מחקר זה נתמך בחלקו על ידי תוכנית המחקר האינטרמורלית של ה-NIH, NIDCD to M.H. (DC000088)

Stria Vascularis Microdissection עבור ריצוף גרעין יחיד או immunostaining

<ol>
	<li>Bosher, S. K., Warren, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+electrochemistry+of+the+cochlear+endolymph+of+the+rat:+a+quantitative+study+of+its+electrical+potential+and+ionic+composition+as+determined+by+means+of+flame+spectrophotometry.">Observations on the electrochemistry of the cochlear endolymph of the rat: a quantitative study of its electrical potential and ionic composition as determined by means of flame spectrophotometry.</a> Proceedings of the Royal Society of London. Series B. 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Cell Physiology. 282 (2), C403-C407 (2002).</li><li>Spicer, S. S., Schulte, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+inner+ear+fibrocytes+according+to+their+ion+transport+related+activity.">Differentiation of inner ear fibrocytes according to their ion transport related activity.</a> Hearing Research. 56 (1-2), 53-64 (1991).</li><li>Wangemann, P., Liu, J., Marcus, D. 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P., Barkway, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Another+role+for+melanocytes:+their+importance+for+normal+stria+vascularis+development+in+the+mammalian+inner+ear.">Another role for melanocytes: their importance for normal stria vascularis development in the mammalian inner ear.</a> Development. 107 (3), 453-463 (1989).</li><li>Ito, T., Nishio, A., Wangemann, P., Griffith, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+irreversible+hearing+loss+is+caused+by+stria+vascularis+degeneration+in+an+Slc26a4-insufficient+mouse+model+of+large+vestibular+aqueduct+syndrome.">Progressive irreversible hearing loss is caused by stria vascularis degeneration in an Slc26a4-insufficient mouse model of large vestibular aqueduct syndrome.</a> Neuroscience. 310, 188-197 (2015).</li><li>Gu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+rare+spindle+and+root+cell+transcriptional+profiles+in+the+stria+vascularis+of+the+adult+mouse+cochlea.">Characterization of rare spindle and root cell transcriptional profiles in the stria vascularis of the adult mouse cochlea.</a> Scientific Reports. 10 (1), 18100 (2020).</li><li>Gow, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deafness+in+claudin+11-null+mice+reveals+the+critical+contribution+of+basal+cell+tight+junctions+to+stria+vascularis+function.">Deafness in claudin 11-null mice reveals the critical contribution of basal cell tight junctions to stria vascularis function.</a> The Journal of Neuroscience. 24 (32), 7051-7062 (2004).</li><li>Chang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virally+mediated+Kcnq1+gene+replacement+therapy+in+the+immature+scala+media+restores+hearing+in+a+mouse+model+of+human+Jervell+and+Lange-Nielsen+deafness+syndrome.">Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome.</a> EMBO Molecular Medicine. 7 (8), 1077-1086 (2015).</li><li>Faridi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+and+phenotypic+spectra+of+KCNE1+deficiency+in+Jervell+and+Lange-Nielsen+Syndrome+and+Romano-Ward+Syndrome.">Mutational and phenotypic spectra of KCNE1 deficiency in Jervell and Lange-Nielsen Syndrome and Romano-Ward Syndrome.</a> Human Mutation. 40 (2), 162-176 (2019).</li><li>Wangemann, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+KCNJ10+protein+expression+abolishes+endocochlear+potential+and+causes+deafness+in+Pendred+syndrome+mouse+model.">Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model.</a> BMC Medicine. 2, 30 (2004).</li><li>Kitajiri, S. -. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+patterns+of+claudins,+tight+junction+adhesion+molecules,+in+the+inner+ear.">Expression patterns of claudins, tight junction adhesion molecules, in the inner ear.</a> Hearing Research. 187 (1-2), 25-34 (2004).</li><li>Jagger, D. J., Nevill, G., Forge, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane+properties+of+cochlear+root+cells+are+consistent+with+roles+in+potassium+recirculation+and+spatial+buffering.">The membrane properties of cochlear root cells are consistent with roles in potassium recirculation and spatial buffering.</a> Journal of the Association for Research in Otolaryngology. 11 (3), 435-448 (2010).</li><li>Ito, T., Kurata, N., Fukunaga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages+in+the+stria+vascularis.">Tissue-resident macrophages in the stria vascularis.</a> Frontiers in Neurology. 13, 818395 (2022).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGFA165+gene+therapy+ameliorates+blood-labyrinth+barrier+breakdown+and+hearing+loss.">VEGFA165 gene therapy ameliorates blood-labyrinth barrier breakdown and hearing loss.</a> JCI Insight. 6 (8), e143285 (2021).</li><li>Shi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+the+cochlear+intrastrial+fluid-blood+barrier+(review).">Pathophysiology of the cochlear intrastrial fluid-blood barrier (review).</a> Hearing Research. 338, 52-63 (2016).</li><li>Gu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+potential+Meniere's+disease+targets+in+the+adult+stria+vascularis.">Identification of potential Meniere's disease targets in the adult stria vascularis.</a> Frontiers in Neurology. 12, 630561 (2021).</li><li>Fischer, J., Ayers, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleus+RNA-sequencing:+how+it's+done,+applications+and+limitations.">Single nucleus RNA-sequencing: how it's done, applications and limitations.</a> Emerging Topics in Life Sciences. 5 (5), 687-690 (2021).</li><li>Korrapati, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+and+single+nucleus+RNA-Seq+reveal+cellular+heterogeneity+and+homeostatic+regulatory+networks+in+adult+mouse+stria+vascularis.">Single cell and single nucleus RNA-Seq reveal cellular heterogeneity and homeostatic regulatory networks in adult mouse stria vascularis.</a> Frontiers in Molecular Neuroscience. 12, 316 (2019).</li><li>Pyle, M. 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למחברים אין מה לחשוף.

לפני הופעתו של ריצוף תא יחיד, חוקרים רבים השתמשו בניתוח רקמות בתפזורת, אשר איפשר רק לנתח תעתיקים ממוצעים על פני תאים. בפרט, תא בודד ו- sNuc-Seq אפשרו לבודד את התעתיק של תא בודד או גרעין יחיד, בהתאמה32. במקרה זה, ניתן לזהות תעתיקי גרעין יחיד עבור תאים שוליים, בינוניים ובזאליים, כמו גם ת?...

השבלול מורכב משלושה תאים מלאים בנוזל, סקאלה וסטיבולי, סקאלה מדיה וסקלה טימפני. סקאלה וסטיבולי וסקלה טימפאני מכילים כל אחד פרילימפה, אשר יש ריכוז גבוה של נתרן (138 mM) וריכוז נמוך של אשלגן (6.8 mM)1. מדיה סקאלה מכיל endolymph, אשר יש ריכוז גבוה של אשלגן (154 mM) וריכוז נמוך של נתרן (0.91 mM)1,2,3. הבדל זה בריכוז היונים יכול להיקרא פוטנציאל אנדוקוכליארי (EP), והוא נוצר בעיקר על ידי תנועה של יוני אשלגן דרך תעלות יונים שונות וצמתים מרווחים בסטריה וסקולריס (SV) לאורך הדופן הצידית של השבלול 4,5,6,7,8,9,10,11 . SV היא רקמה הטרוגנית, מאוד וסקולרית, שמרפדת את האספקט המדיאלי של הדופן הצידית של השבלול ומכילה שלושה סוגי תאים עיקריים: תאים שוליים, תאי ביניים ותאי בסיס12 (איור 1).
תאי שוליים מחוברים על ידי צמתים הדוקים כדי ליצור את המשטח המדיאלי ביותר של SV. הממברנה האפיקלית פונה לאנדולימפה של מדיה סקאלה ותורמת להעברת יוני אשלגן לתוך האנדולימפה באמצעות ערוצים שונים, כולל KCNE1/KCNQ1, SLC12A2 ו-Na+-K+-ATPase (NKA)5,10,13,14. תאי ביניים הם תאים פיגמנטיים השוכנים בין תאים שוליים לתאי בסיס ומאפשרים העברת אשלגן דרך SV באמצעות KCNJ10 (Kir 4.1)15,16. תאי הבסיס נמצאים בסמיכות לדופן הצידית של השבלול וקשורים קשר הדוק עם פיברוציט של הרצועה הספירלית כדי לקדם מחזור אשלגן מהפרילימפה12. פתולוגיה של SV הייתה מעורבת בהפרעות אוטולוגיות רבות17,18. מוטציות בגנים המתבטאים בסוגי תאי SV העיקריים, כגון Kcnq1, Kcne1, Kcnj10 ו-Cldn11, עלולות לגרום לחירשות ולתפקוד לקוי של SV, כולל אובדן EP 19,20,21,22,23. בנוסף לשלושת סוגי התאים העיקריים, ישנם סוגי תאים אחרים פחות נחקרים ב-SV, כגון תאי ציר 22, תאי שורש12,24, מקרופאגים 25, פריציטים 26 ותאי אנדותל 27, שיש להם תפקידים לא מוגדרים לחלוטין הקשורים להומאוסטזיס יוני וליצירת EP 28.
בהשוואה לריצוף רנ"א בתפזורת, ריצוף רנ"א חד-גרעיני (sNuc-Seq) מספק מידע על הטרוגניות התא, ולא ממוצע של mRNA על פני קבוצת תאים29, ויכול להיות שימושי במיוחד כאשר חוקרים את SV30 ההטרוגני. לדוגמה, sNuc-Seq הפיק ניתוח שעתוק המצביע על כך שייתכן שיש תפקיד לתאי ציר ושורש ביצירת EP, אובדן שמיעה ומחלת מנייר18. אפיון שעתוק נוסף של סוגי תאי SV השונים יכול לספק לנו מידע רב ערך על הפתופיזיולוגיה העומדת בבסיס מנגנונים ותת-סוגים שונים של תנודות שמיעה הקשורות ל-SV ואובדן שמיעה. הקציר של מבני האוזן הפנימית העדינים הללו הוא בעל חשיבות עליונה לניתוח רקמות אופטימלי.
במחקר זה מתוארת גישת המיקרודיסקציה לגישה ולבידוד של כלי הדם הסטריה משברלול העכבר הבוגר עבור sNuc-Seq או immunostaining. דיסקציה של SV עכבר בוגר נדרשת כדי להבין סוגים שונים של תאי SV ולאפיין עוד יותר את תפקידם בשמיעה.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >10-&#181;m filter (Polyethylenterephthalat)</td>
			<td >PluriSelect</td>
			<td >#43-50010-01</td>
			<td >Filter tissue during sNuc-Seq</td>
		</tr>
		<tr >
			<td >18 x 18 mm cover glass</td>
			<td >Fisher Scientific</td>
			<td >12-541A</td>
			<td >Cover slip to mount SV</td>
		</tr>
		<tr >
			<td >30-&#181;m filter (Polyethylenterephthalat)</td>
			<td >PluriSelect</td>
			<td >#43-50030-03</td>
			<td >Filter tissue during sNuc-Seq</td>
		</tr>
		<tr >
			<td >75 x 25 mm Superfrost Plus/Colorforst Plus Microslide</td>
			<td >Daigger</td>
			<td >EF15978Z</td>
			<td >Microslide to mount SV on</td>
		</tr>
		<tr >
			<td >C57BL/6J Mice</td>
			<td >The Jackson Laboratory</td>
			<td >RRID: IMSR_JAX:000664</td>
			<td >General purpose mouse strain that has pigment more easily seen in the intermediate cells of the SV.</td>
		</tr>
		<tr >
			<td >Cell Counter</td>
			<td >Logos Biosystems</td>
			<td >L20001</td>
			<td >Used for cell counting</td>
		</tr>
		<tr >
			<td >Chalizon curette 5&#39;&#39;, size 3 2.5 mm</td>
			<td >Biomedical Research Instruments</td>
			<td >15-1020</td>
			<td >Used to transfer SV</td>
		</tr>
		<tr >
			<td >Chromium Next GEM single Cell 3&#39; GEM Kit v3.1</td>
			<td >Chromium</td>
			<td >PN-1000141</td>
			<td >Generates single cell 3&#39; gene expression libraries</td>
		</tr>
		<tr >
			<td >Clear nail polish</td>
			<td >Fisher Scientific</td>
			<td >NC1849418</td>
			<td >Used for sealing SV mount</td>
		</tr>
		<tr >
			<td >Corning Falcon Standard Tissue Culture Dishes, 24 well</td>
			<td >Corning</td>
			<td >08-772B</td>
			<td >Culture dish used to hold specimen during dissection</td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Invitrogen</td>
			<td >D1306, RRID: AB_2629482</td>
			<td >Stain used for nucleus labeling</td>
		</tr>
		<tr >
			<td >Dounce homogenizer</td>
			<td >Sigma-Aldrich</td>
			<td >D8938</td>
			<td >Used to homogenize tissue for sNuc-seq</td>
		</tr>
		<tr >
			<td >Dumont #5 Forceps</td>
			<td >Fine Science Tools</td>
			<td >11252-30</td>
			<td >General forceps for dissection</td>
		</tr>
		<tr >
			<td >Dumont #55 Forceps</td>
			<td >Fine Science Tools</td>
			<td >11255-20</td>
			<td >Forceps with fine tip that makes SV manipulation easier</td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >ThermoFisher</td>
			<td >16000044</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Glue stick</td>
			<td >Fisher Scientific</td>
			<td >NC0691392</td>
			<td >Used for mounting SV</td>
		</tr>
		<tr >
			<td >GS-IB4 Antibody</td>
			<td >Molecular Probes</td>
			<td >I21411, RRID: AB-2314662</td>
			<td >Antibody used for capillary labeling</td>
		</tr>
		<tr >
			<td >KCNJ10-ZsGreen Mice</td>
			<td >n/a</td>
			<td >n/a</td>
			<td >Transgenic mouse that expresses KCNJ10-ZsGreen, partiularly in the intermediate cells of the SV.</td>
		</tr>
		<tr >
			<td >MgCl2</td>
			<td >ThermoFisher</td>
			<td >AM9530G</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Mounting reagent</td>
			<td >ThermoFisher</td>
			<td >#S36940</td>
			<td >Mounting reagent for SV</td>
		</tr>
		<tr >
			<td >Multiwell 24 well plate</td>
			<td >Corning</td>
			<td >#353047</td>
			<td >Plate used for immunostaining</td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td >ThermoFisher</td>
			<td >AAJ216183</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Nonidet P40</td>
			<td >Sigma-Aldrich</td>
			<td >9-16-45-9</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Nuclease free water</td>
			<td >ThermoFisher</td>
			<td >4387936</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Orbital shaker</td>
			<td >Silent Shake</td>
			<td >SYC-2102A</td>
			<td >Used for steps of immunostaining</td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >ThermoFisher</td>
			<td >J61196.AP</td>
			<td >Used for steps of immunostaining and dissection</td>
		</tr>
		<tr >
			<td >RNA Later</td>
			<td >Invitrogen</td>
			<td >AM7021</td>
			<td >Used for preservation of SV for sNuc-Seq</td>
		</tr>
		<tr >
			<td >Scizzors</td>
			<td >Fine Science Tools</td>
			<td >14058-09</td>
			<td >Used for splitting mouse skull</td>
		</tr>
		<tr >
			<td >Tris-HCl</td>
			<td >Sigma-Aldrich</td>
			<td >15506017</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Trypan blue stain</td>
			<td >Gibco</td>
			<td >15250061</td>
			<td >Used for cell counting</td>
		</tr>
		<tr >
			<td >Tween20</td>
			<td >ThermoFisher</td>
			<td >AAJ20605AP&#160;</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Zeiss STEMI SV 11 Apo stereomicroscope</td>
			<td >Zeiss</td>
			<td >n/a</td>
			<td >Microscope used for dissections</td>
		</tr>
	</tbody>
</table>

dissection of adult mouse stria vascularis for single-nucleus sequencing or immunostaining

הפוטנציאל האנדוקוכליארי, אשר נוצר על ידי כלי הדם stria, הוא חיוני כדי לשמור על סביבה תורמת mechanotransduction תאי שיער מתאים ובסופו של דבר שמיעה. פתולוגיות של כלי הדם stria יכול לגרום לירידה בשמיעה. דיסקציה של הסטריה וסקולריס הבוגרת מאפשרת לכידה ממוקדת של גרעין יחיד ולאחר מכן ריצוף גרעין יחיד וצביעת חיסון. טכניקות אלה משמשות לחקר פתופיזיולוגיה של סטריה וסקולריס ברמת התא היחיד. 
ריצוף גרעין יחיד יכול לשמש בהגדרה של ניתוח שעתוק של כלי הדם stria. בינתיים, צביעת מערכת החיסון ממשיכה להיות שימושית בזיהוי אוכלוסיות ספציפיות של תאים. שתי השיטות דורשות דיסקציה נכונה של stria vascularis כתנאי מוקדם, אשר יכול להוכיח להיות מאתגר מבחינה טכנית.

The cochlea consists of three fluid filled chambers, the scala vestibuli, scala media, and scala tympani. The scala vestibuli and scala tympani each contain perilymph, which has a high concentration of sodium (138 mM) and a low concentration of potassium (6.8 mM)1. The scala media contains endolymph, which has a high concentration of potassium (154 mM) and a low concentration of sodium (0.91 mM)1,2,3. This difference in ion concentration can be referred to as the endocochlear potential (EP), and is primarily generated by the movement of potassium ions through various ion channels and gap junctions in the stria vascularis (SV) along the lateral wall of the cochlea4,5,6,7,8,9,10,11. The SV is a heterogenous, highly vascularized tissue that lines the medial aspect of the lateral wall of the cochlea and contains three main cell types: marginal, intermediate, and basal cells12 (Figure 1).
Marginal cells are connected by tight junctions to form the most medial surface of the SV. The apical membrane faces the endolymph of the scala media and contributes to potassium ion transport into the endolymph using various channels, including KCNE1/KCNQ1, SLC12A2, and Na+-K+-ATPase (NKA)5,10,13,14. Intermediate cells are pigmented cells that reside between marginal and basal cells and facilitate potassium transport through the SV using KCNJ10 (Kir 4.1)15,16. Basal cells lie in close proximity to the lateral wall of the cochlea and are closely associated with fibrocytes of the spiral ligament to promote potassium recycling from the perilymph12. Pathology of the SV has been implicated in numerous otologic disorders17,18. Mutations in genes expressed in the major SV cell types, such as Kcnq1, Kcne1, Kcnj10, and Cldn11, can cause deafness and SV dysfunction, including the loss of EP19,20,21,22,23. In addition to the three major cell types, there are other less-studied cell types in the SV, such as spindle cells22, root cells12,24, macrophages25, pericytes26, and endothelial cells27, that have incompletely defined roles involving ionic homeostasis and the generation of EP28.
In comparison to bulk RNA-sequencing, single-nuclei RNA-sequencing (sNuc-Seq) provides information about cell heterogeneity, rather than an average of mRNA across a group of cells29, and can be particularly useful when studying the heterogenous SV30. For example, sNuc-Seq has produced transcriptional analysis that suggests there may be a role for spindle and root cells in EP generation, hearing loss, and Meniere&#39;s disease18. Further transcriptional characterization of the various SV cell types can provide us with invaluable information on the pathophysiology underlying different mechanisms and subtypes of SV-related hearing fluctuation and hearing loss. The harvest of these delicate inner ear structures is of paramount importance to optimal tissue analysis.
In this study, the microdissection approach to access and isolate the stria vascularis from the adult mouse cochlea for sNuc-Seq or immunostaining is described. Dissection of the adult mouse SV is required to understand various SV cell types and further characterize their role in hearing.

מחבר זרקור: דיסקציה של Stria vascularis עכבר בוגר עבור ריצוף גרעין יחיד או immunostaining  

דיסקציה של Stria vascularis עכבר בוגר עבור ריצוף גרעין יחיד או immunostaining

The stria vascularis functions to generate the endocochlear potential and regulate ion homeostasis in the cochlea. The auditory development and restoration program seeks to understand the underlying mechanisms connecting dysfunctional ion homeostasis with hearing and stability disorders, which are diseases where either fluctuation in or sudden hearing loss occurs. Work in my lab has established the transcriptional landscape of the stria vascularis and has contributed to our understanding of its importance in hearing.We have used these data to draw parallels to poorly-understood hearing and stability disorders, including Meniere's disease. This protocol enables the dissection and isolation of whole-mount stria vascularis for further molecular studies including immunohistochemistry. Given that stria vascularis cell types are intricately intertwined with one another, the use of nuclear isolation and single-nucleus RNA sequencing has facilitated our understanding of the distinct transcriptional machinery involved.

אנו מציגים שיטה לבודד את SV לשימוש עבור sNuc-Seq או immunostaining. האנטומיה הרלוונטית (איור 1) של השבלול ביחס ל-SV יכולה לעזור למשתמשים להבין טוב יותר את הארגון של SV ואת השלבים של פרוטוקול הדיסקציה.
כל שלב של מיקרו-דיסקציה זו של SV מעכבר P30 מפורט בווידיאו המשויך, ותצלומי בזק...

Watch this Scientific Journal Video about Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining at JoVE.com

הסטריה וסקולריס חיונית ליצירת הפוטנציאל האנדוקוכליארי. כאן, אנו מציגים את הדיסקציה של העכבר הבוגר stria vascularis עבור ריצוף גרעין יחיד או immunostaining.

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell ...

הסטריה וסקולריס פועלת ליצירת הפוטנציאל האנדוקוכליארי ולוויסות הומאוסטזיס היונים בשבלול. תוכנית ההתפתחות והשיקום השמיעתי מבקשת להבין את המנגנונים הבסיסיים המקשרים בין הומאוסטזיס יונים לא מתפקד לבין הפרעות שמיעה ויציבות, שהן מחלות שבהן מתרחשת תנודה או אובדן שמיעה פתאומי. העבודה במעבדה שלי ביססה את נוף השעתוק של הסטריה וסקולריס ותרמה להבנתנו את חשיבותה בשמיעה.השתמשנו בנתונים אלה כדי ליצור הקבלות להפרעות שמיעה ויציבות שאינן מובנות היטב, כולל מחלת מנייר. פרוטוקול זה מאפשר דיסקציה ובידוד של כלי דם סטריה שלמים למחקרים מולקולריים נוספים, כולל אימונוהיסטוכימיה. בהתחשב בכך שסוגי תאי סטריה וסקולריס שזורים זה בזה באופן מורכב, השימוש בבידוד גרעיני וריצוף RNA של גרעין יחיד הקל על הבנתנו את מנגנון השעתוק הייחודי המעורב.

dissection-adult-mouse-stria-vascularis-for-single-nucleus-sequencing

Research

JoVE Journal

Biology

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

1.3K Views.  National Institutes of Health. הסטריה וסקולריס פועלת ליצירת הפוטנציאל האנדוקוכליארי ולוויסות הומאוסטזיס היונים בשבלול. תוכנית ההתפתחות והשיקום השמיעתי מבקשת להבין את המנגנונים הבסיסיים המקשרים בין הומאוסטזיס יונים לא מתפקד לבין הפרעות שמיעה ויציבות, שהן מחלות שבהן מתרחשת תנודה או אובדן שמיעה פתאומי...

Video: מחבר זרקור: דיסקציה של Stria vascularis עכבר בוגר עבור ריצוף גרעין יחיד או immunostaining  

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. 
Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

Mouse Inner Ear Extraction and Stria Vascularis Dissection

To begin, identify the inner ear within the temporal bone of a euthanized mouse. Scrape away the cranial nerves using number 55 forceps. Then use number 55 forceps to dissect out the bulla and capsule, detaching them from surrounding bone.Remove any remaining soft tissue from the inner ear surface. For SV dissection, use number 55 forceps to hold the specimen by the vestibular portion with the cochlear facing up. Pierce through the cochlear bone at the apex using the same forceps.Scrape along the apical turn, applying gentle force to break away small pieces of the outer bone layer. Gently lift the bone and detach it from the lateral wall. Using number 55 forceps remove cochlear bone pieces from the apical and middle turns.Gently pushing down the lateral wall, pry and remove the bone wall pieces towards the middle turn to expose the lateral wall of the apical and middle turns. Push the apical turn lateral wall aside to expose the spiral ganglion. Detach the lateral wall of the apical and middle turns from the spiral ganglion along the outer hair cell layer.Then remove pieces of spiral ganglion from inside the cochlear. Detach the cochlear from the vestibular portion of the temporal bone by inserting the forceps into the round and oval window and pushing down towards the vestibule to have better access to the lateral wall of the basal turn. After the cochlear is detached remove the vestibular portion of the inner ear.Prioritize preserving as much of the strayer as possible. Continue removing til the bone of the middle turn is detached from the SV.Next, remove pieces of the remaining cochlear bone covering the basal turn. This can be accomplished by pushing the lateral wall layer under the bone to detach it.Pry and gently pull the tissue to remove the farthest basal part of the lateral wall being careful not to damage the soft tissue below. With the basal part of the lateral wall detached, separate the lateral wall from the remaining cochlear by tracing the forceps along the lateral wall. Gently brush the forceps between the bone and the remaining lateral wall until it reaches the apex.Then move the lateral wall to a fresh PBS. Lay the lateral wall flat revealing the SV as a darker layer of tissue on the internal side of the lateral wall. Gently pry the SV layer away from the lateral wall.Push aside the detached SV and advance the forceps along the lateral wall attempting to detach the SV as one long ribbon, trying not to squeeze the SV.Use a tissue scoop to collect the fragile tissue. Cochlear were dissected from adult mouse aged greater than or equal to postnatal day 30. Inner ear was extracted from the surrounding temporal bone.SV was exposed after the removal of bone covering the middle turn. The SV was fully separated from the lateral wall after dissection.

מיצוי אוזן פנימית של עכבר ודיסקציה של Stria Vascularis

Preparation of Stria Vascularis Single-Nucleus Suspension

Homogenize the dissected stria vascularis tissue from mouse ear in a two-milliliter Dounce homogenizer, with 10 to 20 strokes on ice. Lyse the tissue on ice for 25 minutes. Then, filter the tissue through a 30-micron filter and spin the filtrate at 500G for five minutes at four degrees Celsius.Remove the supernatant and resuspend the cell pellet in one milliliter of nuclei wash and resuspension buffer. Next, filter the cells through a 10-micron filter and centrifuge at 500G for five minutes at four degrees Celsius. Remove the supernatant and resuspend in 50 microliters of nuclei wash and resuspension buffer.After Trypan Blue staining, dilute five microliters of the cell suspension into 50 microliters of 1X PBS and count the cells. To prepare single nuclei captures, load the sample with the desired nuclear density onto the chip.

הכנת Stria Vascularis Single-Nucleus Suspension

Immunostaining of Mouse Stria Vascularis and Tissue Mounting

Prepare for immunostaining by dissecting the stria vascularis from the mouse and add the tissue to a 24-well plate containing 200 microliters of 4%paraformaldehyde in 1X PBS. Incubate the tissue for 20 minutes at room temperature. perform two short washes of 1X PBS on an orbital shaker at low RPM.Remove the PBS, then perform permeabilization and blocking for a minimum of one hour at room temperature in 300 microliters of PBST solution. Next, stain the tissue with the primary antibody overnight at four degrees Celsius. Then add a secondary antibody and incubate for two hours at room temperature on an orbital shaker at low RPM.Cover the plate to protect the fluorescently tagged secondary antibody from light. Next, prepare a larger microslide by placing two small streaks of glue along the 25 mm axis, just smaller than the 18 by 18 millimeter glass cover slip. Place one drop of mounting reagent between the glue streaks.Using number 55 forceps, gab as little tissue as possible on one end of the SV, and transfer it from PBS to the mounting reagent. Place one end of the cover slip on the slide where one streak of glue was placed. Then gently release the cover slip to avoid creating air bubbles.Seal the mount by dabbing a drop of transparent nail polish on each corner of the mount. Label the specimen on the glass cover slip, away from the visualization field. Visualize the SV tissue under a dissection microscope to ensure it is lying flat and not near any air bubbles.If the SV tissue is not oriented correctly, try to push out air bubbles or carefully remove the smaller glass cover slip and reposition the SV.A whole mount of SV from P30 mouse was prepared and confocal imaging was performed. ZsGreen expression was observed in the intermediate cells, GSIB4 labeled endothelial cells, and DAPI labeled the nuclei.