמיעתית יכולות לשקם מידה מסוימת של שמיעה לחולים עם SNHL חמור עד עומק, השימוע שסופק על ידי מכשירים אלה עדיין שונה מאוד משימוע &quot;טבעי&quot;, במיוחד במהלך ניסיונות להבין דיבור ברעש או להאזנה למוסיקה. בעוד שתנועת תאי השיער נחשבת מזה זמן רב לתוצאה העיקרית של אירועי השמיעה הטראומטיים ( למשל, חשיפה לרעש חזק), יש ראיות גוברות לכך שהסינפסות המשדרות מידע מתאי שיער לעצב השמיעתי הן לפחות פגיעות לטראומה אקוסטית 3 , 4 , 5 </sup<html> &gt; 6 . מאחר וספים אודיומטריים אנושיים, תקן הזהב הנוכחי להערכת תפקוד השמיעה, אינם מנבאים נזק תאגידי ספציפי באוזן הפנימית, נדרשים כלים מעודנים יותר כדי לזהות ניוון תאי מוקדם ככל האפשר וליזום טיפול הולם. טיפולים תרופתיים מבטיחים לאובדן שמיעה נבדקים לעתים קרובות על תרבויות תאים הומוגניות במבחנה , אך מערכות כאלה אינן מדויקות במדויק את מיקרו-הסביבה של השבלול. תאי Cochlear ידועים כדי להפריש גורמים trophic המשפיעים על סוגי תאים אחרים בתוך שבלול 8 , 9 , תהליך חיוני vivo כי הוא איבד כאשר איבר של קורטי 10 , 11 או ספירל גנגליון נוירונים (SGNs) 12 מתורבתים בבידוד או כאשר סמנים מולקולריים מנותחיםEf &quot;&gt; 13. עם זאת, במחקרים vivo כי ייתכן שיהיה צורך אימות של נתונים במבחנה להקים טיפולים חדשים, אישית עבור אובדן שמיעה במרדף של&quot; רפואה דיוק &quot;דורשים משאבים משמעותיים וזמן.זה רלוונטי במיוחד כאשר שוקלים כמה מאמץ נדרש כדי לבצע מושלמת אוזן באמצע או חלון עגול זריקות הממברנה עם בדיקות השמיעה ולאחר מכן לנתיחה של מכלול שבלול כולו.ההקרנה היעילה של תרכובות מבטיח בתרבויות vivo לשעבר organotypic המכונה explants cochlear מספק חלופה כלכלית ואמינה 14 , 15 , 16 , 17 . מאמר זה מפרט פרוטוקול שבאמצעותו ניתן ליצור, לשמור ולהעריך explants cochlear מטופלים. יישומים ספציפיים למודל זה מודגשים, כולל השימוש בו בהקרנהשל תרכובות טיפוליות פוטנציאליות והערכה השוואתית של וקטורים ויראליים לריפוי גנטי. גישה vivo exant לשעבר מאפשר לחוקרים לדמיין את ההשפעות של טיפול נתון על אוכלוסיות תאים שונים באתרם , להקל על זיהוי של סוג ספציפי תאים מנגנוני ואת חידוד הבאים של טיפול ממוקד. בסך הכל, טכניקה זו מספקת מודל ללמוד את vivo cochlea לשעבר תוך שמירה על חוצה שיחה בין סוגי תאים שונים מאוד להתקיים בתוך cochlea.

<table><tbody><tr><td>Ampicillin, Sodium Salt</td><td>Invitrogen</td><td>11593-027</td><td></td></tr><tr><td>anti-CtBP2 antibody, mouse(IgG1)</td><td>BD Transduction Laboratories</td><td>612044</td><td></td></tr><tr><td>anti-Myo7A antibody, rabbit</td><td>Proteus Biosciences</td><td>25-6790</td><td></td></tr><tr><td>anti-NF-H antibody, chicken</td><td>EMD Millipore</td><td>AB5539</td><td></td></tr><tr><td>anti-PSD95 antibody, mouse(IgG2a)</td><td>Antibodies Inc.</td><td>75-028</td><td></td></tr><tr><td>anti-TuJ1 antibody, mouse</td><td>BioLegend</td><td>801202</td><td></td></tr><tr><td>Cell-Tak Cell and Tissue Adhesive, 5 mg</td><td>Corning</td><td>354241</td><td></td></tr><tr><td>CELLSTAR 15 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile</td><td>Greiner Bio-One</td><td>188161</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 100 x 20 mm</td><td>Greiner Bio-One</td><td>664160</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 35 x 10 mm, 4 inner rings</td><td>Greiner Bio-One</td><td>627170</td><td></td></tr><tr><td>CELLSTAR Cell Culture Dish, 60 x 15 mm</td><td>Greiner Bio-One</td><td>628160</td><td></td></tr><tr><td>CELLSTAR 50 mL Centrifuge Tubes, Conical bottom, Graduation, Sterile</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr><tr><td>Clear Nail Polish</td><td>Electron Microscopy Sciences</td><td>72180</td><td></td></tr><tr><td>Clear Wall Glass Bottom Dishes (Glass 40mm), PELCO&amp;reg;, Sleeve/20, 50 x 7 mm</td><td>Ted Pella Inc.</td><td>14027-20</td><td></td></tr><tr><td>Coverslips, Round, Glass, 10 mm diameter, Thickness #1, 0.13 - 0.16mm</td><td>Ted Pella Inc.</td><td>260368</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>Distilled water, 500 mL</td><td>Thermo Fisher Scientific</td><td>15230-162&amp;nbsp;</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM), high glucose, pyruvate, no glutamine, 500 mL</td><td>Thermo Fisher Scientific</td><td>10313-039</td><td></td></tr><tr><td>Dumont #4 Forceps</td><td>Fine Science Tools</td><td>11241-30</td><td></td></tr><tr><td>Dumont #55 Forceps (Dumostar)</td><td>Fine Science Tools</td><td>11295-51</td><td></td></tr><tr><td>Ethyl alcohol (EtOH), Pure, 200 proof, anhydrous, &amp;ge;99.5%</td><td>Sigma-Aldrich</td><td>459836-1L</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), qualified, USDA-approved regions, 500 mL</td><td>Thermo Fisher Scientific</td><td>10437-028&amp;nbsp;</td><td>Aliquot in 50 mL tubes and store in -20&amp;deg;C freezer</td></tr><tr><td>goat anti-chicken-647 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21469</td><td></td></tr><tr><td>goat anti-mouse(IgG)-568 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-11004</td><td></td></tr><tr><td>goat anti-mouse(IgG1)-568 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21124</td><td></td></tr><tr><td>goat anti-mouse(IgG2a)-488 secondary antibody</td><td>Thermo Fisher Scientific</td><td>A-21131</td><td></td></tr><tr><td>goat anti-rabbit-488 secondary antibody</td><td>Thermo Fisher Scientific</td><td>R37116</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, sterile, EmbryoMax Ultra Pure Water, 500 mL</td><td>EMD Millipore</td><td>TMS-006-B</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS), calcium, magnesium, no phenol red, 500 mL</td><td>Thermo Fisher Scientific</td><td>14025-092</td><td></td></tr><tr><td>Instrument Tray with Lid Stainless Steel</td><td>Mountainside Medical</td><td>TechMed4255</td><td></td></tr><tr><td>Micro (dissecting) knife &amp;ndash; angled 30&amp;deg;</td><td>Fine Science Tools</td><td>10056-12</td><td></td></tr><tr><td>Microscope slides, VistaVision, color-coded, 75 x 25 mm (3 x 1&quot;), 1 mm thick, white, pack of 72</td><td>VWR</td><td>16004-382</td><td></td></tr><tr><td>N-2 Supplement (100X), 5 mL</td><td>Thermo Fisher Scientific</td><td>17502-048</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;, Sodium Bicarbonate 7.5% solution, 100 mL</td><td>Thermo Fisher Scientific</td><td>25080-094</td><td></td></tr><tr><td>NaOH, sodium hydroxide solution, 1 L</td><td>Thermo Fisher Scientific</td><td>SS266-1</td><td></td></tr><tr><td>Normal Horse Serum (NHS)</td><td>Invitrogen</td><td>16050130</td><td></td></tr><tr><td>Operating scissors</td><td>Roboz Surgical Instruments Co.</td><td>RS-6806</td><td></td></tr><tr><td>Paraformaldehyde (PFA), Reagent Grade, Crystalline</td><td>Sigma-Aldrich</td><td>P6148</td><td>Prior to use: Establish Standard Operating Procedures based on protocols available online</td></tr><tr><td>PBS, pH 7.4, 500 mL</td><td>Thermo Fisher Scientific</td><td>10010-023&amp;nbsp;</td><td>Autoclave prior to use</td></tr><tr><td>Phalloidin, Alexa Fluor 568&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A12380</td><td></td></tr><tr><td>Prep Pad, Non Sterile&amp;nbsp;</td><td>Medline</td><td>05136CS</td><td></td></tr><tr><td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 mL</td><td>Eppendorf</td><td>022363719</td><td>Autoclave prior to use</td></tr><tr><td>Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 mL</td><td>Eppendorf</td><td>022363204</td><td>Autoclave prior to use</td></tr><tr><td>Scalpel Blades - #15</td><td>Fine Science Tools</td><td>10015-00</td><td></td></tr><tr><td>Scalpel Handle - #4</td><td>Fine Science Tools</td><td>10004-13</td><td></td></tr><tr><td>Stemi 2000-C Stereo Microscope</td><td>Zeiss&amp;nbsp;</td><td>000000-1106-133</td><td></td></tr><tr><td>TCS SP5 confocal microscope</td><td>Leica</td><td>N/A</td><td></td></tr><tr><td>Triton-X (non-ionic surfactant)</td><td>Integra</td><td>T756.30.30</td><td></td></tr><tr><td>VectaShield antifade mounting medium for fluorescence</td><td>Vector Laboratories, Inc.</td><td>H-1000</td><td></td></tr><tr><td>Zipper Bag, Reclosable, 4 x 6&#039;&#039; - 2 mm thick</td><td>Zipline</td><td>0609132541599</td><td></td></tr></tbody></table>

neonatal murine cochlear explant technique as an in vitro screening tool in hearing research

אמנם יש התקדמות בולטת במחקר השמיעה בעשורים האחרונים, עדיין אין תרופה עבור אובדן שמיעה Sensorineural (SNHL), מצב זה בדרך כלל כרוך נזק או אובדן של מבנים מכאנסנסורי עדין של האוזן הפנימית. מתוחכם מבחני חוץ גופית vivo לשעבר צמחו בשנים האחרונות, מה שמאפשר את ההקרנה של מספר גדל והולך של תרכובות טיפוליות פוטנציאליים תוך מזעור משאבים מאיצים המאמצים לפתח תרופות עבור SNHL. למרות תרבויות הומוגניות של סוגי תאים מסוימים ממשיכים לשחק תפקיד חשוב במחקר הנוכחי, מדענים רבים כיום להסתמך על תרבויות organotypic מורכבים יותר של האוזן הפנימית Murine, הידוע גם בשם explants cochlear. שימור מבנים תאיים מאורגנים בתוך האוזן הפנימית מקנה הערכה באתרו של מרכיבים שונים של תשתית השבלול, כולל תאי שיער פנימיים וחיצוניים, נוירונים גנגליונים ספירליים, נוירוניםItes, ותאים התומכים. כאן אנו מציגים את הכנה, תרבות, טיפול, immunostaining של explants מון neonatal cochlear. הכנה זהירה של explants אלה מאפשרת זיהוי של מנגנונים אשר תורמים SNHL ומהווה כלי רב ערך עבור קהילת המחקר השמיעה.

בעוד פרוטוקולים רבים מתמקדים באיבר של explants קורטי, טכניקה זו מנסה לשמר את האנטומיה של התור כולו cochlear, כולל SGNs. זה נותן לחוקרים את ההזדמנות לנתח את ההשפעות של טיפול נתון על neurites ו somata של SGNs בנוסף האיבר של קורטי. ביצוע דיסקציה שמשמרת חלק מהמודולוס, כפי שמ...

Watch this Scientific Journal Video about Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research at JoVE.com

טכניקת השתלת שבלול של עכברים של ילודים ככלי הקרנה חוץ גופית בחקר שמיעה | רפואה | וִידֵאוֹ

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

המטרה של פרוטוקול זה היא להדגים את הכנה, תרבות, טיפול, immunostaining של explants מון neonatal cochlear. הטכניקה יכולה להיות מנוצל כמו כלי בדיקה במבחנה במחקרי שמיעה.

Neonatal Murine Cochlear Explant טכניקה כמו<em&gt; במבחנה</em&gt; כלי הקרנה במחקר שמיעה

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

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Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

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17.5K Views.  Massachusetts Eye and Ear.  המטרה של פרוטוקול זה היא להדגים את הכנה, תרבות, טיפול, immunostaining של explants מון neonatal cochlear. הטכניקה יכולה להיות מנוצל כמו כלי בדיקה במבחנה במחקרי שמיעה. 

Video: Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

תרבות של עוברי Explants שבלול העכבר וג&#39;ין העברה על ידי Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Developmental Biology

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

יצירת תחושה של הקשבה: סוללה מבחן IMAP

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

אסטרטגיה לזהות מוטציות דה נובו בהפרעות נפוצות כגון אוטיזם וסכיזופרניה

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

תרבות ראשיים electroporation פלסמיד של האיבר Murine של קורטי.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

לטווח ארוך זמן פקיעת הדמיה של Explants שבלול העכבר

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Neuroscience

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

גישת העכבר עגול החלון לototoxic סוכן משלוח: מהיר וטכניקה אמינה גרימת ניוון תא שבלול

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

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 ...

Whole Neonatal Cochlear Explants as an In vitro Model

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is characterized by the cumulative and irreversible loss of sensory hair cells and auditory nerves in the cochlea. Entire and vital cochlear explants are one of the fundamental tools in hearing research to detect hair cell loss and to characterize the molecular mechanisms of the inner ear cells. Many years ago, a protocol for neonatal cochlear isolation was developed, and although it has been modified over time, it still holds potential for improvement. 
This paper presents an optimized protocol for isolating and culturing whole neonatal cochlear explants in multi-well culture chambers that enables the study of hair cells and spiral ganglion neuron cells along the entire length of the cochlea. The protocol was tested using cochlear explants from mice and rats. Healthy cochlear explants were obtained to study the interaction between hair cells, spiral ganglion neuron cells, and the surrounding supporting cells. 
One of the main advantages of this method is that it simplifies the organ culture steps without compromising the quality of the explants. All three turns of the organ of Corti are attached to the bottom of the chamber, which facilitates in vitro experiments and the comprehensive analysis of the explants. We provide some examples of cochlear images from different experiments with live and fixed explants, demonstrating that the explants retain their structure despite exposure to ototoxic drugs. This optimized protocol can be widely used for the integrative analysis of the mammalian cochlea.

Whole Neonatal Cochlear Explants as an In vitro Model

The current protocol updates previous protocols and incorporates relatively straightforward approaches for culturing high-quality cochlear explants. This provides reliable data acquisition and high-resolution imaging in live and fixed cells. This protocol supports the ongoing trend of studying inner ear cells.

צמחי שבלול שלמים של ילודים כמודל במבחנה

Untreated hearing loss imposes significant costs on the global healthcare system and impairs individuals' quality of life. Sensorineural hearing loss is ...

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 ...

Neonatal Murine Cochlear Explant טכניקה כמו

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תרבות של עוברי Explants שבלול העכבר וג'ין העברה על ידי Electroporation