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

用于单核测序或免疫染色的血管纹显微切割

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.

作者聚焦： 用于单核测序或免疫染色的成年小鼠血管纹解剖  

解剖成年小鼠血管纹以进行单核测序或免疫染色

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  

小鼠内耳提取和血管纹夹除术

首先，识别安乐死小鼠颞骨内的内耳。用55号镊子刮掉颅神经。然后用55号镊子解剖大疱和囊，将它们与周围的骨头分离。从内耳表面去除任何残留的软组织。对于 SV 夹层，使用 55 号镊子将标本固定在前庭部分，耳蜗朝上。使用相同的镊子刺穿顶端的耳蜗骨。沿着顶端转弯刮擦，轻轻用力撕开外骨层的小块。轻轻提起骨头并将其从侧壁上分离。使用55号镊子从顶端和中间转弯处去除耳蜗骨片。轻轻向下推侧壁，向中间转弯撬开并取出骨壁碎片，露出顶端和中转的侧壁。将顶端转侧壁推到一边，露出螺旋神经节。从顶端的侧壁和中间转弯沿外毛细胞层从螺旋神经节分离。然后从耳蜗内取出螺旋神经节碎片。通过将镊子插入圆形和椭圆形窗口并向前庭向下推，将耳蜗与颞骨的前庭部分分离，以便更好地进入基底转弯的侧壁。耳蜗分离后，切除内耳的前庭部分。优先保护尽可能多的流浪者。继续移除，直到中间转弯的骨头与 SV 分离。接下来，去除覆盖基底转弯的剩余耳蜗骨碎片。这可以通过将侧壁层推到骨头下方以将其分离来实现。撬开并轻轻拉动组织以去除侧壁最远的基底部分，注意不要损坏下面的软组织。分离侧壁的基部后，通过沿侧壁追踪镊子将侧壁与剩余耳蜗分开。轻轻刷骨和剩余侧壁之间的镊子，直到它到达顶点。然后将侧壁移动到新的PBS。将侧壁平放，露出侧壁内侧的深色组织层。轻轻地将 SV 层从侧壁上撬开。推开分离的SV，沿侧壁推进镊子，试图将SV分离为一条长带，尽量不要挤压SV。使用纸巾勺收集脆弱的组织。从年龄大于或等于出生后第30天的成年小鼠身上解剖耳蜗。从周围的颞骨中提取内耳。SV在去除覆盖中间转弯的骨头后暴露出来。解剖后SV与侧壁完全分离。

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 ...

科研

生物学

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.

血管纹单核悬浮液的制备

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.