Name: Mouse Inner Ear Extraction and Stria Vascularis Dissection
Uploaded: 2023-04-21T12:50:00
Description: 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

Microdissection de stries vasculaires pour le séquençage d’un seul noyau ou l’immunocoloration

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.

Pleins feux sur l’auteur : Dissection de stria vascularis de souris adultes pour le séquençage d’un seul noyau ou l’immunomarquage  

Dissection de la strie vasculaire de souris adulte pour le séquençage mononucléaire ou l’immunomarquage

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  

Extraction de l’oreille interne de souris et dissection vasculaire de Stria

Pour commencer, identifiez l’oreille interne dans l’os temporal d’une souris euthanasiée. Grattez les nerfs crâniens à l’aide de forceps numéro 55. Utilisez ensuite la pince numéro 55 pour disséquer la bulle et la capsule, en les détachant de l’os environnant.Retirez tous les tissus mous restants de la surface de l’oreille interne. Pour la dissection SV, utilisez la pince numéro 55 pour tenir l’échantillon par la partie vestibulaire avec le cochléaire tourné vers le haut. Percez l’os cochléaire à l’apex à l’aide des mêmes pinces.Grattez le long du tour apical, en appliquant une légère force pour briser de petits morceaux de la couche osseuse externe. Soulevez doucement l’os et détachez-le de la paroi latérale. À l’aide de forceps numéro 55, retirez les morceaux d’os cochléaire des tours apical et moyen.En poussant doucement le long de la paroi latérale, soulevez et retirez les morceaux de paroi osseuse vers le virage du milieu pour exposer la paroi latérale des tours apical et moyen. Poussez la paroi latérale apicale de tour de côté pour exposer le ganglion en spirale. Détachez la paroi latérale des spires apicales et médianes du ganglion en spirale le long de la couche externe de cellules ciliées.Ensuite, retirez des morceaux de ganglion en spirale de l’intérieur du cochléaire. Détachez le cochléaire de la partie vestibulaire de l’os temporal en insérant la pince dans la fenêtre ronde et ovale et en poussant vers le bas vers le vestibule pour avoir un meilleur accès à la paroi latérale du tour basal. Une fois la cochléaire détachée, retirez la partie vestibulaire de l’oreille interne.Donnez la priorité à la préservation de la plus grande partie possible de l’égarement. Continuez à retirer jusqu’à ce que l’os du virage du milieu soit détaché du SV. Ensuite, retirez les morceaux de l’os cochléaire restant recouvrant le tour basal. Cela peut être accompli en poussant la couche de paroi latérale sous l’os pour le détacher.Tirez et tirez doucement le tissu pour enlever la partie basale la plus éloignée de la paroi latérale en prenant soin de ne pas endommager les tissus mous en dessous. Avec la partie basale de la paroi latérale détachée, séparez la paroi latérale de la cochléaire restante en traçant les pinces le long de la paroi latérale. Brossez doucement la pince entre l’os et la paroi latérale restante jusqu’à ce qu’elle atteigne l’apex.Déplacez ensuite la paroi latérale vers un nouveau PBS. Posez la paroi latérale à plat révélant le SV comme une couche de tissu plus foncée sur le côté interne de la paroi latérale. Retirez doucement la couche SV de la paroi latérale.Écartez le SV détaché et avancez les pinces le long de la paroi latérale en essayant de détacher le SV comme un long ruban, en essayant de ne pas serrer le SV. Utilisez une cuillère de tissu pour recueillir le tissu fragile. Cochléaire ont été disséqués chez des souris adultes âgées de plus ou d’un jour égal au 30e jour postnatal. L’oreille interne a été extraite de l’os temporal environnant.SV a été exposé après l’enlèvement de l’os recouvrant le virage du milieu. Le SV a été complètement séparé de la paroi latérale après dissection.

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 ...

Recherche

Biologie

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.

Préparation de la suspension mononucléaire de 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.