Name: Mouse Inner Ear Extraction and Stria Vascularis Dissection
Uploaded: 2023-04-21T12:50:00
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mouse inner ear extraction and stria vascularis dissection

Microdissezione della stria vascolare per il sequenziamento di un singolo nucleo o l'immunocolorazione

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.

Autore in primo piano: Dissezione di stria vascolare di topo adulto per il sequenziamento di un singolo nucleo o l'immunocolorazione  

Dissezione della stria vascolare del topo adulto per sequenziamento di singoli nuclei o immunocolorazione

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.

Watch this Scientific Journal Video about Mouse Inner Ear Extraction and Stria Vascularis Dissection at JoVE.com

Mouse Inner Ear Extraction and Stria Vascularis Dissection  

Estrazione dell'orecchio interno del topo e dissezione della stria vascolare

Per iniziare, identifica l'orecchio interno all'interno dell'osso temporale di un topo eutanasia. Raschia via i nervi cranici usando il forcipe numero 55. Quindi utilizzare la pinza numero 55 per sezionare la bulla e la capsula, staccandole dall'osso circostante.Rimuovere qualsiasi tessuto molle residuo dalla superficie dell'orecchio interno. Per la dissezione SV, utilizzare una pinza numero 55 per tenere il campione dalla porzione vestibolare con la cocleare rivolta verso l'alto. Forare l'osso cocleare all'apice usando la stessa pinza.Raschiare lungo la curva apicale, applicando una forza delicata per rompere piccoli pezzi dello strato osseo esterno. Sollevare delicatamente l'osso e staccarlo dalla parete laterale. Utilizzando la pinza numero 55 rimuovere pezzi di osso cocleare dalle curve apicali e centrali.Spingendo delicatamente verso il basso la parete laterale, fare leva e rimuovere i pezzi della parete ossea verso la curva centrale per esporre la parete laterale delle curve apicale e centrale. Spingere la parete laterale di rotazione apicale da parte per esporre il ganglio a spirale. Staccare la parete laterale delle curve apicali e centrali dal ganglio a spirale lungo lo strato esterno delle cellule ciliate.Quindi rimuovere pezzi di ganglio a spirale dall'interno della coclea. Staccare la cocleare dalla porzione vestibolare dell'osso temporale inserendo la pinza nella finestra rotonda e ovale e spingendo verso il basso verso il vestibolo per avere un migliore accesso alla parete laterale della svolta basale. Dopo che la cocleare è stata staccata, rimuovere la porzione vestibolare dell'orecchio interno.Dare priorità alla conservazione del maggior numero possibile di vagabondi. Continuare a rimuovere fino a quando l'osso del giro centrale non viene staccato dall'SV. Quindi, rimuovere i pezzi dell'osso cocleare rimanente che coprono la svolta basale. Questo può essere ottenuto spingendo lo strato di parete laterale sotto l'osso per staccarlo.Fare leva e tirare delicatamente il tessuto per rimuovere la parte basale più lontana della parete laterale facendo attenzione a non danneggiare il tessuto molle sottostante. Con la parte basale della parete laterale staccata, separare la parete laterale dalla restante cocleare tracciando la pinza lungo la parete laterale. Spazzolare delicatamente la pinza tra l'osso e la parete laterale rimanente fino a raggiungere l'apice.Quindi spostare la parete laterale su un PBS fresco. Posare la parete laterale piatta rivelando la SV come uno strato più scuro di tessuto sul lato interno della parete laterale. Sollevare delicatamente lo strato SV lontano dalla parete laterale.Spingere da parte l'SV staccato e far avanzare la pinza lungo la parete laterale tentando di staccare la SV come un lungo nastro, cercando di non schiacciare la SV. Utilizzare uno smisurino di tessuto per raccogliere il tessuto fragile. I cocleari sono stati sezionati da topi adulti di età maggiore o uguale al giorno 30 postnatale. L'orecchio interno è stato estratto dall'osso temporale circostante.SV è stato esposto dopo la rimozione dell'osso che copre la curva centrale. L'SV è stato completamente separato dalla parete laterale dopo la dissezione.

mouse-inner-ear-extraction-and-stria-vascularis-dissection

Research

JoVE Journal

Biology

Mouse Inner Ear Extraction and Stria Vascularis Dissection

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

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

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Biologia

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.

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.

Preparazione della sospensione mononucleare della Stria vascularis

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.